Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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 4 UNM participants are scheduled to receive the LVS vaccination on
3/18/2008.
c. 1 UNM participant and 2 LBERI participants declined the vaccination. 2 UNM
participants are not yet medically cleared.
d. Depending on medical clearance, 2 LBERI and 1 UNM staff and scientists may be
vaccinated on 4/1/08.
e. 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 received UNM IRB approval for 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 (4) and LBERI (33) are offering the LVS vaccinations up to 9 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
Within 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
6. Deliverables completed
32 LBERI scientists and staff have received the LVS vaccination between 9/11/07 and 1/9/08.
Page 1 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
7. Quality of performance
Excellent
8. Percentage completed
58%
9. Work plan for the next month
a. Complete health screenings for the next group of 2 UNM participants and 2 LBERI
participants.
b. Make travel arrangements for medically eligible participants to enter LVS Vaccination
program at USAMRIID on 4/1/08 – possible second 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
4 UNM 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 SCHUS4) bioaerosol technique optimization experiments were
conducted in February 2008.
A new working stock of SCHUS4 was produced from a 48h Chamberlain’s broth culture. The
calculated titer of the new stock is 2.3 x 108 CFU/mL.
Cumulative data from the LVS bioaerosol studies were presented on a poster entitled,
“Optimization of Bioaerosol Generation Techniques for Francisella tularensis” at the annual
American Society for Microbiology Biodefense Meeting in Baltimore on 2/25/2008. Significant
positive feedback was received.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
It is unclear as to specifically how the Aeromist and Collison nebulizers affect SCHUS4
bioaerosols with respect viability upon animal inhalation and lung deposition. This is currently
being addressed in MS4.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
96% (NOTE: 96% was reported to NIAID on 3/20/08, but it should have been at 98% BG)
9. Work plan for upcoming month
Continue Milestone completion report.
10. Anticipated travel
Page 2 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Drs. Sherwood, LeClaire and Hobbs will attend the NIAID/UNM/DVC/FDA meeting on 3/11to
3/13/08 in Bethesda MD at NIAID.
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
Additional mouse bioaerosol challenges against SCHUS4 were conducted on 1FEB08 to
compare the relative virulence in mice of SCHUS4 grown in Chamberlains broth versus growth on
solid BCGA. LBERI chose a dose range of 10 and 1000 SCHUS4 for the Feb 1, 2008 challenge.
This follow-on mouse bioaerosol pathogenicity study comparing low and high doses of SCHUS4
cultured either in 48h Chamberlain’s broth or on 72h BCGA was completed. Rick Lyons
cautioned LBERI that mice are so sensitive to SCHUS4 that detecting changes in virulence
based on SCHUS4 growth conditions may be difficult Morbidity/mortality data are shown in
Figure 1:
Francisella tularensis SCHU S4-exposed BALB/c mice
Morbidity/Mortality Data
11
10
Number of Mice
9
8
7
6
5
4
3
2
1
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Study Day (Exposure = Day 0)
Group 1: Low dose 72h BCGA
Group 2: High dose 72h BCGA
Group 3: Low dose 48h Chamberlains
Group 4: High dose 48h Chamberlains
Figure 1. Morbidity/mortality data for F. tularensis SCHUS4-exposed BALB/c mice. Mice were exposed to
either a low or high aerosol dose of SCHUS4 harvested from either fresh 48h Chamberlain’s broth or 72h
BCGA cultures.( See Table 1 for the measured low and high dose determinations)
Results demonstrated 100% lethality in 4-5 days when challenged with a high dose of SCHUS4
regardless of the growth method. These findings are consistent with observations noted on the first
group of mice exposed similarly to a high dose of SCHUS4 diluted from a frozen stock vial. Though
Page 3 of 46
15
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
mortality was not 100% in the low dose groups, results were similar between the two culture methods.
Taken together, these data indicate that there is no measurable difference in SCHUS4 virulence when
challenge material is prepared using three different methods (frozen stock, grown in Chamberlains and
grown on BCGA solid media).
In order to confirm bacterial deposition, one mouse from each group (n=4 total) was immediately
necropsied after exposure so that the lungs could be removed for microbial culture. These data are
presented in Table 1:
Table 1. Mouse Pathogenicity from Aerosolized F. tularensis SCHUS4
Group
Media
Dose
(CFU)
Animal
No.
Lung Wt
(g)
Avg CFU
count
Diln
CFU/mL
CFU/Lung
%
Deposition
1
2
3
4
BCGA
BCGA
CB
CB
661
22700
437
24900
15965
15943
15959
15951
0.165
0.183
0.223
0.186
0.0
1.3
0.0
3.3
10
100
10
10
0.0
133.3
0.0
33.3
0
291
0
73
0.0%
1.3%
0.0%
0.3%
These data demonstrate a surprisingly low deposition of 0% to 1.3% (approximately 5% was
expected). Significant contamination was noted on many of the plates and this may have
contributed to the low calculations (i.e., “true” SCHUS4 growth was impeded by the overwhelming
presence of contamination). These results led to the question as to the fate of the bacteria
following inhalation; specifically, does the Collison damage SCHUS4 to the point of low
deposition and/or culturability following inhalation? Comparison testing with the Collison and the
Aeromist are currently being designed.
Electronic data from these studies are stored in the following location: \\Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY07-083 and -089
(TUL-04)\1FEB08 Mouse exposure
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Aerosol delivery of SCHUS4 to mice resulted in a lower lung deposition than expected. The
SCHUS4 maybe damaged by the Collison nebulizer. Prior data indicated that the Aeromist
nebulizer may be more gentle. A direct comparison of Collison and Aeromist bioaerosol delivery
to mice will be performed, including lung deposition measurements.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month
Further investigate SCHUS4 deposition in a BALB/c mouse aerosol exposure model. The
Aeromist and Collison will be compared using similar doses as conducted for the previous two
mouse inhalation challenges. Challenge bacterial suspensions will be made from fresh 48h
Chamberlain’s broth cultures seeded with the new working stock of SCHUS4. A total of 20 mice
Page 4 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
will be challenged in the following groups: (1) low dose Collison (2) high dose Collison (3) low
dose Aeromist (4) high dose Aeromist. All lung homogenates will be cultured on selective media
(as specified by UNM) to reduce/prevent contamination and allow more accurate determination of
the SCHUS4 lung depositions.
Data obtained from this study will further our knowledge of potential outcomes for the upcoming
NHP bioaerosol exposures.
