Sub-NIAID Tech Call
1 December 2009
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1

#2
#8
#9
#10
#11
#12/13
#21
#29
Active
Active
Active
Active
Active
Active
Active
Active
Vaccinations of study personnel
LVS vaccination protection of aerosol Schu4 confirmed in primates
Aerosol SOP developed for GLP transition
Efficacy testing of vaccine candidates
(LBERI)- no work in November
In Vivo GLP NHP model efficacy SOPs and efficacy testing of vaccine candidates
Assays for detecting relevant immune responses in animals and humans
Correlates of protection- in vitro assay or other readout of effector function of Ft developed for multiple species
Analysis of T cells from lymph nodes and T cell epitopes- no work in November
Slide 2

UNM/LBERI Staff are vaccinated with current Tularemia vaccine at USAMRIID
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 3


4 people from Lovelace received the current Tularemia vaccination from USAMRIID.
Slide 4


Five more people are scheduled to receive the vaccination in
January 2010.
Slide 5

L VS Vaccinated NHP Challenged with SCHU S4
Round 1 Vaccination Practice/Challenge (n=3 scarification; n=2 subcutaneous)
Round 2 Vaccination/Challenge (n=3 by scarification; n=3 by subcutaneous route; n=4 previously vaccinated; 2 SC, 2 ID)
SCHU S4 Challenge 500 CFU
Round 3 Vaccination/Challenge (Vaccination with Highest Dose of LVS attainable by scarification and s.c.)
SCHU S4 Challenge 1000 CFU
Round 4 Vaccination/Challenge (Vaccination with Lot 16 n=3; Lot 17 n=3;
Lot 20 n=8; Lot 4 n=8)
SCHU S4 Challenge 1000 CFU
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 6


Describe the natural history of aerosol delivered SCHU S4 infection in
NHPs that have been previously vaccinated with LVS
– Compare two different methods of vaccination (scarification and subcutaneous)
– Compare 4 different LVS lots as vaccines (all delivered by subcutaneous route)

Endpoints
– histopathology
– bacterial CFUs of internal organs (lung, spleen, liver, kidneys, and lymph nodes)
– records of clinical symptoms post-infection
– clinical chemistry and hematology during infection
Slide 7


Animals were challenged with ~ 1000 LD50 SCHU S4 by aerosol

Moribund animals were necropsied and tissues plated for bacteriology and preserved for histology.

Non-moribund animals continue to be observed daily.

Note: aerosol and microbiology data are in the process of being QC’d, thus the numbers presented here are tentative until QC is complete.

IgG anti-LVS plasma antibody levels were measured

Attempts were made to correlate LVS-induced immune response to protection from SCHU S4 aerosol-induced mortality
Slide 8

Animal Group
ID
Control
Lot 16*
Lot 17a**
Lot 17b***
Lot 20**
Lot 4*
# of NHPs
3
3
8
8
2
1
Dose Volume
(µL)
120
120
113
113
132
120
Average CFU
Administered
NA
7.09E+05
8.96E+07
3.21E+07
8.54E+06
1.09E+09
* - avg of pre- and post-vaccination conical concentrations used for calculations
** - post-vaccination concentration used for calculations
*** - avg of 2 readable counts for post-vaccination concentrations used for calculations
Slide 9

LVS Lot Number # NHP in group # NHP dead/euth Percent Mortality
Controls
16
17
20
4
3
3
3
8
8
3
1
1
5
2
100
33.3
33.3
62.5
25
Average presented dose across all NHP was 1471 CFU.
Minimum presented dose was 119 CFU and maximum presented dose was 3766. Group averages are below.
Group/Vaccine Lot
Control
Lot 16
Lot 17
Lot 20
Lot 4
Average Presented
Dose
1242
2026
1515
1353
1452
Slide 10

