Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal
models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by Milestone
Active milestones: 2, 7, 8, 9, 10, 11(UNM &LBERI), 12/13 (UNM &LBERI), 14, 17, 18, 19, 21(UNM
&LBERI),
29 (UNM &LBERI), 35 (UNM/ASU), 36, 49, 52, 53.
Completed milestones: 1, 3, 4, 5, 6, 16, 25, 26, 27, 28, 32, 33, 34, 39, 40, 43, 48, 50, 51
Inactive milestones: 15, 20, 22, 23, 24, 30, 37, 38, 54
Milestones terminated after initiation: 41, 42, 44, 46, 55, 56, and 57 (MSCR will be written)
Milestones terminated before initiation: 43 (Cerus) 45, 47, 58, and 59 (MSCR will not be written)
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/2005
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. Three UNM and possibly 6 LBERI scientists will request vaccinations in 2009.
b. USAMRIID tentative vaccination date is August 2009, pending FDA approval
c.UNM, True, LBERI, USAMMDA and USAMRIID fully executed the CRDA modification #1 to
extend the termination date from 6/29/09 to September 29, 2010.
4. Significant decisions made or pending
a. USAMRIID tentatively will resume offering vaccinations to UNM and LBERI in August 2009 if
FDA approval is given.
b. UNM (4) and LBERI (33) are vaccinated; UNM and LBERI will offer the LVS vaccinations to 9
more scientists to total up to 46.
c.Dr. Lyons received UNM IRB re-approval to allow blood draws on the vaccinated LBERI and
UNM scientists after their LVS vaccinations
5. Problems or concerns and strategies to address
a. Nine scientists could be vaccinated in 2009 if USAMRIID receives FDA approval for the new
Tularemia vaccination protocol.
b. USAMRIID may restart LVS vaccinations in August 2009 pending FDA approval
6. Deliverables completed
a. A total of 37 participants (33 LBERI and 4 UNM participants) have received the LVS vaccination
since 9/11/07.
b. 37 participants have terminated from the USAMRIID SIP, after completing the one year health
follow-up at UNM EOH.
7. Quality of performance
Excellent
8. Percentage completed
Page 1 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
76% of the scientific work is complete
9. Work plan for upcoming month
a. Schedule LVS vaccinations for 9 remaining LBERI and UNM scientists in August 2009, if
USAMRIID has FDA approval to reopen the SIP.
Milestone 5
Milestone description: Small species tested for sensitivity to LVS & generation of immunity
against a pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: 4/30/2009
3. Work performed and progress including data and preliminary conclusions
No new lab work done this month
MS 5 MSCR for mouse and associated procedures were written and are being revised
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c.Rat model completed
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming month
Complete milestone completion reports for the mouse, rat, and guinea pigs
Manuscript for rat model was accepted with revisions, and has been resubmitted for review.
Milestone 7
Milestone description: SCHU S4 ED50 in primates determined from selection of challenge
dosing
Institution: LBERI
1. Date started: 2/25/08
2. Date completed: In progress.
3. Work performed and progress including data and preliminary conclusions:
Histopathology analysis was continued.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
LD50 is 1.26 CFU of SCHU S4. The LD99 is 4.23 CFU.
Page 2 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
7. Quality of performance
Good.
8. Percentage completed
95% of the scientific work is complete.
9. Work plan for upcoming month
a. Respiratory rates and temperatures previously reported will be modified as per the format
provided by Kristin DeBord.
b. The draft pathology report will be completed by the end of May 2009 and the final pathology
report will be completed by the end of June 2009.
Milestone 8
Milestone description: LVS vaccination protection of aerosol Schu S4 validated in primates
Institution: LBERI
1. Date started: 8/15/2008
2. Date completed: In progress.
3. Work performed and progress including data and preliminary conclusions
a. LBERI observed no protection against SCHU S4 challenge, in prior LVS vaccinated NHPs.
NIAID/UNM/LBERI postulated that the LVS vaccination doses may have been too low. To
address the low LVS vaccination dose question, LBERI performed 3 different
resuspension/dilution methods to titer the DVC Lot 16 LVS. Previously, LBERI had titered
multiple log lower LVS CFU than UNM and DVC reported.
i.Method 1- UNM method for DVC LVS Lot 16 growth using sterile water from LBERI.
Lyophilized material was resuspended in 1mL of filter- sterilized 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. BCGA plates were made at LBERI because the
commercial vendor would not sell the plates to UNM or LBERI. Plates were incubated
at 37˚C with no CO2. Plates were observed and
colonies counted at 48, 72, 96, and 120 hrs.
Page 3 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
48h
Vol
Plate Plate Plate
Agar/Lot
Plated
Dilution
Mean
Mean CFU/mL
1
2
3
mL
1.00E+04
0.05
0
0
0
0.0
0.00E+00
CHAB
1.00E+05
0.05
0
0
0
0.0
0.00E+00
Lot
1.00E+06
0.05
0
0
0
0.0
0.00E+00
752793
1.00E+07
0.05
0
0
0
0.0
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
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
BCGA
Lot
12MAR090745
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
Page 4 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
72h
Agar/Lot
Dilution
Vol Plated
mL
CHAB
Lot
752793
Chocolate
Lot E1409062
BCGA
Lot
12MAR090745
Plate
1
Plate
2
Plate
3
Mean
Mean CFU/mL
1.00E+04
0.05
0
0
0
0.0
0.00E+00
1.00E+05
0.05
0
0
0
0.0
0.00E+00
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
1.00E+04
0.05
3
7
5
5.0
1.00E+06
1.00E+05
0.05
1
0
0
0.3
6.67E+05
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
1.00E+04
0.05
84
0
4
29.3
5.87E+06
1.00E+05
0.05
2
1
0
1.0
2.00E+06
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
96h
Agar/Lot
Dilution
Vol
Plated
1.00E+04
Plate
1
Plate
2
Plate
3
Mean
Mean CFU/mL
0.05
0
0
0
0.0
0.00E+00
1.00E+05
0.05
0
0
0
0.0
0.00E+00
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
1.00E+04
0.05
3
7
5
5.0
1.00E+06
1.00E+05
0.05
0
1
0
0.3
6.67E+05
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
1.00E+04
0.05
0
4
85
29.7
5.93E+06
1.00E+05
0.05
2
1
0
1.0
2.00E+06
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
mL
CHAB
Lot
752793
Chocolate
Lot E1409062
BCGA
Lot
12MAR090745
Page 5 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
120h
Agar/Lot
CHAB
Lot
752793
Chocolate
Lot E1409062
BCGA
Lot
12MAR090745
Vol Plated
mL
Plate
1
Plate
2
Plate
3
Mean
Mean CFU/mL
1.00E+04
0.05
2
6
7
5.0
1.00E+06
1.00E+05
0.05
2
4
1
2.3
4.67E+06
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
1.00E+04
0.05
3
7
5
5.0
1.00E+06
1.00E+05
0.05
0
1
0
0.3
6.67E+05
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
1.00E+04
0.05
4
85
0
29.7
5.93E+06
1.00E+05
0.05
0
1
7
2.7
5.33E+06
1.00E+06
0.05
0
0
0
0.0
0.00E+00
1.00E+07
0.05
0
0
0
0.0
0.00E+00
Dilution
Table 1. DVC Lot 16 LVS vial was resuspended in 1.0 mL filter-sterilized water from LBERI and dilutions
were performed in the same stock of filter-sterilized water. Dilutions were plated in triplicate on CHABS,
Chocolate, and BCGA plates. Plates were incubated at 37 C without CO2 and colonies were counted at
48, 72, 96, and 120 hours.
Data storage: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY07\FY07-083 (TUL-08)\LVS Growth Testing\DVC LVS Lot 16 Comparison Data
(“UNM Method” worksheet)
Based on the data reported in Table 1, LBERI results were not similar to those
observed by UNM. Additionally the 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. There were significant number of new colonies on BCGA
between 48 and 72 hrs. LBERI hypothesizes that these represented late-growth
bacteria rendered fragile during the lyophilization and resuspension process. Lastly
SCHU S4 does not present similar growth patterns.
i. Method 2- UNM method for DVC LVS Lot 16 growth using sterile water from UNM.
