LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
5 May 2009
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
Milestones
#2
Active
Vaccinations of study personnel- no work
this month
#4
Active
Confirmation of aerosol in vivo in NHP
efficacy studies in primates
#7
Active
SCHU S4 LD50 in primates
#8
Active
LVS vaccination protection of aerosol Schu4
validated in primates
#9
Active
Aerosol SOP developed for GLP transition
#11
Active
In Vivo GLP model efficacy SOPs developed
in one small species and primate 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
Slide 2
MS#8 – Flow Diagram
MS 8: 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 at highest LVS
dose by scarification or s.c.)
Red: completed
Green: in progress
Blue: steps in the milestone
SCHU S4
Challenge
1000 CFU
Slide 3
Milestone #8 - Objective and Endpoints


Describe repeat LVS vaccination and SCHU S4 aerosol challenge in
NHPs. Two different methods of vaccination will be compared
(scarification and subcutaneous) at a single high LVS vaccination dose.
Endpoints include histopathology and bacterial CFUs of internal organs
(lung, spleen, liver, kidneys, and lymph nodes), records of clinical
symptoms post-infection, and clinical chemistry and hematology during
infection.
Slide 4
Milestone #8 – April 2009 Accomplishments
DVC LVS Lot 16 Growth Characterization
•
•
LBERI performed 3 different resuspension/dilution methods.
Method 1- UNM method for DVC LVS Lot 16 growth using sterile
water from LBERI.
Lyophilized material was resuspended in 1mL of filtersterilized water from LBERI and dilutions (10-4 through 10-7)
were performed in the same LBERI water used for
resuspension.
Dilutions were plated in triplicate on CHAB (UNM provided),
Chocolate, and BCGA agars.
Plates were incubated at 37˚C with no CO2.
Plates were observed and colonies counted at 48, 72, 96,
and 120 hrs.
Slide 5
DVC LVS Lot 16 Growth Characterization
48h
72h
CHAB
Lot
752793
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Vol
Plated
mL
0.05
0.05
0.05
0.05
Chocolate
Lot E1409062
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
0
0
0
0
0
0
0
0
0
0
0
0
0.0
0.0
0.0
0.0
0.00E+00
0.00E+00
0.00E+00
0.00E+00
Chocolate
Lot E1409062
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
3
1
0
0
7
0
0
0
5
0
0
0
5.0
0.3
0.0
0.0
1.00E+06
6.67E+05
0.00E+00
0.00E+00
BCGA
Lot
12MAR09
-0745
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
4
0
0
0
0
0
0
0
2
0
0
0
2.0
0.0
0.0
0.0
4.00E+05
0.00E+00
0.00E+00
0.00E+00
BCGA
Lot
12MAR090745
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
84
2
0
0
0
1
0
0
4
0
0
0
29.3
1.0
0.0
0.0
5.87E+06
2.00E+06
0.00E+00
0.00E+00
Agar/Lot
Dilution
Plate
1
Plate
2
Plate
3
Mean
0
0
0
0
0
0
0
0
0
0
0
0
0.0
0.0
0.0
0.0
0.00E+00
0.00E+00
0.00E+00
0.00E+00
CHAB
Lot 752793
1.00E+04
1.00E+05
1.00E+06
1.00E+07
mL
0.05
0.05
0.05
0.05
Mean CFU/mL
Agar/Lot
Dilution
Vol Plated
Plate
1
Plate
2
Plate
3
Mean
Mean CFU/mL
0
0
0
0
0
0
0
0
0
0
0
0
0.0
0.0
0.0
0.0
0.00E+00
0.00E+00
0.00E+00
0.00E+00
96h
120h
CHAB
Lot
752793
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Vol
Plated
mL
0.05
0.05
0.05
0.05
Chocolate
Lot E1409062
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
3
0
0
0
7
1
0
0
5
0
0
0
5.0
0.3
0.0
0.0
1.00E+06
6.67E+05
0.00E+00
0.00E+00
Chocolate
Lot E1409062
BCGA
Lot
12MAR09
-0745
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
0
2
0
0
4
1
0
0
85
0
0
0
29.7
1.0
0.0
0.0
5.93E+06
2.00E+06
0.00E+00
0.00E+00
BCGA
Lot
12MAR090745
Agar/Lot
Dilution
Agar/Lot
Plate
1
Plate
2
Plate
3
Mean
Mean CFU/mL
0
0
0
0
0
0
0
0
0
0
0
0
0.0
0.0
0.0
0.0
0.00E+00
0.00E+00
0.00E+00
0.00E+00
Vol Plated
mL
Plate
1
Plate
2
Plate
3
Mean
Mean CFU/mL
CHAB
Lot 752793
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
2
2
0
0
6
4
0
0
7
1
0
0
5.0
2.3
0.0
0.0
1.00E+06
4.67E+06
0.00E+00
0.00E+00
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
3
0
0
0
7
1
0
0
5
0
0
0
5.0
0.3
0.0
0.0
1.00E+06
6.67E+05
0.00E+00
0.00E+00
1.00E+04
1.00E+05
1.00E+06
0.05
0.05
0.05
4
0
0
85
1
0
0
7
0
29.7
2.7
0.0
5.93E+06
5.33E+06
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
Dilution
Slide 6
Data Interpretation
DVC LVS Lot 16 Growth Characterization





