Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, 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, 11, 12/13(UNM/LBERI), 19, 21, 26, 27, 28, 35(ASU/UNM), 41,
42, 44, 46, 49, 50, 52
Completed milestones: 1, 25, 32, 33, 34, 16, 39, 40, 43, 48, 51
Inactive milestones: 6, 7, 8, 9, 10, 14, 15, 17, 18, 20, 22, 23, 24, 29, 30, 36, 37, 38, 45,
47, 53, 54
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. LBERI has 32 staff and scientists vaccinated as of 1/9/2008.
b. First group of 7 UNM participants are being scheduled to receive the LVS vaccination on
3/18/2008; SIP informed consent is complete and prehealth screenings are in progress
c. USAMRIID will accept up to 10 vaccinees for the 3/18 vaccination date. Depending on
medical clearance, up to 3 LBERI staff and scientists may join the 3/18 group.
d. UNM and USAMRIID are actively using the LVS vaccine web database to track Risk
Assessment form submission and acceptance, Informed Consent submission and
acceptance, Health screening appointments, planned dates for receipt of LVS
vaccinations, dates of medical clearance, and travel arrangements
4. Significant decisions made or pending
a. UNM and LBERI are using their biobubbles as additional physical protective equipment
b. Dr. Lyons has received UNM IRB approval pending minor revisions to the consent form
and protocol, to allow blood draws on the vaccinated LBERI and UNM scientists after
their LVS vaccinations. The LBERI and UNM scientists and staff will be offered the
opportunity to volunteer to donate bloods for the development of immunoassays,
approximately 2 months after receiving the LVS vaccination.
c. UNM (7) and LBERI (32) are offering the LVS vaccinations up to 7 more scientists to total
46; USAMRIID will continue to provide the LVS vaccinations over the next 3-4 months.
d. The CRDA with USAMRIID is valid for 2 years, ending June 2009.
5. Problems or concerns and strategies to address
a. Within less than 1 month, UNM may have access to the blood of UNM and LBERI
scientists who have been vaccinated with LVS at USAMRIID. UNM’s IRB has been
approved pending minor language modifications in the consent form and protocol.
Page 1 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
6. Deliverables completed
32 LBERI scientists and staff have received the LVS vaccination between 9/11/07 and 1/9/08.
7. Quality of performance
Excellent
8. Percentage completed
50%
9. Work plan for the next month
a. Complete health screenings for the next group of 7 UNM participants.
b. Make travel arrangements for medically eligible participants to enter LVS Vaccination
program at USAMRIID on 3/18/07 – first group of UNM participants. Participants will be
at USAMRIID for 2 days following the vaccinations.
c. Maintain excellent communications with UNM EOHS, LBERI and USAMRIID
10. Anticipated travel
LVS vaccination participants will be traveling to USAMRIID on 3/17/08..
11. Upcoming Contract Authorization (COA) for subcontractors
UNM received a signed COA letter for COA 15 on 9/11/07.
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
No Francisella tularensis (LVS or SCHU S4) bioaerosol technique optimization experiments were
conducted in January 2008.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Data continue to indicate that the Collison and Aeromist nebulizers are the optimal choices for
SCHU S4 bioaerosol generation. Based solely on cost effectiveness and ease of use, we are
strongly considering the Aeromist nebulizer for all future F. tularensis bioaerosols. The primary
question now is what culture approach should be used for NHP challenges. This remains to be
discussed.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
96%
9. Work plan for upcoming month
Continue Milestone completion report.
10. Anticipated travel
ASM Biodefense Meeting Feb 24-27 in Baltimore, MD. Abstract entitled, “Optimization of
Bioaerosol Generation Techniques for Francisella tularensis” accepted for poster presentation.
LBERI will use non-TVDC funds for this travel per Dr. Sherwood. Drs Brasel and Sherwood will
email their poster to Barbara Griffith by 2/12 to UNM for review by NIAID as well.
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Page 2 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
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:
. LBERI has been performing experiments to demonstrate the virulence of SCHU S4 delivered in
bioaerosols to mice prior to proceeding with bioaerosol delivery of SCHU S4 to NHP. As reported
in December 2007, LBERI had challenged naïve mice with frozen stocks of SCHU S4 (~1500
dose) and Chamberlains broth-grown LVS (~10,000 dose). All the SCHU S4 challenged mice
had died by day 5 while approximately 55% of the LVS challenged mice had died by day 11.
BCGA-grown SCHU S4 had not been performed to show virulence in mice because it was
hypothesized that growth methods would have little or no impact on virulence. Based on these
mouse data, SCHU S4 challenges of NHP were performed in December 2007. Animals were
challenged with SCHU S4 grown on solid BCGA as LBERI had data showing better spray factors
and viability for the SCHU S4 grown on BCGA relative to growth in Chamberlains broth. Also,
LBERI had some difficulties culturing SCHU S4 in Chamberlains broth. To LBERI’s surprise, the
NHP did not die until at least day 13 after being challenged with an approximate 2500-4000 CFU
dose SCHU S4 grown on BCGA. Additional mouse bioaerosol challenges against SCHU S4
were conducted on 1FEB08 to compare the relative virulence in mice of SCHU S4 grown in
Chamberlains broth versus growth on solid BCGA. LBERI chose a dose range of 10 and 1000
SCHU S4 for the Feb 1,2008 challenge. Rick Lyons cautioned LBERI that mice are so sensitive
to SCHU S4 that detecting changes in virulence based on SCHU S4 growth conditions may be
difficult. UNM requested the inclusion of available data from these mouse challenges in this, the
January data summary report:

44 naïve BALB/c mice were challenged with SCHU S4
a. Groups 1 and 2 (n=11 mice per group) were challenged with SCHU S4 harvested
from a 72h BCGA culture
i.
The target deposited dose for Group 1 was 10 CFU
ii.
The target deposited dose for Group 2 was 1000 CFU
b. Groups 3 and 4 (n=11 mice per group) were challenged with SCHU S4 harvested
from a 48h Chamberlain’s broth culture
i.
The target deposited dose for Group 3 was 10 CFU
ii.
The target deposited dose for Group 4 was 1000 CFU
c. One mouse from each group was sacrificed shortly (approximately 10 minutes)
following aerosol challenge. Lungs were removed from these animals (n=4),
processed and cultured onto BCGA to estimate deposition per group.
d. Bioaerosol data are shown in Table 1 below. Electronic data have been filed in
the following folder: \\Saturn\absl3\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY07-083 and -089 (TUL04)\1FEB08 Mouse exposure
Page 3 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Table 1
Group
Challenge Material
Target
Deposited
Dose
Aerosol
Concentration
Calculated
inhaled
volume
Delivered
dose
Deposited
dosea
CFU
CFU/L
L
CFU
CFU
1
72h BCGA SCHU
S4
10
2.85E+03
0.23
661
46
2
72h BCGA SCHU
S4
1000
9.87E+04
0.23
22712
1590
3
48h Chamberlains
10
1.90E+03
0.23
437
31
4
48h Chamberlains
1000
1.08E+05
0.23
24924
1745
aAssumes
7% deposition at a 1-3 m diameter particle size
e.
f.
Data demonstrate that mice were challenged as planned. Groups 1-4 were
delivered 661, 22,712, 437, and 24,924 CFU, respectively. Based on previous
studies conducted at LBERI for all particles in the size range of 1-3µm, it can be
assumed that the deposited fraction was 7%. Using this as a conversion factor,
the calculated deposited doses for Groups 1-4 were 46, 1,590, 31 and 1,745
CFU, respectively; these values were very close to the target deposited doses.
Table 2 below presents data from mouse lung cultures processed immediately
following bioaerosol challenge. Electronic data have been filed in the following
folder: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07-083 and -089 (TUL-04)\1FEB08
Mouse exposure:
Table 2
Group
Challenge Material
Target
Deposited
Dose
Plate Counts
Dilution
Factor
CFU
1
2
3
Mean
Tissue
Weight
Total
Deposition
Grams
CFU
1
72h BCGA SCHU
S4
10
0
0
0
0.0
10
0.16
0
2
72h BCGA SCHU
S4
1000
2
2
2
2.0
10
0.16
43
3
48h Chamberlains
10
0
0
0
0.0
10
0.16
0
4
48h Chamberlains
1000
0
3
7
3.3
10
0.16
72
g.
Data demonstrate that depositions do not correspond to the calculated
depositions presented in the first table. This was likely due to the overwhelming
contamination present on all of the culture plates. It should be noted that
contamination was not due to the media, but rather the actual necropsy
procedure. Measures are currently being taken to remedy contamination issues
for mouse lung necropsies.
Page 4 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
h.
Groups 2 and 4 mice are beginning to show signs of morbidity and mortality. Inlife observations are continuing as planned and will be reported in the February
data report due on 3/7/08.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
SCHU S4 challenge dose preparation (i.e., choice of growth methods [frozen versus BCGA
versus Chamberlains]) and delivery approaches (i.e., Aeromist versus Collison) have yet to be
decided upon for future NHP exposures. These issues will be addressed following the mouse
challenge experiments currently in progress.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
22%
9. Work plan for upcoming month
a. Decide on SCHU S4 exposure approaches for next set of NHPs. Focus will primarily be
on the culture method used for the SCHU S4 challenge material (i.e., fresh
Chamberlain’s versus fresh BCGA versus frozen stock).
b. LVS-vaccinated NHPs will continue to be bled as a source of cells for Milestone 12/13;
however, no work is anticipated on these NHPs until they are challenged with aerosolized
SCHU S4.
10. Anticipated travel
Bob Sherwood, Ross LeClaire, Julie Wilder and Chuck Hobbs will be attending the 3/11 to
3/13/08 NIAID/DVC/UNM/FDA meeting in Washington DC
11. Upcoming Contract Authorization (COA) for subcontractors
None
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
Characterization of the Fischer 344 rat model is currently being done under Milestone 11, as
the efforts on the Fischer 344 rat model are shifting toward GLP model efficacy
4. Significant decisions made or pending
We have decided to use a standard dose of 106 SCHU, with an expected lung deposition of
approximately 2.5 x105, for all future rat challenge experiments. We expect all naïve rats to
die from this dose but all vaccinated rats to survive.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
Page 5 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
c.