MS #8 – Survival Time by Group and Animal and Presented Dose
LVS lot and Animal
Number
Control NHP
A07624
30015
A06872
Lot 16
30003
29975
Presented dose (CFU)
1268
1823
636
Date of Exposure
11-Nov-09
12-Nov-09
13-Nov-09
29992
2872
1667
1540
11-Nov-09
12-Nov-09
13-Nov-09
Lot 17
A07872
A07754
A07699
1860
1897
787
11-Nov-09
12-Nov-09
13-Nov-09
Lot 20
30048
29976
A06488
A07683
29996
29991
30014
28461
1479
2510
2250
828
2148
304
728
575
11-Nov-09
11-Nov-09
11-Nov-09
12-Nov-09
12-Nov-09
13-Nov-09
13-Nov-09
13-Nov-09
Lot 4
30017
A07756
30019
29987
30039
A07720
A07842
1257
1669
1785
1511
3766
119
1090
11-Nov-09
11-Nov-09
12-Nov-09
12-Nov-09
12-Nov-09
13-Nov-09
13-Nov-09
A07738 416 13-Nov-09
Note: survival times were rounded to the nearest hour
Time survived postexposure's end (hrs)
~ 110 to 117.5
108
109
229
432+ (alive)
408+ (alive)
171
432+ (alive)
408+ (alive)
197
456+ (alive)
456+ (alive)
432+ (alive)
288
151
245
171
456+ (alive)
456+ (alive)
171
432+ (alive)
432+ (alive)
408+ (alive)
408+ (alive)
127 Slide 11

LVS lot and Animal
Number
Control NHP
A07624
30015
A06872
Lot 16
30003
29975
29992
Lot 17
A07872
A07754
A07699
Lot 20
30048
29976
A06488
A07683
29996
29991
30014
28461
Lot 4
30017
A07756
30019
29987
30039
A07720
A07842
A07738
Qualitative
Presented dose (CFU) Study Day of Death
Bacteremia
1268
1823
636
2872
1667
1540
1860
1897
787
1257
1669
1785
1511
3766
119
1090
416
1479
2510
2250
828
2148
304
728
575
Found dead am day 5
4
4
9
NA
NA
7
NA
NA
NA
NA
7
NA
NA
NA
NA
5
8
NA
NA
NA
12
6
10
7
Yes, day 4
Yes, day 4
Yes, day 4
No as of day 9
No as of day 14
No as of day 14
Yes, day 7
No as of day 14
No as of day 14
No as of day 8
No as of day 14
No as of day 14
No as of day 14
No as of day 12
Yes, day 4
Yes, day 10
No as of day 7
No as of day 14
Yes, day 6
No as of day 7
No as of day 14
No as of day 14
No as of day 14
Yes, as of day 4, 14
No as of day 5
Note: A07842 is still clinically ill. Term sac is scheduled for the end of this week. How do we wish to proceed? Term sac must occur by Dec. 8 due to B3 shutdown.
Slide 12

LVS lot and
Animal
Number
Presented dose (CFU)
Study Day of Death
Lung TBLN Spleen Liver
Mesenteric
LN
Control NHP
A07624 1268 Found day 5 3.01E+08 3.94E+06 3.11E+06 9.03E+05 3.20E+05
Lot 16
30015 1823
A06872 636
4
4
5.24E+08 4.72E+08 9.17E+07 6.29E+05
1.75E+09 1.38E+09 2.68E+07 5.36E+05
1.25E+04
3.88E+05
30003 2872
29975 1667
29992 1540
NA
NA
2.19E+08 1.72E+06 2.81E+06 2.35E+05 1.45E+07
Lot 17
A07872 1860
A07754 1897
A07699 787
Lot 20
NA
NA
1.97E+08 5.31E+04 9.64E+03 4.57E+03 BLD
1.43E+08 2.75E+04 9.21E+03 6.30E+03 BLD 30048 1479
29976 2510
A06488 2250
A07683 828
29996 2148
29991 304
30014 728
28461 575
NA
NA
NA
5.10E+07 3.37E+05
1.78E+07 BLD
BLD BLD
1.31E+02 5.49E+01
3.66E+08 1.58E+03 2.60E+05 9.03E+04
BLD
BLD
BLD
5.62E+08 4.74E+06 2.04E+05 1.26E+04 4.56E+02
Lot 4
30017 1257
A07756 1669
30019 1785
29987 1511
30039 3766
A07720 119
A07842 1090
A07738 416
NA
NA
NA
NA
NA
NA
3.45E+08 1.90E+05 2.32E+03 6.61E+02
2.89E+07 BLD 4.96E+02 BLD
4.63E+02
BLD
Slide 13