LBERI results were not similar to those observed by UNM.
Colony counts were lower than previously noted using LBERI
standard methods (resuspension in 1.4 mL sterile water, 1%
peptone dilution blanks, and larger volume plated and incubated
for only 48 hours).
The delayed growth period observed with CHAB was consistent
with what LBERI has historically observed.
Significant number of new colonies on BCGA between 48 and 72
hrs. Hypothesized that these represented late-growth bacteria
rendered fragile during the lyophilization and resuspension
process.
SCHU S4 does not present similar growth patterns.
Slide 7
Milestone #8 – April 2009 Accomplishments
DVC LVS Lot 16 Growth Characterization
•
Method 2- UNM method for DVC LVS Lot 16 growth using sterile
water from UNM.
Lyophilized material was resuspended in 1mL of filtersterilized water provided by UNM and dilutions (10-4 through
10-7) were performed in the same water provided by UNM
used for resuspension.
Dilutions were plated in triplicate on CHAB (UNM provided),
Chocolate, and BCGA agars.
Plates were incubated at 37˚C with 5% CO2.
Plates were observed and colonies counted at 96 and 120
hrs.
Slide 8
DVC LVS Lot 16 Growth Characterization
96h
Agar/Lot
Dilution
CHAB
Remel Lot 757337
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Vol Plated
mL
0.05
0.05
0.05
0.05
Chocolate
Hardy Lot E1409062
1.00E+04
1.00E+05
1.00E+06
1.00E+07
BCGA
LRRI Lot
12MAR09-0745
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Plate 1
Plate 2
Plate 3
Mean
Mean CFU/mL
5
1
1
0
3
1
1
0
8
0
0
0
5.3
0.7
0.7
0.0
1.07E+06
1.33E+06
1.33E+07
0.00E+00
0.05
0.05
0.05
0.05
10
1
0
0
6
0
0
0
6
0
0
0
7.3
0.3
0.0
0.0
1.47E+06
6.67E+05
0.00E+00
0.00E+00
0.05
0.05
0.05
0.05
1
0
0
0
2
0
0
0
1
0
0
0
1.3
0.0
0.0
0.0
2.67E+05
0.00E+00
0.00E+00
0.00E+00
120h
Agar/Lot
CHAB*
Remel Lot 757337
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Vol Plated
mL
0.05
0.05
0.05
0.05
Chocolate
Hardy Lot E1409062
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
10
0
0
0
6
0
0
0
6
0
0
0
7.3
0.0
0.0
0.0
1.47E+06
0.00E+00
0.00E+00
0.00E+00
BCGA
LRRI Lot
12MAR09-0745
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
1
0
0
0
2
0
0
0
1
0
0
0
1.3
0.0
0.0
0.0
2.67E+05
0.00E+00
0.00E+00
0.00E+00
Dilution
Plate 1
Plate 2
Plate 3
Mean
Mean CFU/mL
2
0
0
0
0
0
0
0
0
0
0
0
0.7
0.0
0.0
0.0
1.33E+05
0.00E+00
0.00E+00
0.00E+00
*Colonies from 96h (pinpoint at 96h) were large,cream-colored, and uncharacteristic of LVS at 120h. They are not included in the 120h data.
Slide 9
Data Interpretation
DVC LVS Lot 16 Growth Characterization

LBERI results were not similar to those observed by UNM but
were similar to those observed in the previous growth
experiment using sterile water from LBERI.
Slide 10
Milestone #8 – April 2009 Accomplishments
DVC LVS Lot 16 Growth Characterization
•
Method 3- DVC method for DVC LVS Lot 16 growth
Lyophilized material was resuspended in 0.25 mL of filtersterilized water and dilutions (10-4 through 10-7) were
performed in sterile saline.
Dilutions were plated in triplicate on CHAB (UNM provided),
Chocolate, and BCGA agars.
Plates were incubated at 37˚C without CO2.
Plates were observed and colonies counted at 96 hrs.
Slide 11
DVC LVS Lot 16 Growth Characterization
96h
Vol Plated
Dilution
Plate 1 Plate 2 Plate 3 Mean
mL
1.00E+00
Insufficient volume to culture
1.00E+01
0.1
>300
>300
>300
>300
1.00E+02
0.1
>300
>300
>300
>300
1.00E+03
0.1
>300
>300
>300
>300
CHAB
Remel Lot 1.00E+04
0.1
>300
>300
>300
>300
754558
1.00E+05
0.1
234
258
183
225.0
1.00E+06
0.1
10
7
0
5.7
1.00E+07
0.1
0
0
0
0.0
1.00E+08
0.1
0
0
0
0.0
1.00E+00
Insufficient volume to culture
1.00E+01
0.01
>30
>30
>30
>30
1.00E+02
0.01
>30
>30
>30
>30
1.00E+03
0.01
>30
>30
>30
>30
CHAB
Remel Lot 1.00E+04
0.01
>30
>30
>30
>30
754558
1.00E+05
0.01
21
21
32
24.7
1.00E+06
0.01
3
4
2
3.0
1.00E+07
0.01
1
0
0
0.3
1.00E+08
0.01
0
0
0
0.0
Agar/Lot
1.00E+00
1.00E+01
1.00E+02
Chocolate 1.00E+03
Hardy Lot 1.00E+04
09062
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+00
1.00E+01
1.00E+02
Chocolate 1.00E+03
Hardy Lot 1.00E+04
09062
1.00E+05
1.00E+06
1.00E+07
1.00E+08
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
Insufficient volume to culture
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
426
366
431
407.7
35
64
82
60.3
6
12
7
8.3
2
6
7
5.0
1
0
0
0.3
Insufficient volume to culture
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
40
50
43
44.3
2
3
2
2.3
1
0
0
0.3
0
0
0
0.0
Mean CFU/mL
TNTC
TNTC
TNTC
TNTC
2.25E+08
5.67E+07
0.00E+00
0.00E+00
TNTC
TNTC
TNTC
TNTC
2.47E+08
3.00E+08
3.33E+08
0.00E+00
TNTC
TNTC
TNTC
4.08E+07
6.03E+07
8.33E+07
5.00E+08
3.33E+08
BCGA Lot
17APR20090840
BCGA Lot
17APR20090841
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+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
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
Insufficient volume to culture
>300 >300 >300
>300
>300 >300 >300
>300
>300 >300 >300
>300
>300 >300 >300
>301
118
136 Contam 127.0
16
19
2
12.3
2
0
0
0.7
0
0
0
0.0
Insufficient volume to culture
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
21
19
18
19.3
3
2
1
2.0
1
0
0
0.3
0
0
0
0.0
TNTC
TNTC
TNTC
TNTC
1.27E+08
1.23E+08
6.67E+07
0.00E+00
TNTC
TNTC
TNTC
TNTC
1.93E+08
2.00E+08
3.33E+08
0.00E+00
TNTC
TNTC
TNTC
TNTC
4.43E+08
2.33E+08
3.33E+08
0.00E+00
Slide 12
Data Interpretation
DVC LVS Lot 16 Growth Characterization

