Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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, 8, 9, 10, 11(UNM &LBERI), 12/13 (UNM &LBERI), 14, 17, 18, 19, 21(UNM
29 (UNM &LBERI), 35 (UNM/ASU), 36, 49, 52, 53
Completed milestones: 1, 3, 4, 5, 6, 7, 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
&LBERI),
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 receive LVS vaccinations in 2009.
b.USAMRIID (United States Army Medical Research Institute for Infectious Disease) tentative
vaccination date is Fall 2009, pending FDA (Federal Drug Administration) approval
4.Significant decisions made or pending
a.USAMRIID tentatively will resume offering vaccinations to UNM and LBERI in Fall 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
d.Dr. Lyons received UNM HRRC approval to allow 150ml blood draws rather than 100ml blood
draws, to increase the number of cells available for correlates of protection assays. UNM
HRRC also approved blood draws 1 month before LVS vaccination as well as up to 6 blood
draws in the 12 months post vaccination. A volunteer donor may donate blood no more than
once per month.
e.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.
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 Fall 2009 pending FDA approval
Page 1 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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.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 (Employee Occupational Health).
7.Quality of performance
Excellent
8.Percentage completed
76% of the scientific work is complete
9.Work plan for upcoming month
i.Schedule LVS vaccinations for 9 remaining LBERI and UNM scientists in Fall 2009, if
USAMRIID has FDA approval to reopen the SIP.
Milestone 8
Milestone description: LVS vaccination protection of aerosol Schu S4 confirmed 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 repeated an LVS vaccination/SCHU S4 challenge study in NHP to determine whether
the DVC Lot 16 LVS vaccine protects against a lethal SCHU S4 aerosol challenge.
b. NHPs were LVS vaccinated via subcutaneous inoculation or scarification in May 2009.
Group1, Group 2, and Group 3 were vaccinated with 1.1 x 107, 1.2 x107 and 1 x 106 CFU
LVS, respectively. The vaccinated NHPs and naïve controls were challenged with
approximately 1000 CFU SCHU S4 (target presented value). NHPs that survived to Day 21
post-challenge were euthanized. Survival data is reported in Table 1.
Page 2 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Vaccination
Cohort
Animal ID
Vaccine
Status
Date; Hour of
Challenge
SCHU S4
Presented
Dose (CFU)
Date/Hour of
Death
Notes
1 (1 x 107 LVS)
A06694
Control
7/8; 9:20 A.M.
865
7/22; 9 a.m.
(Day 14; 336 h)
Died during
morning
observations
1 (1 x 107 LVS)
A06873 (F)
Scarified
7/8; 9:45 A.M.
1240
7/15; 9 a.m.
(Day 7; 167 h)
Euthanized
morning of 7/15
1 (1 x 107 LVS)
A07386 (F)
Scarified
7/8; 10:40 A.M.
1260
7/18; 9 a.m.
(Day 10; 238.5
hours)
Found dead at
morning obs
1 (1 x 107 LVS)
A07395 (F)
S.C.
7/8; 10:00 A.M.
1410
7/29; Day 21 –
Terminal SAC
Euthanized on Day
21 - SURVIVOR
1 (1 x 107 LVS)
A07418 (F)
S.C.
7/8; 11:00 A.M
791
7/29; Day 21 –
Terminal SAC
Euthanized on Day
21 - SURVIVOR
2 (1 x 107 LVS)
A06882
Control
7/8; 11:30 A.M.
2140
7/14; 7:30 p.m.
(Day 6; 152 h)
Found dead at
evening obs
2 (1 x 107 LVS)
A06674 (M)
Scarified
7/8; 11:50 A.M.
3740
7/14; 8:40 p.m.
(Day 6; 153 h)
Found moribund;
collapsed after
evening obs;
received
anesthesia and
seized before
euthanasia
administered
2 (1 x 107 LVS)
A07682 (F)
Scarified
7/8; 1:30 P.M.
1690
7/14; 9:05 p.m.
(Day 6; 152.5
h)
Euthanized evening
of 7/14
2 (1 x 107 LVS)
A06675 (M)
S.C.
7/8; 12:50 P.M.
2610
7/14; 7:45 p.m.
(Day 6; 151 h)
Found moribund on
bottom of cage;
died before
anesthesia for
euthanasia
administered
2 (1 x 107 LVS)
A07566 (F)
S.C.
7/8; 2:00 P.M.
1250
7/14; 2:30 p.m.
(Day 6; 144.5
h)
Died during
observation period
3 (1 x 106 LVS)
A07427
Control
7/10; 10:10
A.M.
1950
7/15; ~7:30
p.m. (Day 5;
129 h)
Euthanized evening
of 7/15
3 (1 x 106 LVS)
A06693 (M)
Scarified
7/10; 10:30
A.M.
1330
7/17; 8 p.m.
(Day 7; 178.5
Euthanized evening
of 7/17
Page 3 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
hours)
3 (1 x 106 LVS)
A07686 (F)
Scarified
7/10; 10:55
A.M.
2080
7/18; 3 p.m.
(Day 8; 196
hours)
Euthanized
afternoon of 7/18
3 (1 x 106 LVS)
A06702 (M)
S.C.
7/10; 11:20
A.M.
500
7/18; 9 a.m.
(Day 8; 189.5
hours)
Euthanized
morning of 7/18
3 (1 x 106 LVS)
A07610 (F)
S.C.
7/10; 11:50
A.M.
519
7/15; ~8:30
p.m. (Day 5;
129 h
Euthanized evening
of 7/15
Table 1. Survival Data. Animals were challenged with a target presented dose of 1000 cfu. Each
vaccination group (scarification and subcutaneous) had 4 animals and the naïve control group had 3
animals.
c.
Complete necropsies with tissues being collected for histology and bacteriological burden
were performed on all animals euthanized or found dead. Tissue burden data is reported
in Table 2. Please note that this data has yet to be reviewed for quality control.
Page 4 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Vaccine
Group
Presented
Dose (CFU)
Day to
Death
A06694
Control
865
A06882
Control
A07427
Animal ID
CFU/g
Spleen
Liver
Mes LN
TBLN
Lung
14
2.16E+06
5.94E+03
3.92E+06
8.43E+06
1.41E+08
2140
6
3.71E+08
2.75E+06
1.33E+06
6.84E+08
9.38E+08
Control
1950
5
1.91E+09
2.37E+07
4.87E+08
1.40E+09
2.34E+09
A06873
Scarified
1240
7
3.03E+07
2.19E+06
3.86E+03
6.49E+06
7.84E+08
A07386
Scarified
1260
10
2.60E+08
1.38E+06
BLD
1.68E+07
7.72E+08
A06674
Scarified
3740
6
1.16E+04
2.28E+03
BLD
6.11E+03
2.28E+08
A07682
Scarified
1690
6
2.08E+03
2.50E+03
9.08E+02
3.34E+05
7.96E+07
A06693
Scarified
1330
7
3.00E+05
4.56E+04
BLD
7.82E+06
1.99E+09
A07686
Scarified
2080
8
1.17E+04
1.63E+04
1.17E+04
8.23E+04
1.96E+08
A07395
S.C.
1410
21*
BLD
BLD
BLD
1.38E+04
1.36E+04
A07418
S.C.
791
21*
BLD
5.95E+01
BLD
1.37E+04
1.38E+04
A06675
S.C.
2610
6
1.24E+05
1.73E+04
5.66E+02
6.65E+05
1.32E+09
A07566
S.C.
1250
6
8.94E+03
3.10E+03
BLD
3.01E+06
1.12E+08
A06702
S.C.
500
8
6.44E+05
6.06E+04
2.34E+07
1.39E+07
2.66E+09
A07610
S.C.
519
5
4.92E+03
2.38E+03
BLD
1.73E+06
1.27E+08
aBLD,
below limit of detection; * Terminal sacrifice
Table 2. Tissue Bacteriological Burden Data. Complete necropsies were performed on all animals
euthanized or found dead. Tissues were collected to determine bacteriological burden. Highlighted
animals survived to terminal sacrifice.
d.
