Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
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, 3, 4, 5, 7, 11, 12/13(UNM/LBERI), 14, 17, 19, 21(UNM/LBERI),
26, 27, 28, 35(ASU/UNM), 49, 50, 52, 55, 56, 57
Completed milestones: 1, 25, 32, 33, 34 (UNM/ASU), 16, 39, 40, 43 (UTSA), 48, 51
Inactive milestones: 6, 8, 9, 10, 15, 18, 20, 22, 23, 24, 29, 30, 36, 37, 38, 53, 54,
58, 59
Milestones terminated after initiation: 41, 42, 44, 46, (MSCR will be written)
Milestones terminated before initiated: 43 (Cerus), 45, 47 (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. 13 LVS vaccinees have volunteered to donate blood for immunoassay
development under the TVDC.
4. Significant decisions made or pending
a. Dr. Lyons received UNM IRB approval to allow blood draws on the vaccinated
LBERI and UNM scientists after their LVS vaccinations. The LVS vaccinated
LBERI and UNM scientists and staff have been offered the opportunity to
volunteer to donate bloods for the development of immunoassays, approximately
2 months after receiving the LVS vaccination.
b. USAMRIID has temporarily halted offering the LVS vaccine as of 4/29/08, until a
new study protocol is activated, possibly by late summer 2008
c. UNM (4) and LBERI (33) are vaccinated; UNM and LBERI could offer the LVS
vaccinations up to 9 more scientists to total up to 46. The CRDA with USAMRIID
is valid for 2 years, ending June 2009.
d. UNM EOHS clarified the USAMRIID annual health screening requirements
5. Problems or concerns and strategies to address
a. One UNM and one LBERI scientist are medically pending. One UNM scientist
may be rescheduled for LVS vaccination. The next LVS vaccinations will not
1 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
occur until USAMRIID’s new protocol is activated approximately late summer
2008
b. The UNM TVDC Project Manager received pricing for the annual health
screening to be performed at the UNM Employee Occupational Health Services
for the LBERI and UNM LVS vaccinees.
c. The 37 LVS vaccinees will not begin annual health screenings until
approximately 9/11/2008.
6. Deliverables completed
A total of 37 participants (33 LBERI and 4 UNM participants) have received the LVS
vaccination since 9/11/07.
7. Quality of performance
Excellent
8. Percentage completed
67% of the scientific work is complete
9. Work plan for the next month
a. Obtain an NIAID contract authorization to allow UNM EOHS to charge for the
annual health screening required by USAMRIID and being performed at UNM for
the LBERI and UNM LVS vaccinees.
b. UNM will be obtaining blood donations from LVS vaccinees for immunoassay
development and reimbursing participants $40/ donation.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
On 6/25/2008, UNM requested a COA to include the UNM EOHS 12 month health
screening cost for the LVS vaccinees.
Milestone 3
Milestone description: Bioaerosol technique selected and optimized
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. Work on the milestone completion report was continued. It will be completed
once the SCHU S4 growth procedures have been finalized and the comparison
of multiple aerosol generators is completed.
b. SCHU S4 bioaerosols were conducted on five days in June 2008: The goal is to
select an aerosol generator that reproducibly generates a targeted and delivered
SCHU S4 aerosol dose. SCHU S4 Bioaerosols from the Aerotech I, Aeromist,
Hospitak and Collison were generated and compared.
a. 6/13/2008
i. Completed SCHU S4 bioaerosol testing (n=8) with the Aerotech I
nebulizer as a potential replacement for Aeromist since it is no
longer being manufactured.
ii. Objectives were to verify the predictability of our new SCHU S4
growth curve and to perform bioaerosols with the Aerotech I (a
potential Aeromist replacement) nebulizer.
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Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
iii. Growth method used for SCHU S4:
1. Inoculate BCGA for colony isolation
2. Incubate for 48h at 37°C
3. Inoculate 100 mL of Chamberlain’s broth in a washed
500 mL baffled flask with 400 uL of suspension. The
suspension is prepared from multiple colonies mixed in
4.5 mL broth and normalized to an OD600 of 0.100.
4. Incubate for 24h at 37°C, 200 rpm, in the dark
5. Read OD600; compare to growth curve and dilute to
appropriate concentrations
iv. Results (stored in the following folder: \\Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC
DATA\FY06\FY06-078_TUL-03\TUL-03
data
files\Aeromist nebulizer\13JUN08) are summarized in Table 1
below:
Table 1. 13JUN08 summary SCHU S4 bioaerosol results using the Aerotech I nebulizer.
Data from Table 1 indicate that use of the new OD curve resulted
in accurate generator suspension predictions.
Post- actual
bioaerosol concentrations were slightly lower than pre- actual
bioaerosol titers, an observation consistently observed regardless
of the nebulizer used. Spray factors were lower and more variable
than desired; this result may have been inherent to the nebulizer.
Taken together, these data clearly demonstrate the fragility of
SCHU S4 as a bioaerosol (specifically when aerosolized using
high-pressure compressed air).
b. 6/18/2008
i. Completed SCHU S4 bioaerosol testing (n=12) comparing the
Hospitak with the Aeromist nebulizers.
ii. Objectives were to verify the predictability of our new SCHU S4
growth curve and to perform bioaerosols with the Hospitak (a
potential Aeromist replacement) and Aeromist nebulizers
iii. Growth method used for SCHU S4:
1. Inoculate BCGA for colony isolation
3 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
2. Incubate for 48h at 37°C
3. Inoculate 100 mL of Chamberlain’s broth in a washed
500 mL baffled flask with 400 L The suspension is
prepared from multiple colonies mixed in 4.5 mL broth
and normalized to an OD600 of 0.100.
4. Incubate for 24h at 37°C, 200 rpm, in the dark
5. Read OD600; compare to growth curve and dilute to
appropriate concentrations
iv. Results (stored in the following folder: \\Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC
DATA\FY06\FY06-078_TUL-03\TUL-03
data
files\Aeromist nebulizer\18JUN08) are summarized in Table 2
below:
Table 2. 18JUN08 summary SCHU S4 bioaerosol results using the Aeromist and Hospitak
nebulizers.
Data from Table 2 further indicate that use of the new OD curve
resulted in accurate generator suspension predictions. Again,
post-bioaerosol concentrations were slightly lower than prebioaerosol titers. Spray factors were lower than desired, though
consistent, with the Hospitak demonstrating slightly better
efficiency than the Aeromist. Aerosol concentrations were low as
well; primates exposed at these concentrations would not have
been challenged appropriately (assuming 3.5L inhaled, calculated
doses would have only reached 14,000 CFU at the high end rather
than the desired 25,000 CFU). As with the 13JUN2008 results,
these data further demonstrate the fragility of SCHU S4 as a
bioaerosol.
4 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
c.
6/19/2008
i. Completed repeat bioaerosol testing of 14MAY08 NHP
exposures (without NHPs; n=3).
ii. Objectives were to repeat the Microbiology and Aerosol
parameters of the 14MAY08 NHP exposures. Since clumping
had occurred (culture was grown to 48 hours in 50 mL
Chamberlain’s broth using a single colony) and predicted
concentration and spray factors were too low the bioaerosol was
repeated under the same conditions to see if it again resulted in
lower CFU values. Additionally bioaerosols were being
performed to determine which phase of growth was most hardy
for aerosolization and data collected to date had suggested at 48
hours the bacteria was in stationary phase of growth (refer to
May 08 monthly report for data on growth curves).
iii. Growth method used for SCHU S4
1. Inoculate BCGA for colony isolation
2. Incubate for 48h at 37°C
3. Inoculate 50 mL of Chamberlain’s broth in a washed 500
mL baffled flask with 1 CFU
4. Incubate for 48h at 37°C, 150 rpm, in the dark
5. Centrifuge at 4100 rpm for 20 min
6. Resuspend pellet in 4 mL of Chamberlain’s broth
7. Read OD600; compare to growth curve and dilute to
appropriate concentrations
iv. Results (stored in the following folder: \\Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY07\FY07-083 and -089 (TUL-04)\19JUN08
mock-NHP exposure) are summarized in Table 3 below:
Table 3. 19JUN08 summary SCHU S4 bioaerosol results using the Aeromist nebulizer.
Data from Table 3 further indicate actual generator suspension
concentrations were approximately 1 log different than the target.
This was observed with the 14MAY2008 NHP exposures and was
concluded to be due to the poor growth curve. Contrary to the
14MAY results, however, spray factors here were acceptable. This
was hypothesized to be due to the absence of clumps in the
growth medium, a phenomenon that was significant for the NHP
exposures and was observed in the 14MAY results.
5 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
d. 6/23/2008
i. Completed SCHU S4 bioaerosol testing (n=12) comparing the
Hospitak (potential replacement for Aeromist) and Collison
nebulizers.
ii. Objectives: To continue verification of the new SCHU S4 growth
curve and to perform head-to-head bioaerosol comparisons
between the Hospitak and Collison nebulizers Since the
Aeromist is no longer available and the Collison is considered
the gold-standard for bioaerosols it was chosen as the standard
to determine if the performance of the Hospitak is better.
iii. Growth method used for SCHU S4
1. Inoculate BCGA for colony isolation
2. Incubate for 48h at 37°C
3. Inoculate 100 mL of Chamberlain’s broth in a washed
500 mL baffled flask with 400 uL of a 4.5 ml suspension
normalized to an OD600 of 0.100
4. Incubate for 24h at 37°C, 200 rpm, in the dark
5. Read OD600; compare to growth curve and dilute to
appropriate concentrations
iv. Results (stored in the following folder: \\Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC
DATA\FY06\FY06-078_TUL-03\TUL-03
data
files\Aeromist nebulizer\23JUN08) are summarized in Table 4
below:
Table 4. 23JUN08 summary SCHU S4 bioaerosol results using the Collison and Hospitak
nebulizers.
Data from Table 4 further indicate that use of the new OD curve
resulted in accurate generator suspension predictions. As before,
post- actual bioaerosol concentrations were slightly lower than pre-
6 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
actual bioaerosol titers. Spray factors were more efficient and
consistent with the Hospitak whereas Collison data were
inconsistent and less efficient than desired (many values were in
the 10-8 range). Aerosol concentrations were well within the
acceptable range when considering a desired challenge dose of
25,000 CFU in 3.5L inhaled; concentrations reached as high as
600,000 CFU/L using the Hospitak. These data demonstrate the
fragility of SCHU S4 as a bioaerosol, but show that desirable NHP
exposures can be achieved.
e. 6/27/08
i. Performed viable cell counts from Chamberlain’s broth
inoculated with SCHU S4 at varying optical densities (0.1, 0.2,
0.3, and 0.4).
ii. Objective was to determine if there was significant variability in
the cfu such that prior growth failures might be explained.
iii. Protocol used for the examination:
1. One BCGA plates was streaked with F. tularensis Schu
S4, and incubated for ~48 hours at 37ºC.
2. A loopful of bacteria was inoculated into 4.5 ml
Chamberlain’s broth to an OD600 of 0.1, 0.2, 0.3, and
0.4.
3. Each of the cultures was diluted ten-fold. Aliquots (100
uL) from each indicated dilution, including the undiluted
culture, was plated in duplicate on BCGA plates.
4. Plates were incubated for ~48 hours at 37°C.
5. Colonies were counted and duplicates are averaged; cfu
were very similar for each duplicate.
Results (stored in the following folder: \\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY06\FY06-078_TUL-03) were summarized in
Table 5 below.
Table 5. 27JUN08 summary examination of viable cell counts of SCHU S4 at varying
optical densities
OD600
0.1
0.2
0.3
0.4
Resulting CFU/ml 2.015e8
5.2e8
9.5e8
1.035e9
f.
6/30/2008
i. Performed SCHU S4 bioaerosol testing (n=12) comparing the
Hospitak (potential replacement for Aeromist) and Collison
nebulizers.
ii. Objectives were to verify the predictability of our new SCHU S4
growth curve and to perform bioaerosols with the Hospitak (a
potential Aeromist replacement) and Collison nebulizers to
achieve reproducibility.
iii. Growth method used for SCHU S4:
1. Inoculate BCGA for colony isolation
7 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
2. Incubate for 48h at 37°C
3. Inoculate 100 mL of Chamberlain’s broth in a new 500
mL baffled flask with 400 L of 4.5 ml suspension
normalized to an OD600 of 0.100
4. Incubate for 24h at 37°C, 200 rpm, in the dark
5. Read OD600; compare to growth curve and dilute to
appropriate concentrations
Results (stored in the following folder: \\Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC
DATA\FY06\FY06-078_TUL-03\TUL-03
data
files\Collison Generator\30JUN08) are summarized in Table 6
below:
Table 6. 30JUN08 summary SCHU S4 bioaerosol results using the Collison and Hospitak
nebulizers.