The mean LVS titer was approximately 2.00 x 108 cfu/mL
(depending on the tested media type)
–
Is within the range observed by DVC (1 to 8 x 108 cfu/ml)
with dilutions into saline.
–
Approximately 5.0 x 107 cfu/vial
 Similar to previous UNM observations


LBERI was over diluting with 1 mL water resuspension and was
most likely killing the bacteria.
The NHPs were receiving logs lower LVS in the vaccinations and
were potentially creating antibody responses to dead bacteria.
Slide 13
Decisions Made based on the
DVC LVS Lot 16 Growth Characterization



For the repeat LVS vaccination and SCHU S4 challenge study,
LBERI will resuspend like Baylor in 0.25 mL sterile water and
then use a “drop” (~60 microliters) of neat material for
vaccination by scarification. This will be the highest dose
possible from the DVC LVS Lot 16.
LBERI will use commercial CHAB plates and count on day 4, so
the UNM TVDC procedure is consistent between UNM and LBERI.
There will be one scarification dose (n=6 NHPs), 1 subcutaneous
dose (n=6 NHP; 0.12 ml of LVS diluted 1:1 with saline), and a
control group (n=3 NHPs).
Slide 14
Milestone #8 – LVS Vaccination
Plans for next month




The IACUC protocol and study protocol will be written for the
repeat vaccination/challenge study.
15 animals will be released from quarantine on May 7. Once out
of quarantine, physical exams and baseline blood draws will be
performed.
The current plan is to vaccinate animals during the week of May
17 (1 control, 2 scarified and 2 s.c. vaccinated/day on 3 separate
days).
SCHU S4 challenge (1000 CFU) will be delivered by aerosol
sometime between day 28 – 45 post LVS vaccination.
Slide 15
Milestone #11 – April 2009 Accomplishments