Blood was collected for bacteriology and clinical chemistry on Day 3 post-challenge and
weekly thereafter. Bacteremia data is reported in Table 3. Please note that this data has
yet to be reviewed for quality control .
Page 5 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Vaccine
Group
Presented
Dose (CFU)
Day to
Death
A06694
Control
865
14
BLD
BLD
TNTC
A06882
Control
2140
6
3.33E+00
N/A
No data
A07427
Control
1950
5
BLD
N/A
TNTC*
A06873
Scarified
1240
7
BLD
N/A
1.75E+02
A07386
Scarified
1260
10
BLD
N/A
No data
A06674
Scarified
3740
6
BLD
N/A
No data
A07682
Scarified
1690
6
BLD
N/A
BLD
A06693
Scarified
1330
7
BLD
N/A
BLD*
A07686
Scarified
2080
8
BLD
N/A
BLD
A07395
S.C.
1410
21*
BLD
BLD
BLD
A07418
S.C.
791
21*
BLD
BLD
BLD
A06675
S.C.
2610
6
BLD
N/A
No data
A07566
S.C.
1250
6
BLD
N/A
No data
A06702
S.C.
500
8
BLD
N/A
TNTC
A07610
S.C.
519
5
BLD
N/A
BLD
Animal ID
Day 3
Day 10
@ Necropsy
Table 3. Bacteremia Data. Blood was collected for bacteriology on Days 3 and 10 post-challenge and
when possible, at time of necropsy.
Data Storage: Raw Data :\\saturn\ABSL3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08B)
Tabulated Data: N:\My Documents\Tularemia Contract\Prime Tech NIAID Calls\LBERI NIAID Tech Call
July 2009 final.ppt
Data interpretation:
i.DVC Lot 16 fails to protect most NHPs from a lethal aerosolized SCHU S4 challenge.
ii.The two survivors were subcutaneously vaccinated suggesting that subcutaneous
vaccination with LVS may provide slightly more protection than scarification.
iii.The control animals died between Days 5 and 15.
iv.The vaccinees died between Days 5 and 10, excluding the two NHPs that survived to
Day 21.
v.Vaccination with LVS by either route appears to lessen the SCHU S4 burden in
organs other than the lung, but does not protect the animals from death.
Statistical analysis has yet to be done on this parameter.
vi.Blood draws on day 3 post-aerosol challenge with SCHU S4 rarely yielded any
bacteremia and may be at a time point too early to be informative.
vii.Bacteremia at necropsy is also not very informative as it is often BLD (below limit of
detection) and is not available if the NHP is found dead.
Page 6 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
4. Significant decisions made or pending
Based on discussions between LBERI, UNM, and NIAID another comparative study will be performed
in which NHPs will be vaccinated subcutaneously with DVC Lot 16 (n=3), DVC Lot 17 (n=3), DVC Lot
20 (n=8), and USAMMDA IND 57 (n=8). The goal is to identify a lot of LVS that protects primates well
and that can be used as a standard vaccine for future comparisons to new vaccine candidates
identified on the TVDC.
5. Problems or concerns and strategies to address
DVC Lot 16 failed to protect most NHPs from morbidity due to aerosolized SCHU S4. Another study
will be performed as outlined above.
6. Deliverables completed
None
7. Quality of performance
Good this month
8. Percentage completed
70% of the scientific work is complete.
9. Work plan for upcoming month
a.Compile daily temperature and respiration data.
b.QC aerosol, microbiology, temperature and respiration data.
c.Write study protocol for 4 vaccine comparison study.
d.Perform physical exams and draw baseline blood from 25 naive NHPs to assess the response
of PBMCs to formalin fixed and heat-killed LVS and SCHU S4 in immunological assays and
clinical chemistry parameters. These 25 NHP will be used on the 4 vaccine comparison study
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
a.The Qualification Plan was submitted to the LBERI Compliance Group for review and approval.
b.Performed a practice SCHU S4 run. It passed the criteria outlined in the most recent Qualification
Plan. The generator suspension concentrations were uncharacteristically low even though 100200 CFU/L were obtained. This would result in presented doses of approximately 350 to 700
CFU in 3.5 L Even though the target presented dose of 1000 CFU was not obtained, the
calculated range would result in the same disease presentation (based on previous NHP studies
conducted under this contract at LBERI).
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
45% of the scientific work is complete.
Page 7 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
9. Work plan for upcoming month
a.Compliance Group will complete review of the Aerosol Qualification Plan.
b.The aerosol qualification will be initiated. We plan on completing at least one day of qualification
bioaerosol runs.
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.No new work was performed during the month of July.
4. Significant decisions made or pending
Animals arrived at LBERI during July. Due to the results of the vaccination/SCHU S4 challenge study
performed under Milestone 8 the animals will be used for an additional vaccination/challenge study
under Milestone 8.
5. Problems or concerns and strategies to address
Testing of new vaccine candidates is dependent on MS8 demonstrating that various DVC LVS lots of
vaccine can protect NHP from SCHU S4 aerosol challenge.
6. Deliverables completed
None
7. Quality of performance
None
8. Percentage completed
2% of the scientific work is completed.
9. Work plan for upcoming month
No work is planned for the month of August.
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.Final clinical chemistry and hematology data for the non-telemetered arm of the study was
plotted and data will be presented in the Milestone 11 MSCR report.
b.NHPs for the telemetered study received their third TB test and physical examinations were
performed.
c.Animals for the telemetered study were released from quarantine and surgeries were performed
to implant the telemeters.
d.Animals for the telemetered study were chair trained and telemeters were implanted surgically
in July 2009
Page 8 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
In-life non-telemetered arm of the SCHU S4 natural history study is completed.
7. Quality of performance
Good
8. Percentage completed
60% of the scientific work is complete.
9. Work plan for upcoming month
a.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 for
the non-telemetered arm of the study. It is expected that LBERI will read the slides and
prepare a histopathology report by the end of September 2009 for the non-telemetered arm
of the study.
b.SCHU S4 challenges are scheduled for August 18th and 19th for the telemetered portion of the
milestone.
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
a.This milestone examines the cellular and humoral immunity associated with effective
vaccination of Fischer 344 rats with LVS
b.Experiment PtrIgG2 (Notebook 138 pages 138-140 and 149) and Experiment PtrIgG3
(Notebook 138, pages 141-148)
i. UNM has been using passive immunization as a model system to
characterize the role of antibodies in protecting rats vaccinated with LVS
against intratracheal SCHU S4 challenge.
ii.
UNM has shown repeatedly that passive immunization of Fischer 344
rats with LVS-immune serum protected them against a low dose intratracheal
SCHU S4 challenge. To exclude the contribution of non-IgG serum
components, IgG was purified from immune serum using the Melon Gel IgG
Purification kit from Pierce and tested for protection against SCHU S4
challenge. A pilot experiment showed that sub-optimally purified immune
IgG protected 3 of 6 rats challenged with 300 SCHU S4 and suggested that
additional experiments were warranted (Fig 1A). A second experiment using
optimally purified immune IgG (conditions were reported in the July 2009
tech report) is in progress and the preliminary results shows that purified
immune IgG offers a similar level of protection as immune serum (Fig 1B).