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 SCHUS4
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
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c. Rat model completed
7. Quality of performance
NA
8. Percentage completed
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
Page 5 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
1. Date started: 1/16/2008
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ptran1 (Notebook 112, page 41,54-66)
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 SCHUS4
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 SCHUS4
(actual lung deposition). The challenge dose was intentionally low to give the
vaccinated serum a chance to protect. 72 CFU of SCHUS4 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 SCHUS4 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 SCHUS4
Groups
Vaccination status
Treatment2
Survival ratio (No.
live/total)3
1
1
s.c. LVS vaccinated
None
6/6
2
Naïve
None
0/6
3
Naïve
Naïve rat serum
2/6
4
Naive
Vac. rat serum
6/6
1
Rats vaccinated s.c. with 5 x 107 LVS
Sera collected 36 d after vaccination and 3 ml transferred i.p.
3
Rats challenged i.t. with ≤ 72 SCHUS4 1 d after serum transfer
2
vii. To determine whether passive and active immunization provided similar level
of protection, we compared the bacterial burden in the lung, liver and spleen
between the two groups 18 days after challenge
viii. As shown in Figure 1, the actively vaccinated rats have cleared the SCHUS4
infection from the spleens and livers and very few bacteria left in the lungs.
The passively immunized rats are also clearing the SCHUS4 infection, but
these rats have higher bacterial burden than the actively vaccinated rats.
These results suggest that active and passive immunization may have
different mechanisms of action
ix. We hypothesize that vaccinated rat serum may control bacterial proliferation
thereby enabling the SCHUS4 infected rats to survive longer and develop an
effective cell-mediated immunity that eventually leads to bacterial clearance
Page 6 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Figure 1. SCHUS4 burden in actively and passively immunized rats 18 days
after i.t. challenge with SCHUS4 .
b. Experiment Ptran3 (Notebook 112, in progress)
i. The purpose of this experiment was to determine whether passive transfer of
vaccinated rat serum is sufficient to protect BALB/c mice against intranasal
SCHUS4 challenge
ii. We showed previously in Experiment Ptran1 that vaccinated rat serum was
sufficient to protect naïve BALB/c mice from a lethal respiratory LVS (9,500
cfu) challenge
iii. Naïve BALB/c mice were injected i.p. with 250 l vaccinated rat serum from
Experiment Ptran1. 1 day later, they were challenged i.n. with ~60 cfu
SCHUS4 or the same dose of LVS used in Ptran 1 (9,500 cfu)
iv. The preliminary data suggest that vaccinated rat serum is not sufficient to
protect naïve mice against SCHUS4 challenge. However, we do not know at
this time if the vaccinated rat serum is functional because the LVS
challenged mice are only starting to die. We will consider this an
interpretable experiment only if the naïve mice that received naïve rat serum
died and those that received vaccinated rat serum lived
Group
1
2
Immune status
Naïve
Naïve
Treatment
Naïve rat serum
Vac. rat serum
Challenge strain
LVS
LVS
5d survival ratio
(No. alive/total)
3/4
4/5
3
i.n. vaccinated
None
SCHUS4†
3/5‡
4
Naïve
None
SCHUS4
0/5
5
Naïve
Naïve rat serum
SCHUS4
0/4
6
Naive
Vac. rat serum
SCHUS4
0/5
† Challenge: ~ 60 SCHUS4
‡ One mouse died 1 day after challenge and therefore was unlikely due to SCHUS4 infection
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
Page 7 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
7. Quality of performance
Good
8. Percentage completed
5%
9. Work plan for upcoming month
a. Repeat passive transfer experiment in mice and rats, looking at protection and
bacterial burden
b. Titrate amount of vaccinated rat serum required for protection
c. Measure the level of protection induced by serum by challenging with increasing
doses of SCHUS4
d. Determine the effect of T cell depletion on passive immunization.
e. Deplete vaccinated rats of CD4 and CD8 T cells as soon as Taconic provides ascites
f. Determine whether vaccinated human serum is sufficient to protect rats against
SCHUS4. This experiment will be performed as soon as sera from vaccinated
human volunteers become available
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment MFT33 The data for this experiment is located in Amanda Dubois’
notebook for the tularemia portion of the program project because this assay is being
developed both the TVDC and the program project
i. PA Darrah et al [Nat Med 13:843-850 (2007)] reported that the frequency of
CD4 T cells expressing IFN, TNF, and IL-2 correlates with the level of
protection against Leishmania major
ii. We would like to determine whether the frequency of multifunctional T cell
can be used as a correlate of protection for our selected small animal model
and human of tularemia
iii. CD4+ T cells expressing IFNγ+, TNFα+ and IL-2+multifunctional will be
detected by multi-color flow cytometry
iv. The assay conditions are currently being optimized using LVS vaccinated
mice and in vitro restimulation of splenocytes with heat killed LVS or media
(None) as a negative control.
v. As shown in Figure 6, we were able to detect multifunctional T cells in the
spleens of LVS-vaccinated mice after in vitro restimulation with heat killed
LVS.
vi. Moreover, we found
1. Holding cells overnight on ice at 4oC preserves LVS-specific
responses better than any other approach attempted
2. The surface staining and the intracellular staining steps can be
combined
Page 8 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
3. A combination of staining antibodies that minimize background and
allows for the detection of LVS specific responses in the spleen
4. The background is higher in the lungs than in the spleen but LVSspecific responses can still be detected
Vacc. #1
Vacc. #2
Fresh, 2 hour stimulation
Vacc. #1
Vacc. #2
Held overnight at 4oC
Figure 2. Frequency of multifunctional T cells in splenocytes from LVS vaccinated mice when using fresh
cells or cells held over night at 4oC
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse proliferation assay, IFN and IL-2 ELISpot, anti-Ft antibody titration
Rat IFN ELISpot, anti-Ft antibody titration
Guinea pig anti-Ft antibody titration 
7. Quality of performance
Good
8. Percentage completed
58%
9. Work plan for upcoming month
a. Determine whether boosting LVS vaccinated mice with live or heat killed LVS or an
active SCHUS4 infection would increase the frequency of Ft-specific T cells
b. Continue to optimize the multifunctional T cell assay in mice
c. Measure the number of multifunctional T cells in the lungs of LVS-vaccinated mice
before and after i.n. challenge
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 9 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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 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 1 for historical data)
2. We would like to confirm this by testing ID and SC vaccinated animals on the
same day
ii.
Figure 2 shows the results; only one of SC vaccinated NHPs (A00659)
responded by secreting IFN, whereas both of the ID vaccinated NHPs
responded to ff LVS; this disproves our suspicion that ID and SC vaccinated
NHPs respond differently in the IFN assay although it was the first time that the
two ID vaccinated NHPs had responded well (See Figure 1 for comparison)
iii.