IFN γ Production by PBMCs isolated from NHPs Vaccinated with DVC Lot
16 LVS
700
600
500
400
300
200
100
0
Media
LVS hk Mid
LVS ff Hi
1667 CFU, alive
1540 CFU, alive
2872 CFU, Day 10
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with LVS antigens (heat-killed (hk) or formalin-fixed (ff)) at a dose of 0.25 x 10 5 /ml (Mid) or 1 x
10 5 /ml (Hi)
- Wells that are TNTC are entered into the database as having 600 spots
SCHU S4 challenge presented dose and day of death shown above data
Slide 14

IFN γ Production by PBMCs isolated from NHPs Vaccinated with DVC Lot
17 LVS
700
600
500
400
300
200
100
0
Media
LVS hk Mid
LVS ff Hi
2179 CFU, alive
787 CFU, alive
1860 CFU, Day 8
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with LVS antigens
(heat-killed (hk) or formalin-fixed (ff)) at a dose of 0.25 x 10 5 /ml (Mid) or 1 x 10 5 /ml (Hi)
- Wells that are TNTC are entered into the database as having 600 spots (high s.d. of
A07754 on day 7 is due to one well entered at 600)
SCHU S4 challenge presented dose and day of death shown above data
Slide 15

IFN γ Production by PBMCs isolated from NHPs Vaccinated with DVC Lot
20 LVS
700
600
Media
LVS hk Mid
LVS ff Hi
2510 CFU,
Alive
2148 CFU,
Day 12
500
400
300
200
100
571
CFU,
Day 8
304
CFU,
Day 7
728
CFU,
Day 11
2250 CFU,
Alive
1479 CFU,
Day 9
828
CFU,
Alive
0
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with LVS antigens
(heat-killed (hk) or formalin-fixed (ff)) at a dose of 0.25 x 10 5 /ml (Mid) or 1 x 10 5 /ml (Hi)
- Wells that are TNTC are entered into the database as having 600 spots (high s.d. of
29996 with LVS ff hi on day 7 is due to one well entered at 600)
SCHU S4 challenge presented dose and day of death shown above data
Slide 16

IFN γ Production by PBMCs isolated from NHPs Vaccinated with
USAMMDA Lot 4 LVS
1669 CFU,
Alive
1090 CFU,
Alive 700
600
500
400
300
200
100
0
Media
LVS hk Mid
LVS ff Hi
1511
CFU,
Alive
1257 CFU,
Alive 1785
CFU,
Day 8
Alive
119
CFU,
416 CFU,
Day 6
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with LVS antigens (heat-killed (hk) or formalin-fixed (ff)) at a dose of 0.25 x 10 5 /ml (Mid) or 1 x
10 5 /ml (Hi)
- Wells that are TNTC are entered into the database as having 600 spots
SCHU S4 challenge presented dose and day of death shown above data
Slide 17

100000
10000
1000
100
10
1
Arbitrary Units
Day 0 Day 7
DVC Lot 16
DVC Lot 17
DVC Lot 20
USAMMDA Lot 4
Day 21
1000000
Titer
100000
10000
1000
Day 0 Day 7
DVC Lot 16
DVC Lot 17
DVC Lot 20
USAMMDA Lot 4
Day 21
Slide 18

