LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
3 November 2009
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
Active Milestones
#2
Active
Vaccinations of study personnel
#8
Active
LVS vaccination protection of aerosol Schu4
confirmed in primates
#9
Active
Aerosol SOP developed for GLP transition
#10
Active
Efficacy testing of vaccine candidates
(LBERI)- no work in October
#11
Active
In Vivo GLP NHP model efficacy SOPs and
efficacy testing of vaccine candidates
#12/13
Active
Assays for detecting relevant immune
responses in animals and humans
#21
Active
Correlates of protection- in vitro assay or
other readout of effector function of Ft
developed for multiple species
#29
Active
Analysis of T cells from lymph nodes and T
cell epitopes- no work in October
Slide 2
MS #2- Vaccinations of Relevant Personnel
UNM/LBERI Staff are vaccinated with current Tularemia
vaccine at USAMRIID
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 3
Milestone #2- October 2009 Accomplishments

4 people from Lovelace have been medically cleared to receive
the current Tularemia vaccination from USAMRIID.
Slide 4
Milestone #2- Plans for next month


On November 18th, 4 people from Lovelace will receive the
Tularemia vaccine at USAMRIID. The vaccination sites will be
reviewed on November 19th and 20th at USAMRIID.
Five more people are scheduled to receive the vaccination in
January 2010.
Slide 5
MS#8 – LVS Vaccinated NHP Challenged with
SCHU S4
LVS 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.)
Red: completed
Green: in progress
Blue: steps in the milestone
SCHU S4
Challenge
1000 CFU
Slide 6
Milestone #8 - Objective and Endpoints

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
Milestone #8 – October 2009 Accomplishments



All 26 cynomolgus macaques were screened for responsiveness to LVS
and SCHU S4 antigens in vitro by IFNγ ELISPOT and proliferation
assays prior to their vaccination with LVS
All 26 NHPs were vaccinated with one of four lots of LVS on 10/7/09
25/26 NHPs were tested on day 7 (actual days 6 – 8), day 14 (days 13 –
15) and day 21 (days 20 – 22) post-LVS vaccination for responsiveness
to LVS and SCHU S4 antigens in vitro by IFNγ ELISPOT and
proliferation
–

1 NHP (A06851) which was designated as “failing to thrive” was
vaccinated with DVC Lot 16 LVS and is destined for terminal
euthanasia on 11/9/09 in order to collect potential positive
control PBMC and spleen cells – the responsiveness of this
animal was tested only prior to LVS vaccination and on day 19
post-LVS vaccination
Plasma was collected from each NHP at each PBMC preparation (Days 0
– 21)
Slide 8
Vaccine Preparation Culture Data: Lot 4
FY09-126 TUL08C Vaccination Plate Counts from 07OCT09
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
Volume Plated
mL
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Lot 4 Conical, pre
Lot 4 Conical, pre
Lot 4 Conical, pre
Lot 4 Conical, pre
Lot 4 Conical, pre
Lot 4 Conical, pre
Lot 4 Conical, pre
Lot 4 Conical, pre
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Lot 4 Conical, post
Lot 4 Conical, post
Lot 4 Conical, post
Lot 4 Conical, post
Lot 4 Conical, post
Lot 4 Conical, post
Lot 4 Conical, post
Lot 4 Conical, post
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Sample ID
Dilution
Factor
Lot 4 Vial
Lot 4 Vial
Lot 4 Vial
Lot 4 Vial
Lot 4 Vial
Lot 4 Vial
Lot 4 Vial
Lot 4 Vial
Lot 4 Vial
Plate 1
>30
>300
>300
>300
>300
>300
148
9
3
Plate Count
Plate 2
>30
>300
>300
>300
>300
>300
173
24
2
Plate 3
>30
>300
>300
>300
>300
>300
152
16
0
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.1
>30
>300
>300
>300
>300
>300
>300
86
>30
>300
>300
>300
>300
>300
>300
120
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.1
>30
>300
>300
>300
>300
>300
>300
83
>30
>300
>300
>300
>300
>300
>300
69
Mean
Mean CFU/mL
>30
>300
>300
>300
>300
>300
158
16
2
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
1.58E+09
1.63E+09
1.67E+09
>30
>300
>300
>300
>300
>300
>300
104
>30
>300
>300
>300
>300
>300
>300
103
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
1.03E+10*
>30
>300
>300
>300
>300
>300
>300
82
>30
>300
>300
>300
>300
>300
>300
78
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
7.80E+09*
* - it is likely, because of the specific instructions by the manufacturer not to vortex the vial, that a clump was
picked up and used for 1:1 dilution in the conical . This would explain why it is higher in concentration than the
vial. The conical suspension was what was given to the NHP.
Slide 9
Vaccine Preparation Culture Data: Lot 16
FY09-126 TUL08C Vaccination Plate Counts from 07OCT09
Lot 16 Vial
Lot 16 Vial
Lot 16 Vial
Lot 16 Vial
Lot 16 Vial
Lot 16 Vial
Lot 16 Vial
Lot 16 Vial
Lot 16 Vial
Lot 16 Vial
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
Volume
Plated
mL
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Lot 16 Conical, pre
Lot 16 Conical, pre
Lot 16 Conical, pre
Lot 16 Conical, pre
Lot 16 Conical, pre
Lot 16 Conical, pre
Lot 16 Conical, pre
Lot 16 Conical, pre
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.1
>30
>300
>300
>300
98
7
0
0
>30
>300
>300
>300
93
8
1
1
>30
>300
>300
>300
81
11
1
0
>30
>300
>300
>300
91
9
1
0
TNTC
TNTC
TNTC
TNTC
9.07E+06
8.67E+06
6.67E+06
3.33E+07
Lot 16 Conical, post
Lot 16 Conical, post
Lot 16 Conical, post
Lot 16 Conical, post
Lot 16 Conical, post
Lot 16 Conical, post
Lot 16 Conical, post
Lot 16 Conical, post
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.1
>30
>300
>300
246
12
2
0
0
>30
>300
>300
300
19
8
0
0
>30
>300
>300
280
0
7
0
0
>30
>300
>300
275
10
6
0
0
TNTC
TNTC
TNTC
2.75E+06
1.03E+06
5.67E+06
0.00E+00
0.00E+00
Sample ID
Dilution
Factor
Plate Count
Mean
Mean CFU/mL
Plate 1
>30
>300
>300
>300
242
17
2
0
0
0
Plate 2
>30
>300
>300
>300
228
21
3
0
0
0
Plate 3
>30
>300
>300
>300
211
37
4
2
0
0
>30
>300
>300
>300
227
25
3
1
0
0
TNTC
TNTC
TNTC
TNTC
2.27E+07
2.50E+07
3.00E+07
6.67E+07
0.00E+00
0.00E+00
The conical suspension was what was given to the NHP..
Slide 10
Vaccine Preparation Culture Data: Lot 17
FY09-126 TUL08C Vaccination Plate Counts from 07OCT09
Lot 17 Post Set One
Lot 17 Post Set One
Lot 17 Post Set One
Lot 17 Post Set One
Lot 17 Post Set One
Lot 17 Post Set One
Lot 17 Post Set One
Lot 17 Post Set One
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Volume
Plated
mL
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Lot 17 Post Set Two
Lot 17 Post Set Two
Lot 17 Post Set Two
Lot 17 Post Set Two
Lot 17 Post Set Two
Lot 17 Post Set Two
Lot 17 Post Set Two
Lot 17 Post Set Two
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.01
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Sample ID
Dilution
Factor
Plate Count
Plate 1
>30
>300
>300
>300
>300
>300
76
11
Plate 2
>30
>300
>300
>300
>300
>300
92
8
>30
>300
>300
>300
>300
206
30
8
>30
>300
>300
>300
>300
180
45
3
Mean
Mean CFU/mL
Plate 3
>30
>300
>300
>300
>300
>300
70
9
>30
>300
>300
>300
>300
>300
79
9
TNTC
TNTC
TNTC
TNTC
TNTC
TNTC
7.93E+08
9.33E+08
>30
>300
>300
>300
>300
186
38
2
>30
>300
>300
>300
>300
191
38
4
TNTC
TNTC
TNTC
TNTC
TNTC
1.91E+08
3.77E+08
4.33E+08
We initially suspended 2 vials of lot 17 for use on 3 NHP, however this amount was
only sufficient for the first 2 animals (set one). A second vial had to be thawed and
used for the third animal (set 2). There was insufficient volume to plate a prevaccination sample, thus only posts are listed.
Slide 11
Vaccine Preparation Culture Data: Lot 20
FY09-126 TUL08C Vaccination Plate Counts from 07OCT09
Sample ID
Dilution
Factor
Volume
Plated
Plate Count
mL
Plate 1
Plate 2
Plate 3
Mean
Mean CFU/mL
Lot 20 Post
1.00E+00
0.01
>30
>30
>30
>30
TNTC
Lot 20 Post
1.00E+01
0.1
>300
>300
>300
>300
TNTC
Lot 20 Post
1.00E+02
0.1
>300
>300
>300
>300
TNTC
Lot 20 Post
1.00E+03
0.1
>300
>300
>300
>300
TNTC
Lot 20 Post
1.00E+04
0.1
>300
>300
>300
>300
TNTC
Lot 20 Post
1.00E+05
0.1
57
66
71
65
6.47E+07
Lot 20 Post
1.00E+06
0.1
4
4
8
5
5.33E+07
Lot 20 Post
1.00E+07
0.1
1
0
0
0
3.33E+07
There was insufficient volume to plate a pre-vaccination sample, thus only the post is
listed.
Slide 12
Amount of LVS Received by NHP
Animal
Group ID
# of NHPs
Dose Volume
(µL)
Average CFU
Administered
Control
3
120
NA
Lot 16*
3
120
7.09E+05
Lot 17a**
2
113
8.96E+07
Lot 17b***
1
113
3.21E+07
Lot 20**
8
132
8.54E+06
Lot 4*
8
120
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 13
IFNγ Production by PBMCs isolated from Nonvaccinated NHPs
IFNg Spots (Mean +/- S.D.)
7
6
5
4
3
Media
LVS hk Mid
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
2
NT
1
0
30015
A06872
A07624
NHP
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with either LVS or SCHU S4
antigens (heat-killed (hk) or formalin-fixed (ff)) at a dose of 0.25 x 105/ml (Mid) or 1 x 105/ml (Hi)
- LVS hk could not be tested at the Hi dose due to lack of enough reagent
- We are currently comparing this lot to two new lots of hk LVS prepared at UNM
No response of unvaccinated NHPs reached more than an average of 6 spots
PBMCs from unvaccinated NHPs failed to proliferate to any LVS or SCHU S4 stimuli as noted by
comparison to media control wells (data not shown)
A06872 was not tested (NT) due to paucity of PBMCs after preparation
Slide 14
700
500
Media
LVS hk Mid
LVS ff Hi
400
SCHUS4 hk Hi
600
300
SCHUS4 ff Hi
200
100
A06851, Day 14
A06851, Day 7
A06851, Day 0
30003, Day 14
30003, Day 7
30003, Day 0
29992, Day 14
29992, Day 7
29992, Day 0
29975, Day 14
NT
29975, Day 7
0
29975, Day 0
IFNg Spots (Mean +/- S.D.)
IFNγ Production by PBMCs isolated from NHPs
Vaccinated with DVC Lot 16 LVS
NHP, Day post-LVS Vaccination
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with either LVS or SCHU S4 antigens (heatkilled (hk) or formalin-fixed (ff)) at a dose of 0.25 x 105/ml (Mid) or 1 x 105/ml (Hi)
- Wells that are TNTC are entered into the database as having 600 spots
Inconsistent response of DVC Lot 16 vaccinated NHPs (29992 > 29975 > 30003)
PBMCs from DVC Lot 16 vaccinated NHPs failed to proliferate to any LVS or SCHU S4 stimuli as noted by comparison to
media control wells until day 14 and then only 2/3 showed some proliferation (data not shown)
A06851 (“failure to thrive”) was not tested (NT) on days 7 and 14
Slide 15
700
600
500
400
Media
LVS hk Mid
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
300
200
A07872, Day 14
A07872, Day 7
A07872, Day 0
A07754, Day 14
A07754, Day 7
A07754, Day 0
A07699, Day 14
0
A07699, Day 7
100
A07699, Day 0
IFNg Spots (Mean +/- S.D.)
IFNγ Production by PBMCs isolated from NHPs
Vaccinated with DVC Lot 17 LVS
NHP, Day post-LVS Vaccination
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with either LVS or SCHU S4 antigens (heatkilled (hk) or formalin-fixed (ff)) at a dose of 0.25 x 105/ml (Mid) or 1 x 105/ml (Hi)
- Wells that are TNTC are entered into the database as having 600 spots
- One well that was TNTC was excluded from analysis on Day 7, A07754, LVS FF hi
Somewhat more consistent response of DVC Lot 17 vaccinated NHPs as compared to DVC Lot 16 vaccinated NHPs
PBMCs from DVC Lot 17 vaccinated NHPs failed to proliferate to any LVS or SCHU S4 stimuli as noted by comparison to
media control wells until day 14 and then only 1/3 showed some proliferation (data not shown)
Slide 16
700
Media
600
LVS hk Mid
500
400
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
300
200
A07683, Day 7
A07683, Day 14
A07683, Day 0
A06488, Day 7
A06488, Day 14
A06488, Day 0
30048, Day 14
30048, Day 7
30048, Day 0
30014, Day 14
30014, Day 7
30014, Day 0
29996, Day 14
29996, Day 7
29996, Day 0
29991, Day 14
29991, Day 7
29991, Day 0
29976, Day 14
29976, Day 7
29976, Day 0
28461, Day 14
0
28461, Day 7
100
28461, Day 0
IFNg Spots (Mean +/- S.D.)
IFNγ Production by PBMCs isolated from NHPs
Vaccinated with DVC Lot 20 LVS
NHP, Day post-LVS Vaccination
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with either LVS or SCHU S4 antigens (heatkilled (hk) or formalin-fixed (ff)) at a dose of 0.25 x 105/ml (Mid) or 1 x 105/ml (Hi)
- Wells that are TNTC are entered into the database as having 600 spots
- One well that was TNTC was excluded from analysis on Day 7, 29996, LVS FF hi; one well that had 0 spots
suggesting that no stimuli had been added was excluded from analysis on Day 14, 29976, LVS FF hi
Somewhat more consistent response of DVC Lot 20 vaccinated NHPs as compared to DVC Lot 16 vaccinated NHPs but
not as consistent as DVC Lot 17 vaccinated NHPs
PBMCs from DVC Lot 20 vaccinated NHPs failed to proliferate to any LVS or SCHU S4 stimuli as noted by comparison to
media control wells at any time point (data not shown)
Slide 17
IFNγ Production by PBMCs isolated from NHPs
Vaccinated with USAMMDA Lot 4 LVS
600
500
400
Media
LVS hk Mid
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
300
200
A07842, Day 7
A07842, Day 14
A07842, Day 0
A07756, Day 14
A07756, Day 7
A07756, Day 0
A07738, Day 7
A07738, Day 14
A07738, Day 0
A07720, Day 7
A07720, Day 14
A07720, Day 0
30039, Day 7
30039, Day 14
30039, Day 0
30019, Day 14
30019, Day 7
30019, Day 0
30017, Day 7
30017, Day 14
30017, Day 0
29987, Day 7
0
29987, Day 14
100
29987, Day 0
IFNg Spots (Mean +/- S.D.)
700
NHP, Day post-LVS Vaccination
All PBMCs were cultured at 200,000 cells/well in triplicate and stimulated with either LVS or SCHU S4 antigens (heatkilled (hk) or formalin-fixed (ff)) at a dose of 0.25 x 105/ml (Mid) or 1 x 105/ml (Hi)
- Wells that are TNTC are entered into the database as having 600 spots
Somewhat less consistent response of USAMMDA Lot 4 vaccinated NHPs as compared to DVC Lot 17 or 20 vaccinated
NHPs
PBMCs from 4/8 USAMMDA Lot 4 vaccinated NHPs showed some proliferative response to either LVS or SCHU S4
stimuli as noted by comparison to media control wells at day 14 post-vaccination (data not shown)
Slide 18
Milestone #8– Plans for next month

Challenge vaccinated NHPs and control NHPs to 1000 cfu SCHU S4 on
11/11, 11/12 and 11/13





Clinical signs, body weights, bacteremia, and clinical chemistry
will be followed closely post-aerosol challenge
Necropsies will be performed on all NHPs found moribund or
dead
Organ burden (lung, liver, spleen, LN, brain stem) and
bacteremia will be assessed from those animals undergoing
euthanasia due to morbidity; all but bacteremia will be
assessed on those found dead
Test all NHP plasma collected pre- and post-LVS vaccination for IgG
anti-LVS activity by ELISA
Analyze Day 21 immunology data (IFNγ ELISPOT and proliferation)
Slide 19
Milestone #9 – Aerosol SOP Development
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 20
Milestone #9 - Objective

Develop a SOP compatible with GLP transition for aerosol delivery of
Schu S4.
Slide 21
Milestone #9- October 2009 Accomplishments


SCHU S4 bioaerosol qualification on hold until aerosol dosing
issue is resolved
24h SCHU S4 growth in Chamberlains; normalized to OD600 of
0.500 ± 0.050
Date
Flask
Time
Dil Factor Mean CFU/mL
log10
45
39.0
1.00E+07
3.90E+08
8.59
55
51
57.7
1.00E+07
5.77E+08
8.76
42
45
47
44.7
1.00E+07
4.47E+08
8.65
0.558
119
78
75
90.7
1.00E+06
9.07E+07
7.96
0.556
95
81
50
75.3
1.00E+06
7.53E+07
7.88
3
0.554
46
110
64
73.3
1.00E+06
7.33E+07
7.87
1
0.497
176
184
189
183.0
1.00E+06
1.83E+08
8.26
0.494
221
206
213
213.3
1.00E+06
2.13E+08
8.33
3
0.498
165
177
200
180.7
1.00E+06
1.81E+08
8.26
1
0.497
22
24
25
23.7
1.00E+07
2.37E+08
8.37
0.494
33
25
18
25.3
1.00E+07
2.53E+08
8.40
0.498
26
19
18
21.0
1.00E+07
2.10E+08
8.32
1
2
3
0.547
34
38
0.549
67
3
0.549
1
1
9-Oct-09
23-Oct-09
30-Oct-09
30-Oct-09
Plate
Mean
h
Norm OD600
2
2
2
2
3
24
24
24
24
Mean
Max
Min
2.44E+08 CFU/mL
5.77E+08 CFU/mL
7.33E+07 CFU/mL
Tech 1 and 2
Tech 2 and 3
Brasel
Slide 22
The spectrophotometer functions as expected
(Data from 9OCT09)
(Data from 23OCT09)
9.00
9.00
8.80
y = 0.4708ln(x) + 8.8705
R² = 0.9568
8.60
8.80
y = 0.4825ln(x) + 8.2182
R² = 0.9037
8.60
8.40
8.40
8.20
8.20
8.00
8.00
7.80
Series1
7.80
Series1
7.60
Log. (Series1)
7.60
Log. (Series1)
7.40
7.40
7.20
7.20
7.00
7.00
6.80
6.80
6.60
0.000
0.100
0.200
0.300
0.400
0.500
0.600
6.60
0.000
0.200
0.400
0.600
= OD normalized (0.500) suspension
Slide 23
Data variability appears to be associated with
inter-technician variability

Practice sprays to be conducted on 4NOV09
–
6 different preparations by 6 different individuals
 Trevor Brasel
 Michelle Valderas
 Bob Sherwood
 Felisha Romero
 Alicia Weldon
 Sara London
Slide 24
Milestone #9- Plan for next month

Continue investigating achievement of target dose
–
Pursue use of flow cytometer methods
Slide 25
MS#11 – GLP Model Efficacy SOPs Developed
in NHPs and Efficacy Testing of Vaccine
Candidates
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 26
Milestone #11- Objectives and Endpoints


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 27

Milestone #11- October 2009
Accomplishments
Nothing was performed during this month as we are waiting on
the telemetry data analysis to be performed.
Slide 28
Milestone #11- Plans for next month

Analyze telemetry data.
Slide 29
Milestone #12/13 – Immune Responses in
Animals and Humans
Immunoassay Development and Comparisons in Animal Models
Choose PBMC
Purification Method
Choose PBMC
Freezing Method
Method chosen:
Purdue ListServ
Cerus
Red: completed
Green: In progress
Yellow: on hold; restart if
necessary
Blue: steps in the milestone
Develop
Immunoassay
methodologies
IFNg
Proliferation
assay
ELISPOT
Microagglutination
assay
Determine
protein:CFU
relationship in
FF and HK LVS
antigens
Plasma
IgG
ELISA
Plasma
IgA
ELISA
Slide 30


Milestone #12/13 – October 2009
Accomplishments
Reagents for the microagglutination assay were received
–
Positive control rabbit anti-F. tularensis sera was resuspended and
aliquoted
–
LVS Lot 9; UNM personnel are staining it with non-acidic hematoxylin
for use in the assay
No work was performed attempting to standardize LVS and SCHU S4 CFU to
protein content
–
The need for this analysis is under discussion with DVC and NIAID
given the availability of standardized FF LVS and SCHU S4 reagents
from DVC
–
However, we may also need to standardize our HK LVS and SCHU S4 to
each other and to the DVC FF preparations;
–
in addition newly prepared HK and LVS antigens at UNM must be
compared to formerly prepared antigens in order to bridge NHP
responses in vitro
–
We are considering a protein gel/Coomassie blue approach
Slide 31
Milestone #12/13 – Plans for next month