We have a concern that ff LVS acts as a mitogen in the IFN ELISPOT
assay due the capacity of non-vaccinated primates to respond to ff LVS by IFN
secretion (see Figure 3 for historical data); we will explore this further by testing
more non-vaccinated NHPs and varying the dose of cells and antigens (less ff
LVS or more hk LVS may reveal specific responses)
Page 10 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
300
SC
250
Media
LVS hk Hi
LVS ff Hi
200
150
ID
Day 203
Day 195
Day 238
Day 237
Day 288
Day 203
Day 195
Day 238
Day 237
Day 288
Day 203
Day 195
Day 238
Day 237
Day 288
A00908,
A00908,
A00908,
A00908,
A00908,
A00937,
A00937,
A00937,
A00937,
A00937,
NT
A00896,
A00896,
A00896,
A00896,
A00896,
NT
Day 203
Day 195
Day 238
Day 237
Day 288
NT
A00868,
A00868,
A00868,
A00868,
A00868,
NT
Day 203
Day 195
Day 238
Day 237
Day 288
NT
0
NT
NT
50
NT
100
A00659,
A00659,
A00659,
A00659,
A00659,
IFNg Spots (Mean +/- SEM)
IFN Secretion by NHPs Vaccinated with LVS via the SC and ID Routes
Figure 1: 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. NT: not tested on those days (i.e. A00659 was not tested
on Days 195, 203 or 237).
IFN Secretion by NHPs Vaccinated with LVS via the SC and ID Routes and Plated on the Same
Day
Cell Mean for IFNg Spots
400
Media
350
ID
LVS hk Hi
300
LVS hk Mid
250
LVS ff Hi
200
LVS ff Mid
SC
150
100
50
0
A00659
A00868
A00896
A00908
Figure 2: 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 (Hi) or 0.25 x 105/ml (Mid). Route of vaccination is shown associated with
Page 11 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
each primate. SC vaccinated NHPs are 442 days post-vaccination; ID vaccinated NHPs are 451 days
post-vaccination.
450
400
Media
LVS hk Hi
LVS ff Hi
350
300
250
200
150
100
A05477
A04367
A04344
A04339
A04274
A04168
A03033
0
A03016
50
A02386
IFNgamma Spots (Mean +/- SEM)
IFN Secretion by Individual Non-LVS Vaccinated NHPs
Figure 3: 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.
3. Data Interpretation
i. ID- and SC vaccinated NHPs do not show a consistent difference in their
ability to secrete IFN after ex vivo LVS stimulation as measured by the
ELISPOT assay
ii. More optimization on tittering the hk- and ff LVS concentrations needs to be
done in the IFN ELISPOT assay to rule out the possibility of non-specific
stimulation and inconsistent responses
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay030608.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 030608.svd; 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), TUL 21 (pps. 135 – 140), TUL 22 (141 – 145),
TUL 23 (146 – 151) and TVDC 2 bound notebook (8935): TUL 29 (pps. 21 -24).
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Concern that IFN ELISPOT assay needs to be optimized further, specifically regarding HK and FF
LVS antigen concentration and potential mitogenic activity of the latter
6. Deliverables completed
None
7. Quality of performance
Good
Page 12 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
8. Percentage completed
95% of scientific work has been completed
9. Work plan for upcoming month
1. Continue to test PBMCs from LVS-vaccinated and non-vaccinated NHPs in the IFN ELISPOT
assay to determine the effect of HK and FF LVS at different concentrations
2. Begin to work out the conditions for the flow cytometric IFN intracellular cytokine assay in
order to determine which cell types in the PBMC population are producing IFN
10. Anticipated travel
Dr. Wilder will attend the TVDC/NIAID/FDA Meeting in Washington, DC, 3/10 – 13/08 and the
Tularemia Meeting in Albany, NY, 3/29 – 4/1/08
11. Upcoming Contract Authorization (COA) for subcontractors
COA#18 approves Dr. Wilder’s travel for the Tularemia Meeting in Albany NY from 3/29 to 4/1/08
Milestone 19-UNM
Milestone description: Interaction between human alveolar macrophages and F. tularensis
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No new work 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
Needs improvement
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
Page 13 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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 SCHUS4
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 SCHUS4 proliferation in infected
macrophages
v. Determine whether T cells from vaccinated rats can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
Ali Scrymgeour, Terry Wu, Rick Lyons and Greg Tegethoff (animal caretaker) will be traveling
to USAMRIID for LVS vaccinations from March 17 to March 20, 2008
11. Upcoming Contract Authorization (COA) for subcontractors
COA#15 authorizes the travel to USAMRIID for the LVS vaccinations
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.
Page 14 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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. Previously we reported that removal of E. coli proteins from IVT lysates by 100kDa
Microcon filtration reduced or eliminated antigen-independent IL2 and INF release from
LVS-immunized T cells. Unfortunately this approach also led to significant loss in yield of
the de novo synthesized polypeptides. To reduce yield losses, we tried using filters with
a higher MW cutoff: 300kDA. Results from this experiment are shown on Fig. 1.
Filtration of undiluted IVT samples through 100kDa and 300kDa filters produced very
similar results (lines 1-4), which is that most of the proteins in the lysate are retained by
the filters. Dilution of the lysates 10-fold did not change filtration through the 100kDa filter
(lanes 7-8), but reversed the outcome with the 300kDa filter (lanes 5-6). Only small
amounts of protein are retained above the filter (retentate), and most pass through into
the filtrate.
Lane:
1
2
3
4
undiluted
300kDa
F
R
100kDa
F
R
5
6
7
8
9
10
10-fold diluted
300kDa
F
R
100kDa
F
R
IVT
L
Fig. 1. Coomassie stained proteins retained (R) and filtered through
(F) the Microcon filters at different conditions. IVT – unfiltered IVT, L
– ladder.
File location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU
gels\FTU HTP IVT Coomassie gels\Pall filters\microcon 100kd and 300
kd wo protein spike 021808 2.jpg
However, T-cell stimulation assay performed at UNM showed that filtration through
300kDa unit does not remove the apparent cross-reacting component(s) from the IVT
lysates (fig. 2).
Page 15 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Fig. 2. ELISPOT assay of the filtrate and retentate fractions.
File location: R:\GeneVac\FTU\Contract\Proteome\UNM data\100kDa
300kDa filtration.jpg
Failure to remove the cross-reactivity by filtration through the 300kDa filter suggests that
the cross-reactive components are not associated with large macromolecular complexes,
such as ribosomes which would be retained above the filter, or there are many different
components to the observed reactivity and some of these are not being retained. If so,
then the observed reduction of reactivity observed by 100kDa-filtration is likely due to an
overall reduction of proteins in the filtrate rather than to removal of a specific fraction.
2. Previously we also reported that IVT lysate spiked with recombinantly-produced FTU
proteins can be efficiently separated from majority of E. coli proteins in IVT reaction by
30% acetone precipitation. Effect of acetone fractionation on the cross-reactive
components was evaluated by ELISpot performed at UNM. Although different fractions
showed different reactivity in ELISpot (fig. 3) they generally correlated with amount of E.
coli proteins in the sample (fig. 4). This is consistent with the filter results.
Fig. 3. ELISPOT analysis of the supernatants and pellets of IVT
lysates after precipitation with a range of acetone levels.
