Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Caryl Trotter confirmed that UNM EOH will begin the annual health screenings
for the 37 LVS vaccinees in September 2008.
b. Heidi Holley, NIAID Contract Specialist, determined that no COA was required for
the annual health screening costs associated with the LVS vaccinees.
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.
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
Page 1 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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
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
68% 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
Will be attending the UNM TVDC annual meeting in Phoenix AZ in October 2008.
11. Upcoming Contract Authorization (COA) for subcontractors
None
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 was continued on the milestone completion report. The draft report will be
completed prior to the next NIAID teleconference scheduled for 9/2/2008.
b. SCHU S4 bioaerosols were conducted on four days in July 2008. The goal is to
select an aerosol generator that reproducibly generates a targeted and delivered
SCHU S4 aerosol dose. SCHU S4 bioaerosols were generated and compared
using the Hospitak and Collison nebulizers (4 Hospitak nebulizers and 3 Collison
nebulizers).
a. 7/1/2008
i. Completed SCHU S4 bioaerosol testing (number of sprays=8)
using the Hospitak and Collison nebulizers.
ii. Objectives were to demonstrate the predictability of the
standardized SCHU S4 growth method and to compare SCHU
S4 bioaerosol efficacy between the Hospitak and Collison
nebulizers.
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 previously
unused, sterile 500 mL baffled flask with 400 uL of
suspension. The suspension is prepared from multiple
Page 2 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
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\Collison Generator\01JUL08 are summarized in Table 1
below:
Table 1. 1JUL08 summary SCHU S4 bioaerosol results using the Hospitak and
Collison nebulizers.
Numerous conclusions can be drawn from the data presented in
Table 1: (1) Generator suspensions (pre-sprays; pre-actuals
column in table 1) were close to the target concentrations
demonstrating accurate predictability of our SCHU S4 growth
method. (2) Post-actual bioaerosol concentrations were slightly
lower than pre- actual bioaerosol titers (an observation consistently
observed regardless of the nebulizer used) demonstrating the
fragility of SCHU S4. (3) Aerosol concentrations (CFU/L) were
slightly higher with the Hospitak. These higher concentrations
were likely due to the increased viability retention associated with
the Hospitak. Of significant interest is that a consistent increase in
aerosol concentration (CFU/L column) was observed with
increased generator suspension (pre-actual); this demonstrates
that aerosol concentrations may be targeted rather than only
generator suspension concentrations being targeted in future
animal challenges. (4) Spray factors were similar to those
observed previously with the Collison, but were lower and more
inconsistent with the Hospitak. This latter observation was likely
inherent to the nebulizer chosen for that day. It is unclear,
however, how to pre-screen for the consistency of the individual
Hospitak nebulizers.
Page 3 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. 7/9/2008
i. Completed SCHU S4 bioaerosol testing (number of sprays=8)
using the Hospitak and Collison nebulizers.
ii. Objectives were to demonstrate the predictability of the
standardized SCHU S4 growth method and to compare SCHU
S4 bioaerosol efficacy between the Hospitak and Collison
nebulizers.
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 previously
unused, sterile 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\Collison Generator\09JUL08 are summarized in Table 2
below:
Table 2. 9JUL08 summary SCHU S4 bioaerosol results using the Hospitak and
Collison nebulizers.
Numerous conclusions can be drawn from the data presented in
Table 2: (1) As before, generator suspensions (pre-sprays; pre
actual column in table 2 above) were close to the target
concentrations demonstrating accurate predictability of our SCHU
S4 growth method. (2) Post- actual bioaerosol concentrations
were slightly lower than pre- actual bioaerosol titers (an
observation consistently observed regardless of the nebulizer
used) demonstrating the fragility of SCHU S4. (3) Aerosol
concentrations (CFU/L) were slightly higher with the Hospitak.
These higher concentrations were likely due to the increased
Page 4 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
viability retention associated with the Hospitak. Of significant
interest is that a consistent increase in aerosol concentration was
observed with increased generator concentration; this
demonstrates that aerosol concentrations may be targeted rather
than only generator suspension concentrations in future animal
challenges. (4) Spray factors were virtually the same for both
nebulizers. This was in contrast to the 1JUL08 observations. It
can be hypothesized that the Hospitak chosen for the July 9
bioaerosols met the criteria (currently an unknown variable)
necessary to result in efficient SCHU S4 spray generation.
c.
7/24/2008
i. Completed SCHU S4 bioaerosol testing (number of sprays=6)
using the Hospitak and Collison nebulizers.
ii. Objectives were to demonstrate the predictability of the
standardized SCHU S4 growth method and to compare SCHU
S4 bioaerosol efficacy between the Hospitak and Collison
nebulizers. Focus was on use of a different Collison unit than
used for previous experiments.
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 previously
unused, sterile 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\Collison Generator\24JUL08 are summarized in Table 3
below:
Page 5 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 3. 24JUL08 summary SCHU S4 bioaerosol results using the Hospitak and
Collison nebulizers.
Numerous conclusions can be drawn from the data presented in
Table 3: (1) As before, generator suspensions (pre-actual) were
close to the target concentrations demonstrating accurate
predictability of our SCHU S4 growth method. (2) Post-actual
bioaerosol concentrations were slightly lower than pre-actual
bioaerosol titers (an observation consistently observed regardless
of the nebulizer used) demonstrating the fragility of SCHU S4. (3)
Aerosol concentrations (CFU/L) were similar between the two
nebulizers. Of significant interest is that the a consistent increase
in aerosol concentration was observed with increased generator
concentration; this demonstrates that aerosol concentrations may
be targeted rather than only generator suspension concentrations
in future animal challenges. (4) Spray factors were similar to those
observed previous with the Collison, but were lower and more
inconsistent with the Hospitak. This latter observation was likely
inherent to the specific Hospitak nebulizer chosen for that day.
These data are similar to those obtained on 1JUL08. The
inconsistencies in the spray factors associated with the Hospitak
may negate its occasional good performance. In contrast to this,
data indicate the Collison may be slightly less efficient than the
Hospitak, but is more consistent based on the spray factors.
d. 7/25/2008
i. Completed SCHU S4 bioaerosol testing (number of sprays=6)
using the Hospitak and Collison nebulizers.
ii. Objectives were to demonstrate the predictability of the
standardized SCHU S4 growth method and to compare SCHU
S4 bioaerosol efficacy between the Hospitak and Collison
nebulizers. Focus was on use of a different Collison unit than
used for previous experiments.
iii. Growth method used for SCHU S4:
1. Inoculate BCGA for colony isolation
2. Incubate for 48h at 37°C
Page 6 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
3. Inoculate 100 mL of Chamberlain’s broth in a previously
unused, sterile500 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\Collison Generator\25JUL08 are summarized in Table 4
below:
Table 4. 25JUL08 summary SCHU S4 bioaerosol results using the Hospitak and
Collison nebulizers.
Numerous conclusions can be drawn from the data presented in
Table 4: (1) As before, generator suspensions (pre- actuals) were
close to the target concentrations demonstrating accurate
predictability of our SCHU S4 growth method. (2) Post- actual
bioaerosol concentrations were slightly lower than pre- actual
bioaerosol titers (an observation consistently observed regardless
of the nebulizer used) demonstrating the fragility of SCHU S4. (3)
Aerosol concentrations (CFU/L) were similar between the two
nebulizers. Of significant interest is that the a consistent increase
in aerosol concentration was observed with increased generator
concentration; this demonstrates that aerosol concentrations may
be targeted rather than only generator suspension concentrations
in future animal challenges. (4) Spray factors were similar
between the two nebulizers.
The data from Tables 1-4 were compiled along with aerosol runs
from 23JUN08 and 30Jun2008 (presented in the Previous
Milestone 3 Monthly Report), and were averaged. Standard
deviations were determined and the results were graphed (see
Figures 1-4 below).
Page 7 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Comparison of Targeted vs. Actual Prespray Concentrations in Six Head
to Head Experiments with the Hospitak and Collison Nebulizers
This graph (Figure 1) represents data collected across the June 23 to July 25 bioaerosols. The
targeted pre-spray concentrations for both the Hospitak and Collison nebulizers are achieved
over the 106 to 109 stepwise log range targeted. Thus, LBERI is able to accurately predict the
Prespray concentration of the bacterial suspension for aerosol generation (i.e. the suspension
placed into the nebulizer). Data is stored in Z:\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY07\FY07-083 and -089 (TUL04)\NHP Pilot Studies
Page 8 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 2. Comparison of Bacterial Recovery after Aerosol Runs in Six Head
to Head Experiments with the Hospitak and Collison Nebulizers
This graph (Figure 2) represents data collected across the June 23 to July 25 bioaerosols. The
percentage of bacteria recovered after aerosol runs (represented as the Postspray as a
percentage of the Prespray) demonstrate that there is no significant difference between the
Hospitak and Collison nebulizers, though the trend is slightly higher in the Hospitak. Data is
stored in Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY07\FY07-083 and -089 (TUL-04)\NHP Pilot Studies
Page 9 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Comparison of Log Impinger Concentrations in Six Head to Head
Experiments with the Hospitak and Collison Nebulizers
This graph (Figure 3) represents data collected across the June 23 to July 25 bioaerosols. The
percentage of bacteria recovered from the AGI demonstrates that there is no significant
difference between the Hospitak and Collison nebulizers, though the trend is slightly higher at
lower generator concentrations in the Hospitak. Data is stored in Z:\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY07\FY07-083 and -089
(TUL-04)\NHP Pilot Studies
Page 10 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 4. Comparison of Bacterial Recovery from the Impinger in Six Head
to Head Experiments with the Hospitak and Collison Nebulizers
This graph (Figure 4) represents data collected across the June 23 to July 25 bioaerosols. The
percentage of bacteria recovered from the AGI as a percentage of the Prespray indicates that
large numbers of bacteria do not survive the aerosolization process and that SCHU S4 in
particular is very fragile. It also demonstrates that there is no significant difference between the
Hospitak and Collison nebulizers. Data is stored in Z:\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY07\FY07-083 and -089 (TUL04)\NHP Pilot Studies
4. Significant decisions made or pending
a. Based on consistent data over the last 2 months, the SCHU S4 growth method
has been finalized to the procedure outlined in each section above.
b. Data from 6 head to head comparisons between the Hospitak and Collison
nebulizers has been compiled and no statistical difference exists between the
Page 11 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
two. Smaller standard deviations in spray factors were observed with the
Collison nebulizer. The Collison, therefore, will be used for all future SCHU S4
bioaerosols.
Data from this milestone are scientifically sound and worth publishing. We will
look to doing so in a reputable journal in the near future.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
99.5% of the scientific work is complete
9. Work plan for next month
a. Complete the Milestone Completion Draft Report
b. Update draft SOPs and move to finalization
10. Anticipated travel
Will attend UNM TVDC annual meeting in Phoenix AZ in October 2008.
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:
Bioaerosol Challenge: A bioaerosol challenge with two additional primates
(A04643 and A04645) was performed on 15JUL08 to confirm that aerosolized
SCHU S4 freshly grown in Chamberlain’s broth is virulent in NHPs. The focus for
study was to ensure lethal disease progression at high presented dose.
Therefore, the generator suspension concentration was increased significantly
from the previous two challenge days (1 x 109 CFU/mL on 7/15/08 vs. 1 x 107
CFU/mL prior dates). The nebulizer used for this study was the Hospitak. The
data (summarized in Table 1) is located in the following folder:
\\Saturn\absl3\Agent
and
Study
Specific
Data
and
Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 and -089 (TUL04)\15JUL08 NHP exposure.
Page 12 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 1: Challenge data for animals A04643 and A04645, naïve cynomolgus macaques
exposed to an aerosol challenge of Francisella tularensis SCHU S4.
F. tularensis
Date
15-Jul-08
Strain
Working
Stock
SCHU
S4
28-Feb-08
Nebulizer
Animal ID
Brand
Operating
Pressure
Output
A04643
Hospitak
10.0 psig
4.75 L/min
A04645
Hospitak
10.0 psig
4.75 L/min
CFU/mL
Target
PreActual
PostActual
CFU/L
Presented
Dose
(CFU)
PreSpray
Factor
PostSpray
Factor
1.00E+09
2.92E+09
1.43E+09
4.87E+04
2.36E+05
5.23E-09
3.42E-08
1.00E+09
3.70E+09
1.45E+09
2.61E+05
1.09E+06
7.05E-08
1.80E-07
Data from Table 1 demonstrates that animals A04643 and A04645 were challenged with
presented doses of 2.36 x 105 and 1.09 x 106 CFU, respectively. Actual generator suspensions
(pre-actual column) were very close to the target of 1.00 x 10 9 CFU/mL demonstrating
consistency with the SCHU S4 growth method. Exposure times were 8.63 and 8.30 minutes for
A04643 and A04645, respectively.