For the non-telemetered portion of the natural history study,
animals were challenged on April 1st and 2nd with a target
presented dose of 1000 cfu.
Each group consisted of 4 animals. Groups correlated with
scheduled terminal sacrifice (Study Days 2, 4, 5, or 6). In three
cases, animals were either found dead or euthanized.
Slide 16
Milestone #11- Non-Telemetered Portion of Natural History:
Overview of Survival and Microbiology Data
Group
1 (day 2
sac)
2 (day 4
sac)
3 (day 5
sac)
4 (day 6
sac)
Animal #
Pres.
Dose
(CFU)
Exposure
Day
Bacteremic
Term
Sac Day
Euth
or
Died Day
Bacteri
a in
Lung
Bacteri
a in
TBLN
Bacteria in
Brain
stem
Bacteria
in
Spleen
Bacteria
in Liver
√
Bacteria
in MLN
28012
183
4/1
No
√-2
√
√
√
A06977
1280
4/1
No
√-2
√
√
√
A06712
1840
4/2
No
√-2
√
√
√
A06834
5030
4/2
√-2
√
√
A06752
2240
4/1
√-4
√
√
√
√
A07129
4200
4/1
No
at
term
only
No
√-4
√
√
√
√
A06243
3320
4/2
No
√-4
√
√
√
√
A03597
4680
4/2
√-4
√
√
√
√
√
A06297
2790
4/1
√-5
√
√
√
√
√
A07126
2050
4/1
√-5
√
√
√
√
√
A07031
3380
4/2
√-5
√
√
√
√
√
A07132
7550
4/2
√-4
√
√
√
√
A06940
5420
4/1
√-5
√
√
√
√
√
A03697
1340
4/1
√-6
√
√
√
√
√
A04840
8460
4/2
√-6
√
√
√
√
√
A04925
5970
4/2
No
at
term
only
at
term
only
Day
4,5
At
term
only
Day 3,
4, 5
Day
3,5,6
At
term
only
Day
5,6
√
√
√
√
√
√-5
√
√
√
Slide 17
Milestone #11- Non-Telemetered Portion of Natural
History: Microbiology Data
Group
1 (day 2
sac)
2 (day 4
sac)
3 (day 5
sac)
4 (day 6
sac)
Animal #
Pres.
Dose
(CFU)
Exposure
Day
28012
183
4/1
Euth or
Died Day
2
A06977
1280
4/1
A06712
1840
A06834
Bacteria
in Lung
Bacteria
in TBLN
7.49E+06
Bacteria
in Brain
stem
8.78E+05
BLD
Bacteria
in Spleen
Bacteria
in Liver
Bacteria
in MLN
1.14E+02
5.09E+01
BLD
2
7.34E+05
4.86E+05
BLD
BLD
BLD
BLD
BLD
4/2
2
1.74E+06
6.30E+06
BLD
1.96E+03
BLD
BLD
BLD
5030
4/2
2
3.72E+06
1.13E+06
BLD
BLD
BLD
BLD
BLD
A06752
2240
4/1
4
6.99E+07
7.69E+07
BLD
8.11E+04
5.63E+03
BLD
5.67E+01
A07129
4200
4/1
4
8.55E+07
1.42E+06
n/a
1.86E+03
2.44E+02
BLD
BLD
A06243
3320
4/2
4
5.44E+07
2.94E+07
BLD
1.91E+05
3.17E+03
BLD
BLD
A03597
4680
4/2
4
8.99E+07
1.00E+07
2.24E+04
2.24E+03
1.39E+02 1.18E+02
A06297
2790
4/1
5
2.47E+08
2.19E+08
BLD
2.56E+06
1.55E+05 2.47E+04 7.00E+02
A07126
2050
4/1
5
1.96E+08
1.94E+08
6.13E+03
4.35E+06
3.55E+04
A07031
3380
4/2
5
5.27E+08
1.08E+08
2.30E+02
7.35E+06
1.72E+05 6.79E+03 4.67E+02
A07132
7550
4/2
4
5.12E+08
3.83E+08
BLD
5.29E+05
2.63E+04
A06940
5420
4/1
5
8.67E+08
5.66E+08
3.03E+05
7.67E+08
1.77E+07 4.35E+05 3.00E+03
A03697
1340
4/1
6
3.81E+08
2.37E+06
BLD
3.32E+06
9.83E+04 1.19E+02 3.67E+03
A04840
8460
4/2
6
6.47E+07
5.30E+06
BLD
2.52E+06
1.56E+04 2.46E+02 3.00E+03
A04925
5970
4/2
5
1.56E+08
1.72E+08
2.63E+03
7.21E+06
9.68E+04 7.31E+02 2.67E+02
BLD
BLD
Bacteria
in term.
blood
BLD
BLD
2.80E+02
9.00E+01
Slide 18
Milestone #11- Non-Telemetered Portion of
Natural History Body Temperatures
Slide 19
Milestone #11- Non-Telemetered Portion of
Natural History Respiratory Rates
Slide 20
Milestone #11- Non-Telemetered Portion of
Natural History Body Weights
Slide 21
Milestone #11- Data Interpretation




The trends in respiratory, body temperatures, and body weights
are very similar across all animals. Similar trends were observed
in Waves 1 and 3 of the ED50 (presented doses above 200 cfu).
No dramatic differences in weight were observed over the 6 day
period.
SCHU S4 cfu were consistently very high in lung and TBLN but
were more variable in the spleen and liver. Similar results were
observed in the ED50.
The time to detectable bacteremia did not correlate with
presented doses.
Slide 22
Milestone #11
Plans for next month
•
Final clinical chemistry and hematology data was obtained last
week. We will plot this data and give results post analysis.
•
Animals for telemetered portion arrive in early June. Quarantine
will end early July and surgeries will be scheduled.
Slide 23
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:
Works for
Con A and
LVS
ELISPOT
Determine
protein:CFU
relationship in
FF and HK LVS
antigens
Plasma
IgG
ELISA
Plasma
IgA
ELISA
Slide 24


Milestone #12/13 – April 2009
Accomplishments
Performed LVS CFU:protein content assay
Constructed a positive control LVS-vaccinated plasma sample
for use in IgG anti-LVS ELISA assays
Slide 25
Milestone #12/13 - Immune Responses in
Animals and Humans
LVS CFU and Protein Content Data