Page 16 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
File location: R:\GeneVac\FTU\Contract\Proteome\UNM data\acetone
prec 20-50.jpg.
Fig. 4. PAGE analyses of IVT proteins fractionated acetone
precipitation.
File location: R:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT
data\FTU gels\FTU HTP IVT Coumassie gels\Acetone prec 20-50
coomassie.jpg
3. As we reported earlier, proteins made by IVT appeared to fractionate differently relative
to recombinant versions of the same proteins. They appear less soluble and precipitate
at lower acetone concentration. To address these differences, we assessed effect of
prolonged incubation on protein stability and folding. IVT reactions were incubated for
the length of time designated above each lane, given in hours.
Page 17 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Fig. 5. Effect of the length of IVT reaction (in hours) on the
stability/degradation of the de novo synthesized proteins.
File location: R:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT
data\FTU gels\FTU HTP IVT 35S gels\IVT 05-60 hrs incubation.jpg
Extended IVT reaction-time did not influence stability. However, we noted that the IVT-made
sub-fragments, such as exemplified by the FTU0721a sample shown above were comprised
of multiple bands. By contrast, the IVT-made full-length proteins, such as FTU1695, were
comprised of a single correctly sized band. We interpret these data to suggest that the
difference between the IVT and recombinant proteins was not their in vivo versus in vitro
production, but rather full length versus partial protein production.
4. We also assessed differences in His-tag accessibility of IVT and E. coli made proteins.
The same FTU1695 gene in an N-terminal His tagging construct was expressed by IVT and
in E. coli. E. coli in vivo derived-protein was purified from inclusion bodies and quantified.
This sample was spiked at different amounts into an IVT lysate. All samples were separated
on the same gel and analyzed using a new His-tag staining reagent. Results are shown in
fig. 6. A correctly sized band with intensity roughly equivalent to 2ug of in vivo recombinantly
generated protein was His-detected in the lane loaded with 14ug of IVT-made protein. These
data indicate that only a portion (2ug out of 14ug) of the subfragment-derived polypeptide
carries an accessible tag relative to recombinant generated protein. Lane 1 is an FTU1695
IVT reaction. Lanes 2 through 6 are lysate samples spiked with the given amount of
recombinant FTU1695.
1 2 3 4
5 6
Page 18 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Fig. 6. Detection of an N-terminal His-tag of FTU1695 in E. coli IVT
made protein versus E. coli recombinant made protein.
File location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU
gels\FTU IVT Western\IVT his tag detection.jpg.
These observations suggest that low solubility, specific degradation products and low
efficiency of His-tag exposure are related to improper folding of the IVT synthesized partial
proteins.
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
The well-known low immunogenicity of FTU antigens is troubling relative to the strong cross reactivities we are seeing in the ELISpot assays. In addition we are assuming that these are
heterologous reactivities. Another concern in understanding the “cross-reactivity” is the
relatively low sensitivity of the ELISPOT assays at this time, in the backdrop of very strong
cross-reactivity. We would like to discuss other possibilities such as E. coli contamination in
the LVS material, or somewhere else…
.
6. Deliverables completed
None
7. Quality of performance
Very good
8. Percentage completed
99%
9. Work plan for upcoming month
Currently we are evaluating effect of a thioredoxin fusion and chaperons additions on
solubility, acetone fractionation and His tag based purification of IVT made proteins.
10. Anticipated travel
Page 19 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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 14 (Notebook 115, pages 57-62)
i. This experiment had two objectives:
1. Determine whether filtering ivt reactions through a 300kD filter would
increase the yield of ivt proteins without increasing the crossreactivity
to E. coli proteins
2. Determine whether the crossreactive material can be removed by
differential acetone precipitation
ii. For objective 1, ASU provided retained and filtrate fractions of ivt reactions
that were filtered through either a 100 kD or a 300 kD MW cutoff filter for us
to analyze by IFN ELISpot. As shown in Figure 3, the filtrate from the 300
kD filter stimulated IFN production, indicating that the filter allowed some
crossreactive materials to flow through. Thus, the 300 kD filter provided no
advantage over the 100kD filter
Figure 3. IFN ELISpot analysis of retain and fitrate fractions of ivt reactions that were
filtered with a 100 or 300 kD filter
iii. For objective 2, ASU provided acetone precipitated pellets and supernatants
of ivt reactions that were treated with various amount of acetone, from 0 to
90%. The pellets and supernatants were tested in a T cell stimulation assay
using LVS vaccinated splenocytes. As shown in figure 4, the amount of
crossreactivity decreased in the supernatant and increased in the pellet with
increasing acetone concentration. At 50% acetone, the amount of
crossreactivity was reduced to near-background level. This suggests that if
Page 20 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
the ivt protein remains in the supernatant in sufficient quantities at 50%
acetone, then this strategy may work for generating the Ft protein library
Figure 4. IFN ELISpot analysis of the supernatant and pellet of ivt reactions after
differential acetone precipitation
b. Experiment Ftc59 study 15 (Notebook 115, pages 63-72)
i. The purpose of this experiment was to determine the sensitivity of the IFN
and IL-2 ELISpots. Specifically, we wanted to know if it is even possible to
use this assay for screening potential vaccine candidates and, if not, how to
improve this assay.
ii. For this question, we turned to the DO11.10 T cell receptor (TCR) transgenic
mice. The CD4 T cells from these mice express a TCR that is specific for
OVA323-339 presented in the context of MHC class II molecule I-Ad.
Approximately 20% of the CD4 T cells in the spleen are specific for this OVA
peptide; for comparison, the frequency of pathogenic specific T cells in a
naïve animal is only 1 in 100,000.
iii. To determine the sensitivity of the IFN and IL-2 ELISpot assays, DO11.10
splenocytes were titrated with naïve splenocytes, comprising from 0.5% to
100% of 200,000 cells/well and then stimulated with OVA protein or peptide.
iv. As shown in Figures 5 and 6, several points are clear.
1. The assay sensitivity increases with increasing antigen concentration
used for restimulation.
Page 21 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
2.
Restimulation with peptide is more sensitive than with protein,
presumably because peptides do not need to be processed to be
presented
3. IL-2 ELISpot is more sensitive than IFN ELISpot, presumably
because all activated T cells produce IL-2 but only a subset of them
secrete IFN
4. The assay sensitivity is low. The sensitivity of the IL-2 ELISpot with
5 M protein is about 0.2% of total splenocytes; it is 1% according to
figure 5, but only 20% of the DO11.10 splenocytes are OVA specific.