Page 9 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Percent survival
100
Normal rat IgG
Immune rat IgG
75
50
25
0
0
10
20
30
Days Post-infection
B
Percent survival
100
75
Immue rat serum
Purified normal IgG
50
Purified Immune IgG
25
0
0
4
8
12
Days Post-infection
Figure 1. Passive immunization with purified immune IgG. IgG was purified from normal or
immune rat sera and passively transferred into naïve Fischer 344 rats (n=6). Transfer of suboptimally purified IgG protected 3 of 6 rats (A) while transfer of optimally purified IgG protected 6
of 6 rats (B).
c.Experiment Pdep-1 (Notebook 138, pages 153-155)
i.UNM has been focused on characterizing the role of humoral immunity in the
defense against SCHU S4 infection in the Fischer 344 rat model. Historical
studies in mice suggested that cellular immunity is also important for this
intracellular pathogen. One approach to study the importance of cellular
immunity is to selectively deplete T cell subsets using cell-type specific
antibodies. Previous experiments showed that weekly injections of 250 g
OX-8 antibody effectively depleted rats of CD8 T cells. Injections of W3/25
or OX-38 antibodies did not result in similar depletion of CD4 T cells, but
Page 10 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
UNM has experimental evidence that the W3/25 antibody functionally
inactivated CD4 T cells.
ii.To determine whether T cell immunity is essential for the survival of passively
immunized rats, Fischer 344 rats were treated with antibodies against CD4
and/or CD8 three day before passive immunization and i.t. SCHU S4
challenge.
iii.Figure 2 shows effective depletion of CD8 T cells. The effect of W3/25
treatment was not examined. The experiment is in progress and the results
will be presented in the September 2009 tech report.
Figure 2. Effective depletion of CD8 T cells. Naïve Fischer 344 rats were
injected i.p. with 1 mg of OX8 (anti-CD8) or isotype control antibodies. 3 days
after treatment, splenocytes were isolated from two rats from each group and
stained for CD8+ T cells.
d.
e.
Experiment Ftc65.9 (Notebook 128, pages 108-109)
i. The T cell depletion strategy will be used to determine whether cellular
immunity is essential for the survival of LVS-vaccinated rats. As a
preliminary experiment, UNM is using rats from a separate line of
experiments that had been infected with SCHU S4 and treated successfully
with the antibiotics levofloxacin. A similar study in mice showed that mice
infected with SCHU S4 and then treated therapeutically with levofloxacin
developed immunity to a subsequent SCHU S4 challenge and therefore may
be considered “vaccinated”. These “vaccinated” rats have been treated with
the T cell-depleting antibodies and challenged with 5 x 104 SCHU S4. The
results will be presented in the September 2009 tech report.
Experiment Ftc90 (Notebook 128, pages 110-111)
i. UNM is providing pre- and post-LVS vaccination sera from Fischer 344 rats
to Susan Twine at National Research Council Canada to look for
Page 11 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
immunoreactive proteins across animal species. Pre-bleeds have been
collected individually from 12 naïve rats and stored in a -80o freezer
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
58%
9.Work plan for upcoming month
a.Humoral immunity
i.Determine the antibody level after passive immunization and just before SCHU S4
challenge
ii.Repeat experiment to determine whether purified immune IgG is protective.
iii.Repeat experiment to determine the importance of T cells in passive immunization
iv.Analyze cytokine profiles associated with passive immunization
v.Determine the kinetics of T cell development in passively immunized rats
vi.Consult with biostatistician on the presentation of results on bacterial growth in
passively immunized rats
b.Cellular immunity
i.Determine the importance of CD4 and/or CD8 T cells in the protection of LVS
vaccinated rats.
ii.Develop procedures for adoptive transfer of immune T cells into naïve rats.
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
No work was performed during the month of July.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Protein assays are being tested and optimized in order to compare LVS and SCHU S4 antigen
preparation CFU to protein content. LBERI and UNM wish to standardize the quantitation of antigen
preparations. UNM has not been able to show a linear relationship between LVS protein and CFU.
Currently UNM is considering a different lysis buffer and/or procedure such as sonication.
For the microagglutination assay, UNM has requested rabbit positve sera as a control from Dr.
Marcelo Sztein at University of Maryland .
UNM has requested LVS lot9 and a protocol for staining the Ft antigen from USAMMDA . Lot 9 was
used in the past by Dr. Sztein for other NIAID studies.
6. Deliverables completed
None
Page 12 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
None in July 2009
8. Percentage completed
91% of the scientific work is complete
9. Work plan for upcoming month
a.Consult with UNM on proceeding with the LVS and SCHU S4 protein:CFU assay. UNM has
not been able to show a linear relationship between LVS protein and CFU.
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 in April.
c.Initiate the development of the microagglutination assay, per the procedure provided by Freyja
Lynn at NIAID. The procedure was from Dr. Marcelo Sztein of the University of Maryland.
Currently LBERI is waiting on the positive control antigen and sera to arrive in order to begin
the development of the assay. The antigen will need to be stained with hematoxylin once it
is received.
Milestone 12/13
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 Ftc86,4 (Notebook 130, pages 102-107), Experiment Ftc86,5 (Notebook
130, pages 108-110), Experiment Ftc86,6 (Notebook 130, 111-114), Experiment
Ftc86,7 (Notebook 130, pages 115-17)
i. An important consideration for development of immunoassays is whether
antigen preparations can be standardized and accurately quantified such that
comparisons between experiments and antigens can be made. UNM’s
approach to address this issue is to relate the protein content of antigen
preparations to bacteria number. In July, UNM performed several
experiments to optimize the conditions for extracting proteins from live LVS
using the B-PER protein extraction reagent from Pierce. The variables
included the number of starting bacteria, the volume of lysis buffer, and the
incubation time. These experiments showed that the amount of protein
extracted increased with the volume of B-PER reagent; however, the
increase in extracted protein did not keep pace with the B-PER volume and
the protein concentration was quickly diluted below detection level. Thus far,
UNM has not been able to show a linear relationship between bacteria
number and protein content. As an alternative approach, UNM will repeat
these experiments with sonication of the bacteria.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
a.UNM has not been able to show a linear relationship between bacteria number and
protein content and suspect that this problem is due to the bacterial lysis conditions.
UNM will attempt to improve protein extract efficiency by sonication of bacteria in
PBS.
6. Deliverables completed
None
Page 13 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
NA
8. Percentage completed
79%
9.Work plan for upcoming month
a.Continue to test the robustness of the IFN ELISpot assay and ELISA for human samples
when they are available. UNM’s priority is to develop the correlate of protection assay
described under milestone 21. UNM is aiming to test plasma/sera from approximately 20
unvaccinated humans and 20 LVS vaccinated humans.
b.Determine whether sonication would be better for protein extraction than achieved with the
B-PER protein extraction reagent. Establish the microagglutination assay to measure
humoral immune responses, per Freyja Lynn’s request
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 of assays and reagents described in MS21
b.Determine the titer of anti-Ft antibodies in sera from convalescent tularemia patients on
Martha’s Vineyard. This is dependent on the work being performed under MS12/13 to
quantify protein content in antigen preparations
c.Quantify the number of IFN producing cells in PBMCs from convalescent tularemia
patients on Martha’s Vineyard. This is dependent on the work being performed under
MS12/13 to quantify protein content in antigen preparations
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
No work was done during this month.
Page 14 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
4.Significant decisions made or pending
None
5.Problems or concerns and strategies to address
No work was done during this month because UNM is developing human and rat assays to selectively
deplete effector subsets in vitro , under Milestone 11.
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
UNM cannot start this milestone until a human assay is developed in MS21.
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 work was done
4.Significant decisions made or pending
UNM will try to prepare a manuscript for publication based on these findings. No additional
experiments with human alveolar macrophages will be performed unless needed for manuscript.