Rat model completed
7. Quality of performance
Good
8. Percentage completed
69%
9. Work plan for upcoming month
None
10. Anticipated travel
Terry Wu, Rick Lyons and Barbara Griffith will attend the NIAID/DVC/UNM/FDA meeting
from 3/11 to 3/13/08.
11. Upcoming Contract Authorization (COA) for subcontractors
Terry Wu and Amanda DuBois will attend the Tularemia Workshop in New York from 3/30 to
4/1/2008 (COA#18 has been assigned)
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 Ptrans1 (Notebook 112, page 41,54-65)
i. The purpose of this experiment was to determine whether serum from LVS
vaccinated rats is sufficient to protect naïve Fischer 344 rats from a lethal
respiratory challenge with SCHU S4
ii. 4 groups of rats were included in this study: naïve, LVS vaccinated, naïve +
normal serum, and naïve + vaccinated serum
iii. Groups of naïve rats were either vaccinated s.c. with 5 x 107 LVS or left
unvaccinated as controls
iv. 36 days after LVS vaccination, sera were collected from both naïve and
vaccinated rats and 3 ml were transferred i.p. into each of 6 naïve rats
v. 1 day after serum transfer, the rats were challenged i.t. with ≤ 72 SCHU S4
(actual lung deposition). The challenge dose was intentionally low to give the
vaccinated serum a chance to protect. 72 CFU of SCHU S4 is still a lethal
dose for naïve rats
vi. As shown in Table 1, vaccinated serum was sufficient to protect naïve rats
from a respiratory SCHU S4 challenge. Surprisingly, naïve serum also
protected 2 of 6 rats. We cannot explain this result yet because all of the rats
were infected similarly with the same inoculum
Table 1. Passive immunization protected naïve rats from respiratory SCHU S4
Groups
Vaccination status
Treatment2
1
2
3
s.c. LVS vaccinated1
Naïve
Naïve
None
None
Naïve rat serum
Survival ratio
(No. live/total)3
6/6
0/6
2/6
Page 6 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
4
Naive
Vac. rat serum
6/6
1
Rats vaccinated s.c. with 5 x 107 LVS
2
Sera collected 36 d after vaccination and 3 ml transferred i.p.
3
Rats challenged i.t. with ≤ 72 SCHU S4 1 d after serum transfer
b.
c.
Experiment Ftc65 (Notebook 112, page 67)
i. The purpose of this experiment is to determine the role of CD4 and CD8 T
cells in LVS-induced protection against SCHU S4
ii. Taconic is currently producing the anti-CD4 (W3/25), anti-CD8 (OX-8) and
isotype (TS2/18.1.1) ascites fluid in mice and is expected to be finished by
the middle of February 2008
iii. We have vaccinated Fischer 344 rats s.c. with 5 x 107 LVS in anticipation of
the ascites fluids. The vaccinated rats will be treated with the ascites fluids
to deplete either CD4 and/or CD8 T cells and then challenged with SCHU S4
i.t.
Experiment Ftc63 (Notebook 104, page 129-133)
i. The purpose of this experiment was to determine whether the LVS vaccine
can induce long term protection against SCHU S4 in rats.
ii. We showed previously that in mice LVS-induced protection disappeared 2
months after vaccination. In contrast, 70-80% of vaccinated rats survived i.t.
challenge with ~ 106 SCHU S4 49 and 60 days after vaccination and 50%
survive 3 x 107 SCHU S4
iii. To determine whether vaccinated rats would remain protected against SCHU
S4 for longer periods of time, LVS vaccinated rats were challenged 112 and
185 days after vaccination
iv. As shown in Table 2, 50% of the vaccinated rats from both groups survived
i.t. challenge with 7.2 x 106 SCHU S4. This survival rate is lower than those
from the earlier experiments, suggesting that the protection may have
reduced slightly. However, we cannot make such a conclusion with certainty
because we did not include a group of rats that were vaccinated more
recently and challenged at the same time for comparison
Table 2. Resistance of vaccinated rats to i.t. SCHU challenge 4
and 6 mo after LVS vaccination
Vaccination status
Time of i.t. SCHU S4
Survival ratio (No.
challenge
alive/total)
(days post vaccination)1
Naïve
1/6
LVS vaccinated
112
2/4
LVS vaccinated
185
3/6
1
Lung deposition 7.2 x 106/rat
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
3%
Page 7 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
9. Work plan for upcoming month
a. Repeat passive transfer experiment in rats
b. Determine whether vaccinated rat serum sufficient to protect naïve mice from
respiratory SCHU S4 challenge
c. Titrate amount of serum required to confer protection
d. Deplete vaccinated rats of CD4 and CD8 T cells as soon as Taconic provides ascites
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13-LBERI
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.
Update on background responses in the IFN ELISPOT and proliferation assays
1. Occasionally we observe high background responses in unstimulated wells in
both the IFNγ ELISPOT and proliferation assays performed with PBMC from nonLVS vaccinated or LVS vaccinated NHP
2. Is high background response associated with RBC content?
i. Figure 1 shows that in the IFN ELISPOT assay, it is not associated with
small changes in RBC content when comparing the number of spots in
the unstimulated wells (media); only non-LVS vaccinated NHPs are
shown in this Figure
ii. Historical data does indicate, however, that high RBC content can cause
background spots in the unstimulated, and all stimulated, wells (see also
Figure 2 below), thus, it is our goal to re-lyse any PBMC preps that have
higher than 2% RBC content
3. Is high background response reproducible? Is it always associated with
individual primates?
i. Figure 2 shows that in the IFN ELISPOT assay, high background is not
associated with any individual primate (each primate shows both low and
high background responses) nor is it reproducible (no primate shows
consistently high or low responses)
ii. Figure 2 does confirm that very high RBC content does cause high
background responses in the IFN ELISPOT assay (see A00659, TUL
27 and A00937, TUL 12)
iii. Figure 2 also shows that RBC content does not completely predict the
background response (compare A00659 and A00868 in TUL 19; each
have 1.3% RBC content but different backgrounds; and A00868 in TUL
17 and TUL 27, each have 1% RBC content but different backgrounds)
iv. Figure 3 shows that high background in the proliferation assay is not
related to RBC content, nor is it associated with any particular primate
Page 8 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
v. Interestingly, comparing the background response of A00908 in TUL 14
shows that a high background in the proliferation assay (Figure 3) does
not necessarily mean that a high background will result in the IFN assay
(Figure 2); similarly, this comparison can be made with A00868 in TUL
27 which shows a high background in the IFN ELISPOT assay (Figure 2),
but not in the proliferation assay (Figure 3)
4. Does the high background prohibit the observation of specific responses?
i. Figure 4 shows a representative primate (A00659) which has shown
variable background responses in the proliferation assay (see Figure 3)
in comparison to its response to LVS stimulation on each of those days;
the data indicate that generally a specific response to LVS can be
observed, although that response may be waning as time goes on and
we are further removed from the LVS vaccination date; it is unclear why
sub-optimal responses were observed on days 117 and 237
ii. Figure 5 shows a representative primate (A00659) which has shown
variable background responses in the IFN ELISPOT assay (see Figure
2) in comparison to its response to LVS stimulation on each of those
days; the data indicate that if the RBC content is too high (TUL 27),
specific responses to LVS can be obscured; it is unclear why sub-optimal
responses were observed on day 288 (TUL 19) for LVS hk stimulation
0.5%
450
400
2%
350
Media
LVS hk Hi
LVS ff Hi
0%
300
0%
250
200
0.8%
150
6.3 %
0.6%
2.2%
100
A05477
A04367
A04339
A04274
A04168
A03033
A03016
0
A04344
0.3%
50
A02386
IFNgamma Spots (Mean +/- SEM)
IFN Secretion by Individual Non-LVS Vaccinated NHPs as Compared to Their RBC Content
Figure 1: PBMCs from non-LVS vaccinated NHPs were plated at 1.33 x 106 cells/ml and stimulated with
either HK or FF LVS (1 x 105 cells/ml) or left unstimulated. The percentage of RBC content in the
individual PBMC preparations is shown above each set of bars.
Page 9 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
IFNg Spots (Mean +/- SEM)
Background IFN Secretion by Individual LVS-Vaccinated NHPs as Compared to Their RBC
Content
200
180
160
9.7%
TUL12
TUL14
TUL15
TUL16
TUL17
TUL18
TUL19
TUL27
7.4 %
140
120
100
80
60
1.0%
1.3%
40
20
0
0.4 %
2.7%
3.5%
A00659
A00868
0.4 %
A00896
0.8 %
1.4 %
A00908
A00937
1.3%
Figure 2: PBMCs from LVS vaccinated NHPs were plated at 1.33 x 106 cells/ml and left unstimulated.
The percentage of RBC content in the individual PBMC preparations is shown associated with each data
point. Individual experiments and NHPs are shown. Days post-LVS vaccination range from 140 (TUL 12)
to 414 (TUL 27).
Small RLU (Mean +/- SEM)
Background Proliferation by Individual Non-LVS Vaccinated NHPs as Compared to Their RBC
Content
700000
500000
TUL11
TUL12
TUL14
400000
TUL15
1.4%
600000
300000
200000
9.7%
TUL16
0.8%
2.8%
TUL17
2.4%
100000
TUL18
TUL19
TUL27
0
A00659
A00868
A00896
A00902
A00908
A00937
Figure 3: PBMCs from LVS vaccinated NHPs were plated at 1 x 106 cells/ml and left unstimulated. The
percentage of RBC content in the individual PBMC preparations is shown associated with select data
points. Individual experiments and NHPs are shown. Days post-LVS vaccination range from 117 (TUL
11) to 414 (TUL 27).
Page 10 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Proliferation by an Individual LVS-Vaccinated NHP over Time
RLU Small (Mean +/- SEM)
1400000
TUL 9
Media
1200000
TUL 19
1000000
TUL 15
LVS hk Hi
LVS hk Mid
LVS ff Hi
800000
LVS ff Mid
600000
TUL 17
TUL 27
400000
TUL 11
200000
0
Day 21
Day 28
Day 117
Day 203
Day 237 Day 288
Day 414
Figure 4: PBMCs from A00659, an NHP vaccinated with LVS via the SC route were plated at 1 x 106
cells/ml and either left unstimulated (media) or stimulated with various doses of heat-killed or formalinfixed LVS (Hi = 1 x 105/ml; Mid = 0.25 x 105/ml). The experiment name is shown associated with select
data points. Mid doses of LVS were not used in TUL 11, TUL 15, TUL 17 and TUL 19.