Lyophilized material was resuspended in 1mL of filter-sterilized 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.
Page 6 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Vol Plated
Plate
Plate 1
Plate 3
2
mL
0.05
5
3
8
0.05
1
1
0
0.05
1
1
0
0.05
0
0
0
Agar/Lot
Dilution
CHAB
Remel Lot
757337
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Chocolate
Hardy Lot
E14-09062
1.00E+04
1.00E+05
1.00E+06
1.00E+07
0.05
0.05
0.05
0.05
10
1
0
0
6
0
0
0
BCGA
LRRI Lot
12MAR090745
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
Mean
Mean CFU/mL
5.3
0.7
0.7
0.0
1.07E+06
1.33E+06
1.33E+07
0.00E+00
6
0
0
0
7.3
0.3
0.0
0.0
1.47E+06
6.67E+05
0.00E+00
0.00E+00
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
E14-09062
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
12MAR090745
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
2
0
0
0
0
0
0
0
0
0
0
0
0.7
0.0
0.0
0.0
Mean
CFU/mL
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.
Page 7 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Table 2. DVC Lot 16 LVS vial was resuspended in 1.0 mL filter-sterilized water from UNM and
dilutions were performed in the same stock of filter-sterilized water. Dilutions were plated in
triplicate on CHABS, Chocolate, and BCGA plates. Plates were incubated at 37 C with CO 2 and
colonies were counted at 96 and 120 hours.
Data storage:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08)\LVS Growth Testing\DVC LVS
Lot 16 Comparison Data (“UNM Method + CO2” worksheet)
As reported in Table 2. 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. The addition of CO2 did not impact LVS
growth.
ii. Method 3- DVC method for DVC LVS Lot 16 growth. Lyophilized material
was resuspended in 0.25 mL of filter- sterilized water, held on ice for 30
minutes and dilutions (10-1 through 10-8) 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.
96h
Agar/Lot
CHAB
Remel Lot
754558
CHAB
Remel Lot
754558
Dilution
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
Vol Plated
mL
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
Plate 1
Plate 2
Plate 3
Mean
Insufficient volume to culture
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
234
258
183
225.0
10
7
0
5.7
0
0
0
0.0
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
21
32
24.7
3
4
2
3.0
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
Page 8 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Chocolate
Hardy Lot
09062
Chocolate
Hardy Lot
09062
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
1.00E+00
1.00E+01
1.00E+02
1.00E+03
BCGA Lot
17APR2009- 1.00E+04
0840
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+00
1.00E+01
1.00E+02
1.00E+03
BCGA Lot
17APR2009- 1.00E+04
0841
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
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
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
4.08E+07
6.03E+07
8.33E+07
5.00E+08
3.33E+08
TNTC
TNTC
TNTC
TNTC
4.43E+08
2.33E+08
3.33E+08
0.00E+00
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
Page 9 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Table 3. DVC Lot 16 LVS vial was resuspended in 0.25 mL filter-sterilized water and dilutions
were performed in sterile saline. Dilutions were plated in triplicate on CHABS, Chocolate, and
BCGA plates. Plates were incubated at 37 C without CO2 and colonies were counted at 96
hours.
Data storage: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08)\LVS Growth Testing\DVC LVS
Lot 16 Comparison Data (“DVC Method” worksheet)
The mean LVS titer was approximately 2.00 x 108 cfu/mL (depending on the tested media type)
which is in the 1 to 8 x 108 range reported by DVC. Based on this data there is approximately
5.0 x 107 cfu/vial. This data is similar to previous UNM observations. Prior to using the DVC
tittering method, LBERI was over diluting the lyophilized DVC Lot#16 LVS vial with 1 mL water
resuspension and was most likely killing the bacteria due to a hypotonic condition. The NHPs
were receiving logs lower LVS in the vaccinations and were potentially creating antibody
responses to dead bacteria.
4. Significant decisions made or pending
a. For the repeat vaccination study, LBERI will resuspend the lyophilized LVS as per the DVC
method. A “drop” (60 microliters) of neat material for vaccination by scarification. This will be
the highest dose possible from the DVC LVS Lot 16.The subcutaneous dose will match the
scarification dose.
b. LBERI will use commercial CHAB plates and grow the LVS for 4 days so the UNM TVDC
procedure is consistent between UNM and LBERI.
c.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).
5. Problems or concerns and strategies to address
None
6.
Deliverables completed
7.
Quality of performance
8.
Percentage completed
9.
Work plan for upcoming month
None
Good
41% of the scientific work is complete.
a. The IACUC protocol and study protocol will be written for the repeat vaccination/challenge
study. The study protocol will be provided to UNM by 5/8/09, for review before submission to
NIAID.
b. 15 animals will be released from quarantine on May 7. Once out of quarantine, physical exams
and baseline blood draws will be performed.
c.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) for a total of 15 NHP in the study.
d. SCHU S4 challenge (1000 CFU) will be delivered by aerosol sometime between day 28 – 45
post LVS vaccination.
Page 10 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 9
Milestone description: Aerosol SOP developed for GLP transition
Institution: LBERI
1. Date started: 8/13/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
No new work was performed during the month of April.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
LBERI needs to determine what will be considered an acceptable range of delivered SCHU S4 in the
aerosol (keeping in mind that pulmonary disease is established at or above 89 CFU).
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
40% of the scientific work is complete.
9. Work plan for upcoming month
a. Month of May
i.Establish a broader acceptable challenge dose range (with input from the UNM TVDC
team and NIAID) and conduct an additional day of mock-qualification runs.
ii.Revise and complete qualification plan for the aerosol and submit to UNM for review.
New data from the mock qualification runs and NHP exposures will be incorporated
into the pre-qualification data. This will alter (i.e., improve) the acceptable criteria
(e.g., spray factors, pre- versus post-bioaerosol values, etc.) and change the current
draft version. All target challenge dose values will change since the challenge dose
has been revised from 500 to 1000 CFU presented
Milestone 10
Milestone description: Efficacy testing of vaccine candidates (LBERI) and Characterization of
selected small animal model (UNM)
Institution: LBERI /UNM
1. Date started: 1/1/2009
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. LBERI has ordered 20 NHPs for the USAMMDA study to arrive on June 1 and they should clear
quarantine in early July.
4. Significant decisions made or pending
a. The testing of the USAMMDA IND 157 vaccine will be delayed until LBERI further tests the
vaccine efficacy of the DVC LVS lot 16 used on the TVDC to date. The repeat DVC LVS
lot#16 vaccination and challenge is described under MS8.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
Page 11 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
7. Quality of performance
Good
8. Percentage completed
2%
9. Work plan for upcoming month
LBERI will receive the 20 NHP on approximately June1 for the USAMMDA LVS vaccination
study.
No new work is planned for the month of May as the experiment comparing USAMMDA IND 157
and DVC Lot 16 has been delayed.
Milestone 11
Milestone description: In vivo GLP NHP model efficacy SOP and efficacy testing of vaccine
candidates
Institution: LBERI
1. Date started: 1/16/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. The natural history study (telemetered and non-telemetered portions) will support the
development of standard operating procedures compliant with GLP that will guide
efficacy testing of multiple vaccine candidates and assure the quality of the work
performed. 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 of SCHU S4.
b. Each group consisted of 4 animals. Groups correlated with scheduled terminal sacrifice
(Study Days 2, 4, 5, or 6). In three cases, animals were found either dead or euthanized
prior to their scheduled sacrifice day.
Page 12 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Group
1 (day
2 sac)
2 (day
4 sac)
Animal
#
Pres.