In-life Study Portion: The animals in Pilot 3, A04643 and A04645, were aerosol challenged
using a Hospitak nebulizer with SCHU S4 grown as described above in Milestone 3. The doses
of viable bacteria presented to each animal were 2.36 x 105 and 1.09 x 106, respectively. Animal
A04643 developed clinical signs within 24 hours of exposure as demonstrated by a respiratory
rate of over 80 respirations per minute (Figure 1), markedly increased from a baseline of 40. By
48 hours post-infection, this animals’ respiratory rate had climbed to 120 respirations per minute.
A04643 vomited twice within that time and its biscuit intake nearly ceased, though fruit and
vegetable enrichment was still ingested. At approximately 70 hours, A04643 displayed severe
weakness and ataxia as well as respiratory distress (gasping), then fell off the perch and died on
the bottom of the cage before personnel trained to perform euthanasia could arrive. Animal
A04645 developed clinical signs within 48 hours of exposure. Its respiratory rate increased to 88
respirations per minute (normal rate = 45, Figure 1), and a non-productive cough was observed.
By 72 hours post-infection, A04645’s respiratory rate had increased to above 80 per minute and
the cough was more pronounced. By 9 pm that evening (study day 3) this animal also developed
marked weakness, ataxia, and respiratory distress (gasping); it fainted while being chaired for
temperature readings. Similar to the other animal, this monkey fell off of the perch and died on
the floor of the cage before the Study Director could be contacted to arrange euthanasia. A04645
also nearly ceased eating biscuits, but did continue to eat fruit and vegetable enrichment. Neither
animal displayed weight loss over 20%, nor did either develop a clear fever. In fact, both animals’
temperatures decreased as the disease progressed to death (Figure 2). Both animals had large
numbers of SCHU S4 within lung and TBLNs (Table 2). The spleen and liver of both were
colonized with fewer numbers than lung and TBLN (2-4 logs less, Table 2), while only small
numbers of bacteria were recovered from blood and mesenteric lymph nodes. This is likely
indicative of primary pulmonary disease with minimal progression to the bacteremic state.
Hence, death was due to pulmonary disease. Lungs from each animal are shown below (Figure
3 and 4). Pathology reports are pending at this time.
Page 13 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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: Blood and tissue culture data for animals A04643 and A04645, 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
Presented
Dose
(CFU)
Necropsy
Datea
Bloodb
Spleen
Liver
TBLNc
Mes LN
Lung
A04643
15-July-08
2.36E+05
18-July-08
1620
5.87E+04
4.15E+04
2.64E+08
9.46E+02
5.53E+08
A04645
15-July-08
1.09E+06
18-July-08
125
8.88E+05
6.26E+04
6.28E+07
2.05E+02
2.35E+08
Tissue Culture
a
both animals died before personnel could reach them to euthanize
b
Blood data presented as CFU/mL; tissue data presented as CFU/g; A04643 blood was taken from chest wall pool after aorta was nicked on
necropsy, A04645 blood was taken from the heart
c
In addition to F. tularensis, one contaminant (one to two colonies per plate) was noted in the liver of A04645, currently being characterized
A.
B.
Figure 1. Post-exposure respiration rate by animal for Pilot 3. A is animal A04643 and B is
animal A04645 Data is stored in Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 and -089 (TUL-04)\NHP Pilot
Studies
Page 14 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
B.
Figure 2. Post-exposure temperatures by animal for Pilot 3. A is animal A04643 and B is
animal A04645. Data is stored in Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 and -089 (TUL-04)\NHP Pilot
Studies
Page 15 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
B.
Figure 3: Lung images from animal A04643. A and B images represent ventral and dorsal
aspects, respectively.
Page 16 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
B.
Figure 4: Lung images from animal A04645. A and B images represent
ventral and dorsal aspects, respectively.
4. Significant decisions made or pending
Dr. Lyons, prior to aerosol exposure of the third cohort of animals, made the decision to
expose them to the highest possible concentration of SCHU S4 achievable without
centrifugation. Thus, 1e9 bacteria were used for the generator suspension (pre-actual).
As both animals died within 3 days post-exposure, LBERI has achieved a lethal aerosol
SCHU S4 dose in NHP and has observed clinical signs and symptoms of primary
pneumonic disease in NHP. LBERI is prepared to move forward with ED50 studies.
5. Problems or concerns and strategies to address
None.
Page 17 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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
The Milestone 4 report is presently being written. Pathology reports are still outstanding
for Pilots 2 and 3.
7. Quality of performance
Good
8. Percentage completed
90% of the scientific work is complete
9. Work plan for next month
a. Work on the Milestone Completion Report will continue.
10. Anticipated travel
11. Will attend UNM TVDC annual meeting in Phoenix AZ in October 2008.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. We have been consulting with Dr. Ron Schrader, a biostatistician at UNM, to
perform statistical analyses on the data collected thus far on the Fischer 344
rat model. The preliminary results of our analyses are located
L:\Lyonslab\Tularemia\Tularemia manuscripts\Rat paper\Stats
i. We would like to make recommendations on the LVS vaccination
route and dose, SCHU S4 challenge dose, and group size
ii. 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.
iii. His analyses 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 were similar and both were
slightly better than i.t. vaccination.
iv. He transformed the discontinuous survival data from unvaccinated
rats challenged i.t. with SCHU S4 into a continuous baseline
proportional hazard regression curve reflecting the survivability at all
doses within the dose range analyzed. He then determined the
impact of i.d., s.c., and i.t. LVS vaccination and SCHU S4 challenge
on this baseline curve.
v. Figure 1 shows the proportional hazard regression curves of
unvaccinated and i.d., s.c., and i.t. vaccinated rats at various SCHU
S4 challenged doses. The curves predicated that 90% of
unvaccinated rats would die from i.t. challenge with 10 5 SCHU S4 but
Page 18 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
only 10% of the s.c. and i.d. vaccinated rats and 20% of the i.t.
vaccinated rats would die from the same challenge dose. Even with
an i.t. challenge dose of 107 SCHU S4, the regression model
predicated only 30% of the i.d. and s.c. vaccinated rats and 50% of
the i.t. vaccinated rats to die. At all doses examined empirically in
the laboratory, i.t. vaccination resulted in lower survival
vi. The proportional hazard regression analyses also allowed us to
determine the group size required to detect a protective effect. If the
level of protection is expected to be as large as that induced by LVS
vaccination, then 6 rats per group is sufficient. If the level of
protection is expected to be smaller than that induced by LVS, then 7
rats per group is sufficient
vii. We are continuing the statistical analyses because the model may
be too conserve with the unvaccinated rats. Specifically, the actual
data from Experiments Ftc23, Ftc31, Ftc 32, Ptran2, Ptran6, and
Ptran7.1 (Fig 2) showed that at least 80% of unvaccinated rats died
from i.t. challenge with 103 SCHU S4, but the statistical model
projected that only 60% would die at this dose.
Figure 1. Proportional hazard regression curves for unvaccinated, i.d., s.c., and i.t. LVS
vaccinated Fischer 344 rats
Page 19 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 2. Survival of unvaccinated rats following i.t. challenge with various doses of SCHU S4.
Each panel shows the results of a single experiment, except the panel on the lower right which
shows the results of the unvaccinated rats from 4 independent experiments.
b. 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
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c. Rat model completed
7. Quality of performance
NA
8. Percentage completed
78%
Page 20 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. We showed in Experiment Ftc46 study 6 (Notebook 116, pages 12-17) and
Experiment Ftc46 study 7 (Notebook 116, pages 18-21) that quantum dots
and the coelentrazine substrate had little impact the virulence of LVS in mice.
We will determine whether they have any effect on the virulence of SCHU S4
in rats
b. Complete sub-milestone completion reports for the mouse, rat, and guinea
pigs
c. Continue consultation with GCRC (General Clinical Research Center)
biostatistician at UNM
10. Anticipated travel
Will attend UNM TVDC annual meeting in Phoenix AZ on October 6 and 7, 2008
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 7
Milestone description: SCHU S4 ED50 in primates determined from selection of
challenge dosing
Institution: LBERI
1. Date started: 2/25/08
2. Date completed: In progress.
3. Work performed and progress including data and preliminary conclusions:
a. The IACUC protocol was re-written and re-submitted for approval on 23 July 08.
Approval is pending. Study schedule, observation forms, and euthanasia score
sheets have been prepared in preparation for this study. LBERI training
personnel have been contacted by the Study Director in preparation for additional
training (see below).
4. Significant decisions made or pending
Twelve NHP will tentatively move into the ABSL3 on 15 September 08; a range of SCHU
S4 exposures are tentatively set for 23 and 24 of September 08.
5. Problems or concerns and strategies to address
Additional technical personnel will be trained on determining respiratory rates in NHP and
signed off on proficiency prior to the ED50 study.
6. Deliverables completed
None.
7. Quality of performance
Good.
8. Percentage completed
12% of the scientific work is complete.
9. Work plan for next month
a. Schedule appropriate personnel and confirm animal ID’s for use on study with
Julie Wilder, taking into account F. tularensis pre-existing antibody responses.
Physical exams, collars and microchipping to be scheduled.
b. Present the ED50 draft study protocol to Rick Lyons at UNM
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated.
Page 21 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 Ftc71 (Notebook 104, pages 152-153)
i. In mice, guinea pigs, non-human primates, and humans, protection
against SCHU S4 is dependent on the dose of LVS used for
vaccination
ii. The purpose of this experiment was to determine whether the dose
of LVS affects the protection against SCHU S4 in Fischer 344 rats
iii. Fischer 344 rats were either left unvaccinated or vaccinated s.c. with
103, 105 or 107 LVS; the 107 dose has been our standard vaccinated
dose. One month after LVS vaccination, the rats were challenged i.t.
with 105 SCHU S4.
iv. As shown in Fig 3, most of the rats died from i.t. challenge with 105
SCHU S4, regardless of the LVS vaccination dose. We did not
expect 8 of the 10 rats vaccinated with 107 LVS to die from this
challenge dose since all rats vaccinated and challenged similarly in
Experiment Ftc23 and Ftc32 survived. The difference between this
and the two previous experiments was that we used newer stocks of
both LVS and SCHU S4 . In this case, it is hard to know if the
vaccination failed or the challenge dose was too high.. So UNM is
repeating the experiment but challenging with multiple doses. UNM
also has several new LVS stocks and a new SCHU S4 stock that we
are testing now.
v.
Figure 3. Effect of LVS vaccination dose on survival of Fischer 344 rats
challenged i.t. with SCHU S4. Fischer 344 rats (n = 9 to 10) were vaccinated s.c.
Page 22 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
with the indicated doses of LVS. One month after vaccination, the rats were
challenged i.t. with 105 SCHU S4
b. To determine the role of CD4 and CD8 T cells in protecting LVS vaccinated
rats against i.t. SCHU S4 challenge, we purchased the W3/25 (anti-CD4) and
the OX-8 (anti-CD8) hybridoma cell lines to produce ascites fluids for in vivo
depletion of CD4 and CD8 T cells. We were not aware at the time that the
antibodies produced by the W3/25 hybridoma cell line only inactivate but
does not deplete CD4 T cells. This prevented us from confirming the CD4 T
cell depletion in Experiment Cdep-2.1. Thus, we purchased the CD4depleting OX-38 hybridoma (mouse IgG2a) and the isotype control 55.6
hybridoma (mouse IgG2a anti-HIV-1 gp120). We will contract with Taconic
to produce ascites fluids from these cell lines
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
21%
8. Work plan for upcoming month
a. Repeat the experiment to determine the effect of LVS vaccination dose on
the protection of rats to SCHU S4 challenge. We will use 2 challenge doses,
104 and 105 SCHU
b. Expand the OX-38 (CD4 T cell depleting antibody) and 55-6 (isotype control
antibody for OX-38). After we have confirmed that the OX-38 antibody binds
to CD4+ splenocytes, then we will send the hybridomas to Taconic for
production of ascites fluid
c. Measure the resistance of Fischer 344 rats against i.t. SCHU S4 challenge
after transfer of 1 ml immune rat serum.
d. Titrate the amount of F. tularensis-specific antibodies in immune sera from
vaccinated mice, rats, and non-human primates. This will help us to
determine the equivalent amount of sera from each species to transfer into
naïve Fischer 344 rats to assess their ability to passively protect rats
e. Compare the sensitivity of NCI and Harlan Fischer 344 rats to i.t. SCHU S4
challenge
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
Page 23 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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-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.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. 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 
7. Quality of performance
NA
8. Percentage completed
60%
9. Work plan for upcoming month
None
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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. 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, 16 additional NHPs were screened, bringing the total
number of NHPs screened to 35.
iii. 24 additional NHPs will be screened in the next several weeks (4 – 8/week).
b. Typical profiles of the response of NHPs to LVS and SCHU S4 antigens are shown in
Figures 1 (IFNγ secretion) and 2 (proliferation)
Page 24 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
ii.
iii.
iv.
v.