Page 11 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
IFNgamma Spots (Mean +/- SEM)
IFN Secretion by an Individual LVS-Vaccinated NHP over Time
300
250
200
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
TUL 27
150
TUL 17
TUL 19
100
50
0
Day 237
Day 288
Day 414
Figure 5: PBMCs from A00659, an NHP vaccinated with LVS via the SC route were plated at 1.33 x 106
cells/ml and either left unstimulated (media) or stimulated with various doses of heat-killed or formalinfixed LVS (Hi = 1 x 105/ml; Mid = 0.25 x 105/ml). The experiment name is shown associated with select
data points. Mid doses of LVS were not used in TUL 17 and TUL 19.
5. Data interpretation
i. As regards the IFN ELISPOT assay, small variations in RBC content do
not affect background levels (Figure 1) but a large RBC content in PBMC
preps is associated with high background (Figure 2)
ii. Background levels in either the IFN ELISPOT or proliferation assays are
not primate specific
iii. High background levels resulting from PBMCs in one assay (IFN
ELISPOT) do not necessarily predict that high background levels will be
observed in the other assay (proliferation); compare Figures 2 and 3
iv. Occasional high backgrounds in the proliferation assay do not normally
preclude us from observing even higher LVS-specific responses (Figure
4)
v. Very high background responses in the IFN ELISPOT assay can
preclude us from observing LVS-specific responses (Figure 5)
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay020608.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 020608.svd, TVDC binder (TUL 9, 11 and
12); TVDC 1 bound notebook (8628): TUL 14 (pps. 30 – 40, 49), TUL 15 (pps. 41 – 48, 50), TUL 16 (pps.
51- 62), TUL 17 (pps. 63 – 70), TUL 18 (pps. 81 – 91), and TUL 19 (pps. 99 – 108) and TVDC 2 bound
notebook (8935): TUL 27 (pps. 3 -9).
b. Update on the comparison of IFN secretion by NHPs vaccinated with LVS via the ID vs.
SC routes
1. We have previously noted that NHPs vaccinated with LVS via the SC route seem
to secrete more IFN than those vaccinated with LVS via the ID route (see
Figure 6 for historical data)
Page 12 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
2. We would like to confirm this by testing ID and SC vaccinated animals on the
same day
ii.
Figure 7 shows the results; although the ID-LVS-vaccinated NHP
(A00937) did secrete less IFN than the other two NHPs (both SC-vaccinated),
the data from A00659 is uninterpretable due to the high RBC content and high
background values and A00868 did not have a good response to either heatkilled or formalin-fixed LVS on this day
iii.
We will repeat this experiment in the coming week
300
Media
LVS hk Hi
LVS ff Hi
SC
250
200
150
ID
100
Day 195
Day 203
Day 237
Day 238
Day 288
Day 195
Day 203
Day 237
Day 238
Day 288
Day 195
Day 203
Day 237
Day 238
Day 288
Day 195
Day 203
Day 237
Day 238
Day 288
A00868,
A00868,
A00868,
A00868,
A00868,
A00896,
A00896,
A00896,
A00896,
A00896,
A00908,
A00908,
A00908,
A00908,
A00908,
A00937,
A00937,
A00937,
A00937,
A00937,
0
Day 195
Day 203
Day 237
Day 238
Day 288
50
A00659,
A00659,
A00659,
A00659,
A00659,
IFNgamma Spots (Mean +/- SEM)
IFN Secretion by NHPs Vaccinated with LVS via the SC and ID Routes
Figure 6: Historical data- PBMCs from LVS vaccinated NHPs were plated at 1.33 x 106/ml and stimulated
with either HK or FF LVS at 1 x 105 cells/ml. Where no bars can be seen associated with particular days,
these NHPs were not tested on those days (i.e. A00659 was not tested on Days 195, 203 or 276).
Page 13 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
IFN Secretion by NHPs Vaccinated with LVS via the SC and ID Routes and Tested on a Single Day
SC, 9.7% RBCs
200
1
1.33
175
150
SC, 1.0% RBCs
125
100
75
50
ID, 2.4% RBCs
A00937, LVS ff Mid
A00937, LVS ff Hi
A00937, LVS hk Mid
A00937, LVS hk Hi
A00937, Media
A00868, LVS ff Mid
A00868, LVS ff Hi
A00868, LVS hk Mid
A00868, LVS hk Hi
A00868, Media
A00659, LVS ff Mid
A00659, LVS ff Hi
A00659, Media
0
A00659, LVS hk Mid
25
A00659, LVS hk Hi
Cell Mean for IFNg Spots
225
Figure 7: PBMCs from LVS vaccinated NHPs were plated at 1.33 x 106/ml or 1 x 106/ml and stimulated
with either HK or FF LVS at 1 x 105 cells/ml (Hi) or 0.25 x 105/ml (Mid). A00937 was not tested at 1.33 x
106/ml and A00659 was not tested at 1 x 106/ml. Route of vaccination and RBC content are shown
associated with each primate.
3. Data Interpretation
i. Although the ID-vaccinated NHP (A00937) did not respond to LVS by IFN
secretion, as expected, there were problems when comparing it to the two SC
vaccinated NHPs due to high RBC content (A00659) or poor overall response
to LVS (A00868); thus this data cannot be interpreted.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay020608.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 020608.svd; TVDC 1 bound notebook
(8628): TUL 14 (pps. 30 – 40, 49), TUL 15 (pps. 41 – 48, 50), TUL 16 (pps. 51- 62), TUL 18 (pps. 81 –
91), and TUL 19 (pps. 99 – 108) and TVDC 2 bound notebook (8935): TUL 27 (pps. 3 -9).
c.
Update on the effect of the Cerus freeze/thaw protocol on IFN secretion as measured by
the ELISPOT assay
1. We need to determine what effect the Cerus freeze/thaw protocol has on IFN
measurements
2. Figure 8 compares one LVS-vaccinated NHP (A00896) when plated fresh and
after 3 aliquots were frozen and thawed; as this NHP is an ID-vaccinated NHP, it
was not expected that it would make much IFN; it is unclear why 1 of the 3
aliquots produced more IFN upon LVS ff stimulation
Page 14 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
3. Figure 9 compares one SC LVS-vaccinated NHP (A00868) when plated fresh
and after a single aliquot was frozen and thawed
160
Media
LVS hk Hi
LVS ff Hi
140
120
100
80
60
Frozen, Aliquot 3
NT
Frozen, Aliquot 2
NT
Frozen, Aliquot 1
0
Fresh, Aliquot 3
20
Fresh, Aliquot 2
40
Fresh, Aliquot 1
IFNgamma Spots (Mean +/- SEM)
The Effect of the Cerus Freeze/Thaw Protocol on IFN Secretion by one NHP Vaccinated with LVS
via the ID Route
Figure 8: PBMCs from one LVS vaccinated NHP (A00896) were plated before (Fresh) and after (Frozen)
undergoing the freeze/thaw process (Cerus protocol). All cells were plated at 1.33 x 106/ml and
stimulated with either HK or FF LVS at 1 x 105 cells/ml (Hi). Fresh cells were tested and then divided into
3 aliquots which were frozen and thawed on the same day 8 weeks later.
Page 15 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
IFNgamma Spots (Mean +/- SEM)
The Effect of the Cerus Freeze/Thaw Protocol on IFN Secretion by one NHP Vaccinated with LVS
via the SC Route
90
Media
LVS hk Hi
LVS ff Hi
80
70
60
50
40
30
20
10
0
Fresh
Frozen
Figure 9: PBMCs from one LVS vaccinated NHP (A00868) were plated before (Fresh) and after (Frozen)
undergoing the freeze/thaw process (Cerus protocol). All cells were plated at 1.33 x 106/ml and
stimulated with either HK or FF LVS at 1 x 105 cells/ml (Hi). Fresh cells were tested and then a single
aliquot containing all the remaining cells was frozen and thawed 8 weeks later.
4. Data Interpretation
i. It appears as though IFN secretion is relatively well-spared by the Cerus freezethaw procedure; although, we need to repeat this experiment to confirm these
findings
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay020608.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 020608.svd; TVDC 1 bound notebook
(8628): TUL 16 (pps. 51- 62) and TUL 17 (pps. 64 – 70
d. Update on IgG anti-LVS ELISA
1. We screened the plasma of several non-LVS vaccinated NHPs in order to insure
that they did not have antibodies to LVS before we exposed two of them to
aerosolized SCHU S4; the data was presented last month (01/08) but is regraphed here on a log scale (Figure 10)
Page 16 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
10000
1000
100
A05477
A04344
A04339
A04168
A03033
A03016
A02386
A00937
A00908
A00902
1
A00896
10
A00868
Cell Mean for IgG anti-LVS Titer
IgG Anti-LVS in Non-LVS Vaccinated NHPs
Figure 10: Plasma from non-LVS vaccinated NHPs was tested for heat-killed LVS reactivity by ELISA.
Titers were determined as the highest dilution tested which had a greater OD 405 than the background
value. Dilutions tested were 1/200, 1/1000, 1/5000, 1/25000 and 1/125,000.
2. Data Interpretation
i. Most of the non-LVS vaccinated NHPs had titers of 200 (the lowest dilution
tested); i.e. they had no reactivity above background
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 020608.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 020608.svd; TVDC 1 bound notebook: TUL
19 (pps. 99 – 108); TUL 21 (pps. 135 – 140) and TUL 22 (pps. 141 – 145); and TVDC binder 1(TUL 8 and
TUL 9).
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
94% of scientific work has been completed
9. Work plan for upcoming month
1. Continue to test PBMCs from ID and SC vaccinated NHPs on the same day to determine if
there are actual differences in IFNγ secretion using both HK and FF LVS as antigens
Page 17 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
2. Prepare poster for the ASM Biodefense 08 meeting in Baltimore, focusing on immunoassay
results obtained from ID and SC vaccination with LVS in NHPs
10. Anticipated travel
Dr. Wilder will attend the ASM Biodefense 08 Meeting in Baltimore, 2/24 – 27/08; however, no travel
funds are requested from the TVDC contract per Bob Sherwood. Dr. Wilder will email her poster to
Barbara Griffith by 2/12 for review by UNM and NIAID as well.