Page 15 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
None
6.Deliverables completed
None
7.Quality of performance
NA
8.Percentage completed
27%
9.Work plan for upcoming month
Compile data to determine how to publish the human alveolar macrophage results and whether
additional experiments are needed
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 develop a correlate of protection assay that differentiates
unvaccinated from vaccinated individuals in multiple species. LBERI used flow cytometry
to detect cell surface markers and secretion of intracellular cytokines from PBMC from
LVS vaccinated NHPs. Surface staining was done for CD3, CD4 and CD8 and the
intracellular staining was for IFNγ, TNFα and IL2. LBERI will develop the flow cytometry
assay to enhance sensitivity for the intracellular staining with the goal of distinguishing
vaccinated from unvaccinated individuals by expression of intracellular cytokines
b.Another attempt at detecting intracellular cytokine expression in NHP PBMCs by flow
cytometry was made.
i.Cryopreserved PBMCs from NHP A06873 ,collected and frozen Day 8 post-LVS
vaccination (1.1 X 107 via scarification), were thawed and stimulated at 4 X 105
cells per well for a total of 6 hours (4 hours with Goligi plug). Cells were
stimulated with either media only; 8 X 103 hkLVS or ffLVS per well; 4 X 104
hkLVS or ffLVS per well; 2 X 105 hkLVS or ffLVS per well; 1 µg PHA or Con A per
well; or 10 ng PMA/100 ng ionomycin per well. As an additional control for
cytokine staining HiCK-1 cells were permeabilized and stained along with the
NHP samples. HiCK cells are human lymphocytes that have been stimulated with
antigen and fixed, and are commercially available from BD Biosciences as a
control for intracellular cytokine staining. They should be positive for IL-2, TNF-a
and IFN-g.
ii.For automatic compensation of the Canto cytometer with FACSDiva software, antimouse Ig Compbeads were single stained with mAb-conjugates: IFN-γ-FITC,
TNF-α-PE, CD3-PerCPCy5.5, CD4-PECy7, IL-2-APC, CD8-APCCy7. Nonstained PBMC were used for voltage gain PMT settings.
a.At the first attempt to run samples, the Canto machine did not pass the
Canto software calibration, but had to be serviced, so the samples,
including compensation setup samples, had to be stored in
paraformaldehyde PFA containing buffer for 3 days which affects the
fluorescence emission, in particular from tandem flurochromes such as
APC-Cy7 and PerCP-Cy5.5.
Page 16 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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.There were some compensation issues (Table 4). A spectral overlap
compensation of more than 100% is not good. The software will still do
the compensation, but the data may be very hard to interpret. IL-2-APC
is a rat-antibody and did not bind to the anti-mouse Ig Compbeads. This
is part of the explanation for the above compensation issues. PerCPCy5.5, as a tandem-flurochrome is not stable for prolonged storage in
PFA containing buffer. Anti-rat Compbeads have been purchased, as
well as a CD3-PerCP conjugate to substitute for the CD3-PerCP-Cy5.5.
Fluorochrome
-% Fluorochrome
Spectral overlap
FITC (IFNγ)
PerCPCy5.5 (CD3)
133.58%
PE (TNFα)
PerCPCy5.5
130.6%
APC (IL-2)
PerCPCy5.5
212.78%
Table 4. Compensation Issues. Automatic compensation of the Canto cytometer with FACSDiva software
was performed by using anti-mouse Ig Compbeads that were single stained with mAb-conjugates: IFN-γFITC, TNF-α-PE, CD3-PerCPCy5.5, CD4-PECy7, IL-2-APC, CD8-APCCy7. A spectral overlap
compensation over 100% is not acceptable.
iii.Despite the above compensation issues, lymphocytes were identified by FSC/SSC
and CD4 and CD8 surface stains were normal (Figure 1).
Figure 1. PBMC gated for lymphocytes by forward scatter (FSC, equivalent to cell size) and side scatter
(SSC, equivalent to cell density or granularity) in red gate and lymphocytes further gated as CD4 positive
or CD8 positive.
iv.FSC/SSC gated lymphocytes were analyzed for IFN-γ and TNF-α (Figure 2). For
hkLVS as stimuli, there seem to be an increasing fraction of double-positive IFNγ+TNF-α+ cells with increasing antigen concentrations. However, it seems a bit
unlikely that 59% of total lymphocytes would be double positive for IFN- and
TNF- at the highest antigen concentration, even though this is a very high
concentration of antigen (0.5 bacteria per PBMC in culture well) at which we
would likely expect some background also in non-immunized individuals. The
background in media-only control is also a bit higher than preferred, but that can
be optimized with more stringent gating and titration of the antibody conjugates.
Page 17 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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 fraction of cytokine positive cells in the PMA/iono control appear too low and
also the HiCK positive control shows lower cytokine staining than expected. This
indicates that we may have a problem with the permeabilization process or the
antibody binding, which needs to be addressed by increasing permabilization
time or changing buffer for permabilization and titration of antibody conjugates.
Media only
4.4
%
1.4
8000 LVS
40 000 LVS
3.8
1.5
200 000 LVS
8.7
59.2
3.7
1.3
Figure 2. FSC/SSC gated lymphocytes analyzed for IFN-γ and TNF-α. The numbers in the Q2 quadrants
are % IFNγ+TNF-α+ out of total lymphocytes. From left to right in the panels are increasing antigen
concentrations during the 6 h stimulation.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Page 18 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
LBERI will troubleshoot the ICS assay, per the work plan outlined below.
6. Deliverables completed
None
7. Quality of performance
Fair
8. Percentage completed
8% of the scientific work is complete
9. Work plan for upcoming month
a. Repeat ICS assay and run FACS Canto immediately after PBMC staining. The
compensation will be set with anti-rat Compbeads for Il-2-APC and CD3-PerCP instead
of CD3-PerCP-Cy55 which appeared to cause large emission spectral overlaps. PerCP
is a less intense fluorochrome than PerCP-Cy5.5 and should be less sensitive to
storage.
b.Titrate CD3-PerCPCy5.5 for possible use at a more optimal staining concentration.
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.The goal of this milestone is to develop correlates of protection for vaccinated humans.
UNM appears to have developed an assay that distinguishes vaccinated and
unvaccinated individuals based the ability of vaccinated human PBMC to produce IFN
and control SCHU S4 growth after pre-stimulation with inactivated LVS antigen
preparations. The numbers of samples are quite low at this point and additional donors
(both LVS-vaccinated and unvaccinated) will be needed to increase the significance of
these findings.
b.Experiment FT-AH-22 ((L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and
Results\Andrew's experiments\FT-AH-22) and FT-AH-26
(L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and Results\Andrew's
experiments\FT-AH-26)
i.UNM tested PBMC from an additional 2 vaccinated and 1 unvaccinated donors and
the results were consistent with previously observed patterns for unvaccinated
and vaccinated donors. Both of the vaccinated PBMC cultures produced IFN 48
h after stimulation with formalin fixed LVS whereas HK-LVS and SCHU S4 had
no detectable effect on the unvaccinated PBMC culture. These results suggest
that IFN production by PBMCs in response to FF-LVS or HK-LVS could serve as
a possible clinical assay for correlate of protection to FT infection
Page 19 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Vaccinated #1.2
40000
Unvaccinated
Vaccinated #2.2
40000
40000
HK-LVS / UNINFECTED
30000
FF-LVS / UNINFECTED
FF-LVS / SCHU4
30000
HK-LVS / SCHU4
HK-LVS / LVS
30000
10000
pg/mL
20000
pg/mL
pg/mL
HK-S4 / UNINFECTED
FF-LVS / UNINFECTED
FF-LVS / SCHU4
20000
10000
0
0
24
48
72
HOURS POST-INFECTION
HK-S4 / LVS
10000
0
-24
HK-S4 / SCHU4
20000
0
-24
0
24
48
72
-24
HOURS POST-INFECTION
0
24
48
72
HOURS POST-INFECTION
Figure 3. IFN production by PBMC from LVS vaccinated and unvaccinated human donors. 2
.5 x 106 Ficoll-purified human PBMCs per well from LVS vaccinated and unvaccinated human
donors were incubated for 48 hours with formalin fixed LVS or heat-killed LVS. 48 h after prestimulation, F. tularensis strain SCHU S4 was added to wells at MOI=1 and incubated for 2
hours, followed by addition of gentamicin to 50ug/mL for 45 minutes, followed by PBS wash
and resuspension in gentamicin-free cDMEM. Supernatants were collected every 24 hours for
Luminex cytokine quantitation. Data points indicate average +/- SD of triplicate infected wells,
and duplicate uninfected wells
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
70 %
9.Work plan for upcoming month
a.Gather more data on IFN secretion and CFU reduction from both vaccinated and unvaccinated
donor
b.Determine whether addition of anti-IFN antibody prevents LVS vaccinated PBMC control of SCHU
S4 growth.
c.Identify cell types associated with IFN production by intracellular cytokine staining
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
No new work was performed during the month of July.