The sensitivity of the IFN ELISpot with 5 M protein is about 1% of
total splenocytes
v. The currently established assays are probably not useful for screening
potential vaccine candidates. However, the assay may be better if we can
increase the T cell frequency and restimulate the cells at higher antigen
concentration
Figure 5. Titration of DO11.10 splenocytes in IFN ELISpot assay to determine assay sensitivity 
Page 22 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Figure 6. Titration of DO11.10 splenocytes in IL-2 ELISpot assay to determine assay sensitivity
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
18%
9. Work plan for upcoming month
a. Determine whether we can increase the T cell frequency by taking splenocytes from
vaccinated mice with active SCHUS4 infection or after boosting with LVS.
b. Restimulate splenocytes with 10M of Ft antigens
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Page 23 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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
In anticipation that milestone 26 is nearly complete, we have pooled PCR primers for 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
30%
9. Work plan for upcoming month
Wait for MS 26 to be completed.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 24 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Milestone 35 - UNM
Milestone description: Array hybridization with mouse RNA from virulent SCHUS4
infection and RT PCR confirmation of candidates
Institution: UNM
1. Date started:
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc64 (Notebook 115, pages 73-83)
i. The purpose of this experiment is to determine the SCHUS4 gene
expression pattern at early time points after intranasal infection with SCHUS4
and to compare that with in vitro SCHUS4 gene expression pattern in culture
ii. This experiment will focus on the early time points because the rapid
pathogenesis of tularemia would most likely require that vaccines target Ft
genes that are expressed early in infection.
iii. By comparing the genes expressed in vivo in mouse lungs (stressful
conditions) and in vitro in liquid Chmberlain’s culture (unstressful conditions),
it may be possible to determine the pathways that get turned on in response
to the stresses of in vivo exposure. It is a great comparative control that will
be helpful when it comes time to publish
iv. BALB/c mice were infected i.n. with 1000 SCHUS4 and after 1, 3, 5, 7, and
24 h, the infected lungs were collected and processed to isolate total
eukaryotic and prokaryotic RNA
v. For the in vitro gene expression, SCHUS4 was inoculated into
Chamberlain’s broth and after 1, 3, 5, 7, and 24 h the bacteria was collected
and processed to isolate total bacterial RNA
vi. The RNA will be shipped to ASU on March 10, 2008
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%
9. Work plan for upcoming month
None
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35
Page 25 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Milestone description: Array hybridizations with mouse RNAs from virulent SCHU4
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

We previously reported that we had anomalous results with amplifications of the second dose
response samples in that the RNA from animals challenged with 106 and 107 organisms did
not amplify well or perform well on the arrays. We performed a repeat LAPT (experiment
LAPT-22) to compare with the previous data (experiment LAPT-20). In LAPT-22 we did not
observe the problems associated amplification loss in the 106 and 107 challenge dose
samples. All samples amplified well in the repeat. We first compared the microarray
intensities between the samples of LAPT-22 (Figure 1 left panel). The correlations between
the doses were not as high as previously measured which were consistently > 0.7. We
further compared the correlations between the two independent amplifications which were
very reasonable between the 103 and 105 doses. Because of the major problems in LAPT20, the lower correlations between the 106 and 107 doses were not unexpected.
Figure 1. Spearman correlations between samples in LAPT-22 (left panel) and between
doses between LAPT 20 and 22 (right panel).

We next performed a Venn diagram analysis to compare the top 500 genes detected in the
two experiments to determine which ones were tested common between the two independent
amplifications of LAPT-20 and LAPT-22 (Figure 2). The results show that approximately 5060 percent of the identified genes are concordant between the two amplifications.
Page 26 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Figure 2. Venn analysis of the top 500 genes detected in the 10 3-105 samples between
LAPT-20 and LAPT-22.

We next identified the intersection of the top 500 genes between the two independent
amplifications using the 103-105 doses (Figure 3A, Left panel). As reported last in the
previous tech report 293 genes are concordant between the doses of LAPT-20. The number
of genes that overlap between challenge doses in LAPT-22 were less at 194. We next
identified the genes that overlap between the two data sets. The 293 genes identified in
LAPT-20 and the 194 in LAPT-22, were cross verified and 141 were identified by both
analyses (Figure 3B).
Figure 3A. Overlap of dose response between
doses of LAPT-20 and LAPT-22

Figure 3B. Identification of genes that
overlap between the LAPT-20 and
LAPT-22
A list of the top 32 genes identified by both LAPT-20 and LAPT-22. This is the list that we will
start verification of expression utilizing quantitative PCR
Page 27 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Table 1. Top 32 gene list identified in LAPT-20 and LAPT-22.

Notebook/File locations …, Notebook 514, LAPT 22, page 174-183.
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-20 (Pooled NM Samples).
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-22 (second run of NM
Samples)
4. Significant decisions made or pending.
Analyses of LAPT samples will be performed by at least two independent amplifications to gather
data.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
19%
9. Work plan for upcoming month

Repeat the amplifications of the pooled samples from 103-106 to assess reproducibility of the
LAPT process.
Page 28 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam


A time course experiment has been completed and RNA is expected the week of March 10 th.
The experimental design is for animals to receive 103 CFU SCHUS4 intranasal challenge and
tissues will be harvested at 1,3,5,7 and 24 hours. Separate SCHUS4 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. We will start the RNA Clean-up and LAPT analysis.
Establish quantitative PCR analyses and initially assess 5 genes for expression levels in
multiple samples. These genes will be picked using the microarray data set to span
potentially high, medium, and low level expression.
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).
Page 29 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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
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.
Page 30 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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
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.
Page 31 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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
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
Page 32 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
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
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
Page 33 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis (SCHUS4)
(iglC, pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHUS4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHUS4), Construct iglD F. tularensis
subsp. tularensis (SCHUS4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHUS4), Construct iglB F. tularensis
subsp. tularensis (SCHUS4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHUS4 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 SCHUS4 itself using constructs that
we believe will allow us to make deletions into SCHUS4.
I.
Cloning:
a.
The sequence analysis of KEK1140 containing the igLD introns C16-30a and C12-255a
constructs, respectively, were confirmed to have the respective cloned introns (30aigLD
and 255aigLD).
b.
In order to delete an entire pathogenicity island (FPI) from SCHUS4 we need to move the
pdpD deletion containing the flip recombinase recognition sites from the pwsK30 plasmid
into pUC118 vector. The new oligos containing the kpn I restriction endonuclease site
were used with pwsk30+pdpD deletion (KEK1188) construct as the template and
generated a PCR product of the expected size of ≈3800 bp (see figure 1). This product
was treated with phenol and chloroform then ethanol precipitated. This pdpD deletion
product was reconstituted in sterile water and kpn I restriction endonuclease was added
with the appropriate reaction buffer then incubated overnight at 37° C. The pUC118
plasmid will also be digested with kpn I restriction endonuclease and once gel purified will
be used with the mentioned PCR pdpD::ErmC::flip kpnI product in a ligation reaction.
The results will be discussed on next report.
Figure 1.
This represents the pdpD::ErmC::Flip gene product that was generated from the pKEK1188 pdpD contruct
made earlier as template with the kpnI pdpD oligos. This product was digested with kpnI restriction
endonuclease and run on a 1% gel. The product should be 3800 bp. Data located in TVD UTSA
Notebook 5, page 110.