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
2. Date started: 7/15/06 (MS12) and 12/06 (MS13)
3. Date completed: Pending
4. Work performed and progress including data and preliminary conclusions
No new work done
5. Significant decisions made or pending
None
6. Problems or concerns and strategies to address
None
7. 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 
8. Quality of performance
Good
9. Percentage completed
56%
10. Work plan for upcoming month
a. Determine whether boosting LVS vaccinated mice with live or heat killed LVS would
increase the frequency of Ft-specific IFN-producing cells
b. Determine when to harvest splenocytes after vaccination and boost for the maximum
IFN response
c. Determine whether boosting with SCHU S4 would be better than with LVS because it
persists longer in mice.
11. Anticipated travel
None
12. 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
Page 18 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
No new work performed
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
9%
9. Work plan for upcoming month
a. Determine kinetics of bacterial proliferation in human alveolar macrophages after F
tularensis infection with and without recombinant IFN
b. Determine kinetics of bacterial proliferation in human monocyte derived macrophages
after F. tularensis infection with and without recombinant IFN
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No new work performed
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Needs improvement
8. Percentage completed
25%
9. Work plan for upcoming month
a. Troubleshoot mouse macrophage killing assay with SCHU S4
b. Develop the macrophage killing assay using T cells from vaccinated Fischer 344 rats
i. Develop procedures for isolating and culturing macrophages from rats
ii. Develop procedures for isolating T cells from naïve and vaccinated rats
iii. Determine the optimal MOI for infecting rat macrophages
iv. Determine the kinetics of LVS and SCHU S4 proliferation in infected
macrophages
v. Determine whether T cells from vaccinated rats can induce infected
macrophages to kill intracellular bacteria
Page 19 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Select and test ORF expression constructs
1. Ten eukaryotic in vitro expression cassettes for five complete FTU (Francisella tularensis)
genes (groES, groEL, IglC, katG, Tul4) and two non-FTU antigen (OVA and CalM3) have
optimized and tested as shown in previous report.
B. Select and test IVT Protocols
1. All protocols for HTP protein production in the E. coli based IVT system have been
developed and optimized. If it is necessary, we will test the protocols established for the
bacterial IVT system in the eukaryotic expression systems.
C. Select and test protocols for protein purification
1. In the previous report, initial T cell experiments with Microcon filtered lysates indicated
that filtration could deplete the E. coli proteins that are causing cross-active stimulation of
immune T cells. This was encouraging; however, filtration only restricted yields to
approximate 10% of the synthesized products. When these samples were used in the
ELISPOT assays, no IFNgamma release was detected. This was likely to have been due
to the low quantities of filtered antigen lysate used for stimulation.
2. Many detergents have been used to improve protein solubility. We anticipated that this
might reduce loss of our sample in the filter membranes. We have tested three different
detergents: Tween-20, Octyl-glucopyranoside (OG) and Dodecyl  maltopyranoside
(DM). Our results in Figure 1 show that detergents have no effect on increasing E. coli
lysate proteins in the filtrate as compared to the yield of polypeptides from the IVT
reaction (lane IVT).
Page 20 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
IVT PBS
1
-FTU
DM
2
1
OG
2
1
Tween 20
2
P
B
S
Fig. 1: Effect of detergents on IVT protein filtration using 100 KDa
membrane Microcon unit.
Legend:
DM-Dodecyl-β-maltopyranoside; OG-Octyl-β-Glucoside;1 – 0.1%; 2-1%
Data Location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT
Coomassie gels\Detergent test for filtration 100kda filter 01-10-08
3. Since the majority of IVT proteins have been found in the retentate, it is possible that the
filter membrane is clogged by the relatively highly concentrated sample. We have tested
dilution of E.coli lysate on effect of IVT protein filtration. Two dilutions, 1/3 and 1/10, have
been tested for IVT protein recovery. The Coomassie gel results in Figure 2 show that
dilutions do not increase amount of FTU IVT proteins in the filtrate. The volumes of lysate
loaded onto gel were held constant, so diluted samples appear less intense. The lack of
improvement from dilution is evident by comparing the relative intensity of the FTU band
to all the others in the lane for lane 1 (undiluted) relative to the diluted samples (lanes 27).
Page 21 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
1
9
2
3
4
5
6
7
8
-FTU
Fig. 2: Effect of dilution on E.coli lysate protein filtration
Legend:
1- No Dilution then filtered, 2) 1:10 Dilution with PBS without filtering, 3) 1:10 Dilution in
0.01% Tween 20 filtered, 4) 1:10 dilution in PBS filtered, 5) 1:3 Dilution with PBS without
filtering, 6) 1:3 dilution in 0.01% Tween 20 filtered, 7)1:3 dilution in PBS filtered. Lanes 8
and 9 are irrelevant.
Data Location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT
Coumassie gels\ Filtration test 01-07-2008 no temp no35S 1
4. E. coli lysate contains macro complexes, namely the translational machinery. These
would contribute to the crowding effect during filtration. These macro complexes can be
removed by precipitation with organic solvents or ammonium sulfate. Our previous results
show that ammonium sulfate precipitation did not separate FTU proteins from the large
complexes in E.coli lysate. The FTU proteins co-precipitate with E. coli proteins.
Therefore, ammonium sulfate precipitation is not suitable for our purposes.
5. We have now tested the possibility of using acetone to separate the large complexes
from the small de novo polypeptides in the lysates. We tested a series of acetone
concentrations, and discovered that between 30-40% acetone is optimal for depletion of
non specific proteins into the pellet and retention of the newly synthesized sample in the
supernatant. Three different concentrations; 30%, 35%, and 40%, are reported here in
Page 22 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
<--FTU
Figure 3.
Fig. 3 Acetone Fractionation
1- 30% Acetone Pellet, 2) 30% Acetone Supernatant, 3) 33% Acetone Pellet, 4) 33%
Acetone Supernatant, 5) 35% Acetone Pellet, 6) 35% Acetone Supernatant, 7) 39% Acetone
Pellet, 8) 39% Acetone Supernatant.
Data Location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT
35S gels\ acetone and time test 01-28-08 FTU 1695
6. We found that temperature significantly affects the amount of protein that precipitates in
the presence of acetone. We have observed that at cold temperature, as 4C, most of the
low molecular weight proteins remain in solution. At room temperature much of the low
molecular weight proteins are pelleted. Results are shown in Figure 4.
Page 23 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Fig. 4 Effect of temperature on acetone precipitation of FTU proteins
Legend:
1- IVT Reaction, 2- Room Temp precipitation, 3- Room temperature Supernatant, 4- 4OC
precipitation, 5- 4OC Supernatant, 6- 1:10 Dilution Room Temp precipitation, 7- 1:10 Dilution
Room temperature Supernatant, 8- 1:10 Dilution 4OC precipitation, 9- 1:10 Dilution 4OC
Supernatant.
Data Location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT
35S gels\IVT Temp and dilution effect on acet ppt 1695
7. We have also tested effect of dilution on acetone precipitation. Three different dilutions
of IVT lysate were precipitated with 30% acetone and compared to undiluted IVT Lysate
of 3 FTU Proteins. The results in Figure 5 show that the dilution has no significant effect
on the yield of synthesized product (FTU polypeptide) relative to total E.coli protein.
Page 24 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Fig 5: IVT Dilution For 30% Acetone Precipitation at 4OC
Legend: Samples volumes were kept constant, and not normalized for radiolabel.
1- IVT No Dilution Precipitation, 2- IVT No Dilution Supernatant, 3- IVT ½ Dilution
Precipitation, 4- IVT 1/2 Dilution Supernatant, 5- IVT 1/4 dilution Precipitation, 6- IVT 1/4
Dilution Supernatant, 7- IVT 1/8 dilution Precipitation, 8- IVT 1/8 Dilution Supernatant
Data Location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT
35S gels\ IVT dillution for acetone ppt
8. We have sent the supernatants from a 4oC acetone precipitation of ten FTU polypeptides
and two unrelated proteins as control (ovalbumin and calmodulin), which were
synthesized in the E.coli IVT system, to UNM for testing in the T-cell assays. (Note these
samples were not filtered)
9. Purification of FTU IVT proteins using His-tag Nickel magnetic beads did not provide a
large quantity of purified protein due to low binding as shown in our previous results. We
have tested a new protocol for purification of IVT FTU proteins which incorporates the
acetone fractionation step. In our preliminary test, recombinantly produced and purified
FTU 1695 and EGFP were added to an E.coli IVT lysate, and then this sample was
acetone precipitated. The supernatant was incubated with Nickel magnetic beads and
Page 25 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
purified using Ni affinity chromatography. This was compared to purification of the same
proteins from unprecipitated IVT lysate (later is historic data which is not shown in this
report). We were able to purify and quantitatively recover the tagged proteins. Also the
purification from acetone-fractionated lysates was significantly more efficient than the
unfractionated lysate. Results are shown in Figure 6.
Fig. 6 Affinity purification of His tagged polypeptides from acetone fractionated
IVT Lysates:
Legend:
1- 35% Acetone Pellet, 2- 35% Acetone Supernatant, 3- 35% Flow through, 4-35% elution, 540% Acetone Pellet, 6- 40% Acetone Supernatant, 7- 40% Flow through, 8-40% elution.
Data Location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT
Coomassie gels\ Acetone ppt purification
10. We have tested the de novo synthesized proteins in our new purification protocols. Ten
FTU IVT polypeptides and ovalbumin were precipitated with 30% acetone and the
supernatants were incubated with Nickel magnetic beads. The beads were washed with
20mM imidazole and eluted with 500mM imidazole. Unfortunately, most of the
polypeptides did not bind to the Ni magnetic beads and could not be purified. Data not
shown
11. Acetone interacts with surface residues of the protein to cause it to precipitate. It is
postulated that interaction with acetone of well folded full length protein would be quite
different than a misfolded one. We have compared acetone fractionation of polypetides
synthesized from full length FTU gene templates relative to those synthesized from FTU
subgene templates. We observe that the fragments precipitate more readily in acetone
than a full length protein. We also note that there are significant quantities of partial
product in the samples synthesized from a subgene template. Results are shown in
Page 26 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Figure 7. FTU 1695 is a full length ORF and appears as a single band. FTU 721A and
FTU 1696Aa are fragments and appear with partial products.