Page 20 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
None
8. Percentage completed
16% of the scientific work is complete
9. Work plan for upcoming month
LBERI is waiting for the UNM team to request the tissues from this animal. The NHP will be exposed
to LVS by bronchoscopy when UNM is ready to test the lymph node tissues in the peptide array assay
again.
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.
Experiment Ftc82,4 (Notebook 139, pages 10-14)
i. The goal of this milestone is to identify polypeptides derived from F.
tularensis that stimulate T cell responses and may be potential vaccine
candidates. UNM identified several potential stimulatory peptides pools in an
initial screen of the Ft polypeptide library from ASU. In order to verify the
results from the first screen, UNM selected 3 positive pools and tested the 21
polypeptides individually with vaccinated, NHP lymph node cells frozen at
the time of the initial screen. UNM was expecting only one or two
polypeptides from each of the three pools to stimulate a positive T cell
response while the remaining polypeptides would be non-stimulatory and
serve as internal negative controls. Instead, all 21 polypeptides appeared to
have stimulated a response above media control (Fig. 4). However,
statistical analyses indicated that the polypeptides in wells A10, D10, G10,
B11 and D11 were significantly more stimulatory than the remaining
peptides.
Page 21 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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 4. Screen for stimulatory F. tularensis polypeptides using non-human primate lymph node cells. 6 x 105 NHP
lymph node cells were incubated overnight with individual polypeptides in an IFN ELISpot plate and then processed
according to the manufacturer’s instructions. Each polypeptide was tested in duplicate and the results of 4 replicates
for the negative control (medium) and positive controls (heat-killed and formalin fixed LVS) are shown
4.Significant decisions made or pending
Perform repeat screen of polypeptide library with 6.0 x 105 frozen lymph node cells per well
5.Problems or concerns and strategies to address
NA
6.Deliverables completed
None
7.Quality of performance
Good
8.Percentage completed
25%
9.Work plan for upcoming month
a.Meet with the ASU group to discuss next step for testing/confirming stimulatory polypeptides
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 this period
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: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
None
7.Quality of performance
Good
8.Percentage completed
33%
9.Work plan for upcoming month
a.Isolate lung RNA from SCHU S4 infected rats 1, 3, 5, 7, and 24 h after infection. ASU
requested a repeat rat kinetics of infection experiment as the RNA from the prior
experiment didn’t amplify with the Ft specific primers at ASU
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
Current status: The early time course kinetic experimental design includes two mouse challenge
level groups (low-dose (~200-500 CFU/lung at 1 hour post challenge) and high dose (~4-5,000
CFU/lung at 1 hour post challenge) and a rat challenge (~2,000 CFU/lung at 1 hour post
challenge). Each challenge dose time course has been performed twice at UNM. Time course
samples were taken at 1, 3, 5, 7, and 24 hours post challenge. There are multiple animals per
sample time, a pool of RNA was prepared for each time point and the pool of RNA was purified
over RNAeasy columns. After purification, the RNA pools are processed using the Linear
Amplification of Procaryotic Transpcript (LAPT) protocol. The amplified RNA is processed into
cDNA, fluorescently labeled, and allowed to hybridize to the Francisella tularensis 70-mer
oligonucleotide microarray printed on slides.
 ASU will create two LAPT samples per time point RNA and perform two independent labelings per
LAPT. ASU has completed the hybridizations of the two low-dose mouse and one rat sample set.
Of the high dose mouse samples, only one of the hybridizations of one LAPT is complete. The
second LAPT amplifications for the high dose mouse samples are complete but the labeling and
hybridizations need to be performed. For the last rat experiment, the LAPT amplifications are
underway and hybridizations will ensue.
Page 23 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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. Experimental design from data sets to slides.
4.Significant decisions made or pending
None
5.Problems or concerns and strategies to address
For the data reported in Milestone 36 below, the genomic signals of the most recent arrays were
very low to non-detectable. Because of this problem ASU analyzed the data of the cDNA signal
without genomic normalization. The cDNA signals were extracted from previous experiments to
average the data from multiple LAPTs and labelings.
The last rat experiment samples failed to amplify in the LAPT process in 3 separate experiments.
The reason for this failure is unclear. ASU verified that the kits and reagents were working by
using a mouse RNA sample that had previously amplified as a positive control and this sample
was included in the second run at the same time as the new rat samples. A second RNAeasy
purification was performed with the rat RNA to ensure that the samples were adequately clean.
Bioanlayzer analysis revealed that the RNA was of good quality and not degraded. Yet, in a third
attempt the LAPT process did not amplify the samples. ASU requested a new set of infected rat
lung RNAs from UNM.
Although technically outside the timeframe of this report, ASU reports on the repair status of the
microarray printer and the first QC runs. The new microarray printer pins were received and the
printer performed well in the construction of arrays for this last remaining hybridizations. The QC
analysis showed that the microarrays are hybridizing and performing as expected and that the
both the genomic and cDNA signals are acceptable.
6.Deliverables completed
None
7.Quality of performance
Fair
8.Percentage completed
90%
Page 24 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
9.Work plan for upcoming month
Printed and QC validated microarray slides are ready for hybridizations. ASU will finish the labeling and
hybridizations of the high-dose mouse experiments. In addition, the LAPT amplifications of the last
rat experiment and the subsequent labelings and microarray hybridizations will be completed when
the new rat lung RNAs arrive.
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
The strategy for gene selection utilizes pattern mapping algorithms to determine any one of
three basic patterns of gene expression (Up, Down, and Flat) over the time course of
infection. ASU assigns the pattern in the GeneSpring analysis software so that any
signals will be low in the uninfected lung (T0) and then progress across the time course
with the defined patterns.
Because of genomic signal acquisition problem described in Milestone 35 above, we
performed analyses of the signals of the cDNA signals alone for all available slides. The
gene lists generated based on the expression patterns were then compared to the
previous gene lists selected with the genomically normalized data sets. The Venn
diagrams shown in Figures 2 and 3 are the respective high-dose mouse challenge and
rat challenge experimental analyses, respectively. Using the gene expression pattern
mapping strategy we selected the top approximately 200 genes that correlated with the
patterns. Using Venn analyses ASU identified the highly restricted gene lists by
eliminating the genes that fell on the intersections of the patterns.
Page 25 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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. Venn analyses of the patternmapped gene selected lists in the high-dose
mouse challenge experiments. The Venn
diagram on the left represents the data
analyzed using the genomic normalized
signals. The Venn diagram on the right
utilizes the data from only the cDNA signals.
Figure 3. Venn analyses of the patternmapped gene selected lists in the rat
challenge experiments. The Venn diagram
on the left represents the data analyzed using
the genomic normalized signals. The Venn
diagram on the right utilizes the data from
only the cDNA signals.
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milesto
nes\36\7-20-2009\Mouse_GN_SC.JPG
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milesto
nes\36\7-20-2009\Mouse_GN_SC.JPG
Using the restricted gene lists, the next comparison utilized Venn analyses to identify the
gene sets that are intersecting between mouse and rat experimental data sets. The
genomically normalized data (GN) and the single color (SC) data were compared
independently. The Up, Down, and Flat data are shown in Figures 4, 5, and 6,
respectively. The identifiers for the genes in the intersection between the two data sets
were displayed to the right of each figure. In all cases, the gene lists between the
genomically normalized data and the single color data were independent and did not
cross identify genes. However, this was not unexpected because of the differences
between the data selections between the GN and SC data. This experiment validates
previous results where we cross compared genomically normalized and single color data
and did not observe comparable gene lists. With the resolution of the microarray slide
production and hybridization problems the acquisition of the remaining data should be
readily obtained.