Dose
(CFU)
Exposure
Day
Euth
or
Died
- Day
Bacteria
in Lung
Bacteria
in TBLN
Bacteria
in Brain
stem
Bacteria
in Spleen
Bacteria
in Liver
Bacteria
in MLN
Bacteria
in term.
blood
28012
183
4/1
2
7.49E+06
8.78E+05
BLD
1.14E+02
5.09E+01
BLD
BLD
A06977
1280
4/1
2
7.34E+05
4.86E+05
BLD
BLD
BLD
BLD
BLD
A06712
1840
4/2
2
1.74E+06
6.30E+06
BLD
1.96E+03
BLD
BLD
BLD
A06834
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
BLD
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
BLD
2.80E+02
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
BLD
9.00E+01
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
3 (day
5 sac)
4 (day
6 sac)
Table 3. Survival and Bacteremia Data. Animals were challenged with a target presented dose of 1000
cfu. Each group consisted of 4 animals which correlated with scheduled terminal sacrifice (Study Days 2,
4, 5, or 6).
Data storage: Raw Data Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY09\FY09-012 (TUL-11)\Survival and Bacteremia
Page 13 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 1. Body Temperatures. The body temperatures of each animal was measured twice daily Study
Days -7 to -1, then thrice daily thereafter.
Data storage: Raw Data Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY09\FY09-012 (TUL-11)\Body Temperatures
Figure 2. Respiratory Rates. The respiratory rates of each animal was measured twice daily Study Days 7 to -1, then thrice daily thereafter.
Data storage: Raw Data Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY09\FY09-012 (TUL-11)\Respiratory Rates
Page 14 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 3. Body Weights. Animal weights were taken on Day -14 and once daily beginning on Study Day
-1.
Data storage: Raw Data Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY09\FY09-012 (TUL-11)\Body Weights
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). There
were no dramatic differences in weight 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. Time to detectable bacteremia did not correlate with presented doses.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
For the telemetered arm of the Natural History Study, NHP need to be larger and physically weigh
more to accommodate the telemeters, thus animals greater than 2.5 kg were requested. Availability of
these larger animals is being investigated in conjunction with orders. These NHP will likely have to be
between approximately 2 and 5 years of age.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
50% of the scientific work is complete.
9. Work plan for upcoming month
a. Final clinical chemistry and hematology data was obtained last week. This data will be
plotted and results will be analyzed.
Page 15 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
b. Animals for telemetered portion arrive in early June. Quarantine will end early July and
surgeries will be scheduled.
c. Histopathology slides for lungs, spleen, liver, kidney, nasal cavity, oropharynx with tonsils,
brain, bone with marrow (rib/femur), lymph nodes (tracheobronchial, retropharyngeal,
submandibular, mesenteric, axillary and inguinal), and representative lesions will be read.
Milestone 11
Milestone description: In vivo GLP model efficacy SOPS developed in one small species
and primate and efficacy testing of vaccine candidates
Institution: UNM
1. Date started: 1/16/2008
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Percent survival
a. Experiment Cdep2 (Notebook 122, page 49-50 and Notebook 123, pages 9-23, 35, 81-83)
i. The purpose of this experiment was to determine whether CD4 and CD8 T cells are
required to protect LVS vaccinated Fischer 344 rats against i.t. SCHU S4 challenge.
Parts of this experiment were reported in the July 2008 technical report, but the
conclusions had not been presented.
ii. Rats were vaccinated s.c. with 5 x 107 LVS and challenged i.t. 28 days later with 5.84 x
104 SCHU S4. Three days before challenge and 4 days after, the rats were injected
i.p. with anti-CD4 (clone W3/25), anti-CD8 (clone OX-8) or both antibodies. As
shown in Fig. 1, depletion of CD4 or CD8 T cells caused some of the vaccinated rats
to die from infection and treatment with both antibodies together completely
eliminated protection. These results suggest that both CD4 and CD8 T cells are
required for protection, but immune CD8 T cells may play a more significant role than
CD4 T cells.
100
80
Isotype control
CD4 (W3/25)
60
CD8 (OX-8)
CD4 + CD8
40
20
0
0
10
20
30
40
Days post challenge
Figure 1. Role of CD4 and CD8 T cells in LVS-induced protection. Fischer 344 rats were
vaccinated s.c. with 5 x 107 LVS and challenged i.t. 28 days later with 5.84 x 104 SCHU
S4. Three days before challenge and 4 days after, the rats were injected i.p. with antiCD4 (clone W3/25), anti-CD8 (clone OX-8) or both antibodies. Survival was monitored
daily
b. Experiment Cdep5 (Notebook 141, pages 35-40)
i. This was a repeat of Experiment Cdep2 described above, except the OX-38 antibody
was used to deplete CD4 T cells instead of W3/25. This change was made because
we could not tell if the minimal effect of CD4 T cell depletion was due to incomplete
Page 16 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
depletion because W3/25 is an inactivating antibody. In contrast, the OX-38 antibody
is a depleting antibody and we have shown that it reduced the CD4 + population in
naïve spleen by 10% in Experiment Cdep4.
ii. Rats were vaccinated with 5 x 107 LVS and challenged 38 days later with 104 SCHU S4.
Vaccinated rats were treated with 100 g OX-8 (anti-CD8), 250 g OX-38 (anti-CD4)
or the combination of the two antibodies 7 days before challenge. As shown figure 2,
when the depletion efficiency was measured 1 day before challenge, the OX-8
treatment completely depleted the vaccinated rats of CD8 T cells, but the OX-38
antibody did not.
iii. Based on the depletion deficiency, it was not surprising that the CD8-depleted rats but
not the CD4-depleted rats showed some clinical signs of illness by 7 days post
challenge. However, none of the CD8-depleted rats died, possibly because a second
antibody treatment was not given to maintain depletion. We have since resumed
antibody treatment twice a week in the hopes that we would still be able to detect an
effect. We will report the results in the next tech report.
iv. These results clearly show that we have not yet optimized the CD4 T cell depletion
procedure.
Figure 2. Depletion of CD4 and CD8 T cells from LVS-vaccinated rats. LVS
vaccinated rats were treated with 100 g OX-8 (anti-CD8), 250 g OX-38 (anti-CD4)
or the combination of the two antibodies. 7 days after treatment, the splenocytes
were isolated and stained for CD4 and CD8. The percentages shown in the upper
left and lower right show the percent of CD8 and CD4 T cells respectively.
Page 17 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
c. Experiment PurIgG-1(L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and
Results\Gopi's experiments\PurIG-1)
i. In Experiment PtrIgG-1, we showed that purified IgG from LVS immune rat serum was
sufficient to protect Fischer 344 rats against i.t. SCHU S4 challenge to the same
extent as immune rat serum. This was a small pilot experiment and purified normal
IgG was not used as a control.
ii. The purpose of this experiment was to purify additional IgG from both immune and
normal rat serum for a well controlled experiment. Using a purification procedure
similar to the one used in PtrIgG-1, we found that the purified product still contained
contaminating proteins. When this product was purified a second time, the yield was
low. We believe these results are consistent with a less than optimal ratio of serum to
purification resin. According the manufacturer of the Melon-Gel purification kit
(Pierce), the ratio is serum to resin is important; too little resin will result in
contaminating proteins in the purified product while too much resin will result in loss of
IgG.
iii. We are in the process of optimizing the serum:resin ratio. The plan is to purify a single
volume of serum with different amounts of resin to determine the ratio that produces
the best combination of purity and yield.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
a. CD4 T cell depletion. We will try a larger dose of antibody and/or more frequent
treatments. If at the end we cannot achieve a more consistent depletion that can be
measured by flow cytometry, then we will 1) use a cocktail of W3/25 and OX-38
antibodies and 2) develop an alternative method to measuring CD4 T cell depletion
b. Purification of serum IgG. We believe the problem is due to the ratio of purification resin
and sera and we have an experiment in progress to test this hypothesis
6. Deliverables completed
a. Showed that humoral immunity can play a role in controlling low dose respiratory SCHU S4
infection
7. Quality of performance
Good
8. Percentage completed
43%
9. Work plan for upcoming month
a. Optimize CD4 T cell depletion procedure and then repeat depletion experiment in LVSvaccinated rats
b. Optimize IgG purification procedure and then repeat the protection experiment
Page 18 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 12/13
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and compared to those in other species.