Set up the microagglutination assay using positive control rabbit-F.
tularensis sera and positive and negative NHP anti-LVS plasma
Continue to explore ways to standardize LVS and SCHU S4 CFU to
protein content
–
Try a protein gel/Coomassie blue approach
Slide 32
MS #21 – Correlates of protection
Establish assays of effector function that detect correlates of
protection
Establish conditions to detect intracellular cytokines in NHP PBMCs
Confirm response in
LVS-vaccinated NHPs
Confirm low response
in non- LVS-vaccinated
NHPs
Slide 33


Milestone #21 – October 2009
Accomplishments
PBMC from NHP A07682 were collected and frozen at day 14 post LVS, 1.2x107 by
scarification.
–
PBMC were thawed and stimulated 48 h with graded concentrations (8000; 4x104
and 2x105/well) of hkLVS or ffLVS, media only as negative control, and PHA or
PMA/ionomycin as positive controls. As an additional positive control for ICS
staining, human lymphocytes (Hick-1) were stained along with the other
samples.
–
During the last 4 h of stimulation, Golgi plug was added for retention of
intracellular cytokines.
All samples were surface stained with CD3-PerCP,CD4-PECy7,CD8-APCCy7 and
intracellularly stained with IFN-g FITC, TNF-a-PE and IL2-APC.
–
50, 000 total events were acquired with the 6 color FACS Canto. The same
settings were used as in a previous experiment with the same six antibody
conjugates.
Slide 34
Milestone #21 – October 2009 Accomplishments
In the ELIspot assay PBMC produce IFN-g as an antigen dependent response at 20 h
of in vitro recall stimulation. Can those cells be detected also by Flow cytometry ICS?
What cell population are they?
Where are the IFN-g+ PBMC in the FSC/SSC plot ?
The positive control human lymphocytes have a clearly
distinguishable IFN-g+ population gated in red in P1.
In the FSC/SSC plot the P1 cells show up in the traditional
lymphocyte gate.
The following slide shows that 48 hours of hkLVS or ffLVS stimulation of PBMC from NHP A07682
does not show IFN-g+ PBMC above media control background. Also the PBMC which appear IFNg+ (red in gate P1) are not within the lymphocyte population in the FSC/SSC plot.
The 48 h time point of restimulation was chosen as human blood T cells from LVS vaccinees were
shown by ICS to produce IFN-g at this time point. However, those donors were more than 4 weeks
post LVS vaccination. IFN-g intracellular staining of NHP PBMC has also been tested previously at 6
and 96 h stimulation with no success in detecting antigen dependent cytokine.
Slide 35
Milestone #21 – October 2009 Accomplishments
2x105 LVS/ well, hk(top), ff(bottom) Media only controls
Slide 36
Milestone #21- Plans for next month


Stimulate NHP PBMCs with LVS and harvest at a time point
similar to the ELIspot assay, ~20h.
Focus on IFN-g for intracellular stain and use the available
detectors for additional surface stain to identify the IFN-g
producing cell populations.
–
e.g. exchange IL-2 and TNF-a stain for NK-cell surface
markers CD56/CD16.
Slide 37
Action Items

Michelle/Bob- provide a due date for completion of the telemetery data
analysis on MS 11. “In-life Data” collection was completed in September 2009.
(Note added after tech call: telemetry data will be analyzed by 11/11/09 and
Michelle will email UNM a draft report by 11/30/09)




Trevor may perform all the OD measurements and dilutions to remove
individual variability and to get the challenge doses as consistent as possible
across the 3 days of challenges on the MS8C study.
Trevor/Micro team: On 11/4, LBERI will do practice sprays by 6 people from 6
different SCHU S4 preps. Will do OD normalization. Each person will make
their own dilutions, spray doses, and cultures. Thereafter, wLBERI ill decide
who will perform each step prior to the challenge dates for MS 8C study
Trevor: LBERI has put MS 9 on hold until the aerosol dosing is more
standardized.
UNM (Terry) /LBERI (Julie) will look at coomassie blue stained protein gel (with
titration series) or a reverse elisa as methods to standardize the antigen
preparations for greater consistency.
Slide 38