Page 26 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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 4. Venn diagrams of the restricted
gene lists for the Up pattern map to identify
genes in both the mouse and rat data sets for
both the genomically normalized (GN) and
single color (SC) data. The gene identifiers of
the genes in the intersections are displayed to
the right side of the each diagram.
Figure 5. Venn diagrams of the restricted
gene lists for the Down pattern map to identify
genes in both the mouse and rat data sets for
both the genomically normalized (GN) and
single color (SC) data. The gene identifiers of
the genes in the intersections are displayed to
the right side of the each diagram.
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milesto
nes\36\7-20-2009\Up.JPG
File locations …
R:\GeneVac\FTU\Contract\Microarray\Mileston
es\36\7-20-2009\Down.JPG
Figure 6. Venn diagrams of the restricted gene lists for the Up pattern map to identify genes in
both the mouse and rat data sets for both the genomically normalized (GN) and single color
(SC) data. The gene identifiers of the genes in the intersections are displayed to the right
side of the each diagram.
File locations … R:\GeneVac\FTU\Contract\Microarray\Milestones\36\7-20-2009\Flat.JPG
4.Significant decisions made or pending
None
Page 27 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Analysis is delayed due to troubleshooting with the microarray printing under ASU MS35.
6.Deliverables completed
None
7.Quality of performance
Fair
8.Percentage completed
20%
9.Work plan for upcoming month
As new data sets are completed, ASU will add these to the current data to average the
experimental groups for each time point. As genes of interest are identified either from
experiments in the TVDC consortium or from literature reports, these will be incorporated into
the comparison gene lists.
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.
2.
3.
Date started: April 1, 2006
Date completed: in progress
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 will allow us to make deletions into SCHU S4.
Currently, UTSA is working on creating two single gene SCHU S4 mutants, NadM and FTT0748.
UTSA uses the KKT1 SCHU S4 strain for experiments where electroporation or cryotransformation is
performed to get the plasmid constructs containing the desired gene targets into the bacteria. Once
the plasmid is in the bacteria; the entire plasmid will be incorporated into the genome of SCHU S4 (as
for pKEK1305 pJC84 T20+NadM) or only the portion directed to the gene target will be incorporated
(as for pKEK1261 “tulatron” FTT0748). The potential correct clone is initially determined by the
antibiotic selection used in the transformation experiment. Typically the gene sequence that is
specific to the target gene will have a sequence cassette, such as kanamycin, which allows for
selection of the clones which integrated at the correct location in their genome. The plasmid which
carries this target gene sequence will have a different “marker” (antibiotic resistance) which will allow
for counter selection to verify the removal of the plasmid from the bacteria. In order to verify potential
correct clones, UTSA performs polymerase chain reactions (PCRs) using genomic DNA isolated from
a potential clone and various oligo primer sets. The oligo primer sets are typically directed to an
antibiotic resistant sequence cassette, gene targets at the 5’ or 3’ end of the desired gene and origin
of replication specific oligos to check for the presence of the plasmid in the bacteria. Once the correct
mutant is identified by PCR results, some PCR products are sent for sequencing for verification.
UTSA then will remove the plasmid from the bacteria and the end result is the final mutant which will
Page 28 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
be used in subsequent experiments that will help determine if this mutant is a good vaccine
candidate. The confirmatory experiments include mouse vaccination/challenge experiments and
macrophage assays.
•Creation of the FTT0748 SCHU S4 mutant.
•Result: The FTT0748 was verified to be correct last month by sequencing and this month
UTSA removed the plasmid, pKEK1261, from the bacteria.
•1st Removal of the plasmid from the FTT0748 SCHU S4 mutant at 37ºC.
•Result: The plasmid was removed successfully from the mutant strain based on its
sensitivity to kanamycin.
•2nd The kanamycin sensitive clones were isolated and then checked by PCR to verify the correct
product size compared to the wild type SCHU S4.
•Results: Figure 1 showed the ≈1700 bp size for the FTT0748 mutant’s pcr products and
supported that the mutant is correct.
•3rd Mice were ordered for an experiment to test the attenuation of the mutant. Will be discussed on
next report.
Figure1.
Figure 1 represents PCR profiles using forward and reverse FTT0748 oligos with various genomic isolates
of kanamycin selected colonies resulting from FTT0748 C7c clone. The correct SCHU S4 FTT0748 mutant
should yield one pcr product of ≈1700 bp (lanes 3, 4, 5 and 6) compared to the wild type profile (lane 2,
KKT1) of ≈800 bp. The size difference represents the intron II sequence introduced by the pKEK1261
FTT0748 “tulatron” construct used to create this mutant. Lanes 4, 5 and 6 represents the mutants that were
determined to be kanamycin sensitive and have lost the plasmid. FTT0748 C7c1, C7c2 and C7c3 are final
mutants. Data located in TVD UTSA Notebook 7, page 113.
•Creation of the NadM SCHU S4 mutant.
•Result indicates a mixed population of clones; the NadM mutant is not isolated.
•1st Electroporation experiment with pJC84_T20 (pKEK1305) with SCHU S4 (KKT1).
•Result: Generated 40 kanamycin resistant colonies but background was detected.
•2nd UTSA patched all of the resulting colonies from the test group onto fresh kanamycin plates,
allowed these to grow and then isolated their genomic DNA.
•Result: Twenty clones were tested with the first screen using the pUC origin of replication
oligos to check for insertion of the plasmid into these clones. Of these twenty tested,
only two (clones 1 and 5) illustrated the pUC origin PCR product as compared by the
control pKEK1305.
•3rd USTA used another set of oligos using a kanamycin oligo paired with a NadM gene specific oligo
(5’end).
Page 29 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
•Result: Figure 2 shows that the test clone used did not generate the expected ≈1300 bp
product. Therefore, UTSA took only clones 1 and 5 and streaked these to generate single
clones on fresh kanamycin plates. Experiments are pending on these resultant single
clones.
Figure 2.
Figure 2 represents pcr profiles generated using two oligo sets, respectively.
Panel A used oligos Kan125 (directed of kanamycin sequence) + NadM Sal I (directed to 5’end of gene)
with respective genomic isolates from the identified strains (or plasmid) in legend. This oligo set should
generate a ≈1300 bp pcr product in the correct NadM construct; the positive controls in this screen are the
F.novicida T20NadM (lane 3) and the plasmid, pKEK1305 (lane4). The negative control, KKT1 (lane 2)
should not yield any pcr product. Lane 5 is a SCHU S4 clone 1 that was found to be kanamycin resistant
and contain the pUC origin of the plasmid as seen in panel B, but did not yield a product. This may be due
to the genomic template; these are low yield isolations and if we have a mixed clone this may dilute template
target; lowering annealing temperature may resolve problem.
Panel B used the pUC origin oligos which should produce a ≈800 bp product in all templates containing the
plasmid pKEK1305 (lane 4). The F. novicida T20 NadM mutant (lane 3) should not yield the pUC origin
product since this is the complete mutant and should not contain the plasmid. Lane 2 is KKT1 and should
not yield a product. Lane 5 is a potential T20 NadM mutant and shows the presence of the plasmid’s pUC
origin. Data located in TVD UTSA Notebook 7, page 116.
•Cloning of the mobRP4 gene into the pKEK1305 (pJC84_T20 NadM) plasmid. UTSA pursued
increasing the number of transformants with this construct (pKEK1305) by introducing the
mobRP4 gene. This mobRP4 gene facilitates movement of genes from plasmids into bacterial
genomic targets.
•Result: In progress, the attempt to amplify the mobRP4 was successful.
•1st UTSA analyzed the mating plasmid pDS132’s mobRP4 gene and generated oligos directed to the
5’ and 3’ ends of this gene to include the start (ATG) and stop (TAA) sites.
•Result: The MobRP4upSal I and MobRP4downSal I oligos were ordered and received.
•2nd UTSA set up PCRs to generate the MobRP4 gene product.
•Result: Figure 3 shows the expected ≈1800 bp MobRP4 gene product when using the
MobRP4upSal I and MobRP4downSal I oligos with the pDS132 plasmid as template.
This is the correct size gene product.
Page 30 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Figure 3 represents the pcr product of the MobRP4 gene generated from the mating plasmid pDS132
(template). The PCR was set up in triplicate; the expected size of this gene is ≈1800 bp and the major
product in these profiles appears to be correct. These will be used for digestion with Sal I and subsequent
cloning into the pJC84 construct, pKEK1305. Data located in TVD UTSA Notebook 7, page 119.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
UTSA is using the pJC84+T20 NadM construct in hopes to generate this NadM mutant in SCHU
S4 by this method. The first electroporation only generated 40 colonies and since the selection
used is low (10 ug/ml). UTSA noted spontaneous kanamycin resistant mutants so a higher
number of total transformants is needed to sort out the spontaneous mutants. UTSA chose to
introduce the MobRP4 gene back into this mating plasmid in order to increase the number of
transformants. UTSA chose introduction by mating to get this plasmid into the SCHU S4 bacteria
instead of using electroporation.
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
96%
9. Work plan for upcoming month
a. 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. This strategy is moving the mutant NadM
gene from F novicida to SCHU S4.
b. Will monitor the mice which will be intranasally inoculated with the FTT0748 mutant and report
on whether the FTT0748 mutant is attenuated in mice.
c. Will continue with the cloning of MobRP4 gene into the pKEK1305 plasmid to allow us to
perform a mating experiment to introduce this plasmid into SCHU S4 bacteria instead of using
electroporation.
Page 31 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Currently UTSA is in the process of creating FTN1487 gene mutants in Fn.U112 and FTT1579 gene
mutants in Ft.Schu S4 to break down the restriction barrier in both strains. We have constructed the
tulatron vector pKEK1226 for insertion mutagenesis of FTN1487 or FTT1579 in U112 or Schu S4.
This method allows retargeting the restriction enzyme and inactivating a specific gene to facilitate
introduction of foreign plasmid DNA into Schu S4 strain. Cryotransformation introduces the tulatron
vector into the bacterial strain and inserts the mutated intron RNA for the FTN1487 or FTT1579 gene.
This insertion disturbs or mutates the specific gene in the bacterial genome. After the insertion, the
temperature sensitive plasmid with Kanamycin marker is removed from the mutant strain by
incubation at 37C instead of 30 and is selected on Kanamycin containing medium. The mutant
strain, which has lost the plasmid, is verified by colony PCR and sequencing.
. Creation of FTN1487 gene mutants in F.novicida U112
. Result: The FTN1487 gene mutant U112 , from which the the plasmid is removed, was verified by
sequencing and colony PCR.
.
1st The gel purified PCR DNA was sequenced using the “FTN1487 gene primer FTN1487 Rev” and
the insertion primer “EBS Universal”.
.
.
2nd Removal of the plasmid from the FTN1487 mutant U112 at 37C.
.
.
Result: It was confirmed that the intron insertion was at 849/850bp of FTN1487 gene in
Fn.U112.
Data location: UTSA TVDC notebook #6, page91 and 92.
Result: The plasmid was removed successfully from the mutant strain based on its
sensitivity to temperature and retention of the Kanamycin marker.
3rd Verification of the mutant strain without the plasmid by colony PCR.
.
Result: PCR indicated that the insertion was in FTN1487 gene of Fn.U112.
Figure1: Gel picture of the colony PCR.
Figure1 legend, results and data location: lane2-7 was PCR amplified with the insertion primer. The 5 colonies
(lane2-4 and 6-7) produced the PCR product the same as the positive control with a band of about 900bp. Lane8-13
Page 32 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
was screened with the FTN1487 gene primers. All 5 colonies (lane8-10 and 12-13) produced the PCR product as
expected for the mutants and the positive control (lane11, about 1890bp). Both sets of PCR verified that the 5
colonies were the expected mutants.
Data recorded on UTSA TVDC notebook #6, page93 for Figure1.
.
4th Designation of the FTN1487 gene mutant U112.
.
.
Result: the mutant strain was designated as KKF378. Frozen stocks of KKF378 were made.
Creation of FTT1579 gene mutants in Ft. Schu S4.
.
.
Result: The FTT1579 gene mutant in Schu S4 was created and the plasmid was removed
1st Cryotransformed the tulatron vector pKEK1226 into wild type Schu S4 (KKT1)
.
.
Result: About 19 potential transformants were observed on Kanamycin selective agar medium.
2nd Screened the potential transformants and the single colonies generated from the
transformants using the colony PCR with the FTT1579 gene primers (FTN1487 For and FTN1487
Rev)
.
Result: about 98 colonies were screened. Only 2 colonies were positive for the insertion in
FTT1579.
Figure 2: Gel picture of the colony PCR
Figure 2 legend, results and data location: Lane8 and 22 were the positive control KKF378 with the band of about
1890bp, whereas the negative control Wt Schu S4 (lane9) produced the PCR product of about 990bp. Only colony3
(lane4) and colony8 (lane11) were screened positive for the insertion with the same band as the positive control
(lane8 and 22), which indicated that colony3 and 8 had the insertion in FTT1579 gene of Schu S4.
Data recorded on UTSA TVDC notebook #6, page95 for Figure2.
.
3rd Confirmed the insertion in colony3 and 8 using colony PCR with the insertion specific primer
.
Result: The PCR using two sets of primers showed evidence of the insertion in FTT1579
gene in both strains.
Page 33 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Figure3: Gel picture of the colony PCR
Figure3 legend, results and data location: lane2-5 was PCR amplified with the FTT1579 gene primers.
Lane3 was the wild type Schu S4 with a band of about 990bp, whereas lane4 (KKF378) produced the PCR product of
about 1890bp. Clony3 (lane2) and colony8 (lane5) presented the same result as the positive control (KKF378).
Lane6-11 showed the PCR result using the insertion primer. Colony3 (lane 6 and 7) and colony8 (lane10 and11)
produced the PCR product of about 900bp same as KKF378 (lane9) compared to the wild type Schu S4 without any
product.
Data recorded on UTSA TVDC notebook #6, page96 for Figure3.
.
4th Removed the plasmid from the FTT1579 mutant Schu S4 at 37C.
.
.
Result: The plasmid was removed successfully from the mutant strain based on its
sensitivity to temperature and retention of the Kanamycin marker.
5thDesignation of the FTT1579 gene mutant Schu S4.
.
Result: the mutant strain was designated as KKT19. Frozen stocks of KKT19 were made.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
KKF378 (FTN1487 mutant Fn.U112)
KKT19 (FTT1579 mutant Schu S4)
7. Quality of performance
Good
8. Percentage completed.
About 68% of scientific work completed.
9. Work plan for upcoming month
i. Verify the frozen stock of KKT19 using the colony PCR with both sets of primers.
ii. Send the gel purified PCR product of KKT19 (using the insertion primer “EBS Universal “and the
gene primer “FTN1487 rev”) for sequencing as a second method to confirm the intron insertion.
iii Design and order the primers for constructing the tulatron vector for insertion mutagenesis of
FTT0523 in Schu S4.
Page 34 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
53A: (1) Evaluate the protective efficacy of intranasal KKT-23 (ΔrecAiglC of SCHU S4)
vaccination against intranasal F. tularensis challenge. (Note book #9, page 38-40). Mice
(C57BL/6) were given intranasally a single dose of KKT-23 (3x106 CFU), rested for 30 days,
and challenged i.n. with 187 CFU of SCHU S4. All mice vaccinated with KKT-23 or mock
treated with PBS succumbed to SCHU S4 pulmonary challenge. This is a similar result to
our previous protective efficacy study using the single iglC SCHU S4 mutant, which provided
no protection against SCHU S4 challenge.
(2) Analyze the antibody profiles of mice i.n. vs orally immunized with KKF235 (Ft subsp.
novicida iglB) (Note book #9, pages 41-43). Blood, feces and bronchoalveolar lavage
fluid (BAL) was collected from the PBS- and KKF235 (104 CFU)- immunized mice at day 28
after vaccination. Anti-KKF235 specific antibody titer in serum (total Ig, IgG1, IgG2a), fecal
samples (IgA, IgM) and BAL fluid (total Ig, IgG1, IgG2a, IgA, IgM) were determined by ELISA.
Antigens, either UV-irradiated KKF235 (106/well) or HEL (Hen Egg Lysozyme, 1µg/well, an
unrelated antigen as control), were coated onto 96-well microplates and reacted with serial
dilutions of sera or undiluted fecal and BAL samples. Goat anti mouse Ig(H+L), IgG1, IgG2a,
IgA and IgM antibody conjugated with horseradish peroxidase were used as the secondary
antibodies to determine antibody isotypes and titers. As shown in Fig. 1, mice immunized i.n.
and orally with KKF235 produced comparable amount of anti-KKF235 total serum antibody.
Isotype (IgG1 and 2a) analyses imply that both Th1 and Th2 immune responses are induced
by i.n. and oral KKF235 immunization. We also assessed KKF235 vaccination induced
mucosal immunity. As shown in Fig. 2, a higher level of KKF235-specific IgA was detected in
the G.I. tract of the orally immunized mice compared to i.n. vaccinated mice, and almost no
IgM was produced for both routes of immunization. Mice vaccinated both i.n. and orally with
KKF235 produce a wide array of specific antibodies in the BAL including high level of total
antibody as well as IgG1, IgG2a, while low amount of IgA and IgM as shown in Fig. 2.
Comparable levels of BAL IgG1 and IgG2a suggest i.n. immunization induces both Th1 and
Th2 response in the lungs. On the other hand, oral immunization with KKF235 might have
induced a dominant Th1-type immunity at the challenge site (lungs). Little to no KKF235specific antibody was detected in mock vaccinated mice. All tested samples showed no
reactivity to the unrelated HEL protein (data not shown). In summary, both i.n. and oral
immunization with KKF235 induce significant KKF235-specific systemic and respiratory
antibody production. Mixed Th1 and Th2 antibody responses were induced by i.n.
vaccination; however, oral vaccination resulted in Th1 predominant response in the lungs and
this might contribute at least partially to the better protective efficacy by oral KKF235
immunization.
Page 35 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
Page 36 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
None
7. Quality of performance
Good
8. Percentage completed
20% of scientific work completed on milestone 53A.
9. Work plan for upcoming month
Evaluate the intramacrophage growth of KKT19, a SCHU S4 restriction enzyme
(FTT_1579) mutant.
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: The cellular response of Fisher 344 rats following LVS vaccination was determined.
(Notebook # 10, pages 67, 72, 78) Groups of F344 rats (3 rats per group) were vaccinated
either orally or intradermally (I.D.) with 107 CFU of LVS in PBS or mock vaccinated orally with
PBS alone and rested for 14 days. Rats were then sacrificed, spleens and cervical lymph
nodes were removed, and single cell suspensions were made. Cells were then incubated in
the presence of UV-inactivated LVS (103, 104, 105 CFU), or the unrelated antigen hen egg
lysozyme (HEL), for 72 hours. Culture supernatants were then collected and analyzed for the
presence of IFN- by cytokine ELISA. As shown in Figure 3, both routes of vaccination
induced a significant IFN- response in both the spleens and cervical lymph nodes. However,
intradermal vaccination generated a much greater systemic response in the spleen whereas
oral vaccination generated a greater response in the cervical lymph nodes. No response was
detected in tissues from mock vaccinated rats, or from cells incubated with HEL. These
results show that LVS vaccination, via both oral and intradermal routes, is able to induce
cellular responses in the F344 rat.
Page 37 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
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
27%
9. Work plan for upcoming month
(1)Cellular response following F. novicida challenge
(2)Bacterial dissemination following F. novicida challenge
Page 38 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
Acronym List Updated 8/12/09
ABSL3: Animal Biosafety Level 3 lab
AM: alveolar macrophages
ASU: Arizona State University
BLD: below limit of detection
BMDM: bone marrow derived macrophages
BP: base pair
BSL2: Biosafety Level 2 lab
BSL3: Biosafety Level 3 lab
C: centrigrade
cDNA: complimentary deoxyribonucleic acid
CDA: confidentiality and disclosure agreement
CDM: Chamberlains defined media
CFU: colony forming units
COA: contract officers authorization
CRDA: Cooperative research and development agreement
C-term: Carboxy terminus
DNA: deoxyribonucleic acid
DVC: Dynport Vaccine Company
ED50: Effective Dose, 50 % dead
ELISA: enzyme-linked immunosorbent assay
ELISpot: enzyme-linked immunospot assay
EOH: Employee Occupational Health
FDA: Federal Drug Administration
FF: formalin fixed
FSC: forward scatter, equivalent to cell size FT or Ft or F. tularensis: Francisella tularensis
GDP: genome directed primers
GI: gastrointestinal
GLP: good laboratory practice
KBMA: killed but metabolically active
HK: heat killed
HTP: high through put
IAA: interagency agreement
IACUC: Institutional animal care and use committee
ICS: Intracellular staining
ID: intradermal
IFNγ: interferon gamma
Page 39 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
IL2: interleukin 2
IN: intranasal
IND: investigational new drug
IP: intraperitoneal
IRB: institutional review board
IRS: immune rat sera
IT: intratracheal
IVT: in vitro translation/transcription
KB: kilobase
L: liter
LAPT: Linear Amplification of Procaryotic Transpcript
LBERI: Lovelace Biomedical and Environmental Research Institute
LD50: Lethal dose 50% dead
LD99: lethal dose, 99% dead
LN: lymph node
LPS: lipopolysaccharide
LRRI: Lovelace Respiratory Research Institute
LVS: Live vaccine strain (tularemia)
NHP: nonhuman primate
MDM: monocyte derived macrophages
mL: milliliter
MLN: mesenteric lymph node
mM: Milli molar
MOI: multiplicity of infection
MS: Milestone
MSCR: Milestone Completion Report
MTA: material transfer agreement
MTS reagent: (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt
N: number
NIAID: National Institute of Allergy and Infectious Disease
NRS: normal rat sera
N-term: amino- terminus
OD: optical density
ORF: open reading frame
PBMC: peripheral blood mononuclear cells
PBS: phosphate buffered saline
PFA: paraformaldehyde
Page 40 of 41
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 7/01/2009 to 7/31/2009
Due Date: 8/15/2009
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
pM: pico Molar
pmol: picomole
PCR: polymerase chain reaction
QA: quality assurance
QC: quality control
qPCR: quantitative polymerase chain reaction
RNA: ribonucleic acid
SC: subcutaneous
SDS: sodium dodecyl sulfate
SCHU S4: virulent strain of Francisella tularensis
SIP: Special Immunizations Program at USAMRIID
SOP: standard operating procedure
SSC: side scatter, equivalent to cell density or granularity
TBLN: tracheobronchial lymph node
Tn: transposon
TNFα: tumor necrosis factor alpha
TVDC: Tularemia vaccine development contract
uL: microliter
UNM: University of New Mexico
USAMMDA: United States Army Medical Materiel Development Activity
USAMRIID: United States Army Medical Research Institute for Infectious Disease
UTSA: University of Texas at San Antonio
WT: wild type
Page 41 of 41