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. LBERI Performed LVS CFU: protein content assay with the goal being to establish the
correlation between LVS CFU and protein content. This is due to the fact that 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 (e.g. fixation or heat
treatment). By measuring the protein content of such preparations, we can relate it to
CFU/ml.
b. We ran the assay during the week of 4/27. LVS was grown overnight in Chamberlain’s broth.
Serial dilutions were made and LVS was plated for CFU determination and aliquots were
tested for protein content and compared against an albumin standard curve. LVS was also
formalin-fixed and heat-killed and aliquots were tested for protein content.
c.Figure 5 shows that although the standard curve (albumin) worked quite well in the assay;
however, there were several problems encountered when measuring the LVS protein
content.
a.Only undiluted (10 ml LVS in broth pelleted and resuspended in 1 ml assay buffer) and
a 1:3 dilution of LVS provided enough protein to fall on the standard curve.
b.Although the LVS was diluted 4-fold, the protein content decreased by 8.3 fold.
c.Heat-killed LVS and formalin-fixed LVS produced far less protein content than
anticipated, suggesting that the lysis buffer or protein preparation was somehow not
efficient or that the formalin inhibited the assay; however, there is no reason to think
that there should be any inhibitory compounds in the HK fraction.
Page 19 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
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
Figure 5. Protein Standard (albumin) Curve. Total LVS protein content was measured by interprolation of LVS lysate
dilution OD600 values on this curve. LVS: 1.74 mg/ml (OD 1.322) = 9.8 x 10 8 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/ml; FF LVS: Below detection (OD 0.07) = 2 x 107/ml
Data storage: Raw Data \\Saturn\Group\Wilder Lab\TVDC\LVS ELISA\LVS ELISA DATA
STATVIEW\022309 30’.xls and ...03182009 30 xls.; and in the TVDC 6 (9616) bound notebook pages 86 –
87 and in the TVDC 7 (9633) bound notebook pages 8 – 9.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
The protein assay (BCA from Pierce) needs to be repeated using less than 10-fold dilutions of LVS
(possibly 2 fold so that more than 2 LVS dilutions will fall on the linear portion of the albumin standard
curve) and trying sonication of the LVS in order to better extract the protein and make the protein
available in the BCA assay.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
90% of the scientific work is complete.
9. Work plan for upcoming month
a. Repeat the LVS CFU:Protein content assay using fresh, FF and HK LVS and interpolating the
OD600 values against an albumin standard curve.
- Make 1:1 dilutions of live LVS rather than 1:9 dilutions
- Sonicate the LVS in lysis buffer to possibly elaborate more protein from the HK and FF
LVS antigen preparations
b. Construct a pooled negative control plasma sample for use in IgG anti-LVS ELISAs; this
would be used in conjunction with the positive control sample reported on last month
Page 20 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc80.2 (Notebook 128, pages 72-74) and Experiment Ftc80.3 (Notebook 128,
pages 78-80)
i. The purpose of experiments in this series is to test the Mabtech human IFN ELISpot
kit. We found in Experiment Ftc80.1 that fresh PBMC from both control and LVS
vaccinated volunteer produced IFN in response to killed LVS antigen
preparations.
ii. In Ftc80.2, we tested dilutions of formalin-fixed and heat-killed LVS from 1:100 to
1:10,000 at a constant cell number of 2.5 x 105 per well. In Ftc80.3, we titrated
cell numbers from 2.5 x 105 to 7.8 x 103 at 1:103 dilution of formalin fixed LVS
iii. As shown in Figure 3, the responses from LVS vaccinated PBMC were always higher
than unvaccinated PBMC, suggesting that the responses were tuli-specific.
iv. The results suggest that a combination of 1:104 dilution of antigen and 1.25 x 105
cells per well would produce the lowest background response from unvaccinated
PBMC. However, since this combination also reduced the assay sensitivity and
since we expect variations among the vaccinated human population, we think
that a slightly higher concentration of both cells and antigen would be better.
Page 21 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
A
B
Titration of FF-LVS
(2.5 x 105cells)
(2.5 x 10 5cells)
200
Unvaccinated
Vaccinated
300
200
100
0
1:102
1:103
1:104
Unvaccinated
Vaccinated
150
100
50
0
Antigen Dilution
C
No. of Spots
No. of Spots
400
Titration of Hk-LVS
1:102
1:103
1:104
Antigen Dilution
Titration of PBMC
(1:103 dilution of FF-LVS)
No. of Spots
100
80
Unvaccinated
Vaccinated
60
40
20
3
10
4
7.
8
x
10
4
1.
6
x
10
4
3.
1
x
10
5
x
10
6.
3
x
1.
3
2.
5
x
10
5
0
Antigen Dilution
Figur
e 3. Optimization of human IFN ELISpot assay. Human IFN ELISpot assay was
optimized by titrating the concentration of formalin fixed LVS (A) or heat killed LVS (B) at
a constant cell number of 2.5 x 105 cell or by titrating the number of cells at a constant
dilution of formalin fixed LVS of 1:103 (C).
b. Experiment Ftc86 (Notebook 130, pages 54-70)
i. The purpose of experiments in this series is to establish a correlation between the
amount of protein in an antigen preparation to the number of bacteria so that we
can normalize responses to different antigen preparations and across
experiments
ii. To establish a standard curve, LVS cultured in Chamberlin’s broth for 24 h was
serially diluted 10-fold, centrifuged, and then resuspended in Chamberlains broth
for CFU determination or lysed in sample buffer containing 62.5 mM Tris, 1%
SDS, 20% -mercaptoethanol and 10% glycerol for protein assay. We did obtain
usable results from the protein assay because the amount of -me in the sample
buffer exceeded the tolerances of the BCA Protein assay kit. We subsequently
consulted with the technical support at Pierce, who recommended the B-PER
reagent for bacterial lysis.
iii. In the second experiment, we repeated the culture as described in the first
experiment and lysed the bacteria in the B-PER reagent. The protein assay
worked, but it results fell below the detection limit after only a single 10-fold
dilution. We will perform a third experiment with a 48 h culture to increase the
starting bacteria number.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
Page 22 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
6. Deliverables completed
a. ELISA for measuring FT-specific antibody titer in vaccinated humans
7. Quality of performance
Good
8. Percentage completed
74%
9. Work plan for upcoming month
a. Repeat optimization of the human IFN ELISpot assay using similar antigen and cell
concentration as Ftc80.2 and Ftc80.3
b. Continue Ftc86 experiments to establish a correlation between number of LVS and protein
content.
Milestone 14
Milestone description: Assays in vaccinated humans validated (sensitivity)
Institution: UNM/LBERI
1. Date started: 2/29/2008
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
No work was done
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
None
7. Quality of performance
NA
8. Percentage completed
5%
9. Work plan for upcoming month
a. All work is contingent on the availability for assays and reagents described in MS21
b. Determine the titer of anti-Ft antibodies in sera from convalescent tularemia patients on
Martha’s Vineyard
c. Quantify the number of IFN producing cells in PBMCs from convalescent tularemia
patients on Martha’s Vineyard
Milestone 17
Milestone description: In vitro assay for analysis of cellular and humoral elements of the
immune response in vaccinated human and animal’s response to F. tularensis established
Institution: UNM
1. Date started: 2/29/2008
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Page 23 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
No work was done during this 6-month period because we are waiting for human and rat assays to be
developed so that we can selectively deplete effector subsets in vitro
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
None
7. Quality of performance
NA
8. Percentage completed
0%
9. Work plan for upcoming month
No work planned
Milestone 18
Milestone description: Role of specific  T cells in protection
Institution: UNM/LBERI
1. Date started: 7/1/08
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No work was done
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
None
7. Quality of performance
NA
8. Percentage completed
5%
9. Work plan for upcoming month
No work planned
Milestone 19
Milestone description: Interaction between human alveolar macrophages and F. tularensis
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No new work done this period
4. Significant decisions made or pending
Terminate work with human alveolar macrophages if all required AM contract work has been
completed
Page 24 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
Characterized the uptake and bacterial replication in alveolar macrophages and the anti-bacteria
activities of alveolar macrophages after cytokine stimulation
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month
Review contract documents to ensure all proposed objectives in this milestone have been completed.
