Transcript LBERI TVDC Tech 070108 Minutes

MS#3 – Flow Diagram
MS 3: Bioaerosol Development
Collison Nebulizer
Aeromist
Micropump
Order & receive
instrument
Order & receive
instrument
Order & receive
instrument
Set up instrument
Set up instrument
Set up instrument
Frozen
LVS
Fresh
LVS
Lyophilized
LVS
Aeromist
Frozen
SCHU S4
Frozen
LVS
Fresh
LVS
Down Select for
SCHU S4 Generator
Fresh
SCHU S4
Red: completed
Green: in progress
Blue: steps in the milestone
Prepare bioaerosol SOP and
write MS completion report
Frozen
LVS
Fresh
LVS
Hospitak
Fresh
SCHU S4
Slide 1
Milestone #3 – Bioaerosol Development
Accomplishments to date


Completed Collison, Sparging Generator, Micropump and
Aeromist LVS testing (fresh vs. frozen)
Performed numerous bioaerosols with fresh and frozen SCHU S4
using the Collison and Aeromist
–


Aeromist proved slightly better; chosen as generator for
all future bioaerosols at annual site visit
Have recently tested the Hospitak nebulizer
–
Aeromist production has ceased and LBERI’s stocks have
diminished
–
Technology identical to Aeromist: disposable
compressed air jet nebulizer
Performed pathogenicity study of LVS and SCHU S4 bioaerosols
in mice
Slide 2
Milestone #3 June 2008 Accomplishments




13-Jun-08- Completed SCHU S4 bioaerosol testing (n=8) with the
Aerotech I (potential replacement for Aeromist) nebulizer
18-Jun-08- Completed SCHU S4 bioaerosol testing (n=12)
comparing the Hospitak with the Aeromist nebulizers
19-Jun-08- Completed repeat bioaerosol testing of 14MAY08 NHP
exposures (without NHPs; n=3)
23-Jun-08- Completed SCHU S4 bioaerosol testing (n=12)
comparing the Hospitak (potential replacement for Aeromist) and
Collison nebulizers
Slide 3
June 13


Objectives: To verify the predictability of our new SCHU S4
growth curve and to perform bioaerosols with the Aerotech I (a
potential Aeromist replacement) nebulizer
Growth method used for SCHU S4
–
Inoculate BCGA for colony isolation
–
Incubate for 48h at 37°C
–
Inoculate 100 mL of Chamberlain’s broth in 500 mL
baffled flask with 400 mL of suspension normalized to an
OD600 of 0.100
–
Incubate for 24h at 37°C, 200 rpm, in the dark
–
Read OD600; compare to growth curve and dilute to
appropriate concentrations
Slide 4
June 13 Results
Slide 5
June 13 Discussion

Generator suspensions
–
Concentrations were accurate
–
Demonstrates accurate growth curve
–
Titer decrease observed in post-spray




Expected: SCHU S4 is fragile
CFU/L
–
Lower than desired, though high enough for animal exposures
–
Inconsistent
Spray factors
–
Less efficient than seen with LVS
–
Inconsistent
Inconsistencies were likely the result of poor Aerotech I operation
–
Nebulizers did not behave as observed previously
–
Rapid fluid loss
–
Reasoning unknown
Slide 6
June 18


Objectives: To verify the predictability of our new SCHU S4
growth curve and to perform bioaerosols with the Hospitak (a
potential Aeromist replacement) and Aeromist nebulizers
Growth method used for SCHU S4
–
Inoculate BCGA for colony isolation
–
Incubate for 48h at 37°C
–
Inoculate 100 mL of Chamberlain’s broth in 500 mL
baffled flask with 400 uL of suspension normalized to an
OD600 of 0.100
–
Incubate for 24h at 37°C, 200 rpm, in the dark
–
Read OD600; compare to growth curve and dilute to
appropriate concentrations
Slide 7
June 18 Results
Slide 8
June 18 Discussion

Generator suspensions
–
Concentrations were accurate
–
Demonstrates accurate growth curve
–
Titer decrease observed in post-spray