Page 34 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
II.
Experiments to generate mutants in Schu4:
a.
Continued screening of our Schu4 vgrG tulatron mutant with primers specific to vgrG
yielded much of the same results (ie. No shift in vgrG size due to intron insertion).
Carefully examination of the original sequence that was sent to Sigma to generate
primers showed that there was an error. Close to 80 base pairs of extra sequence
upstream of the start codon of vgrG was sent. Therefore, the primers that target
basepairs 30/31 and 81/82 do not target their respective sites within vgrG, but instead
target 30/31 and 81/81 within the sequence sent to Sigma. Primers 30/31 do not target
vgrG but instead target DNA upstream of vgrG. Luckily primers 81/82 target 3 bases into
the vgrG. This is why we saw no shift in size when we use start and stop primers to
vgrG. The start primer anneals just past where the intron should be.
b.
With this knowledge we decided to screen the 81/82 clones for insertion of the intron with
a primer that anneals to the gene just upstream of vgrG, FTT1346, in combination with a
primer that anneals to the stop codon of vgrG. In a wildtype strain, PCR with these two
primers should give a PCR product of ~900 base pairs. However, if the intron has indeed
inserted at base pair 3 and 4 of vgrG, then the PCR product should be ~1500 base pairs.
With this in mind we screened 12 genomic prepped clones with these sets of primers
(FTT1346 fwd NdeI and vgrG rev RI). Figure 2 shows that clones 1-3,5,6,11, and 12
have this shift we are looking for. As seen before there is a mixed population within the
same clone of wild type vgrG and shifted vgrG. I took these same clones and did PCR
using primers specific to the intron (EBS universal) and specific to vgrG (vgrG rev RI).
This set of primers will tell us if this shift is due to the intron. Figure 3 shows the results
of this PCR screen and that indeed, the intron has inserted into vgrG due to the
appearance of a ~500 base pair PCR product in the clones that is absent in wildtype
Schu4 genomic prep (lane 1). The PCR product of clone one was sent off for sequencing
and indeed the sequence verifies that the intron has inserted into vgrG and this insertion
is between bases 3 and 4. Clone 1 will be streaked on a fresh plate to isolate single
colonies. These new clones will then be screened again with the same set of primers in
hopes of seeing a pure population of shifted vgrG, not a mix of wild type and shifted. As
has been the case with previous tulatron mutants, obtaining a single population can take
3 or more cyclings. Data is located in TVDC notebook 1, pages 26-27.
Figure 2.
1
2
3
4
5
6
7
8
9 10
11
12
13 14
2000
1600
1000
PCR showing a 1000 bp band in all samples, including Wt Schu4 (lane 2). An additional band
showing a shift in the size of vgrG (the band below the 1600 bp marker as shown and labeled in
the figure) was seen in lanes 3-5, 7,8,13 and 14. Data is located in TVDC notebook 1, pages 26-27
Page 35 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Figure 3.
1
2
3
4
5
6
7
8
9
10
11 12 13 14
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
ladder
S4
clone1
clone2
clone3
clone4
clone5
clone6
clone7
clone8
clone9
clone10
clone11
clone12
PCR showing an amplification of PCR product in the potential mutants (lanes 3-14), but not in Wildtype
Schu4 (lane 2). These primers anneal to the intron and to vgrG, giving a PCR product only if the intron
has inserted into vgrG.
b.
Isolated genomic DNA from seven of each of the respective igLD tulatron transformants
30a IgLD (3D) and 255a IgLD (2D) groups. These genomics were used as templates
with intron specific oligos (EBS Universal and EBS2) and igLD gene specific (igLD NcoI
rev and igLD NdeI for) oligos. Only the 255a IgLD group gave a positive result. Figure 4
shows the results from the polymerase chain reactions for the 255a IgLD group. Data
located in TVD UTSA Notebook 5, page 109.
Figure 4.
This represents two PCR screens using 255a igLD tulatron transformants’ genomic
preparations as templates in respective reactions (lanes 3-9). The 255a igLD tulatron
transformants are designated as 2D followed by their respective clone number. Panel A
represents the PCR products generated when using EBS Universal oligo (intron specific) with
IgLD Nco I for (gene specific) oligo. The expected size of ≈400 base pairs (bp) will indicate that
the IgLD intron is in the SCHUS4 chromosome. Panel B represents the PCR products
generated when using EBS2 255/256s oligo (intron specific) with IgLD Nco I rev (gene specific)
oligo. The expected size is ≈1500 bp and would indicate that the igLD is in the SCHUS4
chromosome. Lane 10 represents a colony pool from transformants 2D1, 2D2, 2D3 and 2D4;
this is a crude isolation which can also be used in PCR screening. In this case, the reaction
did not work for the pool because the first step had a melting time for 1 minute instead of 3
minutes. Lane 2 is the SCHUS4 strain used in the transformation experiment and is considered
Page 36 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
wild type which should not generate a product with either of the oligo sets used in this screen.
Data located in TVD UTSA Notebook 5, page 109.
c.
We wanted to verify that the intron was inserted in both genes, igLD1 and igLD2 so we
did a PCR using the igLD gene specific oligos that lie outside to the intron location. The
correct mutant should yield a 800 bp shift compared to the wild type igLD profile. Figure
5 shows that these clones are either mixed populations or the intron integrated only is in
one gene copy for the 2D igLD tulatron group. Whereas, the 3D igLD tulatron group did
not yield any positive potential igLD mutants.
Figure 5.
Legend.
1.
2.
3.
4.
5.
6.
7.
8.
1 Kb Ladder
KKT ∆Blac2
2D1
2D2
2D3
2D4
2D5
2D6
10.
11.
12.
13.
14.
15.
16.
17.
SCHUS4
3D1
3D2
3D3
3D4
3D5
3D6
3D7
This represents PCR products generated when using genomic templates from the 255a igLD
tulatron group (2D, panel A) and the 30a igLD tulatron group (3D, panel B). The oligo set used
with these genomic templates were igLD NdeI for and igLD NcoI rev. The lanes labeled 2 and 10
are controls which will illustrate the wild type expected size when these oligos are used (≈1100
bp). Panel A represents the 2D group and shows multiple PCR product one of which is the
expected mutant size of ≈1900 bp; panel B represents the 3D group and only generated a single
PCR product equal to the wild type profile. Lane 1 is 1 Kb ladder from Invitrogen. Data located in
TVD UTSA Notebook 5, page 104.
d.
Isolated genomic DNA from igLC mutants generated in earlier report labeled as KKT5 (TIgLC) and KKT6 (T-igLC single) clones, in addition, to wild type SCHUS4 and KKT1 the
Blac2 mutant. Prepared some digestions with EcoRI and BamHI enzymes, respectively,
to use in preparing a 0.5% agarose gel to use to transfer DNA to nitrocellulose paper.