Milestone 21
Milestone description: Correlates of protection: in vitro assay or other readout of effector
function of Ft developed for multiple species.
Institution: LBERI
1. Date started: 4/8/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. The goal of this milestone is to identify an assay that can be used in multiple species and that
identifies a read out of vaccination state that correlates with protection from SCHU S4
induced disease. Currently LBERI thinks that production if IFN gamma might be such an
assay. LBERI is developing an intracellular cyotkine staining assay that will measure the
amount of IFN gamma being secreted by individual cells after ex-vivo stimulation with LVS or
SCHU S4 antigens. LBERI 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.
b. PBMCs were stimulated for 85 hours before addition of brefeldin A for the final 4 hours.
c. PBMCs were viable and surface staining with anti-CD3, anti-CD4 and anti-CD8 was readily
detectable.
d. No greater detection of IFNγ, IL-2 and TNFα was apparent in LVS-stimulated cells above the
levels detected in unstimulated cells (very low levels in all samples).
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
85 hours of stimulation is likely too long to see intracellular cytokine staining; we need to test
shorter incubation periods with LVS (approximately 20 – 48 hours) and also include Con A as a
positive control.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
3% of the scientific work is complete
9. Work plan for upcoming month
a. Repeat ICCS assay and include a positive mitogen control (Con A); use PBMCs from newly
vaccinated NHPs (for MS8)
b. Test 20 – 48 hours of stimulation with LVS
Page 25 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 21
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment FT-AH-17 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and
Results\Andrew's experiments\FT-AH-17)
i. In previous experiments, we showed that stimulation of human PBMC with TNF and
IFN reduced SCHU S4 growth in culture. Since these were proof-of-principle
experiments to show that under optimal conditions human PBMC can control SCHU
S4 growth, we added fresh cytokines daily from 2 days before infection until the cells
were harvested 3 days after infection. Since this level of cytokine may not be
achievable by immune PBMCs responding to SCHU S4 in vitro, we wanted to know if
a single dose of cytokines 2 days before infection would be sufficient to control SCHU
S4 growth. We also wanted to repeat a previous experiment to determine whether
pre-stimulation of immune PBMC with killed LVS antigen as a specific antigen would
be sufficient to control SCHU S4 growth
ii. In this experiment, PBMCs were pre-stimulated with heat-killed LVS at MOI=10,
formalin-fixed LVS at MOI=10, or recombinant human IFN and TNF at 100U/ml for
48 hours before infection with SCHU S4 at MOI=1.
iii. As shown in Fig. 4, a single dose of IFN and TNF, but not heat killed or formalin
fixed LVS, at 48 h prior to infection was sufficient to activate both vaccinated and
unvaccinated PBMC to control SCHU S4 growth.
iv. In this experiment, when SCHU S4 growth was calculated as fold increase (CFU at
72 h / CFU at time 0), there was a significant difference in SCHU S4 growth between
unvaccinated PBMC (12,400x) and LVS-vaccinated PBMC (600x). We are analyzing
previous experiments to find out if this holds true for other comparisons between
vaccinated and unvaccinated PBMC
Page 26 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Unvaccinated
Vaccinated
8
8
7
7
6
6
total CFU / well
total CFU / well
Vacc PBMCs: ctrl
Vacc + IFNg + TNFa
Vacc + HK-LVS
5
5
4
4
3
3
2
Vacc + FF-LVS R1
2
0
72
Hours post-infection
0
72
Hours post-infection
Figure 4. Control of SCHU S4 growth by PBMC with 48 pre-stimulation with cytokines
or killed LVS antigens. 2.5 x 106 PBMCs were stimulated with heat-killed LVS (MOI=10),
formalin-fixed LVS (MOI=10) or recombinant human IFN and TNF (100U/ml). After 48
hours, PBMCs were infected with Schu4 at MOI=1. At 3 hours and 72 hours postinfection, cells were lysed in 0.1% sodium deoxycholate and the total bacteria in the cell
lysates was determined. Points and error bars indicate mean +/- standard deviation
from 2 replicate wells at 3 hours post-infection, and 6 replicate wells at 72 hours postinfection
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
60 %
9. Work plan for upcoming month
a.Review previous experiments comparing LVS vaccinated and unvaccinated PBMC to
determine whether the vaccination status can be determined by the fold-increase of SCHU
S4 growth over 72 h.
b.Determine the minimum amount of recombinant IFN and TNF required to control SCHU S4
growth
c. Determine method to maximize IFN and TNF production by PBMC
Page 27 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 29
Milestone description: Analysis of T cells from lymph nodes & T cell epitopes
Institution: LBERI
1. Date started: 8/7/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. No new work was performed during the month of April.
b. UNM transferred 35 vials of DVC LVS Lot#16 to LBERI in April 2009
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8.
Percentage completed
16% of the scientific work is complete
9. Work plan for upcoming month
a. We are planning to boost the LVS immunity of the one primate remaining that was vaccinated
with LVS in October 2008 by sub-cutaneous inoculation. The boost will occur by injecting
0.12 ml of DVC Lot #16 LVS diluted 1:1 with saline via the sub-cutaneous route.
Milestone 29
Milestone description: Analysis of T cells from NHP lymph nodes and T cell epitopes
Institution: UNM
1. Date started: 10/1/08
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. When we screened ASU’s F. tularensis peptide library, we found several potential
stimulatory peptides but most of them were not reproducible in the replicate well or the
2nd tissue (spleen and lymph node). Since there was some concern about our analysis
method and the possibilities of false positive/negative, we sent our raw data to Phil
Stafford, the statistician at ASU, for more statistical analyses
b. Experiment Ftc82 (Notebook 139, pages 8-9)
i. The purpose was to determine how many viable NHP lymph node cells and
splenocytes can be recovered from liquid nitrogen and be used to screen individual
peptides from ASU’s peptide library
ii. We recovered 61% viable splenocytes and 53% viable LN cells. There are enough
splenocytes for 656 wells and LN cells for 360 wells
4. Significant decisions made or pending
a. We will identify a few pools of stimulatory peptides and test individual stimulatory peptides
from these pools with LN cells and splenocytes frozen from Experiment Ftc82
5. Problems or concerns and strategies to address
NA.
6. Deliverables completed
Page 28 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
None
7. Quality of performance
Good
8. Percentage completed
15%
9. Work plan for upcoming month
a. Test frozen LN cells and splenocytes for responsiveness to HK and FF-LVS
b. Test individual stimulatory peptides from the stimulatory pools identified by Phil Stafford
with LN cells and splenocytes frozen from Experiment Ftc82
Milestone 35
Milestone description: Array hybridization with mouse RNA from virulent SCHU S4
infection and RT PCR confirmation of candidates
Institution: UNM/ASU Johnston
1. Date started: 8/1/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
No work done
4. Significant decisions made or pending
No work done
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month
a. We will provide ASU with lung RNAs from SCHU S4 infected mice (2 experiments) and rats (1
experiment). A higher dose of SCHU S4 (1000 to 2000 CFU) at zero time point will be used in
these kinetic experiments.
Milestone 35
Milestone description: Array hybridizations with mouse RNAs from virulent Schu 4 infection &
RT PCR confirmation of candidates.