F. tularensis
Date
Strain
30Jun08
SCHU
S4
Working
Stock
28-Feb08
Nebulizer
Growth
Protocol
CFU/mL
Operating
Pressure
Output
Target
PreActual
PostActual
CFU/L
Brand
Spray
Factor
Collison
25.4 psig
7.4978 L/min
1.00E+06
8.60E+05
3.85E+05
3.69E+01
9.58E-08
Hospitak
10 psig
4.4980 L/min
1.00E+06
8.65E+05
4.65E+05
1.94E+02
4.16E-07
Hospitak
25.4 psig
7.4978 L/min
1.00E+07
1.07E+06
5.70E+06
1.45E+03
2.54E-07
Collison
10 psig
4.4980 L/min
1.00E+07
1.02E+06
5.05E+06
1.06E+03
2.1E-07
Collison
25.4 psig
7.4978 L/min
1.00E+08
7.50E+07
4.90E+07
3.70E+03
7.54E-08
Hospitak
10 psig
4.4980 L/min
1.00E+08
8.95E+07
5.30E+07
1.35E+04
2.55E-07
Hospitak
25.4 psig
7.4978 L/min
1.00E+09
9.65E+08
5.95E+08
1.22E+05
2.05E-07
Collison
10 psig
4.4980 L/min
1.00E+09
1.05E+09
4.25E+08
2.61E+04
6.15E-08
3
Data from Table 5 further indicate that use of the new OD curve
resulted in accurate generator suspension predictions. As before,
post-actual bioaerosol concentrations were slightly lower than preactual bioaerosol titers. Spray factors were consistent within the
same nebulizer but were more efficient when the Hospitak was
used. Aerosol concentrations were well within the acceptable
range when considering a desired challenge dose of 25,000 CFU
in 3.5L inhaled. These data demonstrate the fragility of SCHU S4
as a bioaerosol, but show that desirable NHP exposures can be
achieved (more so with the Hospitak).
4. Significant decisions made or pending
a. The SCHU S4 challenge material preparation will be redefined upon completion
of addition bioaerosol studies with the goal of achieving a reliable reproducible
challenge dose.
8 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
b. The Aerotech I will not be used in future SCHU S4 bioaerosol studies because it
is no longer manufactured.
c. All future SCHU S4 bioaerosol study days will include at least one “control” run
using the Collison 3-jet nebulizer.
d. All future SCHU S4 bioaerosols will incorporate new, unused flasks for bacterial
proliferation; flasks will be discarded after one time use.
e. 06JUN08 experiments will be repeated on 2 additional days to collect more
independent head to head data to decide if the Collison or Hospitak will be used
for future NHP bioaerosol challenges.
5. Problems or concerns and strategies to address
a. The Aeromist nebulizer is no longer manufactured. Preliminary data show that
the Hospitak nebulizer is similar to the Aeromist and Collison nebulizer. Data
collected to date demonstrate the fragility of SCHU S4 as a bioaerosol but show
that the nebulizers are comparable in performance. Two additional bioaerosols
will be performed to demonstrate reproducibility of AGI concentration through
statistical analysis that either Hospitak or the Collison nebulizer can be use in
future bioaerosols.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
99.0% of the scientific work is complete
9. Work plan for next month
a. Continue (with the potential to complete) the Milestone Completion Draft Report
b. Complete and submit SOP drafts used on MS3
c. Perform additional bioaerosols comparing the Hospitak and Collison nebulizers:
a. 1JUL2008
b. 9JUL2008
d. Analyze cumulative data from Hospitak/Collison bioaerosol runs and logically
choose the appropriate nebulizer to use in future NHP SCHU S4 aerosol
challenges based on statistics from 4 independent sprays on different days
looking at 106 to 109 CFU/mL target nebulizer concentrations.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions:
A bioaerosol challenge with two additional primates (A05254 and A05262) was
performed on 14MAY08 to confirm that aerosolized SCHU S4 freshly grown in
Chamberlain’s broth is virulent in NHPs. Initial methods and data were
9 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
presented in the previous report. Updated results are presented below. These
data are located in the following folder: \\Saturn\absl3\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY07\FY07-083
and -089 (TUL-04)\14MAY08 NHP exposure.
Table 7: Blood and tissue culture data for animals A05254 and A05262, naïve cynomolgus
macaques exposed to an aerosol challenge of Francisella tularensis SCHU S4.
FY07-083 Naïve Cynomolgus Macaque Francisella tularensis SCHU S4 Bioaerosol Challenge Data
Animal ID
Challenge
Date
Challenge
Dose
(CFU)
Nx Datea
A05254
14-May-08
Unknown
A05262
14-May-08
Unknown
Tissue Culture
Bloodb
Spleen
Liver
TBLNc
Mes LN
Lung
23-May-08
BLD
3.12E+03
1.28E+02
1.88E+04
BLD
1.48E+05
11-Jun-08
BLD
BLD
1.18E+02
BLD
3.50E+04
BLD
a
Animal A05254 was euthanized on Study Day 9; A05262 was euthanized on Study Day 28
b
Blood data presented as CFU/mL; tissue data presented as CFU/g; BLD, below limit of detection
c
In addition to F. tularensis, one contaminant was noted in the TBLN of A05254
The exact challenge dose is unknown since no culturable bacteria were
recovered from the impinger. Though colony counts were not available, it is
estimated based on the pre- and post- spray data and plethysmography data that
the challenge dose for these two animals was approximately 1000 CFU., which is
below the 25,000 CFU targeted dose. Animal A05254 was euthanized due to a
moribund state at day 9 whereas A05262 survived initial challenge and was
euthanized at the end of the study (28 days post-challenge). Cultures from
A05254 were indicative of a septicemic disease with the highest titers observed
in the TBLN and lung. In contrast to this, SCHU S4 was only present in the liver
and mesenteric LN of A05262 demonstrating clearance and recovery from the
aerosol challenge. Survival was likely due to the low challenge dose (a
concentration hypothesized to be near the LD50 under these conditions). This is
the first time that we have observed bacteria in the mesenteric lymph nodes.
10 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Figure 1: Gross Pathology lung images from animal A05262 euthanized on
Study Day 28. The top and bottom images represent ventral and dorsal aspects,
respectively. It was concluded that this animal was entering a resolving phase of
the disease and would have likely cleared the SCHU S4 presence completely
and returned to a more normal lung appearance had it not been euthanized on
Study Day 28. These lungs are representative of chronic pneumonia, a result
observed in all SCHU S4-exposed NHPs to date.
4. Significant decisions made or pending
Due to the issues with the two animal challenges (Dec 2007 and May 2008), an
additional challenge will be performed once the SCHU S4 growth is optimized and the
challenge material preparation defined and delivers a reproducible challenge dose. This
is tentatively planned for mid July 2008. Following completion of the Hospitak and
Collison comparisons (see Milestone 3), a decision will be made as to what generator to
use for the next primate challenges.
5. Problems or concerns and strategies to address
Optimization of SCHU S4 bioaerosols has proven difficult. Because LVS and SCHU S4
behave significantly different as bioaerosols, results from one microorganism cannot be
linearly transferred to the other.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
52% of the scientific work is complete
11 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
9. Work plan for next month
a. Schedule and/or perform bioaerosol challenge with two additional primates to
confirm that aerosolized SCHU S4 freshly grown in Chamberlain’s broth is
virulent in NHPs. Before challenge the primates, the chosen primates will have
been screened for background titer. The challenge delivered dose will be 25,000
CFU of viable SCHU S4. The generator to be used will depend on finalized
results from Milestone 3. The primates will be observed daily for up to 28 days for
clinical signs of illness. NHP will be offered enriched vegetable/fruit food, as a
clinical measure of health status. NHP will not be euthanized unless moribund
as defined by the LBERI veterinarian. Post mortem will be identical to the NHP
study performed in May 2008.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 5 - UNM
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: pending
3. Work performed and progress including data and preliminary
conclusions
a. Experiment Ftc70 study 1 (Notebook 115, pages 98-100)
i. We have repeatedly observed occasional survivors in SCHU S4
challenge experiments when the other rats in the group challenged
with the same dose died. We do not know if this is due to individual
variation in susceptibility or to technical errors in delivering the
inoculum; we have been using a non-surgical inoculation method
that involves inserting a flexible catheter down the trachea and have
occasionally seen the catheter end up in the esophagus instead.
ii. To address the possible technical problem, we plan to do a side-byside comparison between the non-surgical inoculation method and a
surgical inoculation that the laboratory has been using for years to
infect mice. The advantage of the surgical method is that we can
see the needle being inserted into the trachea. In several surgical
attempts with the rats, we have had contamination problems that we
had not encountered before with the non-surgical i.t. We are now
troubleshooting this problem by using longer needles and refining
our techniques.
1. During one of our weekly internal TVDC meetings, Bob
Sherwood indicated that he has a lot of experience using the
surgical i.t. inoculation method in rats. We had a practice
session under his guidance. The procedure was very
straightforward and the use of a ball-tipped needle could
also be applied to non-surgical inoculation as a refinement.
12 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Additional practice sessions are required for the UNM
technical team to perfect the procedure.
iii. To address the possibility that Fischer 344 rats may have individual
variation in susceptibility to SCHU S4 infection, we are consulting
with Dr. Ron Schrader, a biostatistician at UNM, to determine the
significance of the data we have collected so far and the design of
future experiments
1. According to Dr. Schrader, the experiments we have
performed thus far are very similar and could be analyzed as
a single data set. By combining the experiments and
thereby the number of samples, the experimental power is
increased.
2. Dr. Schrader’s analyses also indicated that LVS vaccination
by s.c., i.d. and i.t. routes all provided significant protection
against i.t. SCHU S4 challenge compared with unvaccinated
rat controls. The protective effects of i.d. and s.c.
vaccinations are similar and both may be slightly better than
i.t. vaccination.
3. We are currently analyzing the data to compare the
resistance of unvaccinated and s.c, i.d., and i.t. LVS
vaccinated rats to i.t. SCHU S4 challenge between. We will
provide NIAID with dose response curves for each of the
four groups.
4. We are also analyzing the data to determine the group size
required to identify statistically significant differences in
future experiments.
b. Experiment Ftc46 study 6 (Notebook 116, pages 12-17)
i. For us to include quantum dots in our experiments to confirm i.t.
delivery of the inoculum into the lungs, we must first be convinced
that it does not affect the virulence of the F. tularensis.
ii. Since the high virulence of SCHU S4 in mice and rats may make any
effect of quantum dots on its pathogenicity difficult to detect, we
decided to test the effect of quantum dots on LVS.
iii. BALB/c mice were infected intranasally with 5 x 104, 2.5 x 105, and 5
x 105 LVS in the presence or absence of 5 pmol quantum dots and
14.5 pmol coelentrazine per rat. These doses were chosen to be
above and below the LD50.
iv. Unfortunately, the challenge doses were clustered too tightly and
mice from all three groups died (Fig. 1). However, it is clear that
quantum dots did not affect the virulence of LVS in BALB/c mice
13 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Figure 1. Quantum dots did not affect the virulence of LVS in BALB/c mice. BALB/c mice (n = 5)
were infected with LVS with and without quantum dots and its substrate. Infected mice were
monitored daily for survival and development of clinical signs of illness.
c.
Experiment Ftc46 study 7 (Notebook 116, pages 18-21)
i. The purpose of this experiment was to determine whether quantum
dots affect the virulence of LVS. This was a repeat of Ftc46 study 6.
ii. Based on the results from Ftc46 study 6, the LVS challenge dose
was adjusted to cover a larger range: 5 x 103, 5 x 104, and 5 x 105
cfu/mouse
iii. As seen in Ftc46 study 6, addition of quantum dots had no effect of
the virulence of LVS in BALB/c mice (Fig. 2). The difference
between the two groups at 5 x 105 LVS was not statistically
significant (P > 0.1)
iv. Based on the results from these two studies, we feel confident that
inclusion of quantum dots does not adversely affect the quality of
infection experiments
Figure 2. Quantum dots did not affect the virulence of LVS in BALB/c mice. BALB/c mice (n = 5)
were infected with LVS with and without quantum dots and its substrate. Infected mice were
monitored daily for survival and development of clinical signs of illness.
d. Characterization of the Fischer 344 rat model is currently being done under
Milestone 11, as the efforts on the Fischer 344 rat model are shifting toward
GLP model efficacy
4. Significant decisions made or pending
We will include quantum dots and substrate in all infection experiments because
quantum dots do not appear to affect the virulence of LVS in mice
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
NA
14 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
8. Percentage completed
75%
9. Work plan for upcoming month
a. Complete sub-milestone completion reports for the mouse, rat, and guinea
pigs
b. Continue development of surgical i.t. for rats
Continue consultation with GCRC (General Clinical Research Center)
biostatistician at UNM
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
On June 26,2008, UNM requested a COA to authorize the purchase of an
anesthesia unit to associate with the Xenogen imaging system, which detects the
quantum dots.
Milestone 7
Milestone description: SCHU S4 LD50 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:
a. A draft protocol was written and submitted to the IACUC.
complete and the comments are being addressed.
IACUC review is
4. Significant decisions made or pending
Confirmation of firm start dates pending completion of Milestone 4.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Fair
8. Percentage completed
8% of the scientific work is complete
9. Work plan for next month
a. Schedule appropriate personnel.
b. Initiate ABSL-3 move-in and challenge dates.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
15 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Milestone 11 - UNM
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. Experiment Ptran6 (Notebook 123, page 1-7, 24, 36-38, 44-48)
i. The purpose of this experiment was to determine the minimum
volume of immune serum required to protect rats against i.t. SCHU
S4 challenge
ii. NCI Fisher 344 rats received 0, 0.4, 1.0, 1.5, or 2.5 ml of immune
NCI rat serum by intraperitoneal injection 1 day before i.t. challenge
with approximately 100 CFU SCHU S4. We showed previously in
Experiment Ptran 1 and 2 that 2.5 ml of normal rat serum protected 2
of 6 and 4 of 6 NCI Fisher 344 rats, respectively, from i.t. SCHU S4
challenge. Thus, the volume of immune serum was titrated from 2.5
ml down to 0 ml in this experiment,
iii. As seen in Fig. 3, as little as 0.4 ml of immune serum enabled NCI
Fisher 344 rats to survive and maintain their weight for 20 days after
i.t. SCHU S4 challenge
iv. Rats that received 1.5 and 2.5 ml of immune serum not only survived
but also gained weight.
v. Table 1 shows the bacterial burden in the lungs, liver, and spleen of
passively immunized rats that survived i.t. SCHU S4 challenge.