Given that each of the two igLC copies are identical at the DNA level; the restriction
endonucleases which cut outside of these genes and generate different fragment sizes
will be the ideal enzyme to use for southern analysis. The predicted fragment sizes that
differentiate the two igLC gene copies with the BamHI enzyme are at 77 Kb (igLC1) and
34 Kb (igLC2) in size, respectively. This may be outside the limitation of the standard
southern blot. I will check to see if one can differentiate these two genes in a traditional
Page 37 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
southern blot and will be reported on next month’s report. Data located in TVD UTSA
Notebook 5, page 107 and 108.
Did some ordering for enzymes and general supplies for ongoing experiments.
e.
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
61%
9. Work plan for upcoming month
b.
c.
d.
e.
Will continue the screening for potential igLD mutants in KKT1 SCHUS4 strain.
Will continue to screen for vgrG mutants in KKT1 SCHUS4 strain.
If time allows, will continue with the pdpD flip pwsk30 contruct cloning into pUC118.
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. May need to do a pulse field gel electrophoresis to
identify clearly each iglC gene disruption. This method uses a different way of separating the
resulting restriction endonuclease fragments that result when using a specific enzyme.
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.
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 (SCHUS4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHUS4) 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 (SCHUS4) 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 (SCHUS4) iglC
mutants (Note book#1 page 148-149): Murine macrophage cell line (J774) were seeded
in a 96-well plate (105/200 μl/well) overnight and infected with the SCHUS4 wild type, or
mutants (iglC, mglA) or the F. novicida (U112) iglC mutant using an inoculum of 10
MOI. Numbers of viable bacteria in macrophages were measured at 3 hr and 24 hr postinfection. Results showed that unlike its parental strain the SCHUS4 iglC mutant had
minimal replication in macrophages (Fig.1). This attenuation in intramacrophage
replication is comparable to that seen in the iglC F. novicida mutant and the mglA of
SCHUS4 mutant
Page 38 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
6
3h
24 h
CFU (Log 10)
5
4
3
2
1
0
WT
mglA
SCHU S4
iglC
iglC
U112
Fig. 1. Intramacrophage survival of iglC mutant. Murine macrophage cell line (J774)
were infected with the iglC mutant or its parental strain (SCHU S4) using an inoculum of
10 MOI. Two previously characterized mutants, mglA of SCHU S4 and iglC of F.
novicida (U112), with minimal growth in macrophage were also assayed for
comparison. Numbers of viable bacteria in macrophages were measured at 3hrs and 24
hrs post-infection.
(2) Evaluate the protective efficacy of F. tularensis SCHUS4 iglC vaccination against
wild type SCHUS4 challenge (Note book #1 pages 146-147 and 150-153): Groups of
BALB/c mice (female, 4-6 weeks) were immunized with 103 CFU of iglC intragastrically
(i.g.) or intradermally (i.d.). Sera and fecal pellets were collected at day 21 after
immunization and assayed for anti-iglC specific antibody titers. Mice immunized with
iglC by either the i.d. or i.g. route induced significant amount of serum antibody as
shown in Fig. 2. Further IgG isotyping analyses of the sera indicated i.g. immunization of
iglC resulted in producing comparable titers of IgG1 and IgG2a, while i.d. vaccination
induced dominant IgG2a (arguable Th-1 bias immunity). Intragastric immunization also
induced measurable anti-iglC specific secretory IgA in the prepared fecal pellet samples
(Fig. 3.).
5000
i.g.
i.d.
Ab Titer
4000
3000
2000
1000
0
Total A b
IgG1
IgG2a
Fig.2. Humoral responses induced by SCHUS4 iglc immunization. Mice were vaccinated
with 1000 CFU of the iglc-null mutant by the intragastric (i.g.) or intradermal (i.d.) route.
Sera were prepared 3-week after immunization, and assayed for anti-iglc specific
antibody.
Page 39 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
0.50
A 414
0.40
0.30
0.20
0.10
0.00
IgA
IgM
IgA
iglC
IgM
Moc k
Fig.3. Mucosal immunity induced by SCHUS4 iglc intragastric immunization. Mice
were vaccinated with 103 CFU of the iglc-null mutant and fecal pellets were collected
3-week after immunization for anti-iglc specific antibody assays. Mice received PBS
are used as mock control.
These mice were challenged intranasally or i.d. with two doses of SCHUS4 3-week after
vaccination. Animals were monitored for survival and weight loss. Here we summarize the
survival data of this ongoing experiment (first week post challenge) in table 1. The completed
survival and weight loss data as well as statistical analyses will be presented in the next
monthly report.
Table1. Protective efficacy of SCHUS4iglC mutant against homologous wild type challenge
Route of
Vaccination
Route of
Challenge
i.n.
i.d.
i.d.
i.n.
i.g.
i.d.
i.n.
Mock
i.d.
Challenge
Dose
(CFU)
20
100
20
100
20
100
20
100
20
100
20
100
D3
D4
Survival Rate
D5
D6
D7
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
2/6
0/6
0/6
6/6
6/6
5/6
5/6
5/6
2/6
6/6
6/6
4/6
0/6
0/6
0/6
4/6
3/6
5/6
5/6
0/6
0/6
3/6
4/6
2/6
0/6
0/6
0/6
2/6
2/6
4/6
5/6
0/6
0/6
2/6
1/6
0/6
0/6
0/6
0/6
Page 40 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
50B. (1) Analyze the antibody profiles of mice intragastrically immunized with F. holarctica LVS
at 8 weeks after vaccination. (Note book #8, pages 7, 15, 28, 55-56). Mice were
vaccinated intragastrically with 103 CFU LVS or mock immunized with PBS alone. At 8
weeks after inoculation, blood was collected and sera were prepared. Some mice
received a second booster dose of 103 CFU LVS I.G. Blood was collected from these
mice three weeks after booster vaccination dose and sera were prepared. Specific antiLVS total antibody titers were determined by ELISA. Antigens, either UV-irradiated LVS
(106/well) or HEL (Hen Egg Lysozyme, 100ng/well, an unrelated antigen as control), were
coated onto 96-well microplates and reacted with serial dilutions of sera. Goat anti-mouse
Ig(H+L) antibody conjugated with horseradish peroxidase was used as the secondary
antibody to determine antibody titers. As shown in Fig. 4 mice immunized with LVS I.G.
retain high total antibody titers 8 weeks after vaccination and mice which received a
second dose of LVS at 8 weeks after initial vaccination had slightly elevated titers. No
LVS-specific antibody was detected in mice mock-vaccinated with PBS. All tested serum
samples showed no reactivity to the unrelated HEL protein.