4000
3500
3000
2500
2000
1500
1000
500
Lot 16
Lot 17
Lot 20
Lot 4
None
0
100 150 200 250 300 350 400 450 500
Hours to Death
Three non-vaccinated controls were the first to succumb (Day 5)
33% mortality in NHPs vaccinated with Lot 16 or 17
62.5% mortality in NHPs vaccinated with Lot 20
25% mortality in NHPs vaccinated with USAAMDA Lot 4
No apparent relationship of low SCHU S4 challenge dose with extended time to death
Slide 19

40000
35000
30000
25000
20000
15000
10000
5000
0
-5000
100 150 200 250 300 350 400 450 500
Hours to Death
Lot 16
Lot 17
Lot 20
Lot 4
None
No apparent relationship of high IgG anti-LVS plasma levels and extended time to death (except possibly with
Lot 4)
Slide 20

Lot 16
Lot 17
Lot 20
Lot 4
None
180
160
140
70
120
100
50
80
30
60
40
10
20
0
-20
100 150 200 250 300 350 400 450 500
Hours to Death
-10
100 150 200 250 300 350 400 450 500
Hours to Death
No apparent relationship of responsiveness to LVS ex vivo by production of IFN γ and extended time to death
Slide 21


Terminal sacrifices will be performed

Data will begin to be analyzed

Tissues will begin to be trimmed
Slide 22

MS #9: Aerosol SOP Development
Develop Qualification Plan for Standard Growth Curve
Perform Standard Growth Curve Qualification
Develop Qualification Plan for Aerosol
Perform Aerosol Qualification
Prepare Aerosol SOP
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 23


Develop a SOP compatible with GLP transition for aerosol delivery of
Schu S4.
Slide 24


Continued optimization of generator suspension preparation procedures for animal challenges. Two approaches were tested:
– Prepare a normalized OD suspension from a 24h
Chamberlains broth culture and assume a starting concentration based on repetitive spread plate colony count data
– Mechanically measure the starting concentration of a 24h
Chamberlains broth culture through the use of flow cytometry and compare to viable culture (i.e., spread plate colony counts)

Data (see following 2 slides) demonstrated similar results for both approaches
Slide 25


24h Chamberlains cultures were normalized to OD600 of 0.500; the allowed error was ± 0.05 for 9, 23, and 30OCT09 and ± 0.01 for
4NOV09
Date
9-Oct-09
23-Oct-09
30-Oct-09
4-Nov-09
Flask Tech ID
2
3
1
1
1
2
3
1
1
1
1
3
1
1
2
1
1
1
2
3
5
5
1
4
5
6
5
5
5
5
Time h
24
24
24
24
Norm OD600
0.547
0.549
0.549
0.558
0.556
0.554
0.497
0.494
0.498
0.493
0.497
0.497
0.493
0.494
0.496
Mean = 2.72 x 10 8 CFU/mL
Max = 5.77 x 10 8 CFU/mL
Min = 5.33 x 10 7 CFU/mL
95
46
176
221
165
36
42
18
1
34
67
42
119
5
40
10
50
64
189
213
200
32
47
37
1
56
17
3
45
51
47
75
81
110
184
206
177
44
39
43
Plate
2
38
55
45
78
10
53
23
Mean Dil Factor Mean CFU/mL
39.0
1.00E+07
57.7
1.00E+07
44.7
1.00E+07
90.7
1.00E+06
75.3
1.00E+06
73.3
1.00E+06
183.0
1.00E+06
213.3
1.00E+06
180.7
1.00E+06
37.3
1.00E+07
42.7
1.00E+07
32.7
1.00E+07
5.3
1.00E+07
49.7
1.00E+07
16.7
1.00E+07
3.90E+08
5.77E+08
4.47E+08
9.07E+07
7.53E+07
7.33E+07
1.83E+08
2.13E+08
1.81E+08
3.73E+08
4.27E+08
3.27E+08
5.33E+07
4.97E+08
1.67E+08 log10
8.59
8.76
8.65
7.96
7.88
7.87
8.26
8.33
8.26
8.57
8.63
8.51
7.73
8.70
8.22
Slide 26