Our goal is to establish the correlation between LVS CFU and protein
content
–
When fixed or heat-killed LVS is prepared, the actual CFU/ml
can only be estimated from the starting (live) material; we
cannot be sure that no loss occurred during preparation
–
By measuring the protein content of such preparations, we can
relate it to CFU/ml
We ran the assay during the week of 4/27
–
LVS was grown overnight in Chamberlain’s broth
–
Serial dilutions were made, LVS was plated for CFU
determination and aliquots were tested for protein content
–
LVS was also formalin-fixed and heat-killed and aliquots were
tested for protein content
Slide 26
Protein Standard Curve and LVS Interpolation
1.6
1.4
1.2
OD600
1
0.8
y = 0.0007x + 0.106
R2 = 0.9927
0.6
0.4
0.2
0
0
500
1000
1500
2000
2500
micrograms/ml
LVS: 1.74 mg/ml (OD 1.322) = 9.8 x 108 CFU/ml ; 0.21 mg/ml (OD 0.254) = 2.45 x 108 CFU/ml;
therefore, 4 fold dilution in bacteria led to a 8.3 fold dilution in protein
HK LVS: Below detection (OD 0.014) = 9.8 x 108 CFU/ml; FF LVS: Below detection (OD 0.07) = 2 x
Slide 27
107/ml
Update on Positive Control NHP Plasma




We wished to construct a positive control NHP plasma that had
high levels of IgG anti-LVS activity for use as a standard curve in
future ELISAs
Plasma from nine LVS vaccinated NHPs was pooled and run on
two separate occasions in the IgG anti-LVS ELISA
We assigned the pooled plasma an arbitrary value of 100,000
units/ml IgG anti-LVS
Aliquots were prepared and frozen for future use
Slide 28
IgG anti-LVS Plasma – Positive Control
Slide 29
Milestone #12/13 - Immune Responses in
Animals and Humans
Plans for next month
Repeat the LVS CFU:Protein content assay
- Make 1:1 dilutions rather than 1:9 dilutions
- Sonicate the LVS in lysis buffer to possibly elaborate more
protein from the HK and FF preparations
Begin to re-titrate the WT and mutant LVS antigens based on
protein content
Construct negative control plasma sample for use in IgG antiLVS ELISAs
Slide 30
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 31
Milestone #21 Accomplishments this month




Attempted to stimulate PBMCs from two LVS-vaccinated NHPs
(scarified in October 2008) with HK LVS to elicit IFNγ production
detectable by intracellular cytokine staining
PBMCs were stimulated for 85 hours before addition of brefeldin
A for the final 4 hours
PBMCs were viable and surface staining with anti-CD3, anti-CD4
and anti-CD8 was readily detectable
No detection of IFNγ, IL-2 and TNFα was apparent in LVSstimulated cells above unstimulated cells (very low levels in all
samples)
Slide 32
Milestone #21 Upcoming work in the next
month
Repeat ICCS assay and include a positive mitogen control (Con
A); use PBMCs from newly vaccinated NHPs (for MS8)
Test 20 – 48 hours of stimulation with LVS
Slide 33
Action Items






Trevor: For LVS titers, will resuspend DVC Lot#16 LVS vials in 0.25ml sterile
water /vial, perform dilutions in saline, per the DVC protocol.
Julie W: presenting MS 8 IACUC amendments on 5/6 to committee and writing
the study protocol for the repeat vaccination/challenge study using highest
possible LVS dose, scarification and subcutaneous routes.
Michelle V: will look for a clinical indicator tied to the telemetered temperatures
in next arm of the natural history study.
Michelle V: MS 11: non-telemetered arm- will plot clinical chemistry and
hematology data in raw counts and percentages of total cells.
Julie W: for antigen prep standard curve (protein vs. CFU), will sonicate the HK
LVS pellets and do 1:1 dilutions to increase detection of protein mass.
Julie W: is making a pooled NHP, non-LVS vaccinated negative plasma
preparation too.
Slide 34