Expected: SCHU S4 is fragile
CFU/L
–
Lower than desired; even lower than 13JUN
–
Achievement of 25,000 CFU delivered dose would not have been possible had NHPs
been present
–
Consistent
Spray factors
–
Less efficient than seen with LVS
–
Consistent within the same nebulizer
–
Data suggest Hospitak is slightly more efficient, though further analyses are required
Reasoning behind overall poor spray factors unknown
–
Implies need to have “control” for each day of bioaerosols; in our case, this would be
the Collison 3-jet nebulizer
Slide 9
June 19


Objectives: To repeat the Microbiology and Aerosol parameters of the
14MAY08 NHP exposures
Growth method used for SCHU S4
–
Inoculate BCGA for colony isolation
–
Incubate for 48h at 37°C
–
Inoculate 50 mL of Chamberlain’s broth in 500 mL baffled flask
with 1 CFU
–
Incubate for 48h at 37°C, 150 rpm, in the dark
–
Centrifuge at 4100 rpm for 20 min
–
Resuspend pellet in 4 mL of Chamberlain’s broth
–
Read OD600; compare to growth curve and dilute to appropriate
concentrations
Slide 10
June 19 Results
Slide 11
June 19 Discussion

Generator suspensions
–
No clumping observed as was seen in May
–
Concentrations were inaccurate

–
Titer decrease observed in post-spray



Expected: SCHU S4 is fragile
CFU/L
–
Lower than desired, but measurable (no CFU were present in the AGI samples from
14MAY08)
–
Achievement of 25,000 CFU delivered dose would not have been possible had NHPs
been present
–
Consistent
Spray factors
–
Less efficient than seen with LVS
–
Consistent
–
Acceptable


Expected due to poor growth curve
Suggests that NHP exposures (and achievement of 25,000 CFU delivered
dose) should have been possible with this method
Data further support SCHU S4 bioaerosol inconsistencies and need for further optimization
Slide 12
June 23


Objectives: To continue verification of the new SCHU S4 growth
curve and to perform head-to-head bioaerosol comparisons
between the Hospitak and Collison nebulizers
Growth method used for SCHU S4
–
Inoculate BCGA for colony isolation
–
Incubate for 48h at 37°C
–
Inoculate 100 mL of Chamberlain’s broth in 500 mL
baffled flask with 400 uL of suspension normalized to an
OD600 of 0.100
–
Incubate for 24h at 37°C, 200 rpm, in the dark
–
Read OD600; compare to growth curve and dilute to
appropriate concentrations
Slide 13
June 23 Results
Slide 14
June 23 Discussion

Generator suspensions
–
Concentrations were accurate
–
Demonstrates accurate growth curve
–
Titer decrease observed in post-spray


Expected: SCHU S4 is fragile
Less in Hospitak than in Collison
–


CFU/L
–
Higher with the Hospitak
–
Very acceptable; NHP exposures at these levels would guarantee high
delivered concentrations
Spray factors
–

Suggests Hospitak is more gentle than Collison;
expected since Aeromist was more gentle than
Collison
More efficient and consistent with Hospitak
Data suggest need to verify these results between the Hospitak and Collison
Slide 15
SCHU S4 Growth Update
6.00E+09
0.600
5.00E+09
0.500
4.00E+09
0.400
3.00E+09
0.300
2.00E+09
0.200
1.00E+09
0.100
0.00E+00
23.5
24.0
24.5
25.0
25.5
26.0
26.5
27.0
Mean OD600
Mean CFU/mL
Francisella tularensis SCHU S4 Growth in Chamberlain's Broth: 24 and 27h PostInoculation
0.000
27.5
Time (h)
24h CFU/mL
27h CFU/mL
24h OD600
27h OD600
Slide 16
SCHU S4 Growth Update (cont’d)
Hour
Mean
OD600
StDev
OD600
Mean
CFU/mL
StDev
CFU/mL
N
24*
0.385
0.094
2.72 x 109
9.44 x 108
6
27*
0.402
0.062
4.66 x 109
8.07 x 108
3
*Hours representative of mid-log phase range using
the normalized OD600 inoculum growth method
Slide 17
Milestone #3 – Bioaerosol Development
Plans for next month


Perform addition bioaerosols using the Hospitak and Collison
–
1 x 106, 1 x 107, 1 x 108, and 1 x 109 CFU/mL
–
30JUN, 1JUL, and 9JUL
Continue MS Completion report
–
95% complete
–
Awaiting finalization of SCHU S4 growth procedures in
Chamberlain’s and choice of bioaerosol method
Slide 18
MS#4 – Flow Diagram
MS 4: NHP Aerosol Confirmation
Aerosol Challenge
Approach
Naïve NHP
Challenges
Vaccinated NHP
Challenges
MS 3
Cohort 1
(n=2)
Vaccinated
NHPs available;
awaiting
completion of
naïve and LD50
challenges
Mouse
Challenges
Cohort 1
Cohort 2
Cohort 3
Cohort 2
(n=2)
Cohort 3
(n=2)
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 19
MS#4 Overview

Mouse studies
–

Naïve NHP studies
–

Purpose is to verify that aerosolized LVS or SCHU S4 are
virulent in mice (concern that aerosol method may alter
virulence)
Purpose is to verify that aerosolized SCHU S4 is virulent
in NHP (concern that aerosol method may alter virulence)
Vaccinated NHP studies
–
Purpose is to demonstrate protection in NHP previously
vaccinated with LVS
–
This study will follow determination of LD50 aerosol dose
Slide 20
Milestone #4 – NHP Aerosol Confirmation
Accomplishments to date


Completed 3 cohorts of mouse bioaerosols
–
Verified LVS and SCHU S4 virulence
–
Demonstrated no difference in SCHU S4 virulence based on 2 growth
methods in this model
–
Demonstrated lung deposition is approximately 1-5% and was greater
when the Aeromist was used as the generator
Completed 2 cohorts of naïve NHP bioaerosols
–
N=2 each
–
Virulence not as expected based on older literature

–
Hypothesized death within 7 days
–
Less than 100% mortality observed to date
–
Morbidity difficult to define
–
Mortality, when present, has occurred at greater than
or equal to 13 days post-aerosol challenge
Awaiting further SCHU S4 bioaerosol characterization and optimization
before next 2 NHPs are challenged
Slide 21
14-May-08 Challenge Blood and Tissue Culture
Data
FY07-083 Naïve Cynomolgus Macaque Francisella tularensis SCHU S4 Bioaersol Challenge
Data
Animal ID
Challenge
Date
Challenge
Dose (CFU)
Nx Date1
Blood3
Spleen
Liver
TBLN
Mes LN
Lung
A052541
14-May-08
Unknown
23-May-08
BLD
3.12E + 03
1.28E +02
1.88E +04
BLD
1.48 E +05
A052622
14-May-08
Unknown
11-Jun-08
BLD
BLD
6.67
BLD
1.45E + 03
BLD
1- Animal A05254 was euthanized on Study Day 9
2- Animal A05262 was euthanized on Study Day 28
3- Blood data presented as CFU/mL; tissue data presented as CFU/g
Note from JW: PBMCs were collected from A05262 and set up in the IFN
ELISPOT assays, however, no spots developed after stimulus with LVS; this
could be due to technical difficulties (blood collected in EDTA, not heparin;
yield was low); spleen cells were collected and frozen and will be set up with
LVS and SCHU S4 antigen preparations in the IFN ELISPOT assay in the
future
Slide 22
A05262: Chronic Pneumonia
ventral
Macaque Lungs-approx day 28
post- exposure
No other significant gross lesions
dorsal
Slide 23
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for next month

Next tentative NHP exposure has been scheduled for 15JUL
–
Dependant upon additional MS#3 bioaerosol runs using the
Collison and Hospitak
–
Bioaerosol consistency is key
Slide 24
MS#7 – Flow Diagram
MS 7: NHP SCHU S4 LD50/ED50
Round 1 (n=4 NHP each dose)
5000 CFU Delivered
500 CFU
50,000 CFU
(Based on virulence study)
Round 2 (n=4 NHP each dose)
x CFU (TBD)
y CFU (TBD)
Round 3 (n=4 NHP each dose)
x CFU (TBD)
Red: completed
Green: in progress
Blue: steps in the milestone
y CFU (TBD)
LD50/ED50
Determination
Slide 25
MS 7 Tentative Endpoints

The endpoints for each set of exposures will be clinical observations,
temperature monitoring, body weight records, gross necropsy, and
viable bacterial blood/tissue cultures
Slide 26
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
Phenotype Blood
and PBMCs
Test whether method
results in loss of B
cells
Red: completed
Green: in progress
Blue: steps in the milestone
Develop
Immunoassay
methodologies
IFN
Proliferation
assay:
Works for
Con A and
LVS
ELISPOT
Plasma
IgG
ELISA
Plasma
IgA
ELISA
IFN
Intracellular
Staining
Slide 27
Update on screening of non-LVS
vaccinated NHPs





We have determined that due to the ability of some non-LVS vaccinated
NHPs to respond to LVS antigens, we need to screen all such NHPs and
choose non- or low-responders for use in future SCHU S4 challenge
studies
In the past two months, 18 NHPs have been screened and they show a
continuum of responsiveness in the proliferation and IFN ELISPOT
assays
The only criteria we have defined as an exclusionary one is the
presence of an IgG anti-LVS titer > 5000 (found in only 1 non-LVS
vaccinated NHP thus far)
15 screened NHPs are currently available for use
40 primates from a new shipment will be screened in the coming weeks
(approximately 4 – 8/week)
Slide 28
Update on testing Cerus Freeze/Thaw Protocol on
IFN ELISPOT Assay Results


We have previously determined that the Cerus Freeze thaw
protocol spares the reactivity of PBMCs in the proliferation
assay (More data provided in the next slides)
In the past two weeks, we have thawed frozen aliquots of cells
from three separate experiments for testing in the IFN ELISPOT
assay
–
All of these used non-LVS vaccinated NHPs
–
The results will be presented in the monthly tech report
–
One experiment resulted in many spots in all the wells for
an unknown reason (2 separate NHPs)
Slide 29
1.2
1
.8
.6
.4
A05997
A05988
A05403
A05262
A05254
A04999
A04713
A04645
A04643
A04308
A04169
0
NT
.2
A02314
Percent recovery of viable cells
(Mean +/- SEM)
Percent Recovery of Viable Cells from Cerus
Freeze/Thaw Protocol
SEM results when multiple vials of the same NHP are
thawed on the same day
Slide 30
0
SCHUS4 ff Mid
SCHUS4 ff Hi
SCHUS4 ff Super
0
600000
400000
200000
SCHUS4 ff Mid
SCHUS4 ff Hi
SCHUS4 ff Super
SCHUS4 hk Mid
SCHUS4 hk Hi
SCHUS4 hk Super
LVS hk Super
Fresh
Frozen
LVS ff Lo
LVS ff Mid
LVS ff Hi
LVS hk Mid
200000
LVS hk Hi
400000
RLU (Mean +/- SEM)
TUL 34
Media
SCHUS4 ff Mid
SCHUS4 ff Hi
SCHUS4 ff Super
SCHUS4 hk Mid
SCHUS4 hk Hi
SCHUS4 hk Super
LVS hk Super
LVS ff Lo
LVS ff Mid
LVS ff Hi
LVS hk Mid
LVS hk Hi
Fresh
Frozen
SCHUS4 hk Mid
SCHUS4 hk Hi
SCHUS4 hk Super
LVS hk Super
LVS ff Lo
LVS ff Mid
Fresh
Frozen
LVS ff Hi
LVS hk Mid
800000
LVS hk Hi
0
Media
RLU (Mean +/- SEM)
600000
Media
RLU (Mean +/- SEM)
Effect of Freeze/Thaw Process on Proliferation
600000
TUL 33
400000
200000
TUL 35
TUL 33: 3 NHPS
averaged; some stimuli
not tested
TUL 34: 4 NHPs averaged
Tul 35: 2 NHPs averaged
Slide 31
MS 12/13: Work upcoming in the next month


Continue to screen naïve NHPs for their reactivity to LVS and
SCHU S4 antigens in our assays
Fully analyze the response of thawed PBMCs in the ELISPOT
assay using data from TUL 33 - 35
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
Intracellular cytokine staining




The first experiment was completed on 5/6/08
PBMCs from 3 LVS-vaccinated NHPs were prepared,
rested overnight and stimulated with either FF- or
HK-LVS or –SCHU S4 for 6 hours (last 2 hours with
Golgiplug reagent to inhibit secretion of cytokines
Cells were stained with either anti-CD3, anti-CD4,
anti-CD8 and a cocktail of anti-cytokine antibodies
(IFNγ, TNFα, and IL-2)
Data show good staining with Anti-CD8 and Anti-CD4
antibodies; spontaneous production of TNFα by
CD8+ cells, but not induced production; no
production of IL-2 or IFNγ
Slide 34
CD8+TNFα+ cells in NHP PBMC Populations
CD8+TNFα+ % of
Total PBMCs
1.2
1
.8
.6
Media
LVS hk Hi
LVS ff Hi
LVS ff Mid
LVS hk Super
.4
.2
0
A00659
A00868
A00908
CD8+ cells are approximately 40% of PBMC
population; CD4+ cells are approximately 38%
Slide 35
IFNγ ELISPOT: Response of LVS-vaccinated
NHPs does not correlate with ICCS results
Cell Mean for IFNg Spots
350
300
250
200
150
100
50
0
A00659
A00868
A00908
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
A00659 and 868 are 523 days post SC-LVS vaccination; A00908 is
532 days post ID-LVS vaccination
Slide 36
MS 21: Upcoming work in the next month

Repeat ICCS assay and include a positive mitogen control (Con
A)
Slide 37
Other Items of Note

MS 4: one LVS- vaccinated NHP (A00896) developed an extremely
aggressive behavior pattern and was becoming a danger to the animal
care technicians
–
We were told by the Veterinary staff that he would not be a good
candidate for an ABSL3 sudy and asked that we use him soon
on a study that would result in his euthanasia
–
On 6/25/08, he was administered 100,000 CFU LVS by
bronchoscopy
–
On 7/7/08, he will be euthanized; we will test his blood and
spleen cells for their reactivity to LVS and SCHU S4 antigens in
our assays; baseline data was obtained for the IFN ELISPOT
assay on 6/25; plasma was also stored
Slide 38
Other Items of Note (cont.)



All previous references to SERUM IgG anti-LVS are incorrect; in
fact, we test PLASMA IgG anti-LVS levels;
In addition, all PLASMA anti-LVS titers previously reported
should be multiplied by three due to the fact that the blood is
diluted 3-fold with PBS before separation over Lymphoprep;
PLASMA is collected from the top layer above the band of
PBMCS
Action: Bob/Michelle be reporting (on 7/7 in the tech report and
on 7/2 by email) the comparison of the initial 0.1 to 0.4 OD to the
CFU grown on plates. Rick wanted the comparison to determine
whether the 0.1 OD is accurate enough to assure sufficient CFU
in the liquid larger culture to generate an FT concentration high
enough for the sprays after 24 hrs of culture.
Slide 39
Action Items





Bob needs to break out the June 18th aerosol data by large culture flasks so “head to
head” data can be reviewed
Kristin will arrange a call with a USAMRIID scientist, Bob Sherwood and UNM to discuss
SCHU S4 growth and sprays.
Bob will look statistically at the aerosol results from the multiple “head to head”
experiments with SCHU S4 cultures being sprayed multiple generators
Bob/Michelle be reporting (on 7/7 in the tech report and on 7/2 by email) the comparison
of the initial 0.1 to 0.4 OD to the CFU grown on plates. Rick wanted the comparison to
determine whether the 0.1 OD is accurate enough to assure sufficient CFU in the liquid
larger culture to generate an FT concentration high enough for the sprays after 24 hrs of
culture.
Julie Wilder will look back at the blood count differential across the NHP used and
screened to date (did manual differential count)
Slide 40