With the exception of a few data points, viable culture data demonstrated that normalized suspensions (OD600 = 0.500) contained approximately 1-5 x 10 8 CFU/mL
– The decision was made to standardize this value to 2.0 x
10 8 CFU/mL for NHP challenges

Culture concentrations are heavily dependant on technician variability
– This will likely never change

This approach was taken on the 11-13NOV09 NHP exposures
– 11NOV09 (Tech 5): 0.494 = 8.90 x 10 7 CFU/mL

Dilution error?
–
12NOV09 (Tech 3): 0.493 = 2.58 x 10 8 CFU/mL
– 13NOV09 (Tech 5): 0.495 = 2.23 x 10 8 CFU/mL
Slide 27


Practice bioaerosols were conducted on 4NOV09
–
The 6 technicians (previous slide) individually prepared his/her own generator suspension and diluted/cultured all samples for his/her respective bioaerosol run

Target aerosol concentration (based on impinger cultures) was
286 CFU/L
– 286 CFU/L x 3.5 L inhaled = 1,000 CFU presented
Slide 28

Date
4-Nov-09
1
4
5
2
3
6
Tech
Norm
OD600
CFU/mL
Target Pre-Actual Post-Actual
Target
CFU/L
Actual
CFU/L
Target Mock Actual Mock
Dose (CFU) Dose (CFU)
Spray
Factor
0.493
0.497
0.497
0.493
0.494
0.496
2.00E+06 1.05E+07 5.43E+06 2.86E+02 1.43E+02 1.00E+03 4.99E+02 2.63E-08
2.00E+06 9.63E+06 4.93E+06 2.86E+02 2.86E+02 1.00E+03 1.00E+03 5.80E-08
2.00E+06 8.97E+06 6.63E+06 2.86E+02 3.76E+02 1.00E+03 1.32E+03 5.67E-08
2.00E+06 3.80E+06 1.49E+06 2.86E+02 9.25E+01 1.00E+03 3.24E+02 6.22E-08
2.00E+06 9.47E+06 7.17E+06 2.86E+02 3.26E+02 1.00E+03 1.14E+03 4.55E-08
2.00E+06 5.67E+06 4.87E+06 2.86E+02 1.64E+02 1.00E+03 5.76E+02 3.38E-08
Slide 29


Practice bioaerosol data demonstrated a similar variability between technicians as observed with the suspension concentrations

All mock doses were close to the target of 1,000 CFU and ranged from 324 to 1,320 CFU
– These are all acceptable and in the pulmonary disease range

This approach was taken on the 11-13NOV09 NHP exposures and presented doses were near the target of 1,000 CFU
– At least one animal from each day demonstrated a presented dose of almost exactly 1,000 CFU
– Deviations from the target dose were due to animal variability which cannot be controlled
Slide 30


24h Chamberlains cultures were treated with BacLight bacterial live/dead stain (Molecular Probes) according to manufacturer’s instructions and processed via culture and flow cytometry
Objective
To correlate counts between viable plating and output from the Partec cytometer
Sample Log dilution
0
Plating CFU/mL Mean
6.62E+08 8.51E+08 8.93E+08 8.02E+08 log10
8.90
Partec Viable CFU/mL Mean
3.57E+08 3.48E+08 3.68E+08 3.58E+08 log10
8.55
7.04E+07 7.88E+07 7.14E+07 7.35E+07 7.87
8.12E+07 8.42E+07 9.58E+07 8.71E+07 7.94
Flask 1
1
2 7.35E+06 6.72E+06 7.98E+06 7.35E+06 6.87
1.08E+07 1.15E+07 1.05E+07 1.09E+07 7.04
3 8.93E+05 1.01E+06 7.77E+05 8.93E+05 5.95
n/a 1.25E+06 1.14E+06 1.20E+06 6.08
% log diff
3.94%
-0.93%
-2.51%
-2.12%
Flask 1
To determine the bacterial concentration from 3 different flask cultures
Flask 2
Flask 3
0
0
0
6.09E+07
3.78E+08
1.13E+09 n/a n/a n/a n/a n/a n/a
6.09E+07 7.78
1.14E+09 1.21E+09 1.31E+09 1.22E+09 9.09
3.78E+08 8.58
5.17E+08 5.80E+08 5.83E+08 5.60E+08 8.75
1.13E+09 9.05
1.77E+09 1.91E+09 1.80E+09 1.83E+09 9.26
-16.72%
-1.99%
-2.30%
Flask 1
To determine if the Partec-indicated concentrations can be used to dilute accurately to 500 CFU/mL (as determined via plate counts)
Flask 2
Flask 3
0
0
0
3.47E+02 3.89E+02 n/a 3.68E+02 2.57
1.05E+02 1.05E+02 1.58E+02 1.23E+02 2.09
3.68E+02 3.26E+02 3.68E+02 3.54E+02 2.55
Slide 31


Results were similar for both approaches

Inherent and technician errors will never be able to be removed completely; with the low target presented dose of 1,000 CFU, minor errors and fluctuations will continue to significantly impact final data
Slide 32


Finalize the generator suspension concentration estimation approach

Update Qualification Plan to reflect changes
Slide 33

GLP Model Efficacy SOPs Developed in NHPs and Efficacy
Testing of Vaccine Candidates
Non-Telemetered Natural History
SCHU S4 Challenge 1000 CFU
Telemetered Natural History Study
Red: completed
Green: in progress
Blue: steps in the milestone
SCHU S4
Challenge
1000 CFU
Slide 34


Describe the natural history of aerosol delivered Schu S4 in the cynomolgus macaque.

Endpoints
Histopathology
Bacterial CFUs
Clinical symptoms
Clinical Chemistry
Hematology

Deliverables will include protocol(s) and documents necessary for an aerosol primate model of Schu S4 compatible with Good
Laboratory Practice (GLP) that will meet FDA standards for product development.
Slide 35


Telemetry data has been analyzed
Slide 36


Full data for every animal will be included in the draft final report; essentially fevers were seen with telemetry as opposed to temperature chips in previous experiments whereas respiration rates did not climb as high as we observed in both this and previous studies (manually determined). This may have been due to telemeter wire placement (on diaphragm and not in pleural cavity).
Slide 37


Draft report will be compiled
Slide 38

Immunoassay Development and Comparisons in Animal Models
Choose PBMC
Purification Method
Method chosen:
Purdue ListServ
Red: completed
Green: In progress
Yellow: on hold; restart if necessary
Blue: steps in the milestone
Choose PBMC
Freezing Method
Develop
Immunoassay methodologies
Cerus
IFN g
ELISPOT
Plasma
IgG
ELISA Proliferation assay
Microagglutination assay
Determine protein:CFU relationship in
FF and HK LVS antigens
Plasma
IgA
ELISA
Slide 39


LVS lot No. 9 (USAMRIID), the antigen used for the microagglutination assay, was stained with hematoxylin according to the SOP provided by USAMRIID. The stained antigen will be transferred from UNM to LBERI and used in the assay.

New lots of HK and FF LVS were tested for their ability to bind IgG anti-LVS antibodies in NHP plasma
Slide 40

Comparison of new lots of HK LVS using the ELISA and a pooled positive control plasma
Heat killed LVS 5.5x10^8/ml Optimization
1,400
1,200
1,000
0,800
0,600
0,400
0,200
0,000
10 5 2,5 1,25 0,63 0,31 0,16 0,08 0,04 0,02
Ag Concentration (x106/ml)
1/1000
1/5000
1/25000
1/125000
Comparison of original HK LVS used for ELISA:
Coated plates at 2.5 x 10 6 /ml
Positive control sera:
1/800 = 0.877 OD
405
1/1600 = 0.542
1/3200 = 0.336
closely
1/6400 – 0.213
1/12800 = 0.144
1/25600 = 0.106
Probably most approximates new
HK LVS 5.2 x 10 8 /ml
Slide 41

Comparison of new lots of FF LVS using the ELISA and a pooled positive control plasma
Formalin fixed LVS 5.5 x10^8/ml Optimization
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
10 5 2,5 1,25 0,63 0,31 0,16 0,08 0,04 0,02
Ag Concentration (x106/ml)
1/1000
1/5000
1/25000
1/125000
In the original comparison of HK and
FF LVS, the FF LVS also did not work well
Slide 42


Set up the microagglutination assay using control positive NHP plasma (containing high levels of IgG anti-LVS by ELISA)

Continue to explore ways of comparing the new HK and FF LVS lots (both from UNM and from DVC) that may help bridge to existing lots that are currently in use in the immunoassays
– Repeat ELISA with DVC lots
– Prepare protein gels
Slide 43

Establish assays of effector function that detect correlates of protection
Establish conditions to detect intracellular cytokines in NHP PBMCs
Establish conditions to detect growth of LVS in
NHP PBMCs
Confirm response in
LVS-vaccinated
NHPs
Confirm low response in non- LVSvaccinated
NHPs
Confirm reduced growth in LVSvaccinated
NHPs
Confirm growth in non- LVSvaccinated
NHPs
Slide 44


No experiments performed on the flow cytometry of vaccinated
NHP’s during November.

New antibodies purchased for identification of NK-cells and blocking of Fc-receptors.
Slide 45


Test 20 h of re-stimulation for IFNg intracellular staining of T cells (CD3+CD4+ and CD3+CD8+) and NK cells (CD3-,CD16+,
CD8+/-). The 20 h time point is similar to the ELIspot restimulation and PBMC from an NHP with a good ELISpot IFNg response will be chosen for this experiment.

The F.t LVS infection and growth assay will be tested for NHP
PBMC (vaccinated versus naïve)
– This assay is similar to that established at UNM with human PBMCs (Andrew Hahn)
Slide 46


Trevor will relay the latest date to euthanize NHP A07842 (lot 4 vaccinated and ill NHP), to Michelle by COB on 12/1/09. (completed)

Michelle will do the IACUC amendments to extend the observations on NHP
A07842 as long as possible, prior to the 12/8/09 BS3 closure. (completed)

UNM TVDC should compare LVS vaccinated/NHP challenges with SCHU S4 grown in Chamberlains vs. grown in Mueller Hinton broth.

Julie Wilder will move the legends out of the middle of the graphs on slides 19,
20 and 21 (completed).

Julie will graph the responsiveness to LVS antigens, by peak time for each
NHP. Perhaps those that peak early have a better protection?

LBERI has frozen cells from most of the vaccinated NHP on MS8C study and could do the multifunctional T cell assay, once the Multifunctional T cell assay is more reproducible

Trevor will add more OD, spray, presented dose data from the Nov 2009 test, to these finalized minutes of the 12/1/09 technical meeting, which is the monthly report. (completed)

Barbara/Juliette are writing a COA to give to NIAID for the purchase of the
Partec (submitted to NIAID 12/409)
Slide 47


On MS 11 Natural History study, Michelle will estimate the time to the final report and finish part A . The veterinary pathologists need the slides for part B.
Michelle will find the part B slides for MS 11.

Barbara will arrange a call for Julie Wilder, Freyja Lynn and Barbara Griffith for a separate call on the LVS antigen bridging data (completed)

UNM will write up a protocol for a growth media comparison for SCHU S4 challenges (Chamberlains vs. Mueller Hinton) USAMMDA may have no lot 4 remaining. Other LVS lots would be used for vaccination.

Ed, Freyja, Patrick: email Barbara and Rick suggestions for the growth media comparison study.

Barbara email Trevor and request that he start optimizing growth and sprays of
SCHU S4 in Mueller Hinton. (email sent)

Barbara email Freyja and Patrick to request LVS lot 17 vials. (awaiting vial number from LBERI 12/8/09)
Slide 48