20000
LVS IG 8 wk
10000
LVS IG 8 wk Plus IG Boos t
50% Binding T iter
Moc k (PBS)
1000
100
LVS
HEL
Fig.4 Total serum antibody responses 8 weeks after LVS IG vaccination. Groups of mice
(12 mice/group) were inoculated IG with 103 CFU LVS. At 8 weeks after vaccination, blood
was collected and sera were prepared. One group received an additional booster dose of
103 CFU LVS IG. Three weeks later, blood was collected from these mice and sera were
prepared. Sera were analyzed to determine titers for anti-LVS specific antibodies.
(2) Analyze the cellular responses to mice vaccinated intragastrically with F. holarctica
LVS at 2 and 4 weeks after vaccination. (Note book #8, pages 43-45) Mice were
vaccinated I.G. with 103 CFU of LVS IG or mock vaccinated with PBS alone. At either 2
or 4 weeks after immunization, spleens were collected, single cells were made and
incubated in the presence of increasing amounts of UV-inactivated LVS (103-105 CFU) for
72 hours. Splenocytes were also cultured in the presence of the unrelated antigen HEL or
media alone as controls. At the end of the culture period, supernatants were collected
and analyzed for IFN-production using the BD OptEIA Mouse IFN- ELISA Set (BD
Biosciences) per manufacturer’s instructions. As seen in figure 5, cells from mice
collected at 2 weeks after LVS vaccination produced high levels of IFN-which increased
when cultured with higher doses of LVS. Little to no IFN- was produced by cells from
Page 41 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
mock vaccinated mice, or by cells cultured with HEL. As seen in Figure 5B IFN- was
also produced by cells collected at 4 weeks after vaccination, although at a lower level
than at 2 weeks after vaccination. Again, little to no IFN- was produced by cells from
mock vaccinated mice, or by cells cultured with HEL.
Mo ck (PBS) I.G.
LVS I.G.
B 8
2 Weeks
A 14
4 Weeks
IFN-  (ng/mL )
12
6
10
8
4
6
4
2
2
ia
EL
ed
M
H
10 5
S
LV
S
10 4
10 3
S
LV
LV
ia
ed
H
M
10 5
LV
S
S
10 4
10 3
LV
S
LV
EL
<31.250
<31.250
Fig. 5 Cellular responses to LVS IG vaccination. Groups of mice (3 mice/group) were
inoculated IG with 103 CFU OF LVS. At either 2 weeks (A) or 4 weeks (B) after
vaccination, spleens were collected, single cells were prepared and incubated in the
presence of LVS, and supernatants were analyzed for IFN- production.
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
62% of scientific work completed on milestone 50A (original plans)NORMALIZED TO 31%
33% of scientific work completed on milestone 50B (intragastric plan)NORMALIZED TO 16.5%
9. Work plan for upcoming month
50A: (1) Continue monitoring the survival and weight loss of the iglC-immunized/SCHUS4challenged mice in the ongoing experiment.
(2) Evaluate the protective efficacy of intragastric F. novicida iglB vaccination against
SCHUS4 intranasal and intradermal challenge in C57BL mice.
50B: (1) Evaluate the protective efficacy of intragastric LVS vaccination against Francisella
type A SCHUS4 intranasal challenge at 8 weeks after either a single vaccination or after
receiving a secondary booster dose. We will initiate this experiment and results are expected
to be reported three months later.
Page 42 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
(2) Analyze granzyme B activity in spleens and lymph nodes at both 2 and 4 weeks after
intragastric inoculation with LVS.
(3) Measure LVS dissemination to target organs after intragastric immunization by PCR.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 52
Milestone description: Create RecA mutants in F. tularensis subsp. tularensis(SCHUS4)
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 SCHUS4, 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.
a.
In the technical report for last month, we reported that colony 7, which had intron RNA
insertion in RecA at 840/841bp in LVS, was streaked onto TSA++ /Kanamycin(30ug/ml)
agar media, and incubated at 30C for at least 4 days to isolate RecA mutant LVS from
wild type LVS carrying the plasmid pKEK1187.
b.
After 5 days incubation at 30C, about 32 single colonies were selected and patched onto
TSA++/Kanamycin plate and incubated at 30C for two days.
c.
The colony PCR was performed with “RecA SchuS4 Rev “and RecA SchuS4 For”
primers flanking the insertion and in RecA gene of LVS. The PCR reaction was set up as
follows:
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
0.4ul
DNA
1.0 ul
At 98C 1minute, 98C 15seconds// 57C 15seconds// 72C 2minutes/ 30
cycles
Page 43 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
d.
Figure1: 1% agarose gel for colony PCR
Lane11 was colony 7 (mentioned in section 3.1) as the positive control and lane12 was wild type LVS as
the negative control. Lane2 to lane 10 were the 9 colonies isolated from colony7 with the PCR product at
about 1500bp. There was no other band present at the same size as wild type LVS (about 630bp) on
lane2 to lane10. It proved that the 9 colonies (lane2-lane10) were the pure recA mutant LVS without
being mixed with wild type LVS carry the plasmid pKEK1187.
e.
Since the plasmid pKEK1140 (the backbone of the Tulatron vector) was a temperature
sensitive plasmid and also resistant to Kanamycin, it could be removed from the strain
after change of the temperature from 30C to 37C and screened for Kanamycin
selection to check the loss of the plasmid. Colony 1 and 3(lane2 and lane4 on Figure1)
were streaked onto TSA++/Ampicillin(100ug/ml) agar plate respectively, and incubated at
37C for about 2-3 days to remove the plasmid from the RecA mutants.
f.
Patched the single colonies after incubation at 37C from section 3.5 onto both
TSA++/Ampicillin(100ug/ml) and TSA++/Kanamycin(30ug/ml) agar plates and incubated
at 37C to determine if the plasmid had been removed from the mutant strain. 12 colonies
were observed sensitive to Kanamycin, so they might be ∆RecA LVS with the plasmid
being removed, but the colony PCR needed to be performed to screen them.
g.
The 3 of 12 colonies were screened by colony PCR. The PCR reaction with “RecA
schus4 for” and “RecA schus4 rev” primers was set up the same as Section 3.4. The
PCR reaction with “RecA SchuS4 For” and “EBS Universal”(in intron RNA) primers was
set up as follows:
ddH2O
34.6 ul
10X Buffer
5.0 ul
MgCl2
2.0 ul
dNTPs
5.0 ul
RecA Schus4 For (25pmol/ul)
1.0 ul
RecA Schus4 Rev (25pmol/ul)
1.0 ul
KOD DNA polymerase
0.4ul
DNA
1.0 ul
At 98C 1minute, 98C 15seconds// 55C 15seconds// 72C 1minutes/ 30 cycles
Page 44 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Figure 2: 1% agarose gel for colony PCR
The colony PCR confirmed that those 3 colonies (sensitive to Kanamycin) were correct (without the
plasmid inside and with intron RNA insertion in RecA of LVS). Lane2 (no band) and lane7(band about
630bