LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
1 June 2010
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
1
Active Milestones
#2
Active
#8
Active
#9
Active
#10
Active
#12/13
Active
#21
Active
#29
Active
Vaccinations of study personnel- no work
in May
LVS vaccination protection of aerosol
Schu4 confirmed in primates
Aerosol SOP developed for GLP
transition- no work in May
Efficacy testing of vaccine candidates
(LBERI)- no work in May
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 May
2
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.) 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
Round 5 Vaccination/2 Broth Challenge (SC Vaccination with Lot 17,
compare CB vs. CAMHB) SCHU S4 Challenge 1000 CFU
Vaccination/Challenge Telemetered Natural History Study SCHU S4
Challenge 1000 CFU
Red: completed
Green: in progress
Blue: steps in the milestone
3
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) – TUL08 A and B
– Compare 4 different LVS lots as vaccines (all delivered by subcutaneous route)
– TUL08 C
– Compare SCHU S4 growth media to see if it has an effect on virulence
(Chamberlain’s broth vs. Mueller-Hinton broth) – TUL08 D
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
4
Milestone #8 – May 2010 Accomplishments
• TUL08D Broth Comparison exposures occurred. Due to Day 14 elevated
CRP and LDH for some animals, study termination will occur 28 days post
exposure.
• TUL08C 4 LVS lot vaccine comparison data was reanalyzed by whether
vaccinated animals survived and had minimal lesions, survived and had
residual lesions, or succumbed. This data is being included in the draft
final report.
• Protocol for the telemetry study in vaccinated NHP was written.
• Protocol amendment for the LD50 study at very low doses was written.
• Tul08A data was, and continues to be, analyzed for inclusion into the final
report.
• NHPs were screened for their ability to secrete IFNγ and proliferate in
response to various LVS and SCHU S4 antigens
– Tested PBMCs on Day 0, 7, 14, and 28
5
Milestone #8 – Data & Interpretation
TUL08C Re-analysis
Key for data presented in the following slides:
• I labeled vaccinated animals that survived challenge to study
termination, possessed minimal interstitial pneumonitis lung lesions and
had clinical chemistry and hematology values that had returned to
baseline or to values minimally above normal pre-exposure values by
study termination, the “minimal lesions” group.
• NHP that were vaccinated and survived to study termination but had
mild pneumonia and clinical chemistry/hematology values that remained
mildly above baseline and did not return to normal by day 56, I labeled
the “residual lesions” group because the status of disease resolution was
uncertain from a pathology perspective at study termination.
• Vaccinated NHP that succumbed to disease are described as deceased
vaccinated, and the control NHP I listed as such.
• Reanalyzing the data in this fashion (independent of vaccine lot) has
given us new insight into how we might potentially categorize vaccine
protection.
6
Milestone #8 – Data & Interpretation
TUL08C Re-analysis, Animal Group Allocation
Lot Number
Control
Control
Control
Lot 16
Lot 17
Lot 20
Lot 20
Lot 20
Lot 20
Lot 4
Lot 4
Lot 4
Lot 16
Lot 17
Lot 20
Lot 4
Lot 16
Lot 17
Lot 20
Lot 20
Lot 4
Lot 4
Lot 4
Lot 4
Animal
Number
Presented
Dose (CFU)
30015
A06872
A07624
30003
A07872
29991
29996
30014
30048
30019
A07738
A07842a
29975
A07699
A07683
30039
29992
A07754
29976
A06488
29987
30017
A07720
A07756
1823
636
1268
2872
1860
304
2148
728
1479
1785
416
1090
1667
787
828
3766
1540
1897
2510
2250
1511
1257
119
1669
Avg Dose/Gp
(Geom.