Figure 1A represents NHPs that secrete IFNγ primarily in response to
high doses of formalin fixed antigens (6/16 NHPs showed this pattern).
Figure 1B represents NHPs that secrete IFNγ in response to heat-killed
antigens in addition to the formalin fixed ones (6/16 NHPs showed this
pattern).
Figure 1C represents NHPs that had a high background response
making it difficult to discern a response to the LVS or SCHU S4 antigens;
although none had RBC contamination higher than 1.6% (3/16 NHPs
showed this pattern).
Figure 2A represents NHPs that had little proliferative response to any of
the LVS or SCHU S4 antigens (4/15 NHPs showed this response).
Figure 2B represents NHPs that proliferated in response to more than
one LVS or SCHU S4 antigen (11/15 NHPs showed this pattern).
A
IFNg Spots (Mean +/- SEM)
160
140
120
100
80
60
40
20
0
28438
28447
28617
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
Page 25 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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
IFNg Spots (Mean +/- SEM)
300
250
200
150
SCHUS4 hk Super
100
SCHUS4 hk Hi
SCHUS4 hk Mid
50
SCHUS4 ff Super
0
SCHUS4 ff Hi
28547
28569
28570
C
300
IFNg Spots (Mean +/- SEM)
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
250
200
150
100
SCHUS4 ff Mid
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
50
SCHUS4 ff Super
SCHUS4 ff Hi
0
28395
28496
28559
SCHUS4 ff Mid
Figure 1: Representative patterns of IFNγ production by non-LVS vaccinated NHPs. 6/16
NHPs secreted IFNγ primarily to formalin-fixed LVS or SCHU S4 antigens (A) whereas 6/16
NHPs secreted IFNγ to other antigens as well (B). 3/16 NHPs demonstrated high
background responses making it difficult to discern whether they responded to the LVS
and SCHU S4 antigens (C). (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 were plated at 1.33 x 106/ml.
Page 26 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
Relative Luciferase Units
(Mean +/- SEM)
45000
A
40000
35000
30000
25000
20000
15000
10000
5000
0
Relative Luciferase Units
(Mean +/- SEM)
28438
28549
80000
B
70000
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
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
60000
LVS ff Mid
50000
LVS ff Lo
40000
LVS hk Super
SCHUS4 hk Super
30000
SCHUS4 hk Hi
20000
SCHUS4 hk Mid
10000
SCHUS4 ff Super
0
SCHUS4 ff Hi
28447
28547
28565
SCHUS4 ff Mid
Figure 2: Representative patterns of LVS- and SCHU S4-stimulated proliferation by nonLVS vaccinated NHPs. 4/15 NHPs proliferated to few or none of the formalin-fixed LVS or
SCHU S4 antigens (A) whereas 11/15 NHPs proliferated to more than one LVS and/or
SCHU S4 antigen (B). (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 were plated at 1 x 106/ml.
Data Interpretation: Once again, it was observed that the highest response in the IFN ELISPOT
assay is usually to LVS FF Hi. The response usually dilutes out when less stimulating antigen is
used (LVS FF Mid or –Lo). A continuum of responses is observed in these non-LVS vaccinated
NHPs.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay080308.svd;
TVDC (3) bound notebook (9225), pp. 41 46; 48– 53; 58 – 63; 68 - 75
Page 27 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
vi.
vii.
Plasma from the 16 non-LVS vaccinated NHPs was tested for IgG antiLVS reactivity by ELISA (Figure 3A); and most had titers less than
100,000.
The definition of titer, however, does not reflect the different properties of
the plasma as shown in Figure 3B and Table 1 (titer = the inverse of the
highest dilution producing: 1) an OD405 value > background and 2) an
OD405 value that is equal to or higher than the next dilution (i.e. in a linear
portion of the curve).
1000000
100000
10000
Cell Mean for OD405
1000
28395
28438
28447
28464
28496
28503
28511
28525
28547
28549
28559
28565
28569
28570
28617
28651
IgG anti-LVS Titer
A
2
1.8
1.6
28464 B
28503
28547
28651
896 Post-boost
896 Pre-boost
1.4
1.2
1
.8
.6
.4
.2
0
800
4000
20000 100000 500000
Plasma Dilution Factor
Page 28 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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: IgG anti-LVS titers from 16 non-LVS vaccinated NHPs are shown in panel A
(above). In Panel B, four non-LVS vaccinated NHPs (28464, 28503, 28547, 28651) are
compared to A00896 (896), an LVS-vaccinated NHP, before (Pre-boost) and 12 days after
(Post-boost) a bronchoscopic boost with 100,000 LVS. All have titers > 20,000 (shown in
Table 1 below).
Table 1: Titer determination of non-LVS and LVS-vaccinated NHPs displayed in Figure 3.
800
4000
20000
100000
500000
28464
0.194
0.099
0.075
0.071
0.071
0.066
28503
0.257
0.112
0.077
0.072
0.071
0.066
28547
0.242
0.105
0.074
0.068
0.07
0.066
28651
0.481
0.161
0.087
0.074
0.072
0.066
896 Pre-boost
0.433
0.139
0.078
0.073
0.07
0.074
896 Post-boost
Posboostboost
1.769
0.531
0.179
0.088
0.072
0.074
NHP
Blank
Data Interpretation: Most (12) of the 16 non-LVS vaccinated NHPs have IgG anti-LVS titers lower
than 100,000. However, similar titers do not always indicate that the plasma has the same
properties (most obvious when comparing 896 post-boost with 28464). These data suggest that
there may be a need to re-think how titer is defined to come up with a way that better expresses
the entire dilution curve.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay080308.svd;
TVDC (3) bound notebook (9225), p. 47; pp. 68 – 75.
c. Continued to test the ability of the Cerus freeze/thaw protocol to spare the
reactivity of IFN secreting cells in the ELISPOT assay.
i. Two more aliquots of frozen non-LVS vaccinated NHPs were thawed and tested
for their ability to secrete IFNγ in response to LVS (Figure 4).
Page 29 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
LVS hk Hi
350
LVS hk Mid
LVS ff Hi
300
LVS ff Mid
250
LVS ff Lo
200
LVS hk Super
SCHUS4 hk Super
150
SCHUS4 hk Hi
100
SCHUS4 hk Mid
50
SCHUS4 ff Super
A06589, Frozen
A06589, Fresh
SCHUS4 ff Hi
A06587, Frozen
0
A06587, Fresh
IFNg Spots (Mean +/- SEM)
Media
400
SCHUS4 ff Mid
Figure 4: IFNγ production by fresh and frozen/thawed 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). All PBMCs were plated at 1.33 x
106/ml.
Data Interpretation: Once again, the results are inconsistent. The freeze/thaw process did not
spare the responsiveness of the cells in A06587 to LVS ff Hi or SCHUS4 ff Super, although it is a
bit difficult to interpret the data based on the fact the background response to media was elevated
in the frozen vs. fresh cells. On the other hand, the response to the various antigens was spared
a little more when considering A06589 although all the responses were quite low even when
considering the fresh cells. As both 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 spared. More
work needs to be done with PBMCs from LVS-vaccinated NHPs.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay080308.svd;
TVDC (2) bound notebook (8935), pps. 88 – 92; TVDC (3) bound notebook (9225), pps. 35 - 40.
c.
Update on A00896, a LVS vaccinated NHP that developed an aggressive behavior
pattern
i.
Since this NHP was not going to be used on a study in the ABLS3, he
was challenged with 100,000 CFU LVS by bronchoscopy.
ii.
On day 12 post LVS bronchoscopy, the NHP was euthanized and blood
and splenocytes were collected.
iii.
Figure 5 compares the ability of PBMCs to secrete IFNγ before and after
LVS boost by bronchoscopy.
iv.
Figure 6 shows the ability of splenocytes to secrete IFNγ after the LVS
boost.
v.
Plasma IgG anti-LVS titer increased from 20,000 to 100,000 after the
LVS boost.
Page 30 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
There were not enough PBMCs to test the proliferative response to LVS
and SCHU S4 antigens on the day of the LVS boost, but when
comparing the PBMCs collected on day 12 post LVS bronchoscopy (day
595) to a previous time point (day 276), no increased response was
noted.
400
350
*
Day 583
Day 595
300
*
*
*
x
x
250
200
*
x
150
x
x
100
LVS hk Super
LVS ff Lo
LVS ff Mid
LVS ff Hi
LVS hk Mid
0
LVS hk Hi
50
Media
IFNg Spots (Mean +/- SEM)
vi.
Figure 5: The ability of PBMCs from an LVS-vaccinated NHP (A00896) to secrete IFNγ
before (Day 583) or after (Day 595) bronchoscopy with 100,000 CFU LVS. All cells plated at
1.33 x 106/ml; * indicates significantly different from both media and the opposite day of
bleed by ANOVA; x indicates significantly different from media.
Page 31 of 82
Tularemia Vaccine Development Contract: Technical Report
500
450
400
1
1.33
N
T
SCHUS4 ff Hi
N
T
SCHUS4 ff Mid
N
T
SCHUS4 ff Super
N
T
SCHUS4 hk Mid
N
T
SCHUS4 hk Hi
LVS hk Super
LVS ff Lo
T
N
T N
C T
LVS ff Mid
LVS ff Hi
LVS hk Mid
LVS hk Hi
T
N
T
C
100
50
0
SCHUS4 hk Super
350
300
250
200
150
Media
IFNg Spots (Mean +/- SEM)
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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: The ability of spleen cells from an LVS-vaccinated NHP (A00896) to secrete IFNγ
before 12 days after bronchoscopy with 100,000 CFU LVS. All cells plated at either 1 or
1.33 x 106/ml; TNTC = too numerous to count; NT = not tested.
Data Interpretation: Challenge of an LVS-vaccinated NHP with LVS by bronchoscopy increases
the ability of the PBMCs to secrete IFNγ in response to LVS and SCHU S4 antigens.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay080308.svd;
TVDC (3) bound notebook (9225), pps. 24 - 34.
4. Significant decisions made or pending
There may be a need to re-assess how IgG anti-LVS titer is determined.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
77% of the scientific work is complete.
9. Work plan for upcoming month
a. Continue to test PBMCs from non-vaccinated NHPs in the IFN ELISPOT and
proliferation assays to determine the effect of HK and FF LVS at different
concentrations.
b. Continue to freeze down extra cells if they are available.
Page 32 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 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. No new work done this month.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
NA
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
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. No new work done this month.
4. Significant decisions made or pending
NA
Page 33 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
NA
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
No new work done this month because the collection was cancelled
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
Page 34 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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-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 PFT3(ABD Primate binder 1 & 2, electronic data D:\My
Documents\NHP studies\PFT3 LVS 6-25-08\PFT3d12 ICC analysis )
i.
This experiment utilized animal A00896, a cynomolgus macaque
vaccinated by intradermal inoculation with 1.5x107 cfu LVS on 11/20/06
and boosted by bronchoscope instillation of 1x105 cfu LVS on 6/25/08.
Lungs, tracheobronchial lymph nodes (TBLN) and spleen were collected
on day 12 after boosting. The purpose of this experiment was to
determine if cells from an LVS-treated NHP could be stimulated in vitro
with HK-LVS to secrete TNF, IL-2, or IFN and if populations of cells
secreting multiple cytokines simultaneously could be detected. Of
particular interest were T-cells capable of secreting high levels of all
three cytokines (multifunctional cells). An increased frequency of
multifunctional cells has been established as a correlate of protection in
other intracellular pathogen infection models and is being explored as a
potential correlate of protection for tularemia vaccines.
ii.
TBLN cells were used for IFN ELISPOT.(See MS#27 for data)
iii.
Lung and spleen single cell suspensions were used in multifunctional
assay to detect intracellular cytokine production. Cells were treated with
media only, anti-CD28 only, anti-CD28+1x107 cfu/ml HK-LVS (LD), or
anti-CD28+2x107 cfu/ml HK-LVS(HD) for a total of 6 hours (last 4 hours
in the presence of Golgi Plug).
a. Spleen assay: Cells were lost at some point in the assay
resulting in insufficient event acquisition.
b. Lung assay:
i. There was a very low frequency of CD4+ multifunctional cells
(CD4+ cells simultaneously secreting TNF, IL-2, and IFN)
in the media only and anti-CD28 treated cells but both doses
of HK-LVS stimulated high levels of multifunctional cells.
(Figure 4)
ii. There was a spontaneous population of CD4+ IFN single
positive cells that was further expanded by specific antigen
stimulation. Overall, the functional profile was similar between
the two doses of HK-LVS. (Figure 5)
iii. CD8+ cells lung cells showed only modest production of IFN
with no increase upon stimulation (data not shown).
Page 35 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 4.Two doses of HK-LVS stimulated high frequency of lung CD4+ multifunctional
cells. Lung tissue was collected from animal A00896, processed to single cell
suspension, and then stimulated as indicated followed by staining with α-CD4 and
irrelevant antibody controls (CD4/Irr Ab) or cytokine-specific antibodies.
Figure 5. Functional cytokine production profile of CD4+ lung cells from animal A00896
treated in vitro with media alone (black bars), α-CD28 (blue bars), α-CD28+LD HK-LVS
(green bars) and α-CD28+HD HK-LVS (red bars). The profiles were similar following
stimulation with each dose of HK-LVS.
b. Experiment PFT4 (ABD Primate Binders 1&2, electronic: D:\My
Documents\NHP studies\PFT4 Naive 7-18-08)
i. In this experiment, TBLN, lung and spleen cells were collected from 2
untreated (naïve) NHP on 7/18/08. The purpose of this experiment was
to determine the specificity of the NHP multifunctional assay by testing if
cells from untreated NHPs could be stimulated in vitro with HK-LVS to
secrete TNF, IL-2, or IFN. In addition, the ability of frozen lung cells
from an LVS-treated NHP to respond to specific antigen stimulation was
Page 36 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
also tested. TBLN cells were frozen, to be used in future IFN ELISPOT
assays.
Fresh lung and spleen cells from naive NHPs as well as frozen lung and
spleen cells from LVS treated NHP (PFT3d12) were used in intracellular
cytokine multifunctional T cell assay performed as above.
a. Spleen assay: cell numbers and events collected were as
expected but very little cytokine production was detected. There
were no differences between splenocytes from naive and LVStreated NHPs or between media only and HK-LVS stimulated
splenocytes (data not shown).
b. Lung assay:
i. Results from tissues from two naive NHP were similar (only
one shown as an example)
ii. Naïve CD4+ lung cells were not stimulated to secrete
cytokines by treatment with HK-LVS. (Figure 6)
iii. Naïve CD8+ lung cells did not produce TNF, IL-2, or IFN
when stimulated with HK-LVS. (data not shown)
iv. Frozen lung cells from LVS treated NHP could be stimulated
with HK-LVS but showed higher levels of CD4+
multifunctional cells as compared to when fresh lung cells
were stimulated (Figure 6)
v. The cytokine production profile of cell populations was similar
between fresh and frozen LVS vaccinated lung cells. There
was a decrease in IFN single positive cells and an increase
in triple positive cells. (Figure 7)
Figure 6. The percentage of lung CD4+ multifunctional cells was below background in
untreated NHP (black bars) but similar between fresh (blue bars) and frozen cells (red
bars) from an LVS vaccinated/LVS boosted NHP (A00896) following stimulation with αCD28+HK-LVS.
Page 37 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. The functional profile of the CD4+ lung cell response was similar between fresh
(blue bars) and frozen (red bars) cells from LVS vaccinated/LVS treated NHP following
stimulation with α-CD28+HK-LVS.
c.
Experiment Ftc30.20b (Notebook 109, pages 137-139)
i. The purpose of this experiment was to determine whether
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 105 or 106
splenocytes from unvaccinated or LVS vaccinated mice for 2 days
iii. In this experiment, addition of 106 naïve splenocytes reduced the
SCHU S4 burden more than in previous experiments that measured
killing of LVS in infected macrophages(Fig 8). This was a surprise
because SCHU S4 is more virulent than LVS and may reflect a
difference between SCHU S4 and LVS infected macrophages, but this
experiment has to be repeated
iv. Nevertheless, these results showed that addition of 105 vaccinated
splenocytes caused a modest but significant reduction in SCHU S4
burden compared with 105 naïve splenocytes. Addition of 106
vaccinated splenocytes caused a more significant reduction in SCHU
S4 burden, but the difference between addition of naïve and
vaccinated splenocytes is at most 1 log in cfu/ml recovered. This is a
relatively small effect.
Page 38 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 murine BMM killing of SCHU S4. Murine
BMM were infected with SCHU S4 at MOI of 1:100 (bacteria:macrophages) and
incubated with 105 or 106 splenocytes from naïve or LVS-vaccinated splenocytes. After
48 h, the bacterial burden was determined.
d. Experiment Ftc61.7 (Notebook 109, pages 163-165) and Experiment
Ftc61.7b (Notebook 109, pages 170-172)
i. The purpose of these experiments was to determine whether T cells
from LVS vaccinated rats can suppress SCHU S4 growth in rat bone
marrow-derived macrophages (BMM).
ii. Rat BMM were infected with SCHU S4 at MOI of 1:100
(bacteria:macrophages) and then incubated with 105 or 106
splenocytes from unvaccinated and LVS-vaccinated rats for 3 days
iii. As shown in Fig 9, addition of 105 vaccinated splenocytes did not
significantly reduce the SCHU S4 burden compared with addition of
105 naïve splenocytes. However, when 106 splenocytes were added,
there was a 10-fold difference between unvaccinated and vaccinated
splenocytes. Although the effect on SCHU S4 growth in BMM was not
as dramatic as on LVS growth in BMM (prior experiments), the fact
that 50 ng/ml recombinant rat IFN further reduced the number of
SCHU S4 recovered suggest that the assay can be improved to
increase the difference between unvaccinated and vaccinated
splenocytes.
Page 39 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. 9. LVS Vaccinated rat splenocytes reduced the number of SCHU S4 recovered
from infected rat BMM. Rat BMM were infected with SCHU S4 at MOI of 1:100
(bacteria:macrophages) and then incubated with naïve or vaccinated splenocytes or
recombinant IFNas indicated After 72 hrs, the bacterial burden was determined.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
In the multifunctional assay, stimulation with HK-LVS did not induce
production of any of the three cytokines examined by CD8+ lung T-cells or
either population of splenic T-cells. In subsequent experiments, ConA, a
positive control mitogen, will be included to confirm that assay conditions
allow for production in response to stimulation and detection of the cytokines
of interest.
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
35%
9. Work plan for upcoming month
a. Tissues from two NHPs infected with 1x105 cfu of LVS will be harvested on
day 28 and day 42 post-exposure. Cells from these animals will be used to
further develop the multifunctional assay and to assess antigen-specific
cytokine responses at these time points.
b. Repeat the murine macrophage SCHU S4 killing assay with naïve and
vaccinated splenocytes.
c. Optimize the rat macrophage SCHU S4 killing assay with naïve and
vaccinated splenocytes, focusing on increasing the difference between naïve
and vaccinated splenocytes. We will first titrate the MOI for infecting rat
macrophages with SCHU S4
Page 40 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 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. No work was done on this Milestone this month.
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
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.
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:
Page 41 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Thioredoxin LEE Construct Design
1. Thioredoxin (Trx) tag facilitates proper protein folding
2. Trx tag will be well exposed due to its large size (11Kda) and folding property
3. Protein purification is done using magnetic beads which are covalently linked
anti-Trx antibody
4. Double tag: Thioredoxin Tag at the N-Terminus for yield and purification
advantages. His Tag at the C-Terminus for full-length visualization of samples
and as an alternative detection method if desired.
5. Two-step assembly protocol.
Fig. 1: Schematic of two-step assembly reaction for expression constructs.
B. Testing protocols for protein purification using anti-Thioredoxin (Trx)
antibodies
1. Test specificity and efficiency of anti-thioredoxin monoclonal and polyclonal antibodies
from different vendors
Page 42 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Four anti-Thioredoxin monoclonal Abs (mAb2A1, 3A1, 8A1, A00180) from two t
manufacturers (Abcam and Genescript) were compared to a polyclonal Ab (pAb
Trx) by immunoblot analysis.
b. In each gel lane 1: Kaleidoscope ladder, lane 2: E.coli IVT lysate without
template, lane 3: purified recombinant Trx, and lane 4: chemluminescent ladder.
c. Gels are probed with Abcam monoclonal trx, Genescript polyclonal and
monoclonal Trx, or Genetech anti-His antibodies as indicated. (data in figure 2)
Figure 2: Comparison of performance of different anti-Trx antibodies by
immunoblot 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
2. These results indicate that the polyclonal (pAbaTrx) and only 1 monoclonal (mAb
A00180) reacts specifically and efficiently with the thioredoxin. The arrow indicates
position of Trx
C. Set up for testing IVT products from new Trx LEE constructs in T cell
assays
1. An anti-trx or anti-his antibody was non-covalently attached to protein G-magnetic beads
via the antibody’s Fc receptor stem.
2. Ni-magnetic beads were used as comparison
3. Trx/His two-tagged polypeptides produced in IVT reactions were incubated with beads
Page 43 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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-α-Thio
and washed.
4. Beads were used to stimulate murine splenocytes.
5. IFN-gamma release was measured in an ELISpot assay (data in Figure 3)
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 3: ELISPOT analysis of Trx-tagged IVT products purified on anti-thio, antihis, or nickel attached magnetic 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
D. Analysis of ELISpot results
1. Left hand panel: Background is only a problem against the LVS-immune splenocytes, as
the cross-reactivity is not seen in these ova-immune cells
2. Right hand panel: We observe significant differences among bead configurations in the
LVS-immune cells. We are testing the anti-trx G bead, Ni bead or anti-His G bead bound
samples in each row to assess level of cross-reactivity remaining in samples after affinity
purification of polypeptides in these 3 different bead-based antigen stimulation protocols
3. Bottom row of LVS panel shows Ni bead samples. As seen previously, there is a high
level of cross-stimulation of LVS-immune T cells with FTU or OVA antigen samples.
4. Middle row of LVS panel: The anti-His G beads displays less background, and
presumably better purification.
5. Top row, LVS panel: the anti-Trx G beads provide the lowest background, and
presumably the highest level of purification.
E. Evaluation of scale-up protocol for thioredoxin fusion FTU polypeptides.
1. We tested the performance of the template design in IVT reactions with a number
of different ORF sequences carried out in HTP formats (Fig. 4).
Page 44 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
Fig. 4. High-throughput cell-free protein synthesis of FTU polypeptides in E.coli
based system
Data locations:
R:\GeneVac\BAN\BAN IVT Data\BAN IVT Gel\BAN IVT 35S gels\BAN IVT Plate 04 Row A and B
Thio Purification Test Supernatant 7-10-08 02
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\
FTU HTP IVT 35S gels\FTU RbG pl 1 Q1 Denaturing purification FT E 3-25-08 01
2. FTU ORFs were constructed into either the original His LEE or the new two-tag
LEE (Trx and His tags). 35S-methionine IVT reactions were performed with
identical conditions and analyzed on gels.
3. The autoradiograph results shown in Fig. 3 demonstrate that the new template
two-tag design (panel 2) both generates higher yields and more robust reaction
success than the His-tag only templates (panel 1).
E. Evaluation of longer LEE constructs.
1. We had selected the shorter LEE design based on the performance of the His
Page 45 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
polypeptide products. Since we now can use the Trx /His fusions, we tested the
possibility of generating longer polypeptides. This would mean fewer samples to
evaluate in T cell assays.
2. We successfully built the longer LEE templates carrying ORFs of ~1000bp, in a HTP
protocol format, as compared to the current constructs carrying 500 to 600bp ORFs.
This is shown in Fig. 5A-C below.
Fig. 5A. LEEs are robustly assembled from a short ORF design, without Trx.
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP
IVT DNA gels\Other\FTU short plate #1 amp2 7-23-07_02
Page 46 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
Fig. 5B. LEEs are robustly assembled from a short ORF design, with Trx.
Data location R:\GeneVac\BAN\BAN IVT Data\BAN Thio LEE Amp Gel\BAN IVT Plate 01 Amp 79-08 02__
Fig. 5C. LEEs are robustly assembled from a long ORF design, with Trx
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT DNA
gels\Thio LEE Construct\Long Plate 1 Columns 1 and 2 LEE test 7-25-08
Page 47 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
3. Conclusion from Fig. 5 results: Trx-His two-tagged LEE constructs were assembled as
efficiently as single-tagged His LEEs.
4. We used these long constructs in IVT reactions to evaluate efficiency and integrity of
longer polypeptides syntheses.
Fig. 6. Radiolabeled IVT reactions using FTU long LEE constructs were analyzed by
autoradiography.
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S
gels\Thio fusion IVT\Thio Long ORF Thio Test 7-30-08 crop
5. These polypeptide yields were calculated as usual and are presented in Table 1 below.
6. We note that the LEE constructs expressing the longer ORFs, shown in red box, yielded
similar quantities of IVT product as compared to the shorter LEE constructs.
Page 48 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 1. Calculated yields of LEEs carrying a range of ORF sizes.
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU
Scintillation results\FTI IVT Thio Fusion tests\FTU Long ORF IVT Test 7-27-08
7. We conclude that longer ORFs can be robustly generated with the context of our new
template two tag (Trx/His) design.
F. Evaluation of the capacities of anti-Trx beads to capture nascent
polypeptides
1. In the pilot studies described above, the Trx antibodies were non-covalently
attached to beads via the Fc stem. To make our purification reagents more
stable and consistent, and to improve binding capacity, we covalently conjugated
the antibodies to the beads, as previously proposed. We now evaluated the
ability of the antibody affinity beads to bind and elute the Trx-fusion proteins.
2. Several IVT reactions were carried out with no template, OVA, and 2 different
FTU ORF expressing templates in the presence of the antibody attached beads.
Beads were washed, and then total bead bound proteins were pH eluted.
3. Samples were electrophoresed and visualized by silver stain (total protein) and
35S detection (de novo polypeptide) in Fig. 7.
4. The results indicate that all 3 antibody samples bind the Trx fusion polypeptides.
Page 49 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
5. However, the mix of Abcam monoclonal antibodies binds with lower efficiency
than either Genescript antibody.
6. Since the polyclonal antibody is a less well-defined reagent, and carries more
irrelevant proteins (as visualized in silver stain) we chose to proceed with the
monoclonal antibody from Genescript.
Fig. 7. Visualizing efficiency of anti-Trx antibody capture of the Trx-fusion
products from IVT lysates
G. Determination of whether bead purified IVT reactions cross-stimulate in
NHP ELISpot assay.
1. IVT lysates without template were incubated with or without anti-Trx antibody
bound beads. These samples were shipped to UNM and evaluated in ELISpot
assays for stimulation of NHP lymph node cells. These results are displayed
din Fig. 8.
Page 50 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Fig. 8. ELISpot analysis of cross-reactivity caused by total IVT
lysate proteins versus IVT lysate proteins after bead purification.
Data location: UNM data located at Ftc59 study 22 (Notebook 115, pages
148-151, 154, 155)
2. Duplicate wells shown in Fig. 7 show that IVT as is (unpurified), shown in
a11,a12 and b11,b12, cross-stimulated NHP cells, as seen with murine cells.
3. However, the bead samples shown in b1 and 2, c1 and 2, d1 and 2, and e1 and
2 indicate that the cross-reacting material does not bind to the beads.
Page 51 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. The formalin fixed LVS antigen added to NHP cells in row g, serves as a
stimulation level reference for this experiment. The LVS antigen was titrated
from neat to a 1:1000 dilution.
H. Second NHP ELISPOT assay.
1. In this experiment, a second monkey was used as source of lymph node cells.
We repeated the E. coli lysate as is samples and also included the NEB
reconstructed IVT samples to evaluate cross-reactivity of its components
against NHP cells (Fig. 9).
Ova antigen
generated in E.
coli lysate, and
bound to antitrx beads.
Amount of Ab
on beads
titrated at 10ul,
20ul, 50ul, in
duplicate
Blank
Titration of
NEB IVT
mix “as is”,
at 5ul thru’
0.005ul in
duplicate
Titration of
E. coli lysate
“as is”, at 5ul
thru’ 0.005ul
in duplicate
FF-LVS titration:
Figure 9: ELISpot analysis of cross-reactivity caused by total IVT lysate proteins
versus NEB pure components.
Data location: UNM data located at Ftc59 study 23 (Notebook 115, pages 156-160)
Page 52 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. The reactivity level in this experiment is about half as judged from the LVS
standards shown in row g.
3. A titration of antibody for bead purification indicates that increasing the amount of
material from 10ul to 50ul does not raise the cross-reactivity level. This is good
because we have previously shown that more antibody on the beads leads to
higher polypeptide yields.
4. The NEB IVT mix does not lead to cross-stimulation of these cells as compared
to the full E. coli lysates. This is in contrast to our result with the murine
splenocytes. We will repeat this to confirm difference between the monkey and
murine cell responses. If repeated, this indicates that we can use the
reconstructed IVT mix without need for polypeptide purification.
4. Significant decisions made or pending
We anticipate closing out MS26 this month.
5. Problems or concerns and strategies to address
None at this time
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
100%
9. Work plan for upcoming month
a. Retest cross-reactivity stimulated by NHP cells with NEB reconstructed
expression mix as compared to full IVT lysate.
b. Test covalently attached anti-trx antibody beads with NHP cells.
c. Test yields generated from stored vs. fresh E. coli lysates.
d. Test sensitivity of frozen NHP cells as compared to fresh samples.
10. Anticipated travel
-Will be attending the CHI Novel Vaccines Conference next week in Boston entitled:
Bridging, Research, Development, and Production.
-Will report back anything relevant to the TVDC program.
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
Page 53 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Ftc59 study 22 (Notebook 115, pages 148-151, 154, 155)
i. The purpose of this experiment was to determine whether the nonspecific reactivity to ivt proteins by LVS vaccinated BALB/c
splenocytes is also observed with lymph node cells from LVS
vaccinated non-human primates
ii. In this experiment, the NHP was vaccinated i.d. with 10 7 LVS on
11/20/06 and then boosted by bronchospcopy with 105 LVS on
6/25/08. The cells from the TBLN was collected on 7/7/08
iii. ASU provided in vitro transcription/translation (ivt) reactions with
DNA templates encoding OVA, Ftu1695 and Ftu1712 or without DNA
template as control. The ivt reactions were purified with monoclonal
or polyclonal anti-thio antibody conjugated to magnetic beads and
sent to UNM as bead-bound proteins in PBS
iv. At UNM, the PBS was replaced with complete RPMI and tested by
IFN ELISpot assay in the presence of 1.5 x 105 LN cells from the
LVS vaccinated and boosted NHP. 90% of each sample was divided
undiluted between two wells and the remaining 10% was diluted 1:10
before being divided between two wells.
v. We used heat killed (wells F1-8) and formalin fixed (wells G1-8) LVS
as positive controls, starting with 4 l of the original preparation
“neat” and serially diluting 10-fold
vi. For comparison, we used Invitrogen’s in vitro transcription/translation
reaction mix without any purification (wells A11-12 and B11-12)
vii. The plate layout and the actual results are shown below
viii. Data interpretation will be presented in ASU section
ivt proteins
bound on G
beads via antithio antibody
1
Monoclonal
B
Monoclonal
C
Polyclonal
D
Polyclonal
E
2
blank
No template
neat
No template
1:10
No template
neat
No template
1:10
Control
F
HK LVS neat
A
Control
G
H
FF LVS neat
1700 ng/ml
anti-CD3
3
blank
4
blank
blank
9
10
No templ
11
12
"as is" neat
OVA neat
Ftu1695 neat
Ftu1712 neat
1:10
"as is" 1:10
OVA 1:10
Ftu1695 1:10
Ftu1712 1:10
OVA
OVA
OVA neat
Ftu1695 neat
Ftu1712 neat
1:10
1:10
OVA 1:10
Ftu1695 1:10
HK LVS
1:100
FF LVS
1:100
17 ng/ml antiCD3
Ftu1712 1:10
Ftu901
Ftu901
HK LVS 1:1000
1:10
1:10
FF LVS
1:1000
Ftu1695
Ftu1695
blank
1:10
1:10
HK LVS 1:10
FF LVS 1:10
170 ng/ml
anti CD3
5
6
7
8
Page 54 of 82
ivt
proteins
bound
on G
beads
via antihis
antibody
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
Ftc59 study 23 (Notebook 115, pages 156-160)
i. The purpose of this experiment was to:
1. Repeat experiment Ftc59 study 22
2. Test different anti-thio antibodies for purification:
3. Compare the Invitrogen ivt reaction mix which has been
used up to now with New England Biolab pure ivt reaction
mix which has been depleted of all proteins not essential for
in vitro transcription/translation
ii. In this experiment, 105 LVS was delivered by bronchospcopy on
6/25/08 to an NHP and the cells from the TBLN were7/23/08. This
differs from Experiment Ftc59 study 22 in that the NHP was not
vaccinated previously. So the NHP for this experiment had been
vaccinated only but had not been vaccinated and boosted.
iii. ASU provided in vitro transcription/translation (ivt) reactions with
DNA templates encoding OVA, Ftu1695 and Ftu1712 or without DNA
template as control. The ivt reactions were purified with one of the
following and sent to UNM in PBS:
1. A cocktail of monoclonal anti-thio antibodies from Abcam
(wells A1-8 and B1-8)
2. A monoclonal anti-thio antibody from Genescreen (wells C18, D1-8, and A-F9 and A-F10)
Page 55 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
v.
vi.
vii.
viii.
3. A polyclonal anti-thio antibody from Genescreen (wells E1-8
and F1-8)
At UNM, the PBS was replaced with complete RPMI and tested by
IFN ELISpot assay in the presence of 1.5 x 105 LN cells. 90% of
each sample was divided undiluted between two wells and the
remaining 10% was diluted 1:10 before being divided between two
wells.
ASU also provided ivt mixes from Invitrogen (wells B-D11 and BD12) and New England Biolab (wells E-G11 and E-G12) without any
purification. UNM tested 0.05l, 0.5l and 5l per well
We used heat killed (wells H1-8) and formalin fixed (wells G1-8) LVS
as positive controls, starting with 4 l of the original preparation
“neat” and serially diluting 10-fold
The plate layout and the actual results are shown below
Data interpretation will be presented in ASU section
1
2
No templ mAb
Abcam
3
4
OVA mAb
Abcam
5
6
Ftu1695 mAb
Abcam
7
8
Ftu1712 mAb
Abcam
9
10
OVA mAb
genescript 10 ul
1:10
No Templ mAb
Genescript
1:10
OVA mAb
Genescript
1:10
Ftu1695 mAb
Genescript
1:10
Ftu1712 mAb
Genescript
1:10
OVA mAb
genescript 20 ul
E
1:10
No Templ pAb
Genescript
1:10
OVA pAb
Genescript
1:10
Ftu1695 pAb
Genescript
1:10
Ftu1712 pAb
Genescript
1:10
OVA mAb
Genescript 50 ul
F
1:10
1:10
1:10
1:10
G
H
FF LVS neat
HK LVS neat
FF LVS 1:10
HK LVS 1:10
FF LVS 1:100
HK LVS 1:100
FF LVS 1:1000
HK LVS 1:1000
1:10
Ftu901 mAb
Genescript
1:10
A
B
C
D
11
12
Blank
5 ul NEB IVT no
templ
0.5 ul NEB IVT no
templ
0.05 ul NEB IVT
no templ
5 ul E coli IVT no
templ
0.5 ul E coli IVT
no templ
0.05 ul E coli IVT
no templ
Blank
Page 56 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
30%
9. Work plan for upcoming month
a.
b.
Determine whether ivt translated B anthracis proteins induce IFN production
by NHP lymph nodes cells. In Ftc59 study 22 and 23, we noticed that the ivt
OVA protein seemed to stimulate more spots than the ivt Ftu proteins. This
raised the question whether it is appropriate to use ivt OVA to measure the
amount of background spots. Perhaps NHPs have higher natural responses
to OVA than Ftu proteins
Determine whether NEB pure ivt reaction mix induce IFN production by
NHP lymph node cells In Ftc59 study 23, we noticed that NEB pure had very
few spots compared with Invitrogen’s ivt mix. It is possible that the NEB pure
mix may be depleted of the crossreactive material
10. Anticipated travel
NA
Page 57 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
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 (inactive)
 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
B.
Generate polypeptide library
C.
Array polypeptide library
4. Significant decisions made or pending.
The decision to proceed into MS28 with implementation of proteome production plan is
anticipated 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
Initiate library production.
10. Anticipated travel
See above
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 58 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 - 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
a. Ftc59 study 22 (Notebook 104, pages 157-158)
i. The purpose of this experiment was isolate RNA from SCHU S4
cultured in Chamberlain’s broth and SCHU S4 from infected mouse
lungs
ii. RNA has been isolated under both conditions and is currently being
tested for sterility. We will analyze the RNA quality and quantity
before sending them to ASU
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
10%
9. Work plan for upcoming month
a. We will prepare 200 g of SCHU S4 genomic DNA per request from ASU
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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 repeated a LAPT amplification of the first time course samples currently at ASU.
These samples have been amplified and are in the process of being labeled for
hybridization to the microarrays.
Page 59 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 had previously reported on optimization of qPCR for one gene (FTT0901 (Tul4)). We
report here that we have extended the validation of our first primer sets for 6 genes
(Table 1). FTT0901, FTT0721, and FTT1712c were determined of interest based on
literature analysis of potential antigens or virulence factor genes. FTT0548, FTT0058,
and FTT0256c were identified by microarray analyses of the first time course experiment
based upon changes in expression over time with a pattern of increasing (FTT0548),
decreasing (FTT0058), or steady (FTT0256c) expression levels.
Table 1. Targeted gene list for initial qPCR validation studies.

Following the study design for the initial validation for FTT0901, genomic SCHU S4
DNA was serially log-fold diluted from 10 ng/l to 0.001 ng/l. The reactions were
run with the remaining 5 primer sets with the following specifications. Melting curve
analysis was performed to ensure the uniformity of amplifications and to identify
primer-dimer formation and cycling protocol was 95 ◦C for 2.00 minutes, 40.0 ◦C for
30 seconds and a 20 minute ramp to 95 ◦C. The quantification protocol included an
initial incubation at 95 ◦C for 5 minutes followed by 40 cycles of 95 ◦C for 20 seconds
and 60 ◦C for 1 minute. Two representative profiles of the 6 genes tested are shown
in Figure 1. The melting curve analysis revealed a single peak for each of the genes.
Across the dose response, there was less that 1.0 degree difference in the peak
melting temperature indicating that amplifications were uniform. The quantitation
plots of the genomic standards revealed a high R2 value of greater than 99. Similar
results were obtained with the other 4 genes listed in Table 1.
Page 60 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Graphical representation of the qPCR reactions for FTT0721 and
FTT0058. The curves at the top of the graph represent melting curve analyses.
The lower plots reveal the regression analysis of the standards.

We next sought to test the primers against a reconstitution sample of SCHU S4 RNA
diluted into an excess of normal mouse lung RNA. SCHU S4 RNA was diluted into
normal mouse lung RNA at log 10 dilutions from 1000 ng/µl to 0.001 ng/µl. After an
initial reverse transcriptase reaction, the cDNAs were quantified for the primer sets
via qPCR using a genomic DNA standard curve. Representative results for the six
primers sets are shown in Figure 3 using FTT0721 primers. In the melting curve
analysis there was a shift in the peak melting temperature when comparing the
genomic standards (dark blue lines) to the reconstitution samples (light blue lines). In
addition, the melting curves show negative pull down peaks at the higher
temperatures. The differences between the melting curves between the samples
may indicate secondary products being amplified and samples will need to be verified
by gel electrophoresis to ensure single products. The negative pull down peaks at
the high temperatures may be the result of using optically clear plates. We are
evaluating different plate formats to determine if these peaks are artifacts. We were
able to obtain reliable standard curves and were able to identify Cycle Threshold (Ct)
values for each of the unknown samples. These results are representative of the
Page 61 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
remaining primer sets with the exception of FTT0256c. We observed technical
problems with the data from this latter primer set and it was not included in the final
analysis.
Figure 3. qPCR analysis for gene FTT0721. This graph is representative for the
other six genes. The cycle time to cross the threshold was plotted for each of the
genomic standards and a linear regression line used to calculate the Ct values for
each of the reconstitution cDNA samples.

We utilized the data from these analyses to create a Ct determination for each of the
cDNA samples. The results, shown in Table 2, reveal that for 4 of the 6 genes
assessed we were able to detect down to 0.01 ng/µl. For samples within the linear
portion of the standard curve, there was an approximate 10 fold increase in the
amount of SYBR Green dye that mirrored the increase in cDNA amounts.
Importantly for each of the 5 genes shown, normal mouse lung cDNA did not reveal a
Ct value after amplification. This reveals that the primer designs do not amplify a
non-specific product.
Page 62 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Ct value calculations of reconstitution cDNA using 5 primer sets and
genomic DNA as a standard curve.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We encountered a problem with purification of SCHU S4 genomic DNA used for microarray
normalization and qPCR standard curves. The UNM samples were alcohol precipitated to
concentrate the DNA at ASU, but the SCHU S4 genomic DNAs would not re-dissolve after
alcohol precipitation. This indicates that the SCHU S4 genomic DNA should be purified a
second time to remove protein or carbohydrate contaminants. New DNA is being prepared
by UNM using a different isolation method
The negative pull-down peaks noticed on the melting curves may indicate an instrument
problem or plate format problem. We performed duplicate analyses on a separate Eppendorf
cycler and did not observe the pull-down peaks. This duplicate assay was run using a white
opaque plate for qPCR analysis instead of the optically clear plates we have been normally
using. We have ordered the white plates to further investigate this as a resolution to the
problem. In addition, we had a service call by Eppendorf to evaluate our cycler function and
it was found to be in good working order.
Page 63 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 shift in primer melting temperatures between the genomic and cDNA samples needs to
be further evaluated. We will be performing duplicate assays to assess the presence of
multiple products by gel electrophoresis.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
40%
9. Work plan for upcoming month




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.
Process the repeat time course samples.
Design and validate primers for genes for normalizing the qPCR reactions (16S
Ribosomal genes)
Assess gene expression levels of 6 current validated primer sets 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 twofold, 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 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:
Page 64 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Performed PCR on various colony pools taken from some of the original clones
generated from the newest cryotransformation with the KEK1162 tulatron VgrG
plasmid construct. These represented 5 colonies per pool and we were able to
screen 33 pools (165 original clones). We used the oligo set FTT1346 fwd NdeI
and VgrG rev EcoRI, which for the correct mutant will yield a PCR product 1600
bp where the wild type product would be 900 bp. Of all these pools we were only
able to see mixed population of VgrG mutant size and wild type size. None of
these sets showed a very strong mutant band in the PCR profile. We were
hoping to find a possible original clone that might have both VgrG genes
simultaneously interrupted without further passaging (data not shown, located in
UTSA TVD Notebook 7, page 20, 22). We took two of these original sets VP22
and VP23 and prepared genomic isolations and ran PCRs using the
aforementioned oligo set, in addition, we ran the EBS Universal with the VgrG
rev EcoRI oligos together. This was able to verify whether these sets had the
VgrG mutant complete and also that we do have a “VgrG intron” in the correct
location (in a VgrG gene, Figure 1).
Figure 1
Figure 1 represents PCR profiles resulting from using genomic templates generated from original
VgrG clones and oligo sets FTT1346 fwd NdeI and VgrG rev EcoRI in panel B, and oligo set EBS
Universal with VgrG rev EcoRI in panel A. Although the picture does not depict it well most of
these clones did show a mutant size PCR 1600 bp fragment and a wild type 900 bp PCR fragment,
lanes 4 thru 13. The strongest mutant band was shown in lane 13, C23-5 original clone. In
addition, in panel B we expect the EBS universal oligo set to yield 800 bp PCR product using the
original V17 clone, lane 3 as the positive control. All of the potential mutants screened here (lanes
4-13) did have the insertion in the correct location. Data located in UTSA TVD Notebook 7, page
25.
b.
We also screened by PCR various single colonies generated from a passage of
V17 original clone; these represent first single clones from the original parent.
These were screened by pools of 5 colonies each and we used the original
aforementioned oligos. We were able to screen V17A thru V17AA pool sets
which represent a total of 35 individual V17 passaged clones. None of these
pools yielded a PCR profile that showed only the mutant product; however, pools
M, O, S, T, U and V illustrated a stronger mutant band profile than seen in other
Page 65 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
PCR groups. Therefore, first we took one of these groups (V17M) and prepared
genomic isolations and ran these as templates with the first set of oligos
mentioned above. This set of 5 colonies did show a stronger PCR mutant size
band but the wild type band was still present (Data not shown; located UTSA
TVD Notebook 7, page 22). Then we took two additional V17 groups (V17S and
V17T) and prepared genomic isolations from these and again did PCR with the
first set of oligos; in addition, we used the EBS universal oligo set to confirm that
these clones still had the “VgrG intron” in the correct location (figure 2).
Figure 2.
Figure 2 represents PCR profiles from some potential VgrG mutants selected from the passaged
V17 original clones Genomic templates from the V17S and T groups were used with primers
specific to the VgrG gene (FTT1346 fwd NdeI and VgrG rev EcoRI, panel A); and the EBS
Universal primer was used with the VgrG rev EcoRI oligo (panel B) to confirm correct location
insertion of the” VgrG intron”. Lane 2 and 14 represents the wild type PCR product profiles for
both sets of primers; in panel A expect 900 bp product and in panel B no product is expected.
Lane 3 is the V17 original mutant profile for both panels. Lanes 4-8 represent resulting PCR
products from the C17S clones 1 thru 5 and lanes 9-13 are the C17T clones 1 thru 5. In panel A
the oligo set FTT1346 fwd NdeI and VgrG rev EcoRI should yield only the 1600 bp band if the VgrG
mutant is complete and both VgrG genes contain the insertion mutation. However, both wild type
and mutant products were evident in all these clones tested. The * marks the V17 clones that we
decided to continue with by passaging in culture further (V17S4 and V17T4 clones). Panel B
verifies that all mutant clones tested continue to have the “VgrG intron” at the VgrG gene location
since the EBS universal is specific to the intron and the expected 800 bp product was evident.
Data located in UTSA TVD Notebook 7, page 24.
Note: the acronym “EBS” is the name of the universal primer designed by Sigma that is specific to
the intron portion of the designed construct. This part always remains the same sequence;
however, the portions specific to our desired gene varies. It stands for “exon binding site”.
c.
Although we do not have the complete VgrG deletion mutant the intensity of the
VgrG mutant PCR product is increasing suggesting that with a few more
passaging of the selected mutants (figure 2, * clones) we will be able to generate
the complete VgrG mutant (that is, were both the VgrG genes are interrupted by
this VgrG intron).
Page 66 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
e.
f.
The igLD SchuS4 mutant was tested for virulence in a mouse experiment and
the mutant was attenuated for virulence. We had 5 mice per inoculation (these
inoculations were by intranasal) test groups with exception of one group (10E6)
which had 6 mice. Our empirically calculated dose inoculations were 4768
(10E3), 4.768 x 10E4, 4.768 x 10E5, and 4.768 x 10E6 (total CFUs). After 35
days all the test mice (igLD inoculated) survived as well as the PBS negative
control set. The positive control (the KKT1 wild-type) did die after five days
with a dose of 128 CFUs. The antibody profiles for this type of infection will be
done by Dr. Arul’s group; in addition, to organ deposition of inoculation (in a
separate experiment). The control for successful inoculation is the wild type
group which is performed with the same protocol as far as dilution preparation
and dose determination by plating serial dilutions on TSA +++ plates and
calculating CFU empirically. Although, sera was taken from these groups of
mice before the challenge was performed. Data will be reported on next
month’s report. Data located in the UTSA TVD Notebook 5, page 143, 144.
We received a new Western Analysis kit and we hope that the new secondary
mouse anti-sera will give a cleaner and stronger igLD immunoblot. We hope
to do the repeated igLD immunoblot this coming month.
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
74%
9. Work plan for upcoming month
b. 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.
c. Will begin the challenge of the igLD inoculated mice and follow their survival.
a. Will continue with screening of the newly isolated second singles from V17S4 and V17T4
clones discussed in figure 2; to search for the correct SchuS4 VgrG mutant via PCR
profiles with inside and outside oligos relative to the expected intron insertion sight.
Some of these generated PCR products will be sent for sequencing to confirm correct
VgrG mutant.
b. 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, I do not plan to attend the meeting in Arizona
11.Upcoming Contract Authorization (COA) for subcontractors
None
Page 67 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 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: Measure intramacrophage (J774) survival of SCHU S4 iglD mutant (Note book
#4 page 159-160): Murine macrophage cell line (J774) were seeded in a 96-well
plate (105/200 μl/well) overnight and infected with the iglD mutant (designated KKT10) or its parental strain (SCHU S4) using an infection dose of 10 or 100 MOI.
Numbers of viable bacteria in macrophages were measured at 3 hr and 24 hr postinfection. As expected, the wild type SCHU S4 replicated rapidly inside macrophages
within a day using MOI of 10 or 100. However, no replication of KKT-10 (SCHU S4
iglD mutant) was observed (either 10 or 100 MOI) during the same incubation time,
indicating high degree of attenuation.
6
3h
CFU / well (log10)
24 h
5
4
3
2
1
SCHU S4
10 MOI
KKT-10
SCHU S4
KKT-10
100 MOI
Fig. 1. Intramacrophage survival of SCHU S4 iglD mutant. Murine macrophage cell
line (J774) were infected with the iglD mutant or its parental strain (SCHU S4) using
an inoculum of 10 or 100 MOI. Numbers of viable bacteria in macrophages were
measured at 3hrs and 24 hrs post infection.
Page 68 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
50B: Monitor Ft subsp. tularensis SCHU S4 replication and dissemination in mice
after intragastric LVS vaccination. (Notebook #8, pages 109, 114-16, 118-21).
BALB/c mice were vaccinated intragastrically with LVS (103 CFU) or mock (PBS)
vaccinated. Mice were rested for three weeks and then challenged intranasally with
130 CFU of F.t. SCHU S4. Lungs, livers and spleens were collected from these mice
at day 1, 3 and 4 after challenge (5 mice per time point). Numbers of SCHU S4
bacteria in each organ were determined by dilution plating. As shown in Fig. 2, there
were low numbers of SCHU S4 bacteria present in the lungs of both LVS and mockvaccinated mice and no detectable bacteria in the spleens and livers at day 1 after
challenge. By day 3 after challenge, there were high numbers of bacteria present in
all organs in mock vaccinated mice while there were still very low levels of bacteria in
the tissues of all LVS vaccinated mice. At day 4 after challenge, at a time point right
before mice normally succumbed to infection, bacterial numbers were as high as 10 8
CFU in the organs of mock vaccinated mice while there remained very few bacteria in
LVS vaccinated mice. These results indicate that intragastric LVS vaccination leads
to decreased ability of SCHU S4 to both replicate at the sight of infection (lungs) as
well as disseminate systemically (liver and spleen).
LVS
M oc k (PBS)
CFU per Organ
1 09
1 09
Lungs
Liver
1 09
1 08
1 08
1 08
1 07
1 07
1 07
1 06
1 06
1 06
1 05
1 05
1 05
1 04
1 04
1 04
1 03
1 03
1 03
1
3
4
Spleen
1
1
3
4
Days A fter SCHU S4 Challenge
3
4
Fig. 2. Kinetics of bacterial growth and clearance in target organs following LVS and mock
(PBS) I.G. vaccination and subsequent F.t. SCHU S4 challenge. Bacterial burdens were
determined in lungs, livers and spleens of individual mice (5 mice per time point). Dashed line
represents the limit of detection.
4. Significant decisions made or pending
None
Page 69 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
77% of scientific work completed on milestone 50A (original plans)
75% of scientific work completed on milestone 50B (intragastric plan)
Normalized average over 1 year is 76% work completed
9. Work plan for upcoming month
50A: (1) Measure humoral responses after KKT-10 (iglD mutant of SCHU S4)
intranasal immunization.
(2) Assays for cell-mediated immune responses after F. novicida iglB oral
immunization using cellular cytokine recall.
50B: (1) Survival after LVS I.G. vaccination and CD4+ T cell depletion/SCHU S4
challenge.
10. Anticipated Travel
Will travel to UNM TVDC annual meeting in Phoenix AZ in October 2008.
11. Upcoming Contract Authorization (COA) for subcontractors
None
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 representing the RecA gene
a. In last monthly report, it was reported that KKT11 (recA mutant Schu S4) was mildly
attenuated with the inoculum about 206CFU and only one mouse survived. On
July11th (Day35 after inoculation), KKT11 group mice (one) and PBS control group
mice (five) were challenged with wild type Schu S4 intranasally to evaluate the
protective efficiency against wild type strain. All of 6 mice died after 5 days.
Table1: The protective efficiency against wild type Schu S4
Group of
Route of
Dose of
Survival rate
mice
challenge
challenge(CFU)
D1 D2 D3 D4 D5 D6
KKT11
I.n.
136
1/1 1/1 1/1 1/1 0/1
PBS
I.n.
136
5/5 5/5 5/5 5/5 1/5 0/5
b. From data shown on table1, the mouse was not protected by KKT11 against wild
type Schu S4 challenge. Although this study didn’t have sufficient numbers of mice
Page 70 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
to draw significant conclusions, the data suggests that similar to other attenuated
mutants we have tested. The recA mutant does not protect against wild type Schu
S4 challenge. The dose for vaccinating the mouse was low (206CFU), but we
followed the procedure for inoculation, especially mixed the inoculum thoroughly for
every mouse before vaccination to make sure each mouse received the same dose
of mutants, so we are confident that the only surviving mouse was vaccinated with
similar dose of recA mutants to the rest mice which were in the same group and died
after vaccination.
Data recorded on UTSA TVDC notebook #6, page42-44 for Table1.
Creation of a LUX operon plasmid in Ft
3.2 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 bacteria. LuxCDABE have been identified as active in the
emission of visible light.
a. In last monthly report, we reported that the effort to clone 7kb Lux operon into both
pKEK843 and pUC118 failed. The low copy plasmid pWSK30 was used as the parent
plasmid to replace pKEK843 or pUC118. 7kb fragment carrying Lux operon from the
plasmid pUTmini5Tnkmlux was ligated into digested pWSK30, and transformed into
E.coli.DH5α. The transformants were selected on LB/X-gal/Ampicillin (100ug/ml) agar
medium for white colonies. About 38 colonies were selected randomly and cultured in
LB/Ampicillin(100ug/ml) liquid medium. The overnight cultures were detected for
bioluminecence
from
lux
operon
using
the
Luminometer
with
pUTmini5Tnkmlux/DH5 for the positive control and pWSK30/DH5 for the negative
control. Colony #24 and #38 were tested to give positive signals. Data was shown) in
Chart1 below
Chart1:
Bioluminecence of pWSK30/Lux operon
80000000
70000000
60000000
RLU
50000000
40000000
Series1
30000000
20000000
10000000
y3
3
y3
1
y3
0
y2
8
y3
8
C
ol
on
C
ol
on
C
ol
on
C
ol
on
y2
5
y2
4
y2
2
y2
1
ro
l
y2
7
C
ol
on
C
ol
on
C
ol
on
C
ol
on
C
ol
on
C
ol
on
C
on
t
e
at
iv
N
eg
Po
s
iti
v
e
C
on
tr
ol
0
Sample
From data shown on Chart1, the reactive light units (RLU) from colony24 and 38 were
much higher than positive control (pUTmini5Tnkmlux), hence they should be carrying lux
operon.
Page 71 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
c.
Even if the new construct does not contain Francisella promoter, the data confirmed
that the fragment carrying lux operon could be cloned into another plasmid and
function properly from lux operon. The mutant plasmid was designated as pKEK1193.
However, our goal was to construct lux operon into the plasmid carrying Francisella
promoter, so pKEK843 containing the Ft promoter was the priority parent plasmid we
needed to work on. Both pUTmini5Tnkmlux providing lux operon and pKEK843
serving as the backbone were digested with restriction enzyme EcoRI and treated
with CIP (Calf Intestinal Alkaline Phosphatase) for the digested pKEK843 to prevent it
from self-ligation. Then these two fragments purified from agarose gel were ligated
and transformed into Ecoli.DH5α using electroporation. The transformants were
selected on LB/Kanamycin (50ug/ml) agar plate at 37°C. About 52 colonies were
patched onto the same type plate and 10 colonies were cultured in LB liquid medium
with 50ug/ml Kanamycin at 37°C for overnight subsequently. 10 liquid cultures were
detected in the Luminometer for bioluminecence from lux operon under the control of
the Ft promoter along with pKEK843 in DH5α for negative control and
pUTmini5Tnkmlux in DH5α for positive control. Colony #7 gave strong positive signal
(data was shown in Chart 2).
Chart 2:
Bioluminecence of pKEK843/lux operon
9000000
8000000
Reactive Light Units
7000000
6000000
5000000
Series1
4000000
3000000
2000000
1000000
y9
y8
y7
y6
y5
y4
y3
y2
y1
y1
0
Co
lo
n
Co
lo
n
Co
lo
n
Co
lo
n
Co
lo
n
Co
lo
n
Co
lo
n
Co
lo
n
Co
lo
n
ro
l
Co
lo
n
Co
nt
e
at
iv
N
eg
Po
s
iti
ve
Co
nt
ro
l
0
Sample
From Chart 2, it was obvious that number of light units from Colony7 were much
higher than Positive Control, and in contrast, other colonies presented similar
signal patterns to Negative Control. Colony7 should be the one which carried lux
operon under the control of Ft promoter.
d. Miniprep DNA was made from colony7 for comparing the size to pKEK843 and
screening.
Figure1: on 1% agarose gel
Page 72 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 size of target construction was about 14.0kb which was about 7kb larger than
parent plasmid pKEK843 (lane2), so colony7 (lane3) was correct, since the Lux
operon is 7kb.
e. The plasmid DNA from colony7 was digested with restriction enzyme XhoI and XcmI
for confirmation. The new construct should be digested into three fragments (about 910kb, 4.5kb and 0.6kb), whereas pKEK843 was supposed to have two fragments
(about 4.2kb and 3.2kb) after being cut with the same enzymes.
Figure2:
Figure3:
From Figure2, colony7 (lane2 and 3) was cut into 3 pieces about the size we
expected, but the control of parent plasmid pKEK843 was not available. Figure3
presents both the mutant (lane2, the same plasmid DNA as lane2 and 3 on figure2)
and parent plasmid (lane3) digested with XhoI and XcmI. The 3 rd band (about 0.6kb)
on lane2 was more visible on electrical file in our lab. The reason was because the
amount of DNA for digestion was not enough and also the agarose gel was run
beyond the usual time so as to separate two fragments on lane3.
Page 73 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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.
Since colony7 was verified by detection of bioluminecence and confirmed by size of
the plasmid as well as digestion with restriction enzymes, it was designated as
pKEK1194.
Data recorded on UTSA TVDC notebook #2, page127-140 for Chart1, Chart2,
Figure1, 2 and 3.
4 Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
pKEK1193 (pWSK30/lux operon) but without the Ft promoter
pKEK1194 (pKEK843/lux operon) with the Ft promoter
7. Quality of performance
Good
8. Percentage completed.
About 32% of scientific work completed.
9. Work plan for upcoming month
i.
ii.
Transform pKEK1194 into U112.
Transform pKEK1194 into LVS.
10. Anticipated travel
Will travel to the UNM TVDC annual meeting in October 2008.
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.
Page 74 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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) Cloning of Listeria monocytogenes (Lm) tularemia vaccine strains. A summary of
vaccine candidates that have been constructed is presented in table #1 below. No new vaccine
candidates were constructed in July.
The work plan proposed for July was not accomplished do to personal holidays and increased
effort on MS 56. The cloning, comparative immunogenicity experiments, and quality control of the
Ft lots will be performed in the following month.
Table 1
Strain
Genetic Background
Antigen Cassette
Status
CRS-100 actAinlB
none
Sequence verified
BH137
ActAN100-Ova
Sequence verified
actAinlB
BH1222
ActAN100-IglC-SL8
Sequence verified
actAinlB
BH2106
ActAN100-KatG-SL8
Sequence verified
actAinlB
BH1228
ActAN100-IglC-SL8
Sequence verified
actAinlBuvrAB
BH1398
ActAN100-KatG-SL8
Sequence verified
actAinlBuvrAB
BH2094
ActAN100-IglC-SL8
Complete
actAinlBuvrABprfAG155S
BH2172
ActAN100-KatG-SL8
Complete
actAinlBuvrABprfAG155S
BH2098
ActAN100-IglC-VacQuad-SL8
Complete
actAinlB
BH2100
ActAN100-IglC-VacQuad-SL8
Complete
actAinlBuvrABprfAG155S
BH2180
ActAN100-IglC-B8R (@ comK)
Complete
actAinlB
BH2182
ActAN100-IglC-B8R (@ comK)
Complete
actAinlBuvrABprfAG155S
BH2184
ActAN100-IglC-B8R
(@
comK)
Complete
actAinlB
ActAN100-KatG-SL8 (@tRNAarg)
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




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)
10. Anticipated travel
Will travel to the UNM TVDC annual meeting in Phoenix AZ on October 6 and 7, 2008
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 75 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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 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 and ICS 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-KatG-SL8 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) To determine whether Lm strains expressing IglC can induce IglC-specific immune
responses, five different strains of mice were vaccinated intravenously with 1106 CFU of
Lm-IglC (∆actA inlB ∆uvrAB prfA* G155S-IglC-SL8; BH2094) and spleens were
removed after seven days. Since IglC-specific immune epitopes have not yet been
identified, a peptide library consisting of 51 15-mer peptides that overlap by 11 amino
acids and span the entire IglC sequence was used as stimulation in ELISpot and
intracellular cytokine staining (ICS) assays. The IglC peptide library was divided into two
separate pools (IglC pool1 and IglC pool2) and also used as individual peptides. Five
strains of mice (Balb/c, C57BL/6, FVB/NJ, C3H/HeJ, and SJL/J) were used in this study
to look for iglC-specific immune responses in animals with different MHC haplotypes (d,
b, q, k, and s2, respectively).
Using the ELISpot assay, after a single vaccination with Lm-IglC, immune
responses to IglC pool2 could be measured in all mouse strains (fig. 1). The IglC pool2specific response was highest in FVBN mice. Each mouse strain also responded to a
pool of LLO peptides that was used as a positive control for the ELISpot assay. LLO is a
secreted virulence determinant from Lm that was been well characterized as an antigen
in Balb/c and C57BL/6 mice. A weak response to IglC pool1 could be measured in
C3H/HeJ mice.
Page 76 of 82
Tularemia Vaccine Development Contract: Technical Report
iglC pool2
400
200
200
0
SJL
C3H
C57BL/6
FVBN
0
400
SJL
600
unstim
LLO pool
C3H
iglC pool1
FVBN
unstim
LLO pool-specific response
600
C57BL/6
800
Balb/c
IFN- SFC per 2e5 splenocytes
IglC responses
Balb/c
IFN- SFC per 2e5 splenocytes
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, 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. Immune responses to IglC after a single immunization with live-attenuated Lm-IglC vaccine
strain BH2094 were measured using ELISpot. IglC pool1 contains peptides 15, 18, 17, 20, 12, 19, 11,
6, 23, 8, 25, 3, 24, 51, 47, 50, 45, 29, 26, 27, 36, 43, 42, 44, 31, 32. IglC pool2 contains peptides 14,
16, 21, 22, 13, 4, 5, 7, 10, 9, 1, 2, 48, 46, 49, 28, 39, 40, 38, 37, 34, 41, 35, 30, 33. Immune responses
against LLO (a strong Lm antigen) were measured as a positive control. Wells without peptides
(unstimulated) were used a negative control NB #2000, pages 11-14.
2) Using multicolor flow cytometry after stimulation with the peptide pools it was possible to
distinguish whether the T cells that were stimulated in each mouse strain were CD4 or
CD8 positive cells. After a single vaccination with Lm-IglC, immune responses to IglC
pool2 were again detectable in all mouse strains using ICS (fig 2). The IglC pool2specific response was highest in FVBN mice, with greater than1% IFN-g+ CD4+ T cells.
In Balb/c and SJL mice, the IglC pool2 responses were lower, at 0.4% and less than
0.1% IFN-g+ CD4+ cells respectively. In C57BL/6 mice, 4% IFN-g+ CD8+ T cells were
measured. Interestingly, Lm-IglC induced similar levels of both CD4+ and CD8+ T cells
specific for IglC in C3H/HeJ mice (0.5%). The LLO pool was used as control and high
levels of CD8+ T cells were seen in Balb/c mice and high levels of CD4+ T cells were
seen in C57BL/6 mice as expected. As seen in the ELISpot assay, all responses to iglC
pool1 were very low (all below 0.15%). These results confirm the ELISpot results and
demonstrate that mice can induce T cell responses against IglC and further demonstrate
that the T cell responses can be CD4, CD8 or both depending on the haplotype of the
mice used.
iglC pool2
0
-2
C3H/HeJ
SJL
-0.5
C3H/HeJ
0.0
2
FVB/N
0.5
C57BL/6
1.0
CD8
4
Balb/c
% IFN- T cells
1.5
FVB/N
SJL
C3H/HeJ
FVB/N
0.00
CD4
CD8
C57BL/6
0.05
2.0
Balb/c
% IFN- T cells
0.10
C57BL/6
6
CD4
CD8
Balb/c
% IFN- T cells
CD4
0.15
-0.05
LLO pool
2.5
Figure 2. Immune responses to IglC pools after a single immunization with live attenuated Lm-IglC vaccine
Page 77 of 82
SJL
iglC pool1
0.20
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
strain BH2094 were measured using ICS NB #2000, pages 11-14. Note that the Y axis scales are different
between the panels in this figure.
Using all of the individual peptides in the IglC peptide library in an ELISpot assay,
the peptides that contain the immunogenic regions of IglC could be identified for each
mouse strain (fig 3). In Balb/C mice peptide 33 stimulated a low response and while
peptide 34 stimulated a high response suggesting that the immune epitope(s) are
contained within the amino acids covered by peptides 33 and 34. In C57BL/6 mice the
immune epitope(s) are contained within the amino acids covered by peptides 34-35.
With FVB/N mice there were two immunogenic regions covered by peptide 24 and
peptides 37-38. C3H/HeJ mice responded to two independent regions spanning peptides
23-24 and peptide 33-36. In SJL mice, responses were found to individual peptides 38,
46, and 50. Interestingly, each mouse strain responded to peptides in the region
spanning peptide 33-38, suggesting that this region of IglC is highly immunogenic.
BH2094 (actAinlBuvrAB prfA*-iglC) in Balb/c mice
180
160
140
Peptide #33, 34
QEYKTDEAWGIMIDL
TDEAWGIMIDLSNLE
IFNg SFC/2e5 cells
120
100
80
60
40
20
51
50
49
48
47
46
45
44
43
42
41
40
39
38
BH2094 (actAinlBuvrAB prfA*-iglC) in C57BL/6 mice
60
50
Peptide #34, 35
TDEAWGIMIDLSNLE
WGIMIDLSNLELYPI
40
30
20
10
51
50
49
48
47
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
0
1
IFNg SFC/2e5 cells
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
9
12
11
8
10
7
6
5
4
3
2
1
0
Page 78 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
BH2094 (actAinlBuvrAB prfA*-iglC) in FVB/N mice
800
Peptide #37, 38
NLELYPISAKAFSIS
YPISAKAFSISIEPT
700
600
IFNg SFC/2e5 cells
500
400
Peptide #24
VLIKSNVRTKIEEKV
300
200
100
49
50
51
51
48
50
47
49
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
9
11
8
10
7
6
5
4
3
2
1
0
BH2094 (actAinlBuvrAB prfA*-iglC) in C3H mice
90
Peptide #33, 34, 35, 36
QEYKTDEAWGIMIDL
TDEAWGIMIDLSNLE
WGIMIDLSNLELYPI
IDLSNLELYPISAKA
80
70
IFNg SFC/2e5 cells
60
50
Peptide #23,24
ITLGVLIKSNVRTKI
VLIKSNVRTKIEEKV
40
30
20
10
48
47
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
BH2094 (actAinlBuvrAB prfA*-iglC) in SJL mice
120
Peptide #37, 38
NLELYPISAKAFSIS
YPISAKAFSISIEPT
100
IFNg SFC/2e5 cells
80
60
Peptide #46
DGLTTSQGSLPVCCA
40
Peptide #50
STDKGVAKIGYIAAA
20
51
50
49
48
47
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Figure 3. Epitope mapping using the ELISpot assay. Splenocytes from each mouse strain were
stimulated with individual peptides from the overlapping peptide library to determine the
immunogenic region of the protein NB #2000, pages 11-14.
By then using individual 15-mer peptides in an ICS assay, we were able to
confirm that these peptides could stimulate an IglC-specific response. We determined
that the response to peptide 34 was mediated by CD4+ T cells in Balb/c mice (fig. 4). In
C57BL/6 mice, we determined that the immune response to peptides 34 and 35 is
IM08-059
mediated
by CD8+ T cells. In FVB/N mice we determined that the strong responses to
peptides 37 and 38 were mediated by CD4+ T cells. As controls, the immunodominant
peptides from LLO were used in Balb/c and C57BL/6 mice and the responses to the
immunodominant peptides was similar in magnitude to the LLO pool. The SL8 peptide
was also used in C57BL/6 mice as a positive control and the responses to this
immunodominant ovalbumin epitope were 25% after a single vaccination.
These ICS and ELISpot results demonstrate that IglC induces a cellular immune
response when secreted from a live-attenuated Lm vaccine strain. We will also
determine whether mice vaccinated with LVS induce an immune response to the same
peptides as those induced by Lm and we will compare the magnitude of the immune
responses. Since CD8+ T cell epitopes are typically between 8 and 10 amino acids in
length, 9-mer peptides that overlap by 8 amino acids and span both peptides 34 and 35
Page 79 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
will be used to better define the C57BL/6 iglC CD8+ epitope. Similar analysis will be
done to define the CD8+ epitope in C3H/HeJ mice.
Balb/c
% IFN- T cells
2
1
Figure 4. Immune responses against individual 15aa IglC peptides were detected by ICS on
splenocytes from mice vaccinated with Lm-IglC NB #2000, pages 11-14..
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
UNM and Anza area negotiating the MTA language to allow sharing of information and
reagents from UCLA.
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
10%
9. Work plan for upcoming month




Anza will order 9mer peptides to identify optimal CD8 T cell epitopes
Anza will vaccinate mice with LVS and Lm to compare the IglC Reponses
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
11. Will travel to the UNM TVDC annual meeting in Phoenix AZ on October 6 and 7,
Upcoming Contract Authorization (COA) for subcontractors
None
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
Page 80 of 82
LLO pool
38
37
unstim
LLO pool
0
LLO 190
LLO pool
LLO 91
unstim
34
0
CD8
SL8
1
CD4
CD8
35
2
CD4
34
3
3
30
28
26
24
22
20
10
8
6
4
2
1.0
0.8
0.6
0.4
0.2
0.0
unstim
CD8
% IFN- T cells
% IFN- T cells
CD4
4
FVBN
C57BL/6
5
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble
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.
We have previously constructed 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 iglC-SL8 cassette
has yet to be performed. Once the expression has been confirmed, biodistribution,
virulence and immunogenicity studies (ICS and ELIspot) will be performed.
No new lab work was performed in July 2008 on MS 57due to the focus on MS 56.
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
Page 81 of 82
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2008 to 7/31/2008
Due Date: 8/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu,
Amanda Dubois, Bob Sherwood, Julie Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman,
Michelle Valderas, Karl Klose, Bernard Arulanandam, Stephen Johnston, Kathryn Sykes,
Mitch Magee, Justin Skoble


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
Will travel to the UNM TVDC annual meeting in Phoenix AZ on October 6 and 7
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 82 of 82