Fig 7: Acetone fractionation comparison of FTU Fragment and full length proteins:
Legend
1
FTU 721A
RT ppt
2
FTU 721A
RT Sup
3
FTU 721A
4C ppt
4
FTU 721A
4C Sup
5
FTU 721A
1:10 Dil RT ppt
6
FTU 721A
1:10 dil RT Sup
7
FTU 721A
1:10 dil 4C ppt
8
FTU 721A
1:10 dil 4C Sup
9
FTU 1695
IVT
10
FTU 1695
RT ppt
11
FTU 1695
RT Sup
12
FTU 1695
4C ppt
13
FTU 1695
4C Sup
14
FTU 1695
1:10 Dil RT ppt
15
FTU 1695
1:10 dil RT Sup
16
FTU 1695
1:10 dil 4C ppt
17
FTU 1695
1:10 dil 4C Sup
18
FTU 1696Aa
RT ppt
19
FTU 1696Aa
RT Sup
20
FTU 1696Aa
4C ppt
21
FTU 1696Aa
4C Sup
22
FTU 1696Aa
1:10 Dil RT ppt
23
FTU 1696Aa
1:10 dil RT Sup
24
FTU 1696Aa
1:10 dil 4C ppt
25
FTU 1696Aa
1:10 dil 4C Sup
Data Location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\
Acetone ppt of FTU fragment protein
Page 27 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
12. We suggest that this difference between full and subgene templates may be caused by a greater
amount of misfolding in the resulting partial protein products.
4. Significant decisions made or pending
None at this time, but decisions on a delivery format for the polypeptide samples is pending.
5. Problems or concerns and strategies to address
To prevent the fragment FTU proteins from precipitating during acetone fractionation and also
improve integrity of the samples, we will test the impact of fusing a thio peptide to the FTU
subproteins during synthesis. This has been shown to enhance proper folding of translated
proteins in vivo. The pBAD-Thio vector from Invitrogen will be used to produce Thioredoxin
peptide FTU fusions. These will be compared to the corresponding unmodified FTU fragment
for solubility, acetone precipitation, and stability.
6. Deliverables completed
None
7. Quality of performance
Very good
8. Percentage completed
99%
9. Work plan for upcoming month
We will test the utility of expressing our polypeptides fused to a thioredoxin peptide. This
fusion has been shown to improve solubility of many proteins. Improved folding would reduce
degradation of misfolded samples. ASU does not expect the thioredoxin to impact the in vitro
cellular stimulation assays performed at UNM.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will use
ASU’s protein fragments in lymph node proliferation assays to define vaccine candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc59 study 12 (Notebook 115, pages 36-43)
i. This experiment had three objectives:
1. Determine whether the size exclusion strategy can be used to
remove cross-reactive materials from the E. coli ivt reaction as
measured by IFN Elispot assay
2. Determine the amount of acetone that can be used in the IFN
Elispot assay without affecting the T cell response. ASU is trying to
remove the cross-reactive material by differential acetone
precipitation and needs to know how much acetone the T cell
culture can tolerate.
Page 28 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
3. Determine the amount of DMSO that can be used in the IFN Elispot
assay without affecting the T cell response. We resuspended the
BALB/c MHC-binding Ft peptides from ASU in 100% DMSO. Since
we would like to use 1-10M peptide for in vitro re-stimulation, which
may add a high concentration of DMSO to the T cell culture, and we
would like to determine the maximum tolerated DMSO concentration
ii. For objective 1, ASU provided three sets of samples: ivt reactions without
template and ivt reactions with templates encoding for ovalbumin and Ftu
1695. These samples were treated with various combinations of 0.01%
Tween-20 to disaggregate large macrocomplexes, PBS, and/or filtration
through a 100kDa cutoff filter as indicated in Figure 1. The results suggest
that filtration through a 100kDa filter was very effective at removing the
cross-reactive material from the E. coli ivt reactions and Tween-20 had no
impact.
iii. For objective 2, we stimulated LVS vaccinated splenocytes with heat killed
LVS and titrated in acetone from 1 to 20% final concentration. We did not
include 40% acetone because, at this concentration, proteins were
precipitating out of the medium. As shown in figure 2, an acetone
concentration of 20% had no effect on IFN production. 20% acetone may
actually be an overkill since we generally add 5 l of ivt reactions to 100 l
cells, resulting in a maximum of 5% acetone
iv. For objective 3, we stimulated LVS vaccinated splenocytes with heat killed
LVS and titrated in DMSO from 1 to 20% final concentration. As shown in
figure 3, even 1% DMSO reduced the IFN production compared with
control. Thus, we have to test lower amounts of DMSO.
Figure 1. Removal of cross-reactive material from E. coli ivt reactions by filtration
through 100kDa cutoff filter
Page 29 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Figure 2. Impact of acetone on IFN production
Figure 3. Impact of DMSO on IFN production
b. Experiment Ftc59 study 8 (Notebook 115, pages 44-50)
i. The purpose of this experiment was to use a genetic immunization strategy
to generate a non-cross-reactive, Ft-specific response that would allow us to
determine 1) the acceptable Elispot assay background and 2) the amount of
ivt proteins necessary for restimulation.
ii. The mice were genetically immunized (GI) three times with a pool of Ft
genes or with the gene encoding for ovalbumin (the DNA construct contains
amino acids OVA323-339, which is presented on H-2d). The GI-mice were then
boosted or not with proteins encoded by the DNA constructs used for
immunization. 18 days after the protein boost, splenocytes from these mice
were restimulated in vitro with (Figure 4):
1. Medium
2. E coli ivt mix (no splenocyte should respond)
3. Heat-killed LVS, formalin-fixed LVS and Ft proteins used for protein
boost (only the mice genetically immunized with the Ft gene pool but
not OVA should respond)
iii. The results showed:
1. There was still a low level cross reactivity to E coli ivt reaction buffer.
However, since we did not include a mouse that was vaccinated with
Page 30 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
LVS for comparison, we do not know whether there was any
improvement to the level of cross reactivity. Moreover, there were
not enough spots for any meaningful comparison to the mice
genetically immunized with OVA, which had even fewer spots.
2. None of the mice genetically immunized with the Ft gene pool with or
without protein boost showed any response to heat killed LVS,
formalin-fixed LVS, or any of the Ft proteins. This result may be
explained by:
a. Ineffective immunization. We cannot rule out this possibility
because we did not re-stimulate the OVA-immunized mice
with OVA protein in this experiment. If the OVA-immunized
mice responded to re-stimulation with OVA protein, then we
would conclude that the genetic immunization was effective
and that the lack of anti-Ft response is specific to the mice
genetically immunized with the Ft genes.
b. Low frequency of Ft-specific T cells. The response to whole
LVS is likely to be polyclonal and therefore many spots
would be seen whereas the response to a single protein is
likely to be monoclonal and thus require many more IFN
producing cells
c. Insufficient protein used for re-stimulation. We used 1 M
proteins for re-stimulation in this experiment, however, it has
been shown that the T cells response to some proteins/
peptide is dose dependent and > 10M has to be used to
detect the response
Figure 4. Plate layout for re-stimulation of genetically immunized mice
c.
Experiment Ftc59 study 13 (Notebook 115, pages 51-54)
i. The purpose of this experiment was to determine whether differential
acetone precipitation effectively removed the cross-reactive material from the
E. coli ivt reactions
ii. ASU provided 12 IVT samples after precipitation with 35% acetone, these
include 10 Ft proteins and ovalbumin and calmodulin as negative controls.
Since we add at most 5 l of each ivt sample to 100 l of cells, the acetone
concentration should not exceed 2%, well under the level that would
precipitate proteins out of the cell culture
iii. These samples were used to re-stimulate splenocytes from LVS vaccinated
mice and mice genetically immunized with OVA. The mice genetically
immunized with OVA were included as a negative control for the LVS
vaccinated mice and to show that they are able to respond to OVA protein to
Page 31 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
address the question of immunization efficacy raised in Experiment Ftc59
study 8.
iv. As shown in Figure 5, LVS vaccinated splenocytes responded to all of the
acetone precipitated samples, including OVA and calmodulin. This
suggested that acetone precipitation was not effective at removing the crossreactive material.
v. The results also suggested that the genetic immunization (without protein
boost) was not effective because the splenocytes from OVA-immunized mice
did not respond to OVA. It is still possible that the immunization was
effective but requires a protein boost to become measureable.
Figure 5. Cross reactivity of acetone precipitated proteins and GI OVA splenocytes not responsive to
OVA in the IFN gamma Elispot assay.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
16%
9. Work plan for upcoming month
a. Boost OVA immunized mice with OVA protein
Page 32 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
b. Test the genetically immunized and boosted mouse splenocytes with a range of
proteins from 1 to 10 M and 2 different sources of OVA, such as commercially
purchased OVA protein
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of polypeptide libraries (Optimize IVT proteinfragment production, Develop IVT protocol for high-throughput production, Validate
immunogenecity of protein-fragments, Full scale production of protein-fragment library,
Purification of protein-fragment library, Array protein-fragment into overlapping pools, Ship
to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome (active)
 Generate complete protein-fragment library (inactive)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)
Institution: ASU-Sykes
3. Date started: 03-01-2007
4. Date completed: Pending
5. Work performed and progress including data and preliminary conclusions
A. Build ORF expression library corresponding to proteome
1. No new lab work this month.
2. In anticipation that milestone 26 is nearly complete, we have pooled PCR primers ORF library
production. We will proceed following decisions on expression system, yield needs, delivery
format, pooling capacity, and purification requirements.
4. Significant decisions made or pending.
The decision to complete the polypeptide purification/optimizations of milestone 26 are pending
but near.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
29%
9. Work plan for upcoming month
Wait for MS 26 to be completed.
Page 33 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 34-UNM
Milestone description: Pilot Studies for the optimization of RNA isolation and hybridization
conditions
Institution: UNM
1. Date started: 03/01/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No new work performed
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming month
We are closing this milestone and moving on to milestone 35
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35 - UNM
Milestone description: Array hybridization with mouse RNA from virulent SCHU S4
infection and RT PCR confirmation of candidates
Institution: UNM
1. Date started: 1/1/2008
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Planning the single dose SCHU S4 infection and kinetics, in conjunction with ASU.
Ordering mice and reagents for SCHU S4 infection experiments
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Page 34 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
None
7. Quality of performance
Good
8. Percentage completed
2%
9. Work plan for upcoming month
a. Test the sensitivity of the microarray analysis by infecting mice with a single SCHU
S4 dose of and isolate RNA at 1, 3, 5, and 7 h after infection. We will also isolate
RNA from SCHU S4 grown in Chamberlain’s broth at the same time points.
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: 06-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

RNA from the new dose response experiment was received, processed with the Qiagen
RNAeasy purification process and then processed for LAPT amplification, labeling, and
microarray hybridization. The results of the hybridizations are shown in Figure 1 representing
the raw signal intensities of the microarrays. Low level signal intensities are shown in blue,
intermediate signal intensities are in yellow and high level signal intensities are in red. The
general trend of expression was approximately the same based on color maps from the 10 1
to 106 samples. The 107 sample had an overall higher level intensity and the color map was
not as consistent with the previous samples. Of note, the amplification level in the 106 and
107 challenge samples dropped from an average of approximately 100 micrograms to 62 and
35 respectively.
Page 35 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Figure 1. Raw signal intensity of microarrays from LAPT-amplified SCHU S4 RNA from
lungs of BALB/c mice infected with various doses of bacteria. Challenge dose is listed
on the first text line below the graph. The second line of text delineates the amplification
level after the LAPT procedure using 10 micrograms of input RNA.
Page 36 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble

To validate the visual assessment of sample expression trends, a Spearman correlation was
performed for the complete gene set on the microarray between the doses (Table 1). The
best correlations were observed between 102 and 105 dose challenge with correlations
ranging from 0.738 to 0.758. The correlations dropped from 105 to 106 and further still
between 106 and 107 challenge doses.
Table 1. Spearman correlations between the challenge doses using the complete gene
set of the microarray data.

To compare the results between the first dose response experiment (LAPT12), we performed
Spearman correlations of the complete data set between comparable challenge doses.
Reasonable correlations were observed with the data sets from 103-105 challenge doses.
Thereafter the correlations sequentially dropped.
Table 2. Spearman correlations between the two dose response challenge experiments
using the complete gene set of the microarray data
Page 37 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble

We next compared the top 500 detectable genes in the 103 – 105 dose challenge samples.
Using a Venn diagram approach we see that in these three samples 293 of the top 500
expressing genes are similarly expressed between the challenge doses (Figure 2 left panel).
A separate comparison was performed to assess gene expression levels of infected mouse
lungs harvested at day 4 post infection (LAPT-5) to the current samples harvested at 4 hours
post infection. The gene data set was from the 293 genes overlapping from the 10 3 and 105
dose response challenge for the 4 hour set. The 4 day dataset was the top 501 genes from 3
mouse lungs harvested 4 days after infection. While 254 genes were in common between
these data sets, there were 39 genes that were detected at 4 hours and not at 4 days. This is
a preliminary indication that there are some genes being regulated early in the course of
disease as compared to late.
Figure 2.

Notebook/File locations …, Notebook 514, LAPT 20, page 143-166.
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-20 (Pooled NM Samples)
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
The dropping correlations of the two high dose challenge samples in this experiment need to be
addressed. We are unsure if this was an isolated technical issue and the LAPT process on all
samples will be repeated.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
13%
9. Work plan for upcoming month

Repeat the amplifications of the pooled samples to see if the lower yields on the 10 6 and 107
samples reproduce.
Page 38 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble

A time course experiment has been planned where animals will receive 104 CFU SCHU S4
intranasal challenge and tissues will be harvested at 1,3,5,7 and 24 hours. Separate SCHU
S4 cultures will be prepared in liquid Chamberlain’s medium and harvested at the same time
points to allow for in vivo to in vivo comparisons.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 41
Milestone description: Optimization of photochemical inactivation and characterization of
KBMA Ft. novicida; determine the amount of S-59 and UVA required to inactivate uvr mutants;
determine extent of metabolic activity of uvr mutants after S-59 and UVA inactivation; determine
the level of virulence attenuation of KBMA uvr strains in mice
Institution: Cerus
1. Date started: 3/2/06
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: We have determined that all the NER-deficient strains of Ft. novicida are only
slightly more sensitive to photochemical inactivation than wild type Ft. novicida. We have
optimized photochemical inactivation conditions at a 3.5 mL scale and a 400mL scale and
produced a lot of KBMA uvrB Ft. novicida for potency testing in MS42. We have
demonstrated that KBMA Ft. novicida are highly attenuated for virulence. Frozen KBMA
uvrB Ft. novicida maintain metabolic activity at –80oC for at least 3 months. Inactivated
NER-deficient strains have a similar degree of metabolic activity as the wild-type Ft. novicida
strain (which is different than has been seen with L. monocytogenes or B. anthracis), and we
have demonstrated that this lack of sensitivity to DNA damage is universal to numerous DNA
damaging agents.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
Nov 16, the vaccines research program was “spun out” of Cerus Corporation. All of the
personnel, intellectual property and other assets related to immunotherapy were transferred
from Cerus to Anza Therapeutics Inc. Cerus and Anza are working to establish a
professional service agreement that will allow the work on the TVDC to proceed using Anza
personnel and that is acceptable to UNM and NIAID. Numerous discussions have been held
between Cerus and Anza representatives and with UNM to discuss possible mechanisms for
continuing with the TVDC contract going forward. Cerus, Anza, and UNM have agreed in
principle to establish a service agreement between Cerus and Anza. However, until this
relationship is approved by NIAID there will be no direct costs charged by Anza to Cerus for
the TVDC. Anza is currently in the process of getting an IACUC established in order to
obtain our OLAW assurance and Anza has submitted APHIS documents to the USDA for
transport of Francisella tularensis strains (application # 07361052).
4. Significant decisions made or pending
All NER mutants (uvrA, uvrB, and uvrA uvrB) of Ft. novicida were equally sensitive to S-59
and had comparable metabolic activity after inactivation. We have chosen to use the uvrB
single mutant for further experimentation. We have selected 40M S-59 and 7J/cm 2 as the
conditions for making 400ml-scale KBMA lots, and have produced a lot of KBMA uvrB Ft
Page 39 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
novicida vaccine that is sterile for further characterization. We have decided to open MS 42 in
order to determine whether KBMA Ft novicida can protect against a lethal wild-type Ft novicida
challenge.
5. Problems or concerns and strategies to address
The 2-fold difference in the concentration of S-59 required for complete inactivation of the
mutants compared to wild type is less than we have observed for other organisms. This
appears to hold true for other methods of induced DNA damage. One possible explanation
for this is that there is a redundant DNA repair mechanism functioning in Ft novicida that may
limit the sensitivity of the NER-deficient mutants to DNA damage and thereby limit the
metabolic activity and potency of KBMA Ft novicida. If there is a redundant repair
mechanism, we may not be able to produce a highly potent KBMA vaccine utilizing
Francisella species as a platform. A new concern is that Cerus may no longer have enough
human resources to complete this milestone in a timely manner.
6. Deliverables completed
400mL-sacle photochemical inactivation process defined
7. Quality of performance
fair progress
8. Percentage completed
85% of scientific work completed on the milestone
9. Work plan for upcoming month
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable. This milestone has been paused due to the Cerus to Anza transition.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 42
Milestone description: Determine whether KBMA F.t. novicida vaccine protects against wildtype F.t. novicida challenge in mice: Vaccination route and regimen optimization, measure
durability of protection
Institution: Cerus
1. Date started: 2/1/07
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: KBMA Ft novicida uvrB vaccine stocks produced in MS41 have been tested in
mice for virulence and protection against a 100 x IP LD50 challenge of Wild-type Ft novicida.
KBMA Ft novicida uvrB were 100% protective when a single dose was administered at or
near the LD50 of the KBMA vaccine (1 x 109 IP, 1 x 108 IV). 100% protection was also
achieved by administration of 1 x 107 KBMA particles IV when the vaccine was given twice
separated by 3 weeks. Depletion of CD4+ T cells prior to the challenge decreased the
survival rate to 80%, depletion of C8+ T cells had no effect, and depletion of both cell
populations resulted in 90% survival. Together, these data demonstrated that CD4 T cells
contribute to a protective immune response in a non-CD8 T cell-dependent manner. These
data suggest that the CD4 T cells may be boosting humoral immunity by stimulating B cells.
This interpretation was supported by an adoptive transfer experiment in which only the hightiter serum from CD8-depleted animals provided any protection against a lethal U112
challenge. Together these data demonstrate that the protection we see after vaccination with
KBMA Ft novicida uvrB correlates with humoral immune responses and explains why the
KBMA vaccine does not perform better than heat killed vaccine. This also makes it nearly
Page 40 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
impossible to rank attenuated Ft novicida mutants by their ability to protect mice against a
lethal challenge. We instead plan to evaluate the ability of KBMA vaccines to induce a potent
CD8 T-cell response to an introduced ovablumin epitope tag and are awaiting the
construction of this strain from UTSA.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
4. Significant decisions made or pending
We have decided to evaluate the potency of the KBMA Ft novicida vaccine by measuring the
CD8 T cell response to an ovalbumin epitope tag.
5. Problems or concerns and strategies to address
Because humoral immunity plays a significant role in protection of mice against a lethal Ft
novicida challenge it is essentially impossible to rank KBMA vaccine candidates that elicit a
potent T cell response using survival after a lethal Ft novicida challenge in MS 43. We have
requested that Karl Klose construct an ovalbumin epitope-fusion protein to facilitate screening
strains of Ft novicida for their ability to elicit a T cell response to this well-defined epitope.
6. Deliverables completed
None
7. Quality of performance
fair progress
8. Percentage completed
25% of scientific work completed on the milestone
9. Work plan for upcoming month
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable. This milestone has been paused due to the Cerus to Anza transition.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 44
Milestone description: Formulation and evaluation of KBMA LVS: establish photochemical
inactivation regimen of selected uvr mutant of LVS and measure metabolic activity and virulence
of KBMA LVS.