Interestingly, the bacterial burden was similar despite the weight
differences between the groups. It is worth noting that the number of
bacteria recovered from the liver of the 2.5 ml group varied more
than the other groups, but the significance of this result is unclear
16 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Figure 3. Weight loss/gain in passively immunized Fischer 344 rats
after i.t. SCHU S4 challenge. Fischer 344 rats from NCI (n = 3) were
injected with the indicated volume of immune NCI rat serum and
challenged 1 day later with ~100 CFU SCHU S4 i.t. Each curve
represents the weight loss/gain of one rat. The asterisks indicate
deaths during the observation period i
Table 1. Effect of serum volume on the bacterial burden in passively
immune Fischer 344 rats 20 days after i.t. SCHU S4 challenge
Bacterial burden in passively immunized rats
challenged with SCHU S4 (cfu)
0.4 ml
1.5 ml
2.5 ml
Lung
4.4 ± 0.7
4.1 ± 0.4
4.3 ± 0.4
Liver
2.5 ± 0.1
2.3 ± 0.1
1.6 ± 1.5
Spleen
3.7 ± 0.8
3.6 ± 0.1
3.3 ± 0.8
b. Experiment Ptran7(Notebook 122, page 57-58, Notebook 123, pages 27-35,
41-43
i. The purpose of this experiment was demonstrate that immune
serum, but not normal serum, protected rats against i.t. SCHU S4
challenge. In the last two experiments, Ptran5 and Ptran6, we
titrated only normal or immune serum, respectively, but not both. In
this experiment, both normal and immune serum were included
17 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
ii. Fischer 344 rats from NCI (n = 7) were injected i.p. with 1 ml of
normal or immune rat serum and challenged 1 day later with SCHU
S4 i.t.. By mistake the concentration of SCHU S4 in the inoculum
was made 10-fold higher than previously used and the actual lung
deposition was estimated to be 1871 cfu.
iii. Table 2 shows that immune serum protected 4 of 6 rats and 6 of 6
rats with normal died of i.t. SCHU S4 challenge.
iv. In light of the fact that immune serum protected all of the rats in
Ptran1 and Ptran2, the observation that 2 of the rats with immune
serum died in this experiment suggest that we may be nearing the
limit of protection by passive immunization
Table 2. Survival of passively immunized Fischer 344 rats
following i.t. SCHU S4 challenge
Treatment
Survival ratio
Percent survival
(No. alive/total)
(%)
Naïve
0/7
0
Normal rat serum
0/7
0
Immune rat serum
4/6
66
c.
Experiment Cdep-2.1 (Notebook 122, page 49-50, Notebook 123, page 9-23)
i. The purpose of this experiment was to determine the role of CD4,
CD8 T cells in the protection of Fischer 344 rats induced by LVS
vaccination
ii. LVS vaccinated rats were treated with ascites fluid to deplete CD4
(W2/25), CD8 (OX-8) or both CD4 and CD8 three days before i.t.
challenge with an estimated lung deposition of 5.8 x 104 SCHU S4
iii. The experiment is in progress and the results will be reported in next
month’s technical report.
3. Significant decisions made or pending
None
4. Problems or concerns and strategies to address
None
5. Deliverables completed
None
6. Quality of performance
Good
7. Percentage completed
19%
8. Work plan for upcoming month
a. Tititrate the serum volume against the SCHU S4 challenge in a matrix with
the goal of determining the level of protection with each serum volume
b. Titrate the amount of F. tularensis-specific antibodies in immune sera
c. Compare the sensitivity of NCI and Harlan Fischer 344 rats to i.t. SCHU S4
challenge
d. Determine whether the dose of LVS used in vaccination affects the level of
protection against i.t. SCHU S4 challenge
e. Repeat experiment to determine the role of CD4 and CD8 T cells in
protecting LVS vaccinated rats
18 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
f.
Expand the OX-38 (CD4 T cell depleting antibody) and 55-6 (isotype control
antibody for OX-38) hybridomas and send them to Taconic for production of
ascites fluid
10. Anticipated travel
The UNM team will travel to Phoenix AZ for the UNM TVDC annual meeting in
October 2008. No COA will be required.
11. Upcoming Contract Authorization (COA) for subcontractors
None
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. No new work done this month.
3. Significant decisions made or pending
None
4. Problems or concerns and strategies to address
None
5. Deliverables completed
Mouse proliferation assay, IFN and IL-2 Elispot, anti-Ft antibody titration
Rat IFN Elispot, anti-Ft antibody titration
Guinea pig anti-Ft antibody titration 
6. Quality of performance
Good
7. Percentage completed
60%
8. Work plan for upcoming month
a. Determine whether LVS vaccinated mice with an active SCHU S4 infection
could be used to increase the sensitivity of the IFN Elispot assay
9. Anticipated travel
None
10. Upcoming Contract Authorization (COA) for subcontractors
None
19 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
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. We have continued to screen non-LVS vaccinated NHPs in both the IFNγ
ELISPOT and proliferation assays in order to avoid choosing any high
responders to serve as LVS-naïve controls
ii. In the past month, one additional NHP (A03822; Figure 1A and B) was
screened, bringing the total number of NHPs screened to 19.
iii. Forty newly arrived NHPs (6/5/08) will be screened in the coming weeks and
months
A
120
100
80
60
40
SCHUS4 ff Mid
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
0
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
20
Media
IFNg spots (Mean +/- SEM)
140
20 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
B
75000
50000
SCHUS4 ff Mid
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
0
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
25000
Media
RLU (Mean +/- SEM)
100000
Figure 1: IFNγ production (A) and proliferation (B) by PBMCs from a single non-LVS
vaccinated NHP (A03822) to LVS and SCHU S4 antigens (ff = formalin fixed; hk = heatkilled; Super = 4 x 105/ml, Hi = 1 x 105/ml; Mid = 025 x 105/ml; Lo = 0.0625 x 105/ml). All
PBMCs were plated at 1.33 x 106/ml in (A) and 1 x 106/ml in (B).
Data Interpretation: Once again, we observe that the highest responsiveness in the IFN
ELISPOT assay is to LVS FF Hi. The response dilutes out when less stimulating antigen is used
(LVS FF Mid or –Lo). In this experiment, the greatest proliferative response was also seen in
wells stimulated with LVS FF Hi. This response also diluted out when less FF LVS was used
(LVS FF Mid or – Lo). Some proliferative response was also observed when PBMCs were
stimulated with the highest concentration of HK SCHU S4 (Super).
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay063008.svd;
TVDC (3) bound notebook (9225), p 14 – 18.
b. We have continued to test the ability of the Cerus freeze/thaw protocol to spare the
reactivity of IFN secreting cells in the ELISPOT assay
ii. In the past two weeks, we have thawed frozen aliquots of cells from 3 separate
experiments for testing in the IFN ELISPOT assay
iii. The percent recovery of the number of cells after the freeze/thaw process is
shown in Figure 2
iii. Data from two of these 3 experiments is shown in Figure 3A and B; in the third
experiment, a large number of spots were observed in all the wells of the plate,
even the unstimulated ones suggesting that we had a technical problem on that
day
iv. Data on the proliferative capacity of the frozen/thawed PBMCs is shown in
Figure 4 A – C.
21 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
1
.8
.6
.4
.2
A05997
A05988
A05403
A05262
A05254
A04999
A04713
A04645
A04643
A04308
A04169
NT
0
A02314
% PBMCs recovered/100
(Mean +/- SEM)
1.2
Figure 2: The proportion of cells recovered after the Cerus freeze/thaw process is shown
relative to the number of cells frozen down 8 week previously. Error bars represent SEM
when more than one aliquot was thawed. NT = not tested. Note: % recovery ranged from
approximately 40% to 100%
22 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
A: TUL 33
IFNg Spots (Mean +/- SEM)
200
150
100
50
0
A04645
A05254
LVS
LVS
LVS
LVS
LVS
LVS
LVS
LVS
LVS
LVS
LVS
LVS
hk Hi, Fresh
hk Hi, Frozen
hk Mid, Fresh
hk Mid, Frozen
ff Hi, Fresh
ff Hi, Frozen
ff Mid, Fresh
ff Mid, Frozen
ff Lo, Fresh
ff Lo, Frozen
hk Super, Fresh
hk Super, Frozen
IFNg Spots (Mean +/- SEM)
B: TUL 34
250
LVS hk Hi, Fresh
LVS hk Hi, Frozen
LVS hk Mid, Fresh
200
LVS hk Mid, Frozen
LVS ff Hi, Fresh
150
LVS ff Hi, Frozen
LVS ff Mid, Fresh
100
LVS ff Mid, Frozen
LVS ff Lo, Fresh
50
LVS ff Lo, Frozen
LVS hk Super, Fresh
0
A02314
A04308
A04713
LVS hk Super, Frozen
Figure 3: IFNγ production by PBMCs from non-LVS vaccinated NHPs to LVS antigens is
shown (ff = formalin fixed; hk = heat-killed; Super = 4 x 105/ml, Hi = 1 x 105/ml; Mid = 025 x
105/ml; Lo = 0.0625 x 105/ml). Fresh and thawed and Frozen PBMCs were plated at 1.33 x
106/ml in 2 separate experiments (A: TUL 33) and (B: TUL 34).
23 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
A: TUL 33
Fresh
Frozen
400000
SCHUS4 ff Mid
SCHUS4 ff Hi
SCHUS4 ff Super
SCHUS4 hk Mid
SCHUS4 hk Hi
SCHUS4 hk Super
LVS hk Super
LVS ff Lo
LVS ff Mid
LVS ff Hi
LVS hk Mid
0
LVS hk Hi
200000
Media
RLU (Mean +/- SEM)
600000
B: TUL 34
400000
SCHUS4 ff Mid
SCHUS4 ff Hi
SCHUS4 ff Super
SCHUS4 hk Mid
SCHUS4 hk Hi
SCHUS4 hk Super
LVS hk Super
LVS ff Lo
LVS ff Mid
LVS ff Hi
LVS hk Mid
0
LVS hk Hi
200000
Media
RLU (Mean +/- SEM)
600000
Fresh
Frozen
24 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
C: TUL 35
Fresh
Frozen
600000
400000
SCHUS4 ff Mid
SCHUS4 ff Hi
SCHUS4 ff Super
SCHUS4 hk Mid
SCHUS4 hk Hi
SCHUS4 hk Super
LVS hk Super
LVS ff Lo
LVS ff Mid
LVS ff Hi
LVS hk Mid
0
LVS hk Hi
200000
Media
RLU (Mean +/- SEM)
800000
Figure 4: Proliferation by PBMCs from non-LVS vaccinated NHPs to LVS and SCHU S4
antigens is shown (ff = formalin fixed; hk = heat-killed; Super = 4 x 105/ml, Hi = 1 x 105/ml;
Mid = 025 x 105/ml; Lo = 0.0625 x 105/ml). Fresh and thawed and Frozen PBMCs were
plated at 1 x 106/ml in 3 separate experiments (A: TUL 33, B: TUL 34 and C: TUL35).
Data Interpretation: The results are inconsistent. In general, the freeze/thaw process did not
spare the responsiveness of the cells in the IFN ELISPOT assay (see Figure 3 TUL 33, A05254
or TUL 34, A04308). On the other hand, the response was spared a little more when considering
TUL 34, A02314. Frozen and thawed PBMCs from A04713 (TUL 34) responded in a different
pattern than the fresh cells. As all of these responses are from non-LVS vaccinated NHPs, it is
difficult to predict what proportion of a high response to LVS antigens would be. This is
particularly true when comparing responses in the proliferation assay as the responses of the
fresh cells to the LVS and SCHU S4 antigens are not high (as expected from non-LVS vaccinated
NHPs). More work needs to be done with LVS-vaccinated PBMCs.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay063008.svd;
TVDC (2) bound notebook (8935), pps. 48 – 63; TVDC (3) bound notebook (9225), pps. 4 - 13.
c.
We have realized that we have been mis-representing PLASMA IgG anti-LVS titers
as SERUM IgG anti-LVS titers in previous technical reports.
d. We have realized that all IgG anti-LVS titers previously reported have been 4-fold
less than their true value. Blood is mixed with 3 volumes of PBS before layering over
Lymphoprep. Plasma is collected from the supernatant after the spin. Therefore, a
previously reported titer of 5000 is actually 20000.
4. Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
25 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
75% of the scientific work is complete. Note, we have just extended this Milestone
completion date until 3/31/09 in order to optimize both the freeze thaw process and the
responsiveness to the LVS and SCHU S4 antigens. Therefore, the percentage
completed has dropped from last month’s report which was 97.5%
9. Work plan for upcoming month
a. Continue to test PBMCs from LVS-vaccinated and non-vaccinated NHPs in the
IFN ELISPOT assay to determine the effect of HK and FF LVS at different
concentrations.
b. Continue to freeze down extra cells if they are available.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
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
a. 1.2 x 108 PBMC were isolated from 90 ml of blood collected from a LVS
vaccinated volunteer and frozen in liquid nitrogen.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
1.5%
9. Work plan for upcoming month
a. Determine the viability of PBMC after a single freeze-thaw cycle.
b. Stimulate PBMC with LVS or mitogen to determine whether antigen-specific
proliferation and IFN production are retained after freeze-thaw
c. Develop SOP for generating macrophages from fresh and frozen PBMC
26 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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 T. 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
a. PBMC was prepared from blood collected from a LVS vaccinated volunteer
and frozen in liquid nitrogen
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
1.5%
9. Work plan for upcoming month
a. Develop SOP for generating macrophages from fresh and frozen PBMC
b. Develop SOP for infecting human monocyte-derived macrophages with LVS
and SCHU S4
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 19-UNM
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
27 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
a. Experiment Ftc36 study 9 (Notebook 115, pages 81-84)
i. The purpose of this experiment was to determine the cytokine
response of human alveolar macrophages infected with LVS or
SCHU S4. The human alveolar macrophages were from a non-LVS
vaccinated volunteer donor.
ii. 106 human alveolar macrophages were infected with LVS or SCHU
S4 at MOI of 1, 10 and 100 (bacteria: macrophages) and, after 24
hours, the culture supernatants were collected and analyzed for
cytokine levels by Luminex multiplex analysis.
iii. As shown in Fig. 4, the cytokine response to LVS and SCHU S4
infection were similar, particularly at MOI of 1 and 100. However, at
MOI of 10, SCHU S4 consistently induced 2-3-fold more IL-1, IL-6,
MIP-1, and TNF. IFN, IL-12p40 and IL-10 were not induced by
LVS or SCHU S4 (data not shown)
Figure 4. Cytokine responses by human alveolar macrophages infected with
LVS or SCHU S4. 106 human alveolar macrophages were plated in 1 ml in a 24well plate and infected with LVS or SCHU S4 at MOI = 1, 10, and 100. After 24
h, the culture supernatants were collected and analyzed by Luminex multiplex
cytokine analysis
b. Experiment Ftc36 study 11 (Notebook 115, pages 135-136 and 142-144)
i. Listed below are several limitations to the human alveolar
macrophage studies.
1. Low average yield ~ 107 cells/patient. This limits the number
of replicates and possibly the assay sensitivity
28 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
ii.
iii.
iv.
v.
2. Fungal and bacterial contamination. Fungal contamination
can be controlled by adding amphotericin B but bacterial
contamination cannot be controlled by adding antibiotics
because they would also kill LVS and SCHU S4
3. Cell loss to washes between steps. In a typical experiment,
the adherent macrophages are washed at least 9 times to
remove non-adherent cells, extracellular bacteria and
gentamicin. In some experiments, this led to significant cell
loss and variability among replicates.
It has been suggested that suspension culture may mimic the
alveolar environment better than adherent culture. Suspension
culture also has the advantage that the cells can be centrifuged
before aspirating the culture supernatant and the wash buffers
thereby reducing cell loss.
The purpose of this experiment was to compare the cytokine
response by adherent and suspended alveolar macrophages
infected with SCHU S4
106 human alveolar macrophages were cultured in 15 ml
polypropylene tubes or in 24-well tissue culture plates in 0.5 ml
RPMI1640 and infected with SCHU S4 at MOI 0.1, 1.0 and 10. 24 h
after infection, the culture supernatants were collected and analyzed
by Luminex multiplex cytokine analysis
As shown in Fig 5, macrophages cultured in tubes produced
noticeably less cytokine than macrophages cultured on tissue culture
plates.
Figure 5. Cytokine responses by human alveolar macrophages cultured in nontissue culture treated tubes or on tissue culture plates.
29 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Needs improvement
8. Percentage completed
13%
9. Work plan for upcoming month
a. Repeat cytokine analysis of human alveolar macrophages cultured in nontissue culture treated tubes and on tissue culture treated plates.
b. Determine the effect of recombinant IFN on intracellular growth of SCHU S4
and LVS.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21-UNM
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 Ftc30.19b-e (Notebook 109, pages 116-118, 122-127, 131-133)
i. The results from Experiment Ftc30.19a indicated that for the murine
macrophage SCHU S4 killing assay :
1. Increasing the phagocytosis incubation time from 1 h to 4 h
improved the consistency across replicates
2. Plating a larger volume (50 l) of cell lysates with the
autoplater improved the consistency across replicates than
plating a smaller volume (10 l) by the microdot technique
3. There was no difference between cell lysis with
deoxycholate and water
ii. Ftc30.19 was repeated 4 times to ensure reproducibility and a
representative result is shown in Fig. 6
iii. In all four experiments, the most consistent results were obtained by
infecting the macrophages for 4 hour, lysing the cells with water, and
plating larger volumes with the autoplater
30 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Figure 6. Optimization of the murine macrophage SCHU S4 killing
assay. Murine bone marrow-derived macrophages were infected with
SCHU S4 at MOI of 1:100 (SCHU S4: macrophages) for 1 h or 4 h. After
48 h, the infected macrophages were lysed with water or 0.1%
deoxycholate and the total number of SCHU S4 in the cell lysate and
culture supernatant was determined. Each bar is a replicate showing the
total number of SCHU S4 in the cell lysate and culture supernatant.
b. Experiment Ftc30.20b (Notebook 109, pages 137-139)
i. The purpose of this experiment was to determine whether
recombinant IFN and splenocytes from LVS vaccinated mice can
suppress SCHU S4 growth in mouse bone marrow-derived
macrophages (BMM).
ii. BMM were infected with SCHU S4 at MOI of 1:100 (bacteria:
macrophages) and then incubated with 50ng/ml recombinant mouse
IFNand 1x106 vaccinated or naïve T cells.
iii. The results showed clearly that IFN reduced the number of SCHU S4
recovered (Fig. 7). However, vaccinated splenocytes had no effect on
the number of SCHU S4 recovered. Since we did not have a control
showing killing of LVS, we do not know if the results with the
vaccinated splenocytes suggest that vaccinated splenocytes are not
capable of inducing SCHU S4 killing or that the vaccinated splenocytes
are not functional in this experiment
31 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Figure. 7. Vaccinated splenocytes failed to induce BMM killing of SCHU S4. Murine
BMM were infected with SCHU S4 at MOI of 1:100 (bacteria: macrophages) and then
incubated with 50ng/ml recombinant mouse IFNand 1x106 vaccinated or naïve T cells.
After 48 h, the bacterial burden was determined.
c.
Experiment Ftc61.6 (Notebook 109, pages 119-121, 128-130)
i. The purpose of this experiment was to determine whether
recombinant IFN and T cells from LVS vaccinated and naïve
Fischer 344 rats can suppress LVS growth in rat bone marrowderived macrophages (BMM).
ii. BMM were infected with LVS at MOI of 1:20 (bacteria: macrophages)
and then incubated with 50 ng/ml (5 ng/well) or 500 ng/ml (50 ng/well)
recombinant rat IFN 1x105 or 1x106 vaccinated or naïve splenocytes
for 3 days
iii. Figure 8 showed clearly that 50 ng/ml IFN reduced the number of LVS
recovered by 3 logs and that 500 ng/ml of IFN completely eliminated
LVS from the culture
iv. More importantly, the number of LVS was reduced by 1-2 logs by the
addition of 105 and 106 vaccinated splenocytes. When 106 naïve
splenocytes were added, some nonspecific activity was detected.
32 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Figure. 8. Vaccinated splenocytes induced rat BMM killing of LVS. Rat BMM were
infected with LVS at MOI of 1:20 (bacteria: macrophages) and then incubated with naïve
or vaccinated splenocytes or recombinant IFNas indicated After 72 h, the bacterial
burden was determined.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
33%
9. Work plan for upcoming month
a. Repeat the murine macrophage SCHU S4 killing assay with naïve and
vaccinated splenocytes, including killing of LVS as a control
b. Perform the rat macrophage SCHU S4 killing assay with naïve and
vaccinated splenocytes
c. Determine the MOI for infecting rat macrophages with SCHU S4
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
33 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
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. Intracellular cytokine staining was performed using 3 LVS-vaccinated NHPs in
May 2008 (TUL 36).
i. PBMCs were prepared, rested overnight, and stimulated with LVS and SCHU
S4 antigens in combination with antibodies specific for two T cell co-stimulatory
antigens (anti-CD28 and anti-CD49d) for 2 hours at 37 degrees before addition of
Golgiplug (Brefeldin A) to inhibit cytokine secretion for an additional 4 hours
ii. Cells were stained with either anti-CD4, anti-CD8 or anti-CD3 in combination
with antibodies specific for cytokines IL-2, IFN and TNFα.
iii. Cell surface staining was good using anti-CD4 or anti-CD8 antibodies, but
staining with anti-CD3 antibody was poor. We have observed this previously.
Although fresh PBMCs stain well with anti-CD3 (TUL 10), cells which have been
cultured overnight do not retain CD3 in a form that is readily detectable (TUL 13).
One problem recently discovered was that the anti-CD3 antibody we used was
expired; we will order a new batch before we stain PBMCs again.
a.
CD8 cells were observed to be producing TNFα, but there was no effect of
the stimuli on its production (Figure 5)
b.
No IFN or IL-2 production was observed by intracellular cytokine staining,
despite the fact that IFN was detectable in the ELISPOT assay using the
same PBMCs on the day of preparation (Figure 6).
34 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
CD8+TNFα+ % of Total
PBMCs
1.2
1
.8
.6
Media
LVS hk Hi
LVS ff Hi
LVS ff Mid
LVS hk Super
.4
.2
0
A00659
A00868
A00908
Figure 5: Intracellular cytokine staining assay: PBMCs were prepared from LVS vaccinated NHPs
and stimulated with anti-CD28, anti-CD49d and the indicated stimuli for 6 hours, the last 4 in the
presence of Brefeldin A (ff = formalin fixed; hk = heat-killed; Super = 4 x 105/ml, Hi = 1 x 105/ml; Mid =
025 x 105/ml). Cells were collected and stained with anti-CD8 and anti-TNFα. A00659 and A00868 are
523 days post SC-LVS vaccination. A00908 is 532 days post ID-LVS vaccination. (Intracellular
cytokine staining assay)
Cell Mean for IFNg Spots
350
300
250
200
150
100
50
0
A00659
A00868
A00908
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
Figure 6: IFNγ production as measured by the IFN ELISPOT assay by PBMCs from LVS vaccinated
NHPs to LVS and SCHU S4 antigens (ff = formalin fixed; hk = heat-killed; Super = 4 x 105/ml, Hi = 1 x
105/ml; Mid = 025 x 105/ml; Lo = 0.0625 x 105/ml). All PBMCs plated at 1.33 x 106/ml. A00659 and
A00868 are 523 days post SC-LVS vaccination. A00908 is 532 days post ID-LVS vaccination. (IFNg
Elispot assay)
35 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Data Interpretation: Although the IFN ELISPOT data showed that the two S.C. vaccinated NHPs
(A00659 and A00868) appear to still have a response to LVS FF (at different doses), as well as a
small, but detectable response to LVS HK (A00659 > A00868) and high doses of SCHU S4 FF,
there was no detectable IFN staining in the intracellular cytokine staining assay. TNFα
production was detectable, only in CD8 cells, but was not dependent upon stimulation with LVS
antigens. The lack of IFN detection by intracellular cytokine staining, and the failure of any of
the stimuli to induce more than background TNFa staining, suggests that the conditions of PBMC
stimulation were not optimum in the intracellular cytokine staining assay (ICCS). In the IFN
ELISPOT assay, the cells are stimulated overnight and the secreted IFN accumulates in the well
for 20 hours before it is measured. The PBMCs were stimulated for only 6 hours in the ICCS
assay. It is possible that we may need to adjust the length of incubation time with the stimuli in
the ICCS assay. We chose a 6 hour incubation period and the culture conditions based on a
published manuscript: Kwissa, M. et al., Journal of Experimental Medicine 204:2733-46, 2007.
Another difference is that the PBMCs were held overnight in the refrigerator on ice for
approximately 16 hours before initiating the stimulation. It is possible that we may need to
stimulate them immediately after purifying them in order to get a good response in the ICCS
assay. We will attempt this, as well as stimulation with Con A, a positive control mitogen, in the
next assay.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay063008.svd;
TVDC (2) bound notebook (8935): TUL 36 (pps. 73 – 81).
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
No stimulus-driven cytokine production was detected by the intracellular cytokine
staining assay. A positive control such as Con A or PMA+ionomycin will be included in
the next assay.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
2% of the scientific work is complete
9. Work plan for upcoming month
Set up another intracellular cytokine assay and include a positive control.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated.
36 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Milestone 26
Milestone description: Confirmation of gene and protein expression (develop HTP
SOPs for ORF library production, protein library production, and protein purification)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Imidazole wash titration: LVS cross-reactivity of E. coli proteins
nonspecifically bound to Ni beads.
As presented in last month’s report, there is a wide diversity and large
amount of proteins sticking to the Ni beads as detected by Mass spec. We
tested whether or not more stringent washes with imidazole might clean up
the bead bound fraction. The 20-500mM imidazole didn’t reduce the
diversity or quantity of proteins sticking to the beads, as detected with
splenocytes from LVS vaccinated mice. (See Figure 1 below)
ii. This demonstrates that the interactions between Ni beads and cross-reacting
proteins are non specific (not related to His ligand), since they are not eluted
with imidazole. On the other hand, the imidizole titration successfully elutes
the OVA polypeptide, as expected for a specific interaction between his and
Ni. This is shown with the 20-500 mM range. At 100mM, most binding is lost
for most Ni interaction. This is consistent with the loss of most Ova
stimulation at 100mM-500mM samples. (Compare enumerations at 100mM
to 500mM for ova samples d8-d10 to no template samples in d3-d5) results
iii. Mass spec analysis of bead-bound proteins showed similar composition after
either 20mM or 500mM wash
i.
37 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
No template
Ova
50% Naïve+ 50% DO11.10
20mM
50mM
100mM
200mM
500mM
20mM
50mM
100mM
200mM
500mM
LVS vaccinated
Figure 1: ELISPOT analysis of ova and FTU polypeptide bound beads washed with
Imidazole titration. Top panel: Ova immune T cells are stimulated with beads
incubated with IVT lysate without a template or with ova polypeptide template.
Bottom panel LVS-immune T cells incubates with same bead samples.
UNM Ftc59 study 20 (Notebook 115, pages 128-132 and 138) L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Ftc59\Ftc59 study 20
UNM Ftc59 study 21 (Notebook 115, 134 and 139)L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Ftc59\Ftc59 study 21
B. Pre-clear IVT lysates with anti-LVS antisera
i.
The objective in this experiment was to confirm that antisera against LVS
proteins also recognized the E. coli proteins that are causing the undesired
IVT lysate T cell reactivity.
ii. Protein G beads were loaded with anti LVS antibodies. Prepared beads
were then incubated with E coli IVT lysate components to remove crossreacting proteins. Supernatant was tested in T cell assays against nonimmune (ova) or LVS immune T cells.
38 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
1
2
3
4
5
50% Naïve+
50% DO11.10
LVS vaccinated
Figure 2: ELISPOT analysis of IVT lysates which have been depleted of E. coli
proteins that cross-react with anti-LVS antisera before the IVT reaction.
Legend 1: No pre clearing of IVT lysate, 2: 10ul, 3: 20ul, 4: 50ul, 5: 100ul protein G
beads used for pre-clearing
UNM Ftc59 study 20 (Notebook 115, pages 128-132 and 138) L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Ftc59\Ftc59 study 20
UNM Ftc59 study 21 (Notebook 115, 134 and 139)L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Ftc59\Ftc59 study 21
iii. In bottom panel (LVS vaccinated T cells: In column F1 lysate was not predepleted with anti-LVS beads. In f2-f4, lysate was depleted. This appears to
reduce background---however, the approach is presumably pulling out E. coli
ribosomes and other expression factors that would be needed for the IVT
reactions. Nonetheless it confirms our presumption that there is specific
cross-reactivity between LVS and E. coli
39 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
C. Post-IVT reaction depletion of E.coli proteins from OVA polypeptide
prep
1
2
3
4
1
2
3
4
50% Naïve+ 50%
DO11.10
LVS Vaccinated
NEB PURE
INVITROGEN
Figure 3: ELISPOT analysis of IVT samples which have been depleted of
proteins that bind anti-E. coli antisera after the IVT reaction.
Legend:
1. IVT “as is”
2: 10ul E.coli IgG bound protein G beads
3: 50ul E.coli IgG bound protein G beads
4: 100ul E.coli IgG bound protein G beads
UNM Ftc59 study 20 (Notebook 115, pages 128-132 and 138) L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Ftc59\Ftc59 study 20
UNM Ftc59 study 21 (Notebook 115, 134 and 139)L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Ftc59\Ftc59 study 21
1. IVT synthesized Ovalbumin polypeptide was incubated with indicated
amounts of protein G beads with attached anti-E.coli IgG. The supernatant
was used in T-cell assay
2. NEB pure appears to be similarly immune-stimulating relative to standard
lysate composition
3. We noted high variability in samples
4. This will be repeated with uniform bead pipetting method
40 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
D. Sensitivity of Rabbit Reticulocyte IVT on Protein G beads in T-cell assay
Ova
FTU901 FTU1695
Ova
FTU901 FTU1695
50% Naïve+ 50%
DO11.10
LVS Vaccinated
IVT as is
Protein G beads
Slide 41
Figure 4: Determining whether binding antigen to beads can make rabbit
retic IVT’s detectable as an antigen source. Top row: splenocytes in
ELISPOT were harvested from ova transgenic mice. Bottom row:
splenocytes harvested from LVS immune mice. Templates in IVT reactions
used to stimulate samples are shown above respective columns. Samples
in h1-h3 were not purified, samples in h4-h6 were purified via hi tag on
protein G beads.
UNM Ftc59 study 20 (Notebook 115, pages 128-132 and 138) L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Ftc59\Ftc59 study 20
UNM Ftc59 study 21 (Notebook 115, 134 and 139)L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Ftc59\Ftc59 study 21
i.
In positive control samples, the bead-bound ova polypeptide stimulated a few
more T cells relative to soluble antigen.
ii. However, numbers are generally low (antigen amount is low) and not
discernable above background in bead-bound IVT negative control sample.
iii. Set-up to test Sensitivity of specific antigens in the T-cell assay
1. Recombinant ovalbumin and FTU proteins were purified (bound
and eluted) from affinity column (Ni) and quantified
2. Four different concentrations prepared from stock and used for Tcell assay were 0.1ug/ml, 1.0ug/ml, 5.0 ug/ml, and 10ug/ml (0.5ng,
5ng, 25ng, and 50ng)
3. 100ul of the eluant was used to bind Ni beads, Protein G beads
with anti-his antibodies, or left in the PBS buffer
41 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Ova
1
2
3
FTU901
4
1
2
3
FTU1695
4
1
2
3
4
PBS ProG Ni
FTU1695
FTU901
Ova
Purified antigen
PBS ProG Ni
50% Naïve+ 50% DO11.10
LVS vaccinated
1: 0.1ug/ml, 2: 1.0 ug/ml, 3: 5.0 ug/ml, 4: 10.0 ug/ml
Slide 20
Figure 5: Establishing the sensitivity of our T cell assay using E. coli IVT produced
ova as a positive control antigen. The coomassie gel in left panel displays purity
of antigen material used to stimulate T cells in Elispot.Top panel displays assay
with ova immune T cells, bottom panel shows assay conducted with LVS immune
T cells. Each IVT reaction antigen was purified on Ni beads (Ni), protein G beads
(protG), or bound to Ni beads then eluted (PBS), as indicated to right of each row.
Each IVT produced sample was tested at 3 concentration in lanes 1 – 3 as
indicated above columns. These results show that the protG beads provide the
highest sensitivity and that 5ug/ml is sufficient to detect ova specific stimulation.
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT Coomassie
gels
UNM Ftc59 study 20 (Notebook 115, pages 128-132 and 138) L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Ftc59\Ftc59 study 20
UNM Ftc59 study 21 (Notebook 115, 134 and 139)L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Ftc59\Ftc59 study 21
42 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
iv. Comparison of antigen (OVA) presentations in T-cell assay
250
PBS
Ni beads
Protein G beads
Number of spots
200
150
100
50
0
0.1
1.0
5.0
10.0
Antigen concentration (ug/ml)
Figure 6: Data from figure 5 represented in a bar graph
1. The protein G beads with attached antibody provide higher
sensitivity than Ni beads in T cell assay.
2. Our assay sensitivity is sufficient to specifically detect
~5ug/ml of antigen
43 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
E. Generate Thioredoxin LEE Constructs for IVT reactions
1.
Thioredoxin Tag at the N-Terminus
2.
His Tag at the C-Terminus
1
2
3
4
5
6
7
8
9
Figure 7: This agarose gel display the LEE templates (lanes 1-9) used in the IVT
reactions. All ORFs are of predicted molecular weight and show efficient linkage
full length expression unit including promoter and terminator.
44 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
F. Evaluating IVT products from new Thioredoxin LEE constructs in T cell
assays
Attachment of anti-thio antibody via Fc using protein G magnetic
beads
G-α-Thio
i.
Ni
G-α-His
No
Template
No
Template
Ova
FTU901 FTU1695
50% Naïve+
50% DO11.10
Ova
FTU901
FTU1695
LVS Vaccinated
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU Scintillation results\FTI IVT Thio Fusion
tests
Figure 8: ELISPOT analysis of Thio tagged IVT products purified on anti-thio
attached beads. These samples were conducted during the pipetting problem at
UNM, so precise comparisons of numbers cannot be done some wells have
considerably more or less beads than others. Nonetheless, ASU noted a few
trends in the data and these are described below.
UNM Ftc59 study 20 (Notebook 115, pages 128-132 and 138) L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Ftc59\Ftc59 study 20
UNM Ftc59 study 21 (Notebook 115, 134 and 139)L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Ftc59\Ftc59 study 21
1. Right hand panel: We observe significant differences between
bead configurations. We are testing the thio G bead, Ni bead or
His G bead bound samples in each row to assess level of crossreactivity remaining in samples after affinity purification of
polypeptides. By 3 different beads based protocols
2. Bottom row of LVS panel has Ni bound samples, and as seen
previously we see much cross-stimulation of LVS-immune T cells
with FTU or OVA antigen samples. H11 is presumably the same
as h9 and h10 but the pipetting accuracy problem had not been
resolved yet here.
3. Middle row of LVS panel: The His G beads provide lower
background
4. Top row, LVS panel: the thio G beads look the cleanest (i.e. has
45 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
the
least background).
5. The nickel NTA (nickel chelated Nitrilotriacetic Acid) is significantly
more “sticky”, with respect to the cross-reacting lysate proteins,
than either the anti-His or Thio antibody.
6. The His antibody is stickier than the Thio antibody
7. Use of the thio tag appears to be the clean approach (no or very
low amounts of nonspecifically bound IVT components.)
ii. Autoradiograph of second batch of thioredoxin fusion FTU IVT
proteins
Figure 9: Radiolabeled lysates were electrophoresed to display nascent polypeptides only.
These bands can be compared to immunoblots with anti-tagged antibodies to assess
specificity of antibody fro tag, relative to other components in the IVT lysate.
1: Ladder, 2; Ova, 3; FTU 721A, 4: FTU 721B, 5: FTU 721C, 6: FTU 1695, 7: FTU 1696Aa, 8;
FTU 1696Ba, 9: FTU 1712, 10: GFP
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\Thio fusion
IVT\Thio Prm IVT test on FTU 9 6-25-08 03 crop
46 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
iii. Total thioredoxin fusion FTU IVT proteins production calculated
from CPM counts
1
2
3
4
5
6
7
8
9
Fragment Name
Trx-LEE Ova
Trx-LEE 721A
Trx-LEE 721B
Trx-LEE 721C
Trx-LEE 1695
Trx-LEE 1696Aa
Trx-LEE 1696Ba
Trx-LEE 1712
GFP
ug prot
8.78
6.66
6.22
6.54
6.28
8.88
9.30
11.61
7.71
Table 1: Calculated yields, based on cpms, of samples displayed in Figure 9.
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU Scintillation results\FTI IVT Thio Fusion
tests
iv. Western blot using polyclonal anti-Thioredoxin antibody: probed
with antibody gel as in #2.
Figure 10: Immunoblot analysis of IVT reactions with anti-thio antibody
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU IVT Western\IVT Thio pAb
LEE FTU9 6-25-08 crop
1. Western and radiograph results match. This indicates that there is
little to no other reactivities of the anti-Thioredoxin antibody with
proteins other than thio specific polypeptides. Additional bands are
partial products, since they are smaller than the expected
migration of full length and also match with the autorad data in fig
9.
2. This is very different than the results we obtained with the anti-His
antibody in which case many other polypeptides were visualized in
the anti-His immunoblot.
47 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
G. Testing protocols for protein purification using Thioredoxin (Trx) Tag
i.
Test specificity and efficiency of anti-thioredoxin monoclonal vs.
polyclonal antibodies for thio fusion polypeptide binding
1. Four anti-Thioredoxin monoclonal Abs from different
manufacturers (mAb2A1, 3A1, 8A1, A00180) were compared to a
polyclonal Ab (pAb Trx) by immunoblot analysis.
2. Each gel contains: 1. IVT lysate without template, 2. purified
Thioredoxin, and 3. chemluminescent ladder.
3. Results show that the polyclonal and only 1 monoclonal (mAb
A00180) reacts effectively with the thioredoxin. The arrow indicates
position of Trx (data below).
1 2 3
1 2 3
1 2 3
1 2 3 1 2 3
1 2 3
Figure 11: Comparison of performance of different anti-thioredoxin antibodies
by western analysis
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU IVT Western\Thio mAb and
pAb test 02 on Ecoli and Trx 6-25-08
ii. Magnetic beads were constructed with a covalently attached anti-Thioredoxin
antibody and binding to thio tag was analyzed by Coomassie stain (below).
iii. These results show that thioredoxin is bound by both the polyclonal and one
of the monoclonal antibody-attached beads, and is highly enriched in the
bead bound fraction (lanes 6 and 8) relative to the supernatant (lanes 2 and
3).
48 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Figure 12: Coomassie gel shows efficiency of fusion protein bound to antibody
bound beads by comparing bead fractions to supernatant fractions
1 : Kalidoscope Ladder, 2: 1/10 polyclonal Ab (pAb) Trx supernatant, 3: 1/10 monoclonal Ab
(mAb) Trx , supernatant, 4: 1/10 Trx purified 100ng/ul, 5: pAb-Trx IVT, 6: pAb-Trx IVT purified, 7:
mAb-Trx IVT, 8: mAb-Trx IVT purified, 9: 100ng Trx purified, 10: 50ng Trx purified
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT Coomassie gels\Thio
IVT\Thi purified with aThio mAb and pAb beads Test 01 6-27-08 crop
iv. Testing methods of binding IVT fusion products to beads. Since we have
moved away from using the His tag, urea denaturation of the IVT samples
during beads binding is not possible. The Trx –antibody reaction (as any
non-covalent antibody interaction) will not occur in denaturing conditions.
Therefore we tested whether folded fusion proteins could be efficiently bound
to the tag and compared these results to adding the beads during the
synthesis step.
1. Incubation of anti-Trx beads simultaneously with IVT reaction
2. IVT proteins were incubated with beads for 2 hours with shaking at
4°C
3. IVT proteins were incubated with beads overnight with shaking at
4°C
4. Binding of IVT proteins were evaluated from silver stain gel,
autoradiograph, and CPM counts of unbound proteins in the
supernatant.
49 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
I. Silver stain gel analysis of IVT reactions
Figure 13: 1: Silver stain analysis of bound beads and supernatants shows purity of
samples and efficiency of fusion tag binding.
Kalidoscope Ladder, 2: 50ng purified Trx, 3: 100ng purified Trx, 4: 200ng purified Trx, 5:
FTU721A IVT incubation with mAb beads, 6: FTU721A 2 hrs incubation with mAb beads, 7:
FTU721A overnight incubation with mAb beads,8: FTU1696Ba IVT incubation with pAb
beads, 9: FTU1696Ba 2 hrs incubation with pAb beads, 10: FTU1696Ba overnight incubation
with pAb beads, 11: FTU721A IVT, 12: FTU721A IVT incubation with mAb beads
supernatant, 13: FTU721A 2 hrs incubation with mAb beads supernatant, 14: FTU721A
overnight incubation with mAb beads supernatant, 15: FTU1696Ba IVT , 16: FTU1696Ba IVT
incubation with pAb beads supernatant, 17: FTU1696Ba 2 hrs incubation with pAb beads
supernatant, 18: FTU1696Ba overnight incubation with pAb beads supernatant
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT Coomassie
gels\Thio IVT\ IVT purified w aThio mAb and pAb beads Test 02 7-01-08 crop
II. Autoradiograph of same samples
Figure 14: 1: Kalidoscope Ladder, 2: 50ng purified Trx, 3: 100ng purified Trx, 4: 200ng
purified Trx, 5: FTU721A IVT incubation with mAb beads, 6: FTU721A 2 hrs incubation with
mAb beads, 7: FTU721A overnight incubation with mAb beads,8: FTU1696Ba IVT incubation
with pAb beads, 9: FTU1696Ba 2 hrs incubation with pAb beads, 10: FTU1696Ba overnight
incubation with pAb beads, 11: FTU721A IVT, 12: FTU721A IVT incubation with mAb beads
supernatant, 13: FTU721A 2 hrs incubation with mAb beads supernatant, 14: FTU721A
50 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
overnight incubation with mAb beads supernatant, 15: FTU1696Ba IVT , 16: FTU1696Ba IVT
incubation with pAb beads supernatant, 17: FTU1696Ba 2 hrs incubation with pAb beads
supernatant, 18: FTU1696Ba overnight incubation with pAb beads supernatant
III. Comparison of Coomassie gel to silver stain identifies which
polypeptides are the nascent IVT products from among the full
lysate and shows relative amount of IVT product bound by beads
vs. unbound (remaining in supernatant).
IV. Incubation of beads during IVT synthesis yields more
polypeptide than adding beads after reaction. Yields of 20% to
40% are much better than we saw with His system.
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\Thio
fusion IVT\Thio fusion purified 721A 1696Ba 07-01-08 02 crop
V. Calculation of unbound protein from anti-thioredoxin magnetic
beads in the supernatant
1
2
3
4
5
6
7
Fragment Name
Trx-LEE 721A IVT
Trx-LEE 721A Incubation with IVT beads
Trx-LEE 721A 2-hour bead incubation
Trx-LEE 721A overnight bead incubation
Trx-LEE 1696Ba IVT
Trx-LEE 1696Ba Incubation with IVT
beads
Trx-LEE 1696Ba 2-hour bead incubation
Trx-LEE 1696Ba overnight bead
incubation
ug prot
8.88
5.45
6.82
7.25
8.08
6.34
7.28
8
7.16
Table 2:
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU Scintillation results\FTI IVT Thio
Fusion tests\Thio_721A and 1696Ba incubation time test on M280 beads
v. Prepare Trx-tag purified FTU polypeptides and Ovalbumin for
Shipment to UNM



Two sets of Ova, FTU 1695, and FTU 1712 with Thioredoxin
fusion IVT were bound on 10ul of beads overnight (1) via
monoclonal Ab Trx, (2) via polyclonal Ab Trx
All beads were shipped in 20ul of PBS buffer to UNM
These will be tested on both murine and NHP T cells.
4. Significant decisions made or pending
We plan to confirm the performance of the new thioredoxin bead purification protocol
in the murine and NHP T cell assays and then make the decision to proceed with
library production in the upcoming month
5. Problems or concerns and strategies to address
See above
6. Deliverables completed
None
51 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
7. Quality of performance
Excellent
8. Percentage completed
99.9%
9. Work plan for upcoming month
Currently we are:
 Generating and testing Thioredoxin LEE assemble in high throughput
format (1/2 96-well plate)
 Testing Thio-LEE ORF IVT protein production in single and HTP format
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will
use ASU’s protein fragments in lymph node proliferation assays to define vaccine
candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary
conclusions
a. Ftc59 study 20 (Notebook 115, pages 128-131) and Ftc59 study 21
(Notebook 115, pages 134 and 139)
i. The purpose of this experiment was to test ivt proteins after various
purifications for crossreactivity. The purification conditions were:
1. Preclearing ivt lysates with anti-LVS antisera
2. Post-ivt reaction depletion of E. coli proteins from OVA
polypeptide prep
3. Binding ivt thio fusion proteins to beads via thio or His tags
ii. A second purpose of this experiment was to test the sensitivity of the
T cell assay.
iii. As in previous experiments, 5 l of each sample was incubated with
2 x 105 splenocytes from LVS-vaccinated mice or a mixture of 105
splenocytes from a DO11.10 transgenic mouse and 105 splenocytes
from a naïve BALB/c in 100 l complete RPMI 1640 medium
overnight at 37oC. The plates were then processed for IFN ELISpot
iv. The results will be presented ASU’s section of this technical report.
v. The most striking observation in these two experiments is the
variability even with the same set of samples. In subsequent
discussion with ASU, it was decided that sample volumes larger than
5 l of must be used because the magnetic beads are heavy and
tend to settle to the well bottom very quickly.


52 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
4. Significant decisions made or pending
Must use volumes larger than 5 l to analyze samples from ASU because the
magnetic beads are heavy and tend to settle to the well bottom very quickly
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
24%
9. Work plan for upcoming month
a.
b.
Test ivt translated proteins from ASU with lymph node cells from LVSvaccinated NHP
Test ivt translated proteins from ASU with murine splenocytes when samples
become available. 
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of polypeptide libraries (Optimize IVT proteinfragment production, Develop IVT protocol for high-throughput production, Validate
immunogenicity of protein-fragments, Full scale production of protein-fragment
library, Purification of protein-fragment library, Array protein-fragment into
overlapping pools, Ship to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome (active)
 Generate complete protein-fragment library (inactive)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)
Institution: ASU-Sykes
3. Date started: 03-01-2007
4. Date completed: Pending
5. Work performed and progress including data and preliminary conclusions
A. Build ORF expression library corresponding to proteome
PCR primers are ready for ORF library production. We will proceed following
decisions on expression system, yield needs, delivery format, pooling capacity, and
purification requirements.
B. Generate polypeptide library
C. Array polypeptide library
53 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
4. Significant decisions made or pending.
The decision to complete the polypeptide purification/optimizations of milestone 26 will be
made this month
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
40%
9. Work plan for upcoming month
Prepare for initiating library production.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35 - UNM
Milestone description: Array hybridization with mouse RNA from virulent SCHU S4
infection and RT PCR confirmation of candidates
Institution: UNM
1. Date started:
2. Date completed: pending
3. Work performed and progress including data and preliminary
conclusions
No new work done
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
6%
9. Work plan for upcoming month
a. ASU requested additional RNA from the lungs of BALB/c mice after i.n.
SCHU S4 infection and from SCHU S4 cultured in Chamberlain’s broth for 1,
3, 5, 7, and 24 h.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
54 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
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: 08-01-2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

We have initiated qPCR testing for known potential antigens of SCHU S4 to establish the
assay. Our assay utilizes SYBR green incorporation into double stranded cDNA products
to determine a critical cycle time to reach the fluorescent threshold of detection. During
each cycle the fluorescent intensity of the product is assessed and plotted over time.
One of the first trials is shown to detect the gene for TUL4 (FTT901) (Figure 1). The key
feature of this graph is the critical fluorescent threshold line (red line across the graph)
which indicates the detection level, above background, where the plot of the fluorescent
intensities is in the linear phase of the amplification. This allows for comparison of copy
numbers of genes between samples. The center of the graph shows a dose-response of
genomic DNA, using 10, 50, or 100 ng of input DNA. Each of the reactions were tested
in triplicate to assess reproducibility.
Figure 1. Graphical representation of the qPCR reaction for FTT901. The red line indicates the
fluorescent intensity of the reaction above the threshold for detection. The curves in the center of
the graph represent a dose response of genomic DNA at 10, 50, and 100 ng per reaction.
Fluorescent intensity is measured at each cycle by the incorporation of SYBR green dye into the
55 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
double stranded cDNA product. Graph
Location…R:\GeneVac\FTU\Contract\Microarray\Milestones\35\qPCR\TEST RUN 6-13-08\
FTU_06-13-08.pdf, page 6


Notebook/File locations …, Notebook 661, FTU qPCR_Trial_2, pages 42-53.
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\qPCR\TEST RUN 6-13-08

Using these samples as a standard, we plotted the cycle times to threshold (Figure 2)
and plotted a linear regression line to assess the fit of the curve. The R2 value of
this line was 0.95 revealing that the cycle threshold line was placed within the linear
portion of the curve.
Figure 2. The cycle time to cross the threshold was plotted for each of the genomic standards
and a linear regression line calculated to fit the data. The R2 value of the line was 0.95. Graph
Location…R:\GeneVac\FTU\Contract\Microarray\Milestones\35\qPCR\TEST RUN 6-13-08\
FTU_06-13-08.pdf, page 6


Notebook/File locations …, Notebook 661, FTU qPCR_Trial_2, pages 42-53.
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\qPCR\TEST RUN 6-13-08
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Low yields were obtained in the LAPT of the last batch of in vitro grown SCHU S4. The
samples will be produced again at UNM. Training of new personnel is ongoing, but good
progress is being made.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
32%
56 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
9. Work plan for upcoming month and next 6 months




The repeat experiment of the time-course challenge of 1000, FTU SCHU S4 in mice
will be performed and the RNA extracts processed through by LAPT.
Finish the labeling and hybridization of amplified RNA of F. tularensis grown in
culture medium and compare the in vitro gene expression results to those identified
in vivo gene expression patterns.
Establish the Q-RT-PCR process for gene expression confirmation.
Assess gene expression levels of known potential antigens (tul4, katG, iglC, groEl) in
the current RNA from the initial dose response and time course experimental sample
set.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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:
We were unable to continue with the cloning of the pdpD deletion containing the
flip recombinase recognition sites (Flp) from the pwsK30 plasmid into pUC118
vector due to work being done in the BSL3 lab to generate the vgrG SchuS4
mutant. We expect that we will have time in the upcoming month to work on this
cloning.
II.
Experiments to generate mutants in Schu4:
a. Prepared a large plasmid preparation of KEK1162 which contains the vgrG intron
construct created using the 81/82 oligo set mentioned in an earlier report. The
concentration of this plasmid was determined and used in a cryotransformation
experiment with SchuS4 strain KKT1 (∆βlac2).
b. Following our standard cryotransformation procedure by using 5 ug KEK1162
plasmid in the experiment, we were able to generate over 800 colonies after two
days of growth at 30ºC. One hundred of these colonies were patched onto fresh
TSA+++ 75 ug/ml kanamycin plates and place at 30ºC. The next day, we
prepared genomic preparations of twenty of these colonies. These were used to
screen for potential VgrG mutants by doing PCR with oligos FTT1364 fwd NdeI
57 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
and VgrG rev EcoRI. The correct SchuS4 VgrG mutant will yield a PCR product
size of 1600 bp while the native profile will yield a product size of 900 bp. The
results of the first twenty selected colonies are shown in figure 1 below.
Figure 1.
1 Kb
Legend:
1 2 13 14 15 16 17 18 19 20 21 22
1. 1 Kb Ladder
2. KKT1
1.5
3. C1 VgrG
13. C11 VgrG
0.5
4. C2 VgrG
14. C12 VgrG
5. C3 VgrG
15. C13 VgrG
6. C4 VgrG
16. C14 VgrG
7. C5 VgrG
17. C15 VgrG
8. C6 VgrG
18. C16 VgrG
9. C7 VgrG
19. C17 VgrG
10. C8 VgrG
20. C18 VgrG
11. C9 VgrG
21. C19 VgrG
1.5
0.5
1 2 3 4 5 6 7 8
9 10 11 12
12. C10 VgrG 22. C20 VgrG
Figure 1 represents PCR products resulting when using various SchuS4 vgrG
transformants’ genomic templates with oligos FTT1346 fwd NdeI and VgrG rev EcoRI.
Lanes 3 - 22 represents VgrG transformants and lane 2 is the wildtype control which will
generate a 900 bp product. Lane 19, clone 17 (C17) VgrG seems to have at least one of
the vgrG genes interrupted explaining the mutant PCR product of 1600 bp and the
wildtype product profile in the same mutant PCR product. Data located in UTSA TVD
Notebook 7, page 13.
b.
Prepare another ten genomic isolations from vgrG clones 21 thru 30 and used
these templates with the same oligo sets mentioned above and did not find a
PCR product profile that yielded only the mutant size of 1600 bp (Data not
shown). Select a few of the 30 initially screen vgrG transformants and performed
PCR with the oligos EBU with VgrG rev EcoRI to confirm that this vgrG intron is
inserted into the correct location in the SchuS4 chromosome (figure 2).
Figure 2.
Legend:
1. 1 Kb Ladder
1 2 3 4 5 6 7 8
1 Kb
2. KKT1
3. C13 VgrG
4. C15 VgrG
1.5
0.5
5. C17 VgrG
6. C21 VgrG
7. C23 VgrG
8. C24 VgrG
Figure 2 represents PCR products generated when using VgrG genomics
templates with oligos EBU (specific to the intron construct) and VgrG rev EcoRI
(specific to the VgrG gene). Lane 2 is the wildtype result, which should not yield
a product since the intron screened here is at the vgrG gene location. Lanes 3-8
are various vgrG transformant clones; all of which did show that the integration of
58 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
the intron is at the vgrG gene location (expected size of 700 bps). Data located
in UTSA TVD Notebook 7, page 15.
The screening for a clone containing more than one VgrG gene interrupted
simultaneously will require screening a greater number of colonies. Therefore,
we resorted to colony lift “pools” to do PCR with using the first set of oligos
mentioned in II b. This will allow us to screen many more clones in a shorter
amount of time for the correct VgrG mutant.
Each colony pool represents
between 7 and 9 colonies and 4 sets out of 17 pools showed a profile similar to
C17 VgrG seen in figure 1 (data not shown). In addition, we believe that during
an actively growing culture the “tulatron” construct (KEK1162) continuously looks
for the correct location to insert its “intron”. Thus, C17 VgrG mutant was
inoculated into 2 ml Chamberlains media with 75 ug/ml kanamycin selection
pressure and grown at 30ºC for 24 hours at a time; passaging this culture three
times before preparing dilutions and plating on TSA+++ 75 ug/ml kanamycin
plates and grown at 30ºC overnight. Some of these resulting passaged colonies
were patched onto fresh TSA+++ 75 ug/ml kanamycin plates and grown at 30ºC.
After 24 hours these C17 single colonies were used by pooled colony picks and
run with the FTT1346 fwd NdeI and VgrG rev EcoRI oligos in a PCR reaction
(figure 3).
c.
Figure 3.
1
1.Kb
Kb
2.0
2 3
4
5 6
Legend:
1. 1 Kb Ladder
2. KKT1
3. V17 (1-3)
4. V17 (4-6)
5. V17 (7-10)
6. V17 (8-16)
1.0
Figure 3 represents pooled colony lifts of various C17 (V17) vgrG potential mutants taken
from a patch TSA+++ 75 ug/ml kanamycin plate. The pools represented mostly 3 clones
each except for V17(8-16) which contain 9 clones. Lane 2 represents the wildtype PCR
product profile of 900 bps. Lanes 3 – 6 represents resulting PCR products from the C17
passaged colony pooled samples collected to run with the oligo set FTT1346 fwd NdeI
and VgrG rev EcoRI. The 1600 bp band is evident in all the pools tested, with the
strongest signals being in lanes 4 and 5; representing clones 7-10. Genomic isolations
will be made from each of these clones (C1-10) to check if one of these produce only the
mutant PCR product. Data located in UTSA TVD Notebook 7, page 16.
d.
e.
Ten of the single clones generated from the passaged culture of C17 (V17)
were used to isolate genomic DNA to use with the oligo pair mentioned in
figure 3 in a PCR experiment. The result showed a similar profile with none
containing only the mutant PCR product (data not shown; located in UTSA
TVD Notebook 7, page 17). Will continue with new passaged V17 clones by
preparing more colony lift pools to examine for the correct VgrG mutant profile.
The igLD SchuS4 mutants KKT9 and KKT10 were prepared for new, repeat
Western analysis experiments. We used our basic western blotting protocol in
UTSA TVD Notebook 5, page 6. The first experiment performed we used a
1:378 mouse anti-igLD serum concentration and varied the secondary HPlabeled secondary antibody (1: 5,000 and 1:10,000). This was done to try and
59 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
f.
g.
increase the signals and reduce the background. This experiment did not
work but, we were able to determine that the lower dilution of the secondary
antibody resulted in higher background.
However, since our positive
(wildtype) protein sample did not illustrate the igLD protein we needed to
repeat experiment. It seems that our mouse anti-igLD serum obtained from a
third party is not very concentrated therefore, decided to use a more
concentrated preparation of this antibody in the next experiment. In this
second experiment, we used a 1:150 dilution of the mouse anti-igLD serum
concentration and a 1:10,000 secondary HP-labeled antibody. This experiment
may have worked but a person opened the developing room door as I was
getting ready to develop the film and this caused my film to be ruined; the
entire film turned black when I ran through the developing machine. I tried to
place another film on this blot but the light emission with this technique has a
limited lifetime and it is totally expended after 1 hour. The second blot did not
show any signal on the positive control (data not shown, located in UTSA TVD
Notebook 5, pages 139-142). This experiment will need to be repeated;
however, we believe these igLD clones to be correct and we decided to
continue with a mouse experiment using high inoculations to test the effect of
this mutant for virulence.
A liquid culture (in Chamberlains medium) of KKT10 igLD was prepared in the
BSL3 lab to use in a mouse experiment. This requires freezing aliquots of this
culture growth and subsequently determining the starting concentration of this
stock empirically. Three sets of titrations were done and the average titration
concentration was used as a reference in the mouse experiment. Our goal is
1E3 (i.e. 1,000); 1E4; 1E5 and 1E6 cfu inoculations, respectively. Each test
group inoculated with KKT10 iglD is comprised of 5 mice (except 1E6, there
are 6 mice in this group). We ran two controls in this mouse experiment: 5
mice were inoculated with PBS (a neutral buffer) and 5 mice were inoculated
with 1E2 of wild type KKT1 SchuS4 strain. After 5 days the wild type SCHU S4
control should die (they did). The PBS control will survive for the entire length
of this experiment of 1 month. Based on calculations of colony forming units
(cfu) counts for the mouse inoculations; it seems we may have used a log
higher for each group (i.e. 1E3 group actually received 1E4 cfu). Will have an
actual number count by next report. After 6 days all test groups are still alive
and do not appear to be sick. Since the control group died and the igLD
(KKT10) test groups have not; this indicates that this mutant is defective for
virulence.
Typically if the mice survive for more than two weeks with no
obvious signs of sickness we usually expect survival for one month. Data
located in UTSA TVD Notebook 5, page 137, 138 and 143.
Did some ordering for enzymes and general supplies for ongoing experiments.
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
72%
60 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
9. Work plan for upcoming month
a. Will continue the characterization of the igLD mutants (KKT9 and KKT10) in KKT1
SCHU S4 strain. This will be done by repeating the western analysis and preparing
PCR products that can be sent for sequencing using various intron specific and igLD
specific oligo sets.
b. Will monitor the survival of KKT10 iglD infected mice in the upcoming month.
c. Will continue with screening of the newly generated VgrG transformants to search for
the correct SchuS4 mutant via PCR profiles with inside and outside oligos relative to
the expected intron insertion site. Some of these generated PCR products will be
sent for sequencing to confirm correct VgrG mutant.
d. If time allows will continue with cloning the pUC pdpD::Flp::ErmC fragment into the
pUC118 plasmid. This should allow a complete pathogenicity island to be removed if
we can generate the pdpD deletion in these mutants using this resulting construct
(pUC pdpD::Flp::ErmC).
10. Anticipated travel
None
11.Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB;
LVS uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4)
pdpD, iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double
mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4)
iglA, iglB strains
Institution: UTSA
1. Date started: 05/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
50A: We have planned to report this month on the intramacrophage growth of the
iglD SCHU S4 mutant, however, the experiment is still ongoing and results will not be
ready in time for this report. In lieu, we report here the histology of lungs from F.
novicida iglB-vaccinated and SCHU S4-challenged mice. (Notebook #4, page 158159). In our last (May, 2008) monthly report, we showed that four out of the ten
C57BL/6 mice receiving intragastric vaccination of iglB (a prime and a booster dose
of 103 CFU) survived SCHU S4 intranasal challenge (50 CFU). Lungs of these
survivors were collected 35 days after challenge, fixed in formalin, and embedded in
paraffin. Thin sections (5m) of the embedded tissues were prepared and stained
with hematoxylin and eosin (Fig.1.)
61 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
4x
10x
20x
Naive
iglB
Fig. 1. Lung histology after F. novicida iglB vaccination and SCHU S4 challenge. Groups
of C57BL/6 mice were vaccinated intragastrically with 10 3 CFU of iglB and challenged
intranasally with SCHU S4 (50 CFU) 3 weeks after the boost. Lungs were collected from
survivors at day 35 after the challenge. Lung sections were stained with hematoxylin and
eosin.
At day 35 post-challenge, lungs of iglB-vaccinated and SCHU S4-challenged mice
appeared mostly devoid of inflammation, although small pockets of mononuclear cell
infiltrates around some bronchioles still could be seen.
50B. Perform histological analyses on tissues at varying time points after LVS oral
immunization and subsequent SHU S4 challenge. (Notebook #8, pages 107-108)
BALB/c mice were vaccinated intragastrically with 103 CFU LVS or mock immunized
with PBS alone. Four weeks later, mice were challenged intranasally with 100 CFU of
SCHU S4. At various time points (45 and 60 days) after challenge, mice were
sacrificed and lungs were collected. Briefly, 10% neutral buffered formalin was
injected into the lungs via the trachea, the lungs were removed and set overnight in
formalin for fixation. Tissues were then embedded in paraffin and sliced into 5m
sections and placed on slides, 3 sections per slide. Every fourth slide was stained
with hematoxylin and eosin and visualized using light microscopy. As reported in
May, 2008, LVS vaccinated mice at 30 days after challenge still displayed minimal
amounts of inflammatory cellular infiltrates. Therefore, we looked at two further time
points to ensure that inflammation had cleared completely. As shown in Fig. 2, at 45
days post-challenge, lungs of vaccinated and challenged mice appeared with minimal
inflammation. However, there were still small pockets of mononuclear cell infiltrates
around some bronchioles. At 60 days post-challenge, immunized-challenged lungs
were completely clear and comparable to lungs of naïve mice. Therefore, while
SCHU S4 i.n. challenged LVS vaccinated mice might display a slightly prolonged
inflammatory response, by 2 months after challenge the lungs of these mice appear
to have completely recovered.
62 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Naive
LVS
45 Days Post
Challenge
LVS
60 Days Post
Challenge
4X
20X
Fig. 2. Lung histology after LVS vaccination and SCHU S4 challenge.
Groups of BALB/c mice were vaccinated I.G. with 103 CFU of LVS or PBS
as a control. Mice were challenged 3 weeks later with 100 CFU SCHU S4
i.n. and lungs were collected at varying time points after challenge. Lung
sections were stained with hematoxylin and eosin.
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
74% of scientific work completed on milestone 50A (original plans)
66% of scientific work completed on milestone 50B (intragastric plan)
Normalizes to 70% across 50A and 50B, over a 1 year period.
9. Work plan for upcoming month
50A: Measure intramacrophage (J774) replication of Ft subsp. tularensis (SCHU S4)
iglD mutant
50B: (1) Survival after LVS I.G. vaccination and SHU S4 challenge at 8 weeks after
immunization
(2) Bacterial dissemination in oral LVS-vaccinated BALB/c mice after SCHU S4
challenge.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
63 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
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
Creation of Schu S4 recA mutant
3.1 To inactivate RecA in Schu S4, we were in the process of constructing a Targetron
vector for targeting and inactivating the RecA gene. The Targetron vector was designed
to be constructed with the intron expression vector pKEK1140 for the backbone, and a
350bp PCR product for the insertion to mutate intron RNA.
a. In last month report, we reported that KKT11 (recA mutant Schu S4) was not
attenuated at the dose of 105 CFU in Balb/c mice, and low dose of KKT11 would be
applied to evaluate the attenuation. About 206 CFU KKT11 were inoculated into 6
Balb/c mice intranasally on June 6th. The control groups were inoculated with wild type
Schu S4 and PBS intranasally at the same time. Only one mouse inoculated with
KKT11 survived after 30 days. The table below presents the details for this experiment.
Table1. Attenuation of Schu S4 recA mutant
Inoculation
Survival Rate
b. F
Route of
Dose
r Inoculum
Inoculation
D1 D2 D3 D4 D5 D6 D7 D8 D30
(CFU)
o
m KKT11
I.n.
206
6/6 6/6 6/6 6/6 6/6 2/6 2/6 1/6 1/6
Wt Schu S4
I.n.
34
5/5 5/5 5/5 5/5 5/5 1/5 0/5
t
PBS
I.n.
5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5
h
e data shown in Table1, we might predict that KKT11 was attenuated very mildly in
mice.
Data recorded on UTSA TVDC notebook #6, page40-41 for Table1.
Creation of a LUX operon plasmid in Ft
A new focus of this milestone is to introduce LUX operon (LuxC, LuxD, LuxA, LuxB and
LuxE) into the plasmid pKEK843 containing Ft groELp promoter to allow the generation
of the bioluminescent LVS and U112. Lux operon is a set of genes which encode the
luciferin-luciferase system in bacterial. LuxCDABE have been identified as active in the
emission of visible light.
a. The ligation product (pKEK843 carrying lux operon) prepared last month was
transformed into the host cells Ecoli.DH5α using electroporation at 25μF, 200Ω and
1.8kV. The transformed cells were incubated in LB liquid medium for an hour with
shaking, and plated onto LB/Kanamycin (50ug/ml) agar medium to incubate at 37°C for
overnight.
b. Only 4 colonies were observed on the plate after overnight culture. Miniprep plasmid
DNA was made from liquid culture of 4 colonies using QIAprep Miniprep Kit. The figure
for the plasmid DNA was shown below.
Figure1: On 1% agarose gel.
3.2
64 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
The correct plasmid with lux operon cloned in the parent plasmid pKEK843 should be
about 14kb. It was obvious that none of 4 colonies was carrying the target plasmid
since miniprep DNA (lane1-3 and lane6) was smaller than the parent plasmid (lane4)
and even less than 1.6kb.
c. Since it didn’t work to clone lux operon into pKEK843, we tried to change the parent
plasmid from pKEK843 to the commercial plasmid PUC118. All the techniques and
reagents were the same as pKEK843. pUTmini5Tnkmlux and PUC118 were digested
with EcoRI. Then the fragment carrying lux operon was ligated into PUC118 and
transformed into DH5α. The potential transformants were selected on LB/Xgal/Ampicillin (100ug/ml) agar medium and miniprep DNA was prepared. Also the liquid
cultures from 19 colonies separately were tested for bioluminescence from lux operon
using the luminometer, but none of them gave positive signal (data not shown). The
figure for miniprep DNA was presented below.
Figure2: On 1% agarose gel.
From the figure above, none of the bands was over 3.0kb, which indicated that they
were not correct.Data recorded on UTSA TVDC notebook #2, page124-126 for Figure1
and 2.
4 Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
Will continue attempts to find a plasmid vector that allow the LUX operon to be cloned
and expressed
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed.
About 28% of scientific work completed.
9. Work plan for upcoming month
i.
Challenge the mice (one survival mouse inoculated with KKT11 and five control mice
inoculated with PBS on June 6th) with wild type Schu S4.
65 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
ii.
iii.
Clone lux operon fragment into the plasmid pWSK30.
Screen the transformants from Section ii.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None.
Milestone 55
Milestone description: Compare Cellular Immunogenicity of Francisella and ListeriaBased Vaccine Platforms. Measure cellular immunogenicity of live-attenuated vaccine
platforms. Compare immunogenicity of KBMA tularemia vaccine platforms
Institution: Cerus/Anza
1. Date started: 4/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary of objectives: We will construct and prepare live and KBMA Listeria monocytogenes
(Lm) vaccines expressing Ft antigens. To directly compare the cellular immunogenicity of Lm
and Ft-based vaccines, each Lm vaccine candidate will express an antigen fused to a model
ovalbumin epitope SIINFEKL (SL8) and these will be compared to Ft vaccines expressing pepOSL8 fusions (provided by UTSA). We will measure the ability of each vaccine to stimulate a CD8
T cell response in vitro using a B3Z assay. We will measure the cytokine responses elicited by
vaccination with each platform in mice, compare the CD8 T cell response to SL8 after prime and
boost vaccinations in mice using intracellular cytokine staining (ICS) and ELIspot assays and
measure the potency of the T cells elicited by use of an in vivo cytotoxicity assay.
We previously demonstrated that iglC-SL8 fusion proteins are expressed to a higher level than
katG-SL8 in the cytosol of macrophages. Live attenuated vaccines expressing either antigen
were able to stimulate the B3Z-Tcell line. The iglC-SL8 fusion protein induced a stronger immune
response in mice than katG-SL8 by ICS and ELISpot. Incorporation of a constitutively active prfA
allele into the chromosome of the live-attenuated LM-igl-C-SL8 vaccine increased
immunogenicity by 2-fold. Inclusion of a much larger tag (containing an additional 4 epitopes
from vaccinia virus) decreased the immunogenicity of the Lm vaccine.
1) Cloning of Listeria monocytogenes (Lm) tularemia vaccine strains. New vaccine
candidates have been constructed (see table #1 below). BH2172 is a recloned version of
BH2096 LmactAinlBuvrABprfAG155SKatGSL8 (that did not stimulate a B3Z cells). A second
integration vector (pPL1) was used to construct bivalent Lm vaccines expressing IglC and KatG.
pPL1 integrates at the comK locus in Lm and confers resistance to chloramphenicol whereas the
other vector used (pINT) confers resistance to erythromycin and integrates at the tRNA Arg locus.
An iglC-B8R antigen expression cassette was inserted into this vector composed of 1) an actA
promoter for prfA-dependent transcription 2) the N-terminal 100 amino acids of ActA to facilitate
secretion of 3) the iglC coding sequence and a single immunodominant B8R epitope tag from
vaccinia virus at the C-terminus. This construct was introduced into the chromosome of 3 strains,
actAinlB, actAinlBuvrABprfAG155S, actAinlBuvrABprfAG155SKatGSL8. The iglCB8R tagged cassette will also be introduced onto the chromosome of BH2172 to make a bivalent
KBMA vaccine strain. Expression levels from each new construct need to be determined.
66 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
figure 1
actAp
Molecular construct at comK:
ActAN100
IglC
B8R
Table 1
Strain
Genetic Background
CRS-100 actAinlB
BH137
actAinlB
BH1222
actAinlB
BH2106
actAinlB
BH1228
actAinlBuvrAB
BH1398
actAinlBuvrAB
BH2094
actAinlBuvrABprfAG155S
BH2172
actAinlBuvrABprfAG155S
BH2098
actAinlB
BH2100
actAinlBuvrABprfAG155S
BH2180
actAinlB
BH2182
actAinlBuvrABprfAG155S
BH2184
actAinlB
Antigen Cassette
none
ActAN100-Ova
ActAN100-IglC-SL8
ActAN100-KatG-SL8
ActAN100-IglC-SL8
ActAN100-KatG-SL8
ActAN100-IglC-SL8
ActAN100-KatG-SL8
ActAN100-IglC-VacQuad-SL8
ActAN100-IglC-VacQuad-SL8
ActAN100-IglC-B8R (@ comK)
ActAN100-IglC-B8R (@ comK)
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8 (@tRNAarg)
* yellow highlighted strains are new this month
Status
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Complete
Complete
Complete
Complete
Complete
Complete
Complete
2) To facilitate further testing of vaccine candidates, we have produced a new 400mL scale lot of
live DVC lot 16 LVS that was produced according to the TVDC SOP (NB# 2001-012). This lot
can be used for both Milestone 56, and 57 to determine the number of T cells elicited against iglC
after administration of sub-lethal doses of LVS and also used as a lethal challenge to determine
whether the Lm vaccines provide protection. A 400mL scale lot of live LVS PepO-SL8 was
produced using same methods (NB# 963-092). This vaccine candidate will be used to vaccinate
mice and SL8 responses will be compared with LM-SL8-expressing strains. A 400mL scale lot of
KBMA BH2094 was produced: LMactAinlBuvrABprfAG155S:ActAN100-IglC-SL8 (NB#2001026B). The exact titer of live lots, log kill for KBMA lot, and Metabolic activity of KBMA lot needs
to be determined.
4. Significant decisions made or pending
Because the vaccinia virus quadrotope tag significantly decreased the immunogenicity of
the iglC vaccine, this tag will not be used for further immunogenicity studies.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
12%
9. Work plan for upcoming month
67 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble






We will construct the bivalent IglC/KatG LM vaccine strain on the KBMA
background
We will evaluate the immunogenicity of the bivalent Lm strain expressing IglCB8R and KatG-SL8 fusion proteins and compare the immunogenicity with each
monovalent strain
We will produce a lot of live Ftn-PepO-SL8
The 400mL-scale lots of vaccine will be undergo quality testing (e.g. cfu analysis
and metabolic activity assays)
The SL8 responses induced by Ft-PepO-SL8 and LM-iglC-SL8 will be compared
We will clone a bivalent vaccine strain that expresses both IglC and KatG.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
Modification 4R2 to subaward agreement has been fully executed by Cerus and UNM.
Milestone 56
Milestone description: Characterize the Cellular Immune Response that Correlates
with Protection Against an LVS Challenge and demonstrate that Cerus Strains of
Live and KBMA Lm-IglC and Lm-KatG Protect Against a SchuS4 Challenge
Institution: Cerus/Anza
1. Date started: 6/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary of objectives: We will measure the T-cell response to IglC induced by live and KBMA
Lm expressing IglC compared with those elicited by Ftn or LVS vaccination. We will produce an
IglC overlapping peptide library (15aa overlapping by 11aa) to identify iglC epitopes that are
recognized by mouse T cells. We will use the IglC peptide library for ELISpot assays to measure
the IglC-specific T cell responses induced after vaccination with live and KBMA Lm-IglC and
compare to live and KBMA Ftn and LVS vaccination. We will demonstrate that the mechanism of
protection induced by Lm vaccines is cellular by depletion of T cell populations and passive
transfer studies, We will demonstrate that strains of Live and KBMA Lm-IglC-SL8 and Lm-KatGSL8 protect against a SchuS4 challenge and we will produce lots of KBMA vaccine and send to
UNM for testing in animal models (mice and rats).
1) The IglC overlapping peptide Library has been synthesized and has arrived at Anza. 51
peptides that are 11 amino acids in length were synthesized and were determined to be
of > 80% purity, which refers to the expected length of the peptide. The peptides arrived
lyophilized as individual peptides and also as a pool of all 51.
2) As described in MS55, a 400mL scale lot of KBMA BH2094 was produced:
LMactAinlBuvrABprfAG155S:ActAN100-IglC-SL8 (NB#2001-026B). This lot needs to
undergo quality testing but can then be used to determine whether KBMA Lm vaccines
can induce an immune response that protects against a lethal Ft challenge.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
68 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
2%
9. Work plan for upcoming month



Mice of various haplotypes (BALB/c, C57BL/6, FVBN, C3H/HEJ, SJL) will be
vaccinated with live BH2094: (actAinlBuvrABprfAG155S ActAN100-IglC-SL8)
and splenocytes will be incubated with peptides to determine whether there are tcells responsive to any iglC epitopes by IFN-g ELISpot
Anza will vaccinate mice with various Lm vaccines to determine whether IglC,
KatG, or both protect against lethal LVS infection
Once MTA is approved, live and KBMA Lm lots can be sent to UNM for
evaluation in SchuS4 challenge model
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
Modification 4R2 to subaward agreement has been fully executed by Cerus and UNM
Milestone 57
Milestone description: Optimization of KBMA Lm Vaccination Route and Regimen.
Institution: Cerus/Anza
1. Date started: 6/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary of objectives: We will compare various routes of administration including IV, IM, IN,
ID and oral. For oral, IN, and ID administration in mice, we will first mutate the inlA gene of
Lm to allow for binding of murine E-cadherin in order to mimic the human interaction (as
described in Wollert et al., Cell, 2007). We will compare the potency of the inlA gain of
function mutants to our traditional platform strain. Routes will be ranked by ability to induce a
cellular immune response using ELISpot, ICS, and in vivo cytotoxicity. We will optimize
dosing regimen of most potent and tolerable route. Lm expressing IglC and/or KatG will be
used to evaluate immunogenicity. Optimized route and regimen will be confirmed by SchuS4
protection studies at UNM.
1) This month we have constructed 8 vaccine candidates that contain the inlA gain of
function mutations (Table 2). The sequence of the wild-type EGDe inlA gene (from the
Lm strain used in the Wollert manuscript) was synthesized and the inlA gene in our
platform strain was replaced (inlAWT) in our live-attenuated and KBMA platform strains as
there are a number of differences in the sequence between the native sequences
between these strains. Two point mutations, S192N and Y369S, were incorporated into
the EGDe inlA sequence (inlAM) and inserted into the chromosome of our live-attenuated
and KBMA platform strains. Into these 4 strains the ActAN100-iglC-SL8 expression
cassette was inserted using the integration vector pINT. Expression analysis of the iglCSL8 cassette has yet to be performed. Once the expression has been confirmed,
biodistribution, virulence and immunogenicity studies (ICS and ELIspot) will be
performed.
69 of 70
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2008 to 6/30/2008
Due Date: 7/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas,
Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes, Mitch Magee,
Justin Skoble
Table 2
Strain
CRS-100
BH2130
BH2164
BH2170
BH2164
BH2132
BH2166
BH2134
BH2168
Genetic Background
actAinlB
actAinlBinlAWT
actAinlBinlAWT
actAinlBinlAM
actAinlBinlAM
actAinlBuvrABprfAG155SinlAWT
actAinlBuvrABprfAG155SinlAWT
actAinlBuvrABprfAG155SinlAM
actAinlBuvrABprfAG155SinlAM
Antigen Cassette
none
none
ActAN100-IglC-SL8
none
ActAN100-IglC-SL8
none
ActAN100-iglC-SL8
none
ActAN100-iglC-SL8
Status
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
2%
9. Work plan for upcoming month


Expression from the iglC-SL8 expression cassette will be confirmed in each of these
strains
Virulence and immunogenicity of inlAWT and inlAM expressing live-attenuated strains
will be evaluated in the coming month.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
Modification 4R2 to subaward agreement has been fully executed by Cerus and UNM
70 of 70