Institution: UNM/ ASU-Johnston
1. Date started: 04-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
 We have been addressing differences in hybridization signal intensities when compared to
previous hybridization results. We investigated changes to the oligos during storage or to the
slides after printing and found no problems with the microarray slides. Several potential
problems were simply technical, e.g. accidental change in hybridization temperature and
problems with microarray scanner. These technical problems have been resolved. The
scanner issues were addressed by sending the scanner back to the original company for a
factory-reset and refurbishment. The scanner has been returned to ASU and is working at
peak performance. At the same time, we investigated the target labeling process to ensure
Page 29 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
that our labeling process did not result in degradation in samples before hybridization. The
results shown in Figure 1 are Bioanalyzer analyses to evaluate RNA or cDNA integrity during
various phases of the processing. The graph in the upper left is a sample of RNA provided
by UNM and processed at ASU by RNAeasy preparation prior to LAPT amplification. The xaxis represents the time in seconds that a sample travels through the microfluidics of the
analysis chamber and is a surrogate for size determinations. We see two prominent peaks
representing the ribosomal peaks of the prepared RNA. This is an expected profile and
indicates that the RNA was well prepared and remains intact without any signs of degradation
prior to labeling. The graph in the upper right represents the size distribution LAPT-amplified
RNA. This is again an expected distribution showing a broad range of amplification sizes
with a peak between 25-30 S. The two graphs on the lower portion of the curve represent the
labeled cDNA with the unamplified sample on the left and the amplified sample on the right.
The key point is that the both the labeled fractions have similar sample distributions with a
peak between 25-30 S. This indicates that there is no degradation during the labeling
reaction and thus any changes in signal are not due to the processing and labeling of the
samples.
Figure 1. Bioanalyzer results of RNA and cDNA analysis before and after fluorescent labeling.
File locations … ASU: Notebook 966, Bioanalyzer, page 72
 While we were troubleshooting the slides/sample production and labeling, we also tested a different
hybridization chamber. In theory, a method to mix the sample and slide during hybridization
should result in better hybridization conditions. Early in milestone 34, we compared the Maui
hybridization chamber system with the static ArrayIt chambers with which we began our studies.
The Maui system has a constant mixing provided by an air-bladder that causes a mixing action
over the slide. The results at that time showed that the Maui did not provide enhanced signal
detection over the static ArrayIt hybridization system and we chose the static system. Since that
time, we acquired an Agilent hybridization oven and slide holder system. The sample is placed in
a slide coverslip with a rubber o-ring which is filled with the hybridization sample and then
clamped to the microarray slide. The process is allows for a small bubble to be formed during the
clamping. The o-ring forms a seal on the face of the microarray slide. The oven has a rotating
chamber holder so that the sample is mixed by rotation during the hybridization incubation. We
tested the capability of the Agilent system to increase hybridization signals on our current array.
A comparative scan of the same sample hybridized in the Agilent oven versus the static ArrayIt
chamber is shown in Figure 2. This slide was scanned on an older Perkin Elmer scanner during
Page 30 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
the repair of our main scanner. Comparing the median signal intensity of the array shows that the
ArrayIt chamber was markedly lower than the Agilent chamber-hybridized slide. We conclude
that the Agilent system does provide enhance signals over the static ArrayIt chamber system.
Figure 2. Comparison of hybridization signals between the Static ArrayIt chamber and the Agilent
rotational mixing chamber.
Notebook/File locations : Notebook 966, Slide QC, page 65.
4. Significant decisions made or pending
Switch from static ArrayIt hybridization chambers to Agilent chambers with rotational mixing.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
83%
9. Work plan for upcoming month
ASU will receive new, infected mouse and rat lung RNA samples in mid May and processing will begin as
samples are received.
Page 31 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 36
Milestone description: Final integration of expression data and informatics analysis
Institution: UNM/ ASU-Johnston
1. Date started: 03-01-2009
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No new array data have been acquired in the past few weeks. New SCHU S4 infected mouse and rat
lung samples are expected from UNM in mid May for processing at ASU. This milestone will continue
when new data become available.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
10%
9. Work plan for upcoming month
Continue to scan literature for virulence or antigenicity-related F. tularensis genes. Will include literature
reported genes in databases for comparisons to proteomic and transcriptome data generated at ASU.
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis (SCHU S4) (iglC,
pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F. tularensis subsp.
tularensis (SCHU S4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F. tularensis subsp.
tularensis (SCHU S4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHU S4 we need to develop tools to generate successful deletions.
Therefore, our focus is two fold, one is cloning experiments to get our target deletions into vectors that we
can use in creating these deletions and experiments with SCHU S4 itself using constructs that we believe
will allow us to make deletions into SCHU S4.
I. Cloning:
a. UTSA screened another twenty pJC84 +T20 NadM DH5α transformants from an earlier
transformation via Pst I or BamHI restriction endonuclease digestions. These results did not yield any
correct plasmids; therefore, we decided to use another enzyme to generate this construct. We
ordered an oligo where the nadM Sal I oligo sequence was used ; however, the restriction site Sal I
was removed and replaced with a Bgl II site. This new oligo will be paired with the nadM Sal I Stop
oligo (used earlier) to generate a new T20 NadM (≈3700 bp) product using the F.novicida NadM
Page 32 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
mutant’s genomic DNA as the template. This new PCR product will be cloned into pJC84 using
BamHI and Sal I restriction endonuclease sites for cloning. The Bgl II and the BamHI restriction sites
generate compatible ends for ligation. We received the new oligo and will set up the PCR reaction;
this is currently in progress. Data located in TVD UTSA Notebook 7, page 90 and 91.
b. The KEK1140 + FTT0748 intron II plasmid was confirmed to be correct by sequencing and we
designated this plasmid as pKEK1261. The FTT0748 gene is considered a putative transcriptional
regulator. The FTT0748 gene was identified by Monack, et.al. as a virulence factor of F. novicida;
and these F.novicida mutants demonstrated higher levels of pro-inflammatory cytokines in infected
macrophages and reduced virulence in mice. Data located in TVD UTSA Notebook 7, page 92. We
proceeded with a cryotransformation experiment with Schu S4 (next section).
II.
Experiments to generate mutants in Schu S4:
a. The last report indicated various potential NadM mutants which were selected for further
passaging to generate more isolated clones. The clones N2F,3-1E3, N3-4B,4-33g5, N3-4B,433h3, N7-6,4-23A2 and N7-6,5-3D4 were used to continue with passaging to generate single
colonies. These single colonies were used to prepare genomic isolations and used in a
polymerase chain reaction using the earlier mentioned oligos:
nadM-NcoI: 5’- cgcgcgccatgggcatgtatgatatttcagtttttataggaagatttcag -3’
nadM-EcoR1: 5’- cggaattcttatagtttcttaccacattcctctaataaaatc -3”
These oligos will yield the mutant profile of ≈1900 bp for the complete correct NadM mutant and
the wild type profile with be ≈1000 bp (Figures 1). We used some name abbreviations to make
the figure easier to read: N2F, 3-1E3 is designated as N2F-3; N3-4B, 4-33g5 is designated as N33g5; N3-4B, 4-33h3 have clones designated at N-33H3A (thru D); N7-6,4-23A is designated as
N4-23A2; and N7-6,5-3D4 is designated as N5-3D4.
Figure 1.
Kb
1 2 15 16 17 18 19 20 21 22 23 24 25 26
2.0
0.7
2.2
Legend
1. 1 Kb Ladder 14. N-33H3D
2. KKT1 15. N7 orig
3. N2F-3 16. N4-23A2
4. N2F-3A 17. N4-23A2A
5. N2F-3B 18. N4-23A2B
6. N2F-3C 19. N4-23A2C
7. N-33g5 20. N5-3D4
8. N-33g5A21. N5-3D4A
9. N-33g5B22. N5-3D4B
10. N-33g5C 23. N5-3D4C
11. N-33H3A 24. N5-3D4D
12. N-33H3B 25. N5-3D4E
13. N-33H3C 26. N7-6, 5-3D
0.4
1
2 3
4 5 6 7
8 9 10 11 12 13 14
Figure 1 represents the PCR profiles of various cycled NadM Schu S4 mutant candidates using the
forward and reverse NadM oligos. The correct Schu S4 NadM mutant should yield only one pcr product
of ≈1900 bp. The wild type profile (lane2) will show ≈1000 bp pcr product. Lanes 3, 7 15, 16, 20 and 26
are previously passaged clones which were run as controls to check banding profiles. The remaining
lanes represent isolated Schu S4 NadM clones that were on cycle 8 or 9. This gel profile is not very
clear; however, it is evident that there are no complete NadM Schu S4 mutants. The comparison with the
respective parent (e.g. lane 20) or a previously cycled clone (e.g. lane 26) with this new test group show
Page 33 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
that a few may have a stronger mutant product and we will continue with passaging some of these
(perhaps, N2F-3A, N3-4B, 4-33h3A, N7-6,4-23A2B, N7-6,5-3D4B, C and D from this test group). Data
located in TVD UTSA Notebook 7, page 89.
b.
c.