Institution: Cerus
1. Date started: 6/18/2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary: using a small-scale inactivation procedure we have determined that the S-59 psoralen
concentration required to inactivate uvrB LVS is 5uM. This is the same concentration at which
we have been able to inactivate WT LVS. The uvrB LVS was also not more sensitive to DNA
damaging agents compared to WT. This suggests that there may be redundant DNA repair
mechanisms in LVS that may be functioning to repair photochemically induced crosslinks.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
4. Significant decisions made or pending
none
5. Problems or concerns and strategies to address
Page 41 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
The uvrB mutant of LVS does not appear to be more sensitive to DNA damage induced by
photochemical inactivation with S-59 and UVA or by other chemical means. This suggests that
the potency of a KBMA uvrB LVS vaccine is likely to be the same as KBMA Wt LVS which failed
to protect mice against lethal a schuS4 challenge (see MS46). These results suggest that we
reevaluate the KBMA tularemia vaccine strategy and we suggest comparing the efficacy of a
KBMA LVS vaccine to a KBMA Listeria monocytogenes vaccine that expresses Ft antigens.
6. Deliverables completed
none
7. Quality of performance
fair
8. Percentage completed
5%
9. Work plan for upcoming month
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable. This milestone has been paused due to the Cerus to Anza transition.
10. Anticipated travel
none
11. Upcoming Contract Authorization (COA) for subcontractors
none
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale LVS
culture conditions, Establish 3L culture scale purification conditions, Optimize 3L scale
photochemical inactivation process, Verify protective immunogenicity of vaccine candidates
produced by optimized large-scale process
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: we have demonstrated that LVS grows robustly in Chamberlains Defined Media
(CDM) and have prepared expanded DVC lot 16 LVS cultures grown in CDM for 36 hours,
and stored at -80oC. We have determined that the minimum concentration of S-59 required
for complete inactivation of DVC lot 16 LVS is 5µM and that photochemically inactivated LVS
maintain metabolic activity for at least 12 hours. We produced a 3L lot of LVS in our
fermentor using .001% Sigma antifoam A in CDM and have demonstrated stability for 4
months at -80o in 2 cryopreservation medias. We have found that the LVS provided by DVC
is greatly attenuated for virulence in mice when administered IP compared to literature
reports. We have demonstrated that LVS replicate rapidly in livers and spleens of mice
immediately following IV injection; however, it appears that there is a lag that specifically
affects growth in the lungs. We have also demonstrated that LVS is nearly avirulent when
administered by the SC route.
We have produced a 400mL lot of KBMA wild-type LVS using 10 uM S-59 and 6 J/cm 2 UVA
for initial proof of concept studies, and for later comparison with NER-deficient uvrB LVS
and we have demonstrated that the metabolic activity of this lot is stable for 3 months. We
have demonstrated that KBMA WT LVS IV LD50 is 6.8x108, which represents a 4-5 log
attenuation compared with live LVS. We have demonstrated that doses of KBMA WT LVS as
low as 1 x107 provide protection against 100 x IP LD50 challenge of live LVS. However, none
of the mice vaccinated with the equivalent doses of HK LVS died either. This is consistent
with protection against an LVS challenge being largely humoral. b We recently attempted to
measure the T-cell response to a CD4 Tul4 epitope in mice vaccinated with live or KBMA
Page 42 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
LVS by intracellular interferon-gamma (IFN-) cytokine staining (ICS) or ELISpot assay, but
were unable to detect an induced response to this epitope. This may be because this epitope
does not bind the MHC molecule with high affinity, or the T cell response elicited by LVS may
actively suppress T cell responses. We recently demonstrated that LVS does not induce IL-6
or MCP-1which are critical hallmarks of a protective inflammatory response. Furthermore,
co-vaccination with LVS decreased the innate inflammatory response to Lm. Administration
of LVS decreased the ability of the elicited T cells to produce the cytokine IL-2. Terry Wu at
UNM completed a protection study with KBMA WT LVS in which neither a (IV or IN) prime
nor a prime and boost (separated by 3 weeks) vaccination regimen with KBMA WT LVS
protected against a lethal SchuS4 challenge in mice. KBMA WT LVS vaccine appears to be
less potent than live attenuated LVS.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
4. Significant decisions made or pending
Because wt Ft novicida is inactivated with S-59 concentrations that are only slightly higher than
uvrB mutant we have been investigating the efficacy of a wild-type KBMA LVS vaccine. Now
that we have received the uvrB mutant we will focus on producing a lot of KBMA uvrB LVS
5. Problems or concerns and strategies to address
The protection seen with the KBMA WT LVS against a lethal LVS challenge is independent of
metabolic activity. This suggests that comparison of various routes, regimens, or formulations will
be difficult to optimize by protective efficacy. The SchuS4 challenge model in mice is more
stringent, but KBMA LVS failed to protect after two doses. It is possible that the rat model may
allow a higher degree of sensitivity. The suppression of the innate inflammatory response and
the suppression of CD4 T cell cytokine production may potentially indicate that LVS is not a
potent inducer of protective T cell responses. We would like to screen for T-cell responses using
the peptides generated by ASU as an alternative method for optimization of vaccine potency or
construct an overlapping peptide library for IglC.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
53% of scientific work completed on the milestone
9. Work plan for upcoming months
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable. This milestone has been paused due to the Cerus to Anza transition.
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
Page 43 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHU S4 we need to develop tools to generate successful deletions.
Therefore, our focus is two fold, one is cloning experiments to get our target deletions into vectors
that we can use in creating these deletions and experiments with SCHU S4 itself using constructs
that we believe will allow us to make deletions into SCHU S4.
I. Cloning:
a. The transformations from the various igLD ligations yielded hundreds of colonies compared to
the re-ligation (with vector only) which yielded only 9 colonies. Ten colonies from each
transformation igLD set; that is, from 1140+igLD30a and 1140+igLD255a, respectively; were
grown in LB broth with 70 ug/ml kanamycin overnight and then UTSA isolated plasmid from these
cultures. These plasmids were subsequently digested with Bgl II restriction endonuclease which
should yield a profile with three DNA bands of 1500 bp, ≈2700 bp and ≈3800 bp sizes for the
correct igLD construct. The parent plasmid, KEK1140 will yield the same DNA pattern except the
largest DNA band will be ≈4000 bp in size. The first ten colonies screened from each set did not
show the correct pattern; however, the next group, clones 11 (C11) through C17 yielded many
correct DNA patterns (see Figure 1 and Figure 2). Data located in TVD UTSA Notebook 5, page
97 and 98.
Figure 1.
This represents a Bgl II digestion profile of 1140+30a igLD clones 11-17 (lanes 4-9,11) and C11
of 1140+255a igLD (lane 12). Lane 3 is parent plasmid KEK1140 Bgl II digested. The correct
clone will yield three DNA band fragments as in the parent plasmid with the largest size fragment
being smaller than its parent at approximately 3800 bp in size. Clones in lanes 5-9 and 11, 12 all
appear to be correct 1140+igLD constructs. Lane 2, 10 and 13 are uncut plasmid and lane 1 is
the 1 Kb ladder from Invitrogen.
Page 44 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Figure 2.
This represents a Bgl II digestion profile of 1140+255a igLD clones 11-17 (lanes 4 -10). Lane 3
is parent plasmid KEK1140 Bgl II digested. The largest DNA band on this profile is the
determining band between the correct clone and the parent vector KEK1140. This band should
be smaller than its parent plasmid and clones in lanes 4-6 and 8-10 appear to be correct
1140+255aIgLD constructs. Lane 2 and 11 are uncut plasmid and lane 1 is the 1 Kb l adder from
Invitrogen.
b. Prepared larger plasmid isolations from clone 16 of 1140+30aIgLD and C12 of 1140+255aIgLD
to send for sequencing to confirm correct construct of IgLD to use for making a mutant in SCHU S4.
c.
In order to delete an entire pathogenicity island (FPI) from Schu 4 we need to move the pdpD
deletion containing the flip recombinase recognition sites from the pwsK30 plasmid into pUC118
vector. Based on sequence analysis of the current pwsk30 pdpD deletion construct UTSA
decided to use Kpn I restriction endonuclease site to clone the necessary DNA fragment into
pUC118 vector. We’ve designed oligos with an introduced Kpn I site to help in this cloning. The
oligos are as follows:
i.
pdpDS4KpnIF: 5’-cggggtacctatcgtaagagcctataagg-3’
ii.
pdpDkpnIR: 5’-cggggtaccgacctaagccagtaaaataagcatac-3’
II. Experiments to generate mutants in Schu4:
a. Previous month’s work has generated hundreds of potential vgrG Schu4 mutants by using the
plasmids pKEK1161 and pKEK1162. pKEK1161 targets vgrG at bases 30 and 31, while
pKEK1162 targets vgrG at bases 81 and 82. Potential mutants were screened by using the
primers vgrG fwd XhoI and vgrG rev EcorI. These primers amplify the entire vgrG gene from start
to stop codon. PCR products of wild type vgrG should yield a 500 bp fragment whereas a tulatron
vgrG mutant would yield a ~1200 bp fragment, due to the insertion of the intron. Four potential
mutants of each set, pKEK1161 and pKEK1162, were screened using these primers (figure 3).
Lanes 2 and 3 show a PCR product of ~500 bp, the expected size of vgrG. Lanes 4-11 also show
a PCR product of 500 bp, without any shift of size due to an insertion of the intron. This indicates
that these clones do not contain the intron. More clones will be screened with these exact primers
in hopes of finding a vgrG mutant. Data located in TVD UTSA Notebook 1, page 25.
Page 45 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Figure 3.
1.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Ladder
Wt schu4
KKT1
clone1 (pKEK1161)
clone 2 (pKEK1161)
clone 3 (pKEK1161)
clone 4 (pKEK1161)
clone 1 (pKEK1162)
clone 2 (pKEK1162)
clone 3 (pKEK1162)
10. clone 4 (pKEK1162)
b. Did cryotransformation experiments using the potential igLD tulatron constructs C16
1140+30aIgLD and C12 1140+255aIgLD.
Used 2.5 ug of total DNA from each plasmid
preparation and cryo-transformed into the KKT1 SCHU S4 strain, respectively. Used TSA+++
plates containing 70 ug/ml kanamycin to select for transformants at 30°C. The negative control
was KKT1 with no DNA was plated on their own kanamycin selective plates. The 30aIgLD
construct yielded 359 colonies and the 255aIgLD construct yielded several thousand colonies.