Mean)
1137
1155
1422
1257
Study Outcome
Unvaccinated Control
Unvaccinated Control
Unvaccinated Control
Deceased, Vaccinated
Deceased, Vaccinated
Deceased, Vaccinated
Deceased, Vaccinated
Deceased, Vaccinated
Deceased, Vaccinated
Deceased, Vaccinated
Deceased, Vaccinated
Deceased, Vaccinated
Residual Lesions
Residual Lesions
Residual Lesions
Residual Lesions
Minimal Lesions
Minimal Lesions
Minimal Lesions
Minimal Lesions
Minimal Lesions
Minimal Lesions
Minimal Lesions
Minimal Lesions
Study Day of Avg Day of
Death
Death
39
39
40
44
42
41
47
45
43
42
40
56
56
56
56
56
56
56
56
56
56
56
56
56
39.3
44.4
56.0
56.0
7
Milestone #8 – Data & Interpretation
TUL08C Re-analysis: CRP levels during disease
Unvaccinated Control CRP-H Summary (Absolute Numbers)
Day Post-Exposure
0
2
4
6
10
14
21
Mean
6.7
118.4
435.3
dead
dead
dead
dead
SD
7.3
59.1
55.0
N
3
3
3
Deceased, Vaccinated CRP-H Summary (Absolute Numbers)
Day Post-Exposure
0
2
4
6
10
14
21
Mean
3.3
59.4
402.9
419.4
362.0
318.9
217.8
SD
2.5
35.2
51.6
73.3
17.7
NA
NA
N
8
8
7
7
3
1
1
Mean
SD
N
Residual Lesions CRP-H Summary (Absolute Numbers)
Day Post-Exposure
0
2
4
6
10
14
3.1
139.8
380.3
333.2
214.0
143.4
0.7
78.1
85.1
52.1
89.4
113.1
4
4
4
4
4
4
21
36.9
13.4
4
Mean
SD
N
Minimal Lesions CRP-H Summary (Absolute Numbers)
Day Post-Exposure
0
2
4
6
10
14
2.5
28.6
256.9
197.2
29.7
27.0
1.1
43.5
132.2
125.8
31.6
45.7
8
8
8
8
8
8
21
7.6
9.8
8
Conclusions:
• CRP levels in control
animals rise rapidly after
exposures and animals
succumb rapidly.
• CRP levels in vaccinees
that succumb rise rapidly
and remain high over time.
• In animals with residual
lesions, CRP levels do not
rise as high as the controls
or vaccinees that
succumbed and return to
near normal by study
termination.
• Animals with minimal
lesions have CRP levels
that are much lower than
the other groups and that
return to near baseline
levels by study termination.
8
Milestone #8 – Data & Interpretation
TUL08C Re-analysis
Conclusions:
• When data is reanalyzed, differences between the groups become more clear. Respiratory rate increase is
more pronounced in the controls and the vaccinees that succumb. It was less pronounced in the animals that
survived whether they had residual or resolved lesions. Statistical analysis is in progress.
• Temperatures increased for all groups, however progression to hypothermia was most impressive in the
control group. Those with minimal lesions returned to baseline (~ 37 degrees)faster than those with residual
lesions. The decrease in temperature associated with vaccinees that succumbed is likely due to animal death
during that time and not a return to baseline. Please note one animal comprises that group beginning day9 49.
Milestone #8 – Data & Interpretation
TUL08C Re-analysis
Conclusions:
• When data is reanalyzed, differences between the groups become more clear. Weights in the minimal lesion
group dip less than 5% from baseline. Control animals weights drop to near 10% loss and then succumb.
Interestingly, those with residual lesions and those vaccinees that succumb track together. Day 49 and after
cannot be compared for those groups because one NHP comprises the deceased vaccinees. Those with
residual lesions have weight increases beginning day 48, but never return to baseline as the minimal lesion
group did. Statistical analysis is in progress.
• Clear differences between groups is now seen in the lungs and other tissues. Statistical analysis is in 10
progress.
Milestone #8 – Data & Interpretation
TUL08D Presented Doses and Deaths
Broth
CB
MHB
Animal # Pres. Dose
A09011
2,823
A09410
339
A09139
1,107
A09088
1,580
A09094
1,741
A09487
1,344
A09164
2,097
A09064
1,455
A09097
971
A09169
2,178
A09079
642
A09518
2,341
A09072
1,439
A09066
1,050
A09068
1,329
A08739
2,150
A06086
2,266
A09523
2,010
A09140
1,630
A09511
1,801
Vx Dose Died (Y/N)
NA
Y
NA
Y
NA
Y
8.75E+08
N
8.75E+08
N
8.75E+08
N
8.75E+08
N
2.10E+08
Y
2.10E+08
N
2.10E+08
Y
NA
Y
NA
Y
NA
Y
8.75E+08
N
8.75E+08
N
8.75E+08
N
2.10E+08
N
2.10E+08
N
2.10E+08
N
2.10E+08
N
Animal #
A09079
A09011
A09139
A09518
A09064
A09072
A09410
A09169
Broth
MHB
CB
CB
MHB
CB
MHB
CB
CB
Days post Exposure
Vx or Ctrl
Day 5
Day 6
Day 7
Ctrl
found dead
Ctrl
found dead
Ctrl
moribund
Ctrl
found dead
Vx
moribund
Ctrl
found dead
Ctrl
found dead
Vx
moribund
Conclusions:
• Control NHP succumbed in the same time
frame whether they were challenged with SCHU
S4 grown in Chamberlain’s broth or Mueller
Hinton Broth.
• The only vaccinees that have succumbed were
those challenged with Chamberlain’s grown
SCHU S4
11
Milestone #8 – Data & Interpretation
TUL08D CRP Levels Post Exposure
Broth
CB
MHB
Animal #
Status
A09011
A09410
A09139
A09088
A09094
A09487
A09164
A09064
A09097
A09169
A09079
A09518
A09072
A09066
A09068
A08739
A06086
A09523
A09140
A09511
Ctrl
Ctrl
Ctrl
Vx
Vx
Vx
Vx
Vx
Vx
Vx
Ctrl
Ctrl
Ctrl
Vx
Vx
Vx
Vx
Vx
Vx
Vx
Pres. Dose
Days Post Exposure
Day 5
Day 7
340.2
Dead
324.3
Dead
487.8
Dead
Day 0
Day 3
2,823
0.8
264.6
339
2.1
302.4
1,107
2.3
403.8
1,580
1.9
309.6
1,741
2.9
258
388.5
1,344
1.8
171.6
247.5
2,097
1.6
240
1,455
2.7
206.7
602
971
1.9
310.8
173.6
2,178
2.8
414.6
429
642
4.1
350.7
339.6
2,341
2.3
438.3
282.6
1,439
3.3
405.6
515.1
1,050
1.9
169.1
348.3
1,329
2.5
120.3
130.5
2,150
0.5
331.2
493.5
2,266
6.3
255.6
440.7
2,010
0.6
143.5
99.6
1,630
0.9
169.3
168.9
1,801
0.9
200.1
248.7
not enough sample to run CRP
341.1
387.6
106.7
338.7
33.1
489.3
Dead
Dead
Dead
395.1
37.8
457.2
372.6
63.5
154.2
42
Day 10
Dead
Dead
Dead
79.9
258.3
155.6
225.3
Dead
7.6
Dead
Dead
Dead
Dead
128.4
265.2
155.9
6.1
5.2
66.3
Day 14
Dead
Dead
Dead
30.5
336.4
252.9
155.1
Dead
6
Dead
Dead
Dead
Dead
25.9
77.3
60.7
40.8
6.9
2.1
8.7
Conclusions:
• CRP levels in
vaccinated NHP that
were challenged with
CB-grown SCHU S4
in general have
elevated CRP levels
by 14 days post
exposure as
compared with the
ones challenged with
MHB-grown SCHU
S4.
• all groups had
high CRP levels
early in disease and
decrease in levels
appears to be more
rapid in those
challenged with
MHB-grown SCHU
S4.
• Analysis is in
progress.
• Because the CRP
levels remained very
high in the CB
group, term is now
28 days post12
exposure.
Milestone #8 – Data & Interpretation
TUL08D Weight Changes
% Body Weight Loss from ~ Day-1
Pres.
Broth Animal # Status
Dose
A09011 Ctrl
2,823
A09410 Ctrl
339
A09139 Ctrl
1,107
A09088
Vx
1,580
A09094
Vx
1,741
CB
A09487
Vx
1,344
A09164
Vx
2,097
A09064
Vx
1,455
A09097
Vx
971
A09169
Vx
2,178
A09079 Ctrl
642
A09518 Ctrl
2,341
A09072 Ctrl
1,439
A09066
Vx
1,050
A09068
Vx
1,329
MHB
A08739
Vx
2,150
A06086
Vx
2,266
A09523
Vx
2,010
A09140
Vx
1,630
A09511
Vx
1,801
Study Day Post Exposure
Day 0
Day 2
Day 4
Day 6
Day 8
Day 10
Day 12
Day 14
Day 16
-5
-6
0
-2
-2
0
2
0
-5
-5
0
-4
-4
-2
-2
-4
-5
-5
-4
-5
-6
-5
-1
0
0
0
0
0
-5
-1
0
-4
-2
0
0
-4
-1
0
0
-5
-6
-8
-6
-3
-5
-3
-3
0
-7
-7
0
-4
-4
-1
0
-8
-4
-1
-4
-5
Dead
-11
Dead
-6
-5
-1
-6
Dead
-4
-11
Dead
Dead
-6
-4
0
-11
-6
0
-1
-5
Dead
Dead
Dead
-7
-7
-7
-12
Dead
-6
Dead
Dead
Dead
Dead
-9
0
-14
-7
-2
-3
-3
Dead
Dead
Dead
-4
-11
-7
-12
Dead
-2
Dead
Dead
Dead
Dead
-8
0
-17
-7
-2
-1
-1
Dead
Dead
Dead
-4
-12
-9
-14
Dead
-4
Dead
Dead
Dead
Dead
-5
0
-10
-6
0
0
-1
Dead
Dead
Dead
0
-15
-9
-12
Dead
-2
Dead
Dead
Dead
Dead
-6
0
-7
-5
0
0
0
Dead
Dead
Dead
0
-15
-9
-10
Dead
0
Dead
Dead
Dead
Dead
NA
NA
NA
NA
NA
NA
NA
Conclusions: In animals that survive challenge, animals with CRP levels that have returned to near normal display less
weight loss by day 14/16 than NHP with high CRP levels. Vaccinated animals exposed to MHB-grown SCHU S4
appear to recover weight faster than those that were exposed to CB-grown SCHU S4.
13
600
Media
LVS ff Hi
SCHUS4 ff Hi
DVC LVS ff 10^7
DVC S4 ff 10^7
500
MHB
400
CB
300
CB
200
MHB
A09523, Day 28
A09523, Day 14
A09523, Day 7
A09523, Day 0
A09164, Day 28
A09164, Day 14
A09164, Day 7
A09164, Day 0
A09094, Day 28
A09094, Day 14
A09094, Day 7
A09094, Day 0
A09068, Day 28
A09068, Day 14
0
A09068, Day 7
100
A09068, Day 0
IFNg Spots (Mean +/- S.D.)
Representative IFN-γ production by NHP PBMCs
stimulated with F. tularensis antigens
LVS FF Hi (1 x 105/ml, UNM) stimulates the most IFNγ production by PBMCs from vaccinees.
DVC LVS FF (1 x 107/ml) does not stimulate as much IFNγ production even though it is used at 100
times the concentration as LVS FF Hi (1 x 105/ml from UNM).
SCHU S4 FF Hi (1 x 105/ml) and DVC SCHU S4 (1 x 107/ml) generally stimulate less IFNγ production
than LVS FF Hi (1 x 105/ml; UNM).
MHB and CB denote animals eventually exposed to SCHU S4 grown in Mueller Hinton and
Chamberlain’s broth respectively. All PBMCs cultured at 200,000/well.
Milestone #8 – Immune Assay Data
Interpretation
• All LVS vaccinees secreted more IFN-γ in response to
FF LVS Hi (UNM prep) post-vaccination as compared
to Day 0 (pre-vaccination)
– Five NHPs had relatively low responses compared to the
other 9 NHPs (3 of the 5 were eventually exposed to SCHU
S4 grown in Chamberlain’s broth; one succumbed to
challenge)
• DVC FF LVS does not stimulate as much IFN-γ
production as the FF LVS from UNM even when
tested at 100x the concentration
15
Milestone #8 – Plans for next month
•
•
•
•
•
Submit the TUL08C draft final report
Continue to work on the TUL08A report
Begin analysis of serum for cytokines from TUL08D
Analyze data from TUL08D collected while in the ABSL-3
Test the F. tularensis antigen-induced IFN-γ production in PBMCs and
splenocytes of SCHU S4 aerosol survivors
16
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
Develop Immunoassay
methodologies
Proliferation
assay
Red: completed
Green: In progress
Yellow: on hold; restart if necessary
Blue: steps in the milestone
IFNg
ELISPOT
Plasma
IgG ELISA
Plasma
IgA ELISA
Determine
protein:CFU
relationship in FF
and HK LVS
antigens
Microagglutination
assay
17
Milestone #12/13 - Objective and Endpoints
• Develop immunoassays that reliably distinguish LVSvaccinated from non-vaccinated NHPs
– Thus far can rely on IgG anti-LVS ELISA for this purpose
• Develop immunoassays that may distinguish NHPs which
survive SCHU S4 aerosol challenge from those that do not
– Thus far have not been able to refine our assays to
function as such
– IgG anti-LVS plasma levels do not correlate with protection
– IFNγ production or proliferation by bulk PBMCs in
response to LVS or SCHU S4 antigens also appear not to
distinguish those NHPs which have been protected from
SCHU S4-induced mortality
18
Milestone #12/13 – May 2010 Accomplishments
• In an effort to quantify the protein
content:CFU ratio of the various F. tularensis
antigen preparations we use to stimulate
PBMCs, we:
– Further tested the effect of sample lysis buffer on
Bradford reagent in the Bradford protein assay
– Used monoclonal anti-F. tularensis antibodies to
detect bound antigen in an ELISA
19
Sample lysis buffer reacts with Bradford Assay
reagent and results in a detectable color change
in the absence of protein
0.9
0.8
0.7
y = 0.014x + 0.5056
R² = 0.9142
AU 595
0.6
0.5
25 µl
standard +
1975 µl SB
0.4
- Further
dilutions
made in PBS
0.3
0.2
0.1
0
0
5
10
15
mg/ml
20
25
30
Blank = PBS + Bradford reagent
Replicate A595
mean
SD
conc: g/ml
Independent dilution of 25 µl PBS into
1
0.764
0.772 0.01131 18.3542
1975 µl Sample buffer and then diluted
2
0.780
1:1 with Bradford reagent:
Use of Sample buffer + Bradford Reagent as the assay
blank reduces the number of points above zero
0.15
0.1
0.05
AU 595
0
0
5
10
15
-0.05
-0.1
-0.15
y = 0.0116x - 0.1804
R² = 0.7496
20
25
30
25 µl
standard +
1975 µl SB
- Further
dilutions
-0.25
mg/ml
made in PBS
Blank = Sample buffer + Bradford reagent
-0.2
Due to the fact that the Sample buffer reacts with the Bradford reagent to cause a
detectable color change and the fact that PBS used as a sample interpolated as 18
µg/ml protein, we do not favor use of this assay to measure the protein content of F.
tularensis antigen preparations.
Anti-fop A antibody detects HK LVS
bound to an ELISA plate
0.8
0.7
a-fopA ELISA
OD 405 nm
0.6
0.5
FF LVS UNM
0.4
HK LVS 5.2 UNM
FF LVS DVC
0.3
FF LVS O- MUT DVC
0.2
DVC FF SS4
0.1
DVC FF SS4 O-MUT
0
Dilution of antigen
UNM antigen stocks are at approx. 5 x 10^8/ml; DVC stocks are at 10^9/ml.
Despite the fact that a higher quantity of the DVC antigens are plated in the ELISA, less
anti-Fop A antibody reacts with those preparations as compared to HK LVS (UNM).
Anti-DNA K antibody detects F. tularensis
antigens bound to an ELISA plate
Anti-DNA K ELISA; 30
minute development
UNM antigen stocks are at approx. 5 x 10^8/ml; DVC stocks are at 10^9/ml.
Despite the fact that a higher quantity of the DVC antigens are plated in the ELISA, less
anti-DNA K antibody reacts with those preparations as compared to the UNM antigens.
Milestone #12/13 – Data Interpretation
• The Bradford assay would be difficult to use to
determine protein content in F. tularensis antigen
preparations due to the interaction of the sample
buffer with the Bradford reagent creating a
significant color change
• Use of an ELISA strategy to detect protein bound to a
plate suggests that HK LVS (UNM) has the highest
protein:CFU ratio; however this assay assumes that
all of the preparations bind to the plate equivalently
24
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 nonLVS-vaccinated
NHPs
Red: completed
Green: In progress
Blue: steps in the milestone
Establish conditions to detect
growth of LVS in NHP PBMCs
Confirm reduced
growth in LVSvaccinated NHPs
Confirm growth in
non- LVS-vaccinated
NHPs
25
Milestone #21 - Objective and Endpoints
• Develop immunoassays of effector function that can detect correlates of
protection from SCHU S4 aerosol-induced morbidity
– Developing two assays for this purpose thus far:
– 1) Intracellular cytokine staining;
• Perhaps IFNγ production by particular cell types will distinguish
NHPs which are protected vs. those that are not
• Perhaps the presence of particular cells that are producing more
than one cytokine (ex. IFNγ, TNFα and IL-2) will predict protection
– 2) inhibition of in vitro growth of SCHU S4 by some cells contained in
the PBMC preparations of protected NHPs vs. those that succumb to
SCHU S4 aerosol
26
Milestone #21 – May 2010 Accomplishments
• One more NHP was tested for intracellular
IFN-γ by flow cytometry
– Day 28 post-LVS vaccination
27
CD4+ and CD8+ cells from PBMCs of an LVS
vaccinee are stimulated to produce IFN-γ by
UNM FF LVS
0.8
0.7
% IFNg+ CD3+CD8- cells
% IFN-g+ CD8 T cells A09523 Day 28 post vacc
% IFN-g+ CD4 T cells A09523 Day 28 post
vacc
0.6
0.5
UNM ffLVS
0.4
DVC ffLVS
0.3
0.2
2
% IFN-g+ of CD3+CD8+ T cells
0.9
1.8
1.6
1.4
1.2
1
UNM ffLVS
0.8
DVC ffLVS
0.6
0.4
0.2
0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
cfu/well
0.1
0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
cfu/well
UNM FF LVS stimulates CD4 and CD8 cells to produce IFN-γ, possibly in a
dose-dependent manner; DVC FF LVS does not.
Milestone #21-Data Interpretation
• FF LVS (UNM) can stimulate production of IFN-γ by
CD4 and CD8 cells in the PBMC population of a
vaccinee
– Could possibly be in a dose-dependent manner but more
data collection is necessary to determine that for certain
– In addition CD14+ (monocytes) and CD56+ cells (CD3+
monocytes or T cells?), but not CD16+ (NK cells) also
respond to FF LVS (UNM)
– DVC FF antigen does not stimulate IFN-γ production by
cells in the PBMC population
29
Milestone #21 - Plans for next month
• Test the ability of PBMCs from naïve NHPs to
produce IFN-γ as detected by the intracellular
staining (ICS) assay
– This data will be critical to the interpretation of
the ICS data from LVS-vaccinated NHPs and will
inform us as to whether the FF LVS if working as a
mitogen (i.e. as many PBMCs from naïve NHPs
would make IFN-γ as those from vaccinees) or a
specific antigen (less PBMCs from naïve NHPs
would make IFN-g compared to PBMC from
vaccinees)
30
Action Items
• Michelle : LBERI will remove the second to last box on slide 3.
NIAID requested that LBERI not run the non telemetered arm
of the vaccination/challenge/telemetered study. (Completed
in minutes 6/7/10).
31
Additional Points
Deliverables completed for each active milestone:
No deliverables were completed during the month of May
List of relevant publications from the past month
None
MSCR status
MS 3: NIAID reviewing MSCR (UNM sent to NIAID on 2/18/2010 )
MS 4: NIAID reviewing MSCR (UNM sent to NIAID on 3/23/2010 )
MS 7: NIAID reviewing MSCR (UNM sent to NIAID on 4/7/2010 )
MS 11: NIAID reviewing MSCR (UNM sent to NIAID on 3/24/2010 )
MS 8 a: LBERI writing and due to UNM 5/14/10
MS 8 b: LBERI writing and due to UNM 6/30/10
MS 8 c: LBERI writing and due to UNM 5/4/10
32