Therefore, this coming month, we will continued to passage some select clones by streaking for
single colonies on a TSA+++ 60ug/ml kanamycin plates which will be grown and 30°C. Isolated
clones from this passage will be screen by PCR to search for the correct ≈1900 bp band without
any wild type band present (≈1000 bp).
Started a cryotransformation experiment using the new intron II FTT0748 construct, pKEK1261.
The Schu S4 strain KKT1 was grown up on TSA +++ plates from frozen stock at 37°C overnight.
These cells were then inoculated into 2 x 5 ml liquid cultures (TSB+++) and grown overnight at
37°C. These overnight cultures were centrifuged into a pellet at 8K rpms for 10 minutes. This
pellet was resuspended in 0.15M rubidium chloride to an O.D. 600 of 50 (determined by making
dilutions of the suspension). These suspended cells were used in a cryotransformation with 4 ug
of pKEK1261 the intron II FTT0748 plasmid. The cryotransformation buffer was used containing
the plasmid and this was mixed with an equal volume (60 ul) of KKT1 resuspended cells into a
sterile microcentrifuge tube. In addition, we prepared a mixture of cell suspension with only
cryotransformation buffer as our negative control. These suspensions were incubated at room
temperature for 10 minutes then quick frozen in liquid nitrogen for 5 minutes. These cells were
allowed to thaw at room temperature for 5 minutes and these cells were immediately placed on
sterile filters which had been placed on top of TSA+++ plates. These cells were allowed to
incubate at 30°C for 5 hours. At this time the cells were resuspended in 0.15M rubidium chloride
and spread on selective TSA+++ 75 ug/ml kanamycin plates and place at 30°C for a few days.
We are currently following this experiment and will report on results on next report. Data located
in TVD UTSA Notebook 7, page 93.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We have not generated the NadM Schu S4 mutant but we will continue to passage potential clones until
we get the next plasmid construct pJC84 + T20 NadM completed. We will then begin experiments with
this construct in hopes to generate this NadM mutant.
6. Deliverables completed
KKF5: igLC1 IgLC2 Schuh4: KKF10: iglD1 igLD2 Schuh4; and KKF13: VgrG1 VgrG2 Schuh4 mutants are
completed Schuh4 strains to date.
7. Quality of performance
Good
8. Percentage completed
93%
9. Work plan for upcoming month
a. Will continue with the screening of the NadM mutant which will require cycling of various clones to
facilitate the effective insertion of the NadM intron into the SchuS4 chromosome.
b. Will continue with second strategy for generating NadM Schu S4 mutant via the pJC84 mating vector
from Dr. Celli lab at the Rocky mountain labs. Once NadM T20 is cloned into pJC84 will do restriction
digestions and send for sequencing for confirmation.
c. Will continue with the FTT0748 Schu S4 mutant; by checking for cryotransformants and screening
these by using FTT0748 gene specific oligos, in addition, to oligos specific to the intron II region of this
mutant in various polymerase chain reactions.
Page 34 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 52
Milestone description: Create RecA mutants in F. tularensis subsp. tularensis(Schu S4)
Institution: UTSA
1. Date started: 9/15/2007
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
3.1 Creation of FTT1579 and FTT0523 gene mutants in Schu S4. FTT1579 functions as a Type III
restriction enzyme, and FTT0523 as the hypothetical protein that is similar to Q89Z57 Type I
restriction enzyme EcoAI specificity protein. Both of FTT1579 and FTT0523 genes limit the entrance
of the plasmid DNA into Schu S4 strain. The goal is to break down the restriction barriers (FTT1579
and FTT0523) of Schu S4. The method allows us to retarget these two restriction enzymes and
inactivate certain gene(s) to facilitate introduction of the plasmid DNA into Schu S4 strain.
3.1.1 In last monthly report, it was reported that 350bp PCR for gene disruption in FTN1487
(FTT1579) was amplified for both insertion sites (849/850bp and 1254/1255bp). The PCR
product was purified using QIAquick Gel Extraction Kit, and digested with the restriction
enzymes XhoI and BsrGI (New England Biolabs) at 37C for overnight. Then the digested
350bp PCR fragment was ligated into the parent plasmid pKEK1140 (digested with the same
enzymes) at 16C overnight using T4 DNA ligase (New England Biolabs). Below is the gel
picture of the digestion reaction.
Figure1: Gel picture of the digestion reaction for both 350bp PCR fragment and pKEK1140.
Figure1 legend, results and data location: Lanes 2 and 3 were 350bp PCR products for insertion site at 849/850bp
digested with XhoI and BsrGI. Lanes 4 and 5 were the PCR fragments for 1254/1255bp insertion site digested with
the same enzymes. Lanes 6 and 7 were the parent plasmid pKEK1140 cut with XhoI and BsrGI and the larger
fragment (close to 8.0kb) was used for cloning of 350bp PCR DNA. Lane 8 was undigested pKEK1140. Data
recorded on UTSA TVDC notebook #6, page 74 for figure1. The data confirmed that the 350bp PCR fragments were
ligated correctly into the pKEK1140 plasmid.
3.1.2The ligation reaction was purified using Phenol and Chloroform, and transformed into
the host cells DH5 using electroporation. Then the transformed cells were cultured in 1ml
LB liquid medium for 1 hour at 37C with shaking and subsequently plated onto LB agar
medium containing 45ug/ml X-gal and 50ug/ml Kanamycin for selection of the transformants.
After overnight incubation at 37C, white colonies were observed on the corresponding agar
medium for both transformation (849/850bp and 1254/1255bp insertion sites).
Page 35 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
3.1.3To screen white colonies mentioned above, the plasmid DNA was purified from those
white colonies using the QIAprep Spin Miniprep Kit, and digested with BglII (New
England Biolabs) at 37 for 3-4 hours.
3.1.4
Figure 2: Gel picture of miniprep plasmid DNA digested with BglII.
Figure2 legend, results and data location: Lane 5 was the parent plasmid pKEK1140 cut with BglII. It was digested
incompletely, so the uppermost band was the undigested pKEK1140. Lane2-4 were the digested plasmid #1-3 for
insertion site at 849/850bp, and lane6-11 were the digested plasmid #4-9 for insertion site at 1254/1255bp. The only
difference between the parent plasmid pKEK1140 and the mutated pKEK1140 (carrying 350bp PCR fragment) was
that the band a little bit larger than 4.0kb on lane5 (the parent plasmid) shifted slightly below 4.0kb on lane2-4 (the
mutated pKEK1140 at 849/850bp insertion site) and lane6-11 (the mutated pKEK1140 at 1254/1255bp insertion site).
Lane2-4 and lane6-11were the correct tulatron vector based on the digestion with BglII, but the sequencing needs to
be done to confirm the tulatron vector. Data recorded on UTSA TVDC notebook #6, page 77 for figure2.
4. Significant decisions made or pending
None
5.
Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed.
About 58% of scientific work completed.
9. Work plan for upcoming month
i. Send the tulatron vector (pKEK1140 carrying 350bp PCR fragment at 849/850bp or
1254/1255bp insertion site, for knockout of FTN1487or FTT1579 gene) for
sequencing.
ii. Transform the tulatron vector (pKEK1140 with 350bp PCR fragment) into FTN U112.
iii. Screen the colonies after the transformation.
Page 36 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 53A
Milestone description Phenotyping and vaccine efficacy demonstration of recA, other subsp.
tularensis mutants.
53.1: phenotyping and immunologic characterization of Ft subsp. tularensis recA and recAiglC
strains, as well as strains generated in milestone #54.
53.2: phenotyping and immunologic characterization of Ft subsp. tularensis recA plus best
attenuating mutation(s) strains (generated in milestone #52), as well as strains generated in
milestone #54.
Institution: UTSA
1. Date started: 04/01/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. Determine the LD50 of F. tularensis recA (KKT-11) and recAiglC (KKT-23) double mutant. (Note
book #9 page 29-31): Groups of C57/B6 mice (male, 4-6 weeks, 5 mice/group) were
intranasally challenged with 75 or 320 CFU of KKT-11 (SCHU S4 recA mutant) and 3X103 ,
3X104, 3X105 or 3X106 CFU of KKT-23 (SCHU S4 recAiglC double mutant). As shown in
Fig. 1, all KKT-11 challenged mice succumbed to the infection by day 6 even with a
challenge dose as low as 75 CFU, indicating high virulence of this recA mutant. On the other
hand, the recAiglC double mutant is highly attenuated as shown in Fig. 2. There is no
mortality observed at any given challenge dose and no significant weight loss of the KKT-23
infected mice. The LD50 of KKT-23 in the intranasal infection model (C57/B6 mice) was
greater than 3X106 CFU. The LD50 of the parental strain (SCHU S4) of F. tularensis has
previously determined to be less than 10 CFU in a similar i.n. infection model.
% Surv iv al
100
80
75 CFU
320 CFU
60
40
20
0
% O riginal Body Weight
0 .0 0
110
105
100
95
90
85
80
0
2 .0 0
2
4 .0 0
6 .0 0
8 .0 0
1 0 .0 0
4
6
8
10
Days after Infection
1 2 .0 0
1 4 .0 0
12
14
Fig.1. Survival of mice infected with SCHU S4 recA (KKT-11) mutant. Groups of C57BL/6
mice (male, 6-week old) were challenged intranasally with either 75 or 320 CFU of KKT-11
and monitored for survival and weight change.
Page 37 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
% Survival
100
80
3x10 3 CFU
3x10 4 CFU
3x10 5 CFU
3x10 6 CFU
60
40
% O riginal Body Weight
20
00
2
4
2
4
6
8
10
12
14
6
8
10
Days after infection
12
14
120
110
100
90
80
0
Fig.2. Survival and weight loss of mice infected with SCHU S4 recAiglC (KKT-23)
double mutant. Groups of C57BL/6 mice (male, 6-week old) were challenged
intranasally with escalating inocula (3x10 3 , 3x104 , 3x105 and 3x106 CFU) of KKT-23
to determine LD50 of this strain.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
5% of scientific work completed on milestone 53A.
9. Work plan for upcoming month
a. Evaluate the J774 phagocytosis of the F. tularensis recA , iglC and recAiglC mutants.
b. Analyze the antibody profiles of mice immunized with the F. tularensis recAiglC mutant at day
28 after vaccination.
Page 38 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 53B
Milestone description: Examining the protective efficacy of LVS and two attenuated SCHU S4
mutant strains via oral vs. intradermal inoculations in the rat model;
50.1: replication of LVS, Schuh4, iglC Schuh4, and one additional attenuated Schuh4 mutant
derived in milestone 49 in rat macrophages .
50.2: protective efficacy of LVS, iglC Schuh4, and one additional attenuated Schuh4 mutant
derived in milestone 49 against Schuh4 intratracheal challenge (oral vs. intradermal
vaccinations in rats)
50.3: antigen specific cellular and humoral responses of rats following vaccination with LVS,
iglC Schuh4, and one additional attenuated Schuh4 mutant derived in milestone 49
50.4: bacterial dissemination and lung pathology of rats following vaccination with LVS, iglC
Schuh4, and one additional attenuated Schuh4 mutant derived in milestone 49
Institution: UTSA
1. Date started: 12/01/2008
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
53B-a: Replication of F. holarctica LVS and F. tularensis SCHU S4 within rat bone marrow
derived macrophages. (Note book # 10, pages 59, 62-3). We have previously reported (Jan 2009,
Mar 2009) on the replicative ability of LVS and SCHU S4 within rat bone marrow derived
macrophages. In these earlier studies, we found that under these culture conditions, both strains
were unable to replicate within these macrophages. In order to further analyze these results, the
experiments were repeated with a few alterations in the conditions. Previous studies were
performed using bacterial cultures from frozen stocks while in this instance, fresh overnight
cultures were used. Also, some bacteria were cultured in the presence of homologous rat serum
to investigate if opsonization was required for enhanced phagocytosis. Bone marrow derived
macrophages were derived from F344 rats, seeded in 96-well culture plates at a density of 2 X
105 cells per well and allowed to adhere over night. Cells were infected with fresh overnight
cultures of either F. holarctica LVS or F. tularensis SCHU S4 at 100 MOI for 2 hours. Cells were
then pulsed with Gentamicin for 1 hour to kill any remaining extracellular bacteria, after which
they were incubated at 37 degrees C in either fresh media, or media containing 10% homologous
rat serum. Cells were lysed at 3, 24, 48, or 72 hours after infection and serial dilutions of lysate
were plated on TSA plates to enumerate intracellular bacteria. Additionally, culture supernatants
from each timepoint were plated to determine if some bacteria were being released in to the
media. As shown in Figure 1, 2-3 logs of both LVS and SCHU S4 were taken up at three hours
after infection. At 24 hours, there was a slight decrease of intracellular LVS and a minimal
increase of intracellular SCHU S4 while 3-4 logs of either bacteria were present within the culture
supernatant. The numbers of intracellular organisms continued to decrease throughout the time
course with only minimal numbers recovered at 72 hours while the numbers of bacteria in
supernatant remained constant. It was also found that the presence of homologous serum did not
have an effect on either bacterial uptake or replication. These results indicate that the difference
in the metabolic status between frozen cultures (minimal uptake and replication) versus fresh
overnight cultures, as reported here, may contribute to the ability of Francisella strains to replicate
in rat macrophages.
Page 39 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 1. Rat BMDM phagocytosis of LVS and SCHU S4. F344 bone marrow derived macrophages
were infected with overnight cultures of either LVS or SCHU S4 at 100 MOI. At indicated time points,
serial dilutions of culture supernatants and cell lysates were plated for bacterial enumeration.
,
53B-b: Determination of LVS-specific serum antibodies following LVS vaccination of F344 rats
(Notebook # 10, pg. 40, 46, 61). Groups of female F344 rats (6 rats per group) were vaccinated
either orally or intradermally with 107 CFU of LVS in PBS or mock vaccinated (PBS alone). Four
weeks later, rats were bled and sera were prepared. LVS-specific antibody titers were determined
by ELISA. As shown in Figure 2, vaccinated rats exhibited high total antibody titers which were
predominantly of the IgG2a isotype while there was no detectable IgG1 response. There was no
significant difference between antibody titers of rats vaccinated orally or intradermally. There was
no binding of sera from mock vaccinated mice nor was there binding to the unrelated antigen,
HEL. The data shows that either route of vaccination induces an appreciable humoral response
which is TH1 biased.
Page 40 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 4/01/2009 to 4/30/2009
Due Date: 5/15/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 2. Serum antibody titers after LVS vaccination of F344 rats. Groups of female F344
rats were vaccinated either orally or intradermally with 107 CFU of LVS in PBS or mock
vaccinated with PBS alone. Four weeks later, rats were bled and serum analyzed for LVS-specific
antibody. Results represented as 50% end-point titers.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
18%
9. Work plan for upcoming month
53B. (1) Survival of F344 rats after LVS vaccination and SCHU S4 challenge.
(2) Measure fecal and respiratory antibody titers in F344 rats following either oral or
intradermal LVS vaccination
Page 41 of 41