After four days the control did yield 7 total colonies on the TSA +++ containing 70 ug/ml
kanamycin which is considered to be spontaneous resistance.
c. Patched 37 individual colonies from the transformation plates for each of the selection plate from
C16 1140+30aIgLD and C12 1140+255aIgLD, respectively, onto fresh TSA+++ 70 ug/ml
kanamycin plates. Will isolate genomic DNA from some of these clones and screen by PCR using
oligos specific to the cloned intron (described earlier) as well as to igLD gene specific oligos which
will help to view a size change due to the insertion of the intron. This will be reported on next
report. Data located in TVD UTSA Notebook 5, page 101 and 102.
e. 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
58%
9. Work plan for upcoming month
a. Will analyze any sequence information received for the 1140 + igLD constructs mentioned above
and also of the vgrG clones which were used in the SCHU S4 transformation experiments.
b. Will continue the screening for potential igLD mutants in KKT1 SCHU S4 strain.
c. Will continue to screen for vgrG mutants in KKT1 SCHU S4 strain.
d. If time allows, will continue with the pdpD flip pwsk30 construct cloning into pUC118.
e. Will work on a Southern blot with the igLC mutant made earlier to further confirm this “intron”
insertion in both gene copies on the FPI.
10. Anticipated travel
Page 46 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
Crystal Lauriano, Ping Chu and Heather Powell will attend the Tularemia Workshop in New York
from 3/30 to 4/1/2008 under COA#18 on TVDC funds.
11.Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS uvrA or
uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD, iglD
strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA, iglB strains
Institution: UTSA
1. Date started: 05/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
50A. (1) Measure intramacrophage (J774) replication of Ft subsp. tularensis (SCHU S4) iglC
and mglA mutants (Note book #4, page 148-149): We have made frozen bacterial stocks
of the wild type SCHU S4, iglC , and mglA and have confirmed the titers of each stock.
The macrophage cell line (J774) has been recovered from a cryo-stock and is propagating in
5% CO2 incubator. We plan to setup the intramacrophage replication experiment within a
week when J774 cells are ready for use.
(The SCHU S4 mglA which has been shown to be attenuated and did not replicate in
macrophage (J774) will be used as a negative control of bacterial replication.)
(2) Evaluate the protective efficacy of intragastric F. tularensis SCHU S4 iglC vaccination
against wild type SCHU S4 challenge (Note book #4, page 146-147): Groups of BALB/c
mice (female, 4-6 weeks) have been immunized with 103 CFU of iglC intragastrically (i.g.) or
intradermally (i.d.). Mice treated with PBS were used as a mock-control. We will collect sera,
and fecal pellets at day 21 after immunization and assay for antibody titers. These mice will
be challenged intranasally or i.d. with two doses of SCHU S4. Animals will be monitored for
survival and weight loss.
50B. Measure bacterial dissemination in various tissues at early time points following
intragastic LVS immunization (Note book #8, pages 20, 24-27, 29-32, 34-35). BALB/c mice
were challenged with LVS (~103 CFU) intragastrically. Lungs and trachea were collected
from the infected mice at 30 min and at days 1, 2, 3 and 5 after challenge (3 mice per time
point). Liver, spleen, cervical and mesenteric lymph nodes were collected from the infected
mice at days 1, 2, 3, 5 and 7 after challenge. Numbers of bacteria in each organ were
determined by dilution plate counting. As shown in Fig. 2A, there were no detectable
bacteria present in either the trachea or lungs within 30 minutes or 1 day post-inoculation,
with gradual increases of viable bacteria recovered from days 2 to 5. This data suggests that
the bacteria may not be accidentally inoculated directly in to the lungs and may be seeding
the respiratory compartment by systemic circulation following intragastric inoculation. As
shown in Fig. 2B, elevated numbers of bacteria were not present in the liver and spleen until
day 5 after inoculation which is consistent with results reported previously (Nov. 2007). There
were minimal bacteria recovered from both cervical and mesenteric lymph nodes at days 5
and 7 after challenge.
Page 47 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
CFU Per Organ
A
10
6
10
10
6
5
10
5
10
4
10
4
10
3
10
3
Trachea
30
Min
CFU Per Organ
B
1
3
2
30
Min
5
Da y s Afte r In o c u l a ti o n
10
6
5
10
5
10
4
10
4
10
3
10
3
10
6
10
6
10
5
10
5
10
4
10
4
10
3
10
3
10
6
10
Liver
CLN
1
2
3
5
7
Lungs
2
1
3
5
Da y s Afte r In o c u l a ti o n
Spleen
MLN
1
2
3
5
7
Day s After Inoc ulation
Fig.2. Kinetic growth and clearance of Ft LVS in target organs after intragastric vaccination. (A) Bacterial burdens
were determined in the trachea and lungs per individual mouse. (B) Bacterial burdens were determined in the liver
and spleen per individual mouse and cervical and mesenteric lymph nodes per group of mice. Dashed lines
indicate level of detection.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
Page 48 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
7. Quality of performance
Good
8. Percentage completed
60% of scientific work completed on milestone 50A (original plans) (NORMALIZED TO : 30%)
25% of scientific work completed on milestone 50B (intragastric plan) (NORMALIZED to 12.5%)
9. Work plan for upcoming month
50A: (1) Measure intramacrophage (J774) replication of Ft subsp. tularensis (SCHU S4) iglC
and mglA mutants
(2) Evaluate the protective efficacy of intragastric F. tularensis SCHU S4 iglC vaccination
against wild type SCHU S4 challenge:
(3) Evaluate the protective efficacy of intragastric F. novicida iglB vaccination against SCHU
S4 intranasal and intradermal challenge in C57BL mice.
(The 50B project focuses on the assays of immune responses in mice which received
intragastric LVS vaccination in the SCHU S4 challenge model. Immunological
characterization of mutants (SCHU S4 iglC and F. novicida iglB) generated under the
contract should still fall within the 50A project, and the basis of new experiments in lieu of the
UVR mutants
50B: (1) Evaluate the protective efficacy of intragastric LVS vaccination against Francisella
type A SCHU S4 intranasal challenge at 8 weeks after either a single vaccination or after
receiving a secondary booster dose.
(2) Analyze cytokine production and granzyme B activity in spleens and lymph nodes at both
2 and 4 weeks after intragastric inoculation with LVS.
10. Anticipated Travel
Crystal Lauriano, Ping Chu and Heather Powell will attend the Tularemia Workshop in New York
from 3/30 to 4/1/2008 under COA#18 on TVDC funds.
Bernard Arulanandam will attend the NIAID/DVC/UNM/FDA Tularemia Workshop from 3/11 to
3/12 in Washington DC.
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
To inactivate RecA in Schu S4, we constructed a Targetron vector for targeting and inactivating the
RecA gene. The Targetron vector was constructed with the intron expression vector pKEK1140 for
the backbone, and a 350bp PCR product to retarget the intron RNA for insertion mutagenesis of
RecA.
3.1 In last month technical report, we reported that the Targetron vector pKEK1187(at 840/841s retarget
site) was transformed into LVS, and the transformants needed to be identified by colony PCR. Two
primers( RecA schus4 for and RecA Schus4 rev) flanking RecA gene in Schu S4 for colony PCR were
ordered and three sets of colony PCR reactions were performed with two primers mentioned above (
flanking RecA gene ) and EBS Universal ( in intron RNA).
3.2 Colony PCR with RecA schus4 for and RecA schus4 rev was set up as follows:
Page 49 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
ddH2O
32.6 ul
10X Buffer
5.0 ul
MgCl2
2.0 ul
dNTPs
5.0 ul
RecA Schus4 for (25pmol/ul)
2.0 ul
RecA Schus4 rev (25pmol/ul)
2.0 ul
KOD DNA polymerase
0.4 ul
DNA
1.0 ul
At 98C 1minute, 98C 15seconds// 57C 15seconds// 72C 2minutes/ 30 cycles
Figure 1: 1% agarose gel for colony PCR.
Lane2 to lane5 were the potential Rec A mutants with the PCR product at about 1500bp, whereas about
630bp yield for wild type LVS (lane 6). Since the two primers flanked RecA gene, we could be sure that
the retargeted intron RNA had been inserted into RecA of LVS. There was a very weak band at about the
same size(600bp) as wt LVS (lane6) on lane5. It was probable that LVS with insertion mixed with LVS
with the plasmid inside but no insertion happening.
3.3 Another two sets of colony PCR were performed to make sure that intron RNA was inserted in RecA
and determine the orientation of the insertion DNA. The colony PCR reactions were set up with two sets
of primers. One was RecA Schus4 for and EBS Universal, and the other was RecA Schus4 rev and EBS
Universal. The procedures were the same as Step3.2.
Figure2: 1% agarose gel for colony PCR.
There were PCR products about 600bp on lane2-lane5, and no specific bands on lane6-lane11. Lane6
was wild type LVS for control with the same primers as lane2-lane5. The primer EBS Universal was in
Page 50 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2008 to 1/31/2008
Due Date: 2/7/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood,
Trevor Brasel, Julie Wilder, Karl Klose, Bernard Arulanandam, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Justin Skoble
RecA gene, whereas the primers RecA Schus4 for and RecA schus4 rev were located in each side of the
intron RNA. From the PCR, we were certain that the insertion had happened, and the primer EBS
universal was in the reverse direction comparing to RecA sequence and the same orientation as the
primer RecA schus4 rev.
3.4 The about 600bp PCR product from colony7 (lane2 on figure2) was gel purified, and the gel purified
DNA was sent for sequencing with the primers EBS Universal and RecA Schus4 for. The sequencing
result confirmed that the insertion was in RecA at 840/841bp in LVS.
3.5 It was possible that two kinds of mutant LVS mixed together. One was LVS with the insertion in RecA,
and the other was LVS only with the plasmid inside, but no insertion happened. So it was necessary to
separate them from each other to obtain the right mutant LVS. Colony7 was streaked onto
TSA++/Kanamycin (30ug/ml) plate, and incubated at 30C for several days until the single colonies came
up. So far, the separated single colonies were not big enough to be patched.
Data recorded on UTSA TVDC notebook #6, page8-10 for figure1 and 2.
4 Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None.
7. Quality of performance
Good
8. Percentage completed.
About 16% of scientific work completed.
9. Work plan for upcoming month
Screen for separated colonies to get pure LVS with insertion in RecA. Get rid of the
plasmid from the mutant LVS. Prepare Schu s4 strain in BSL3 lab for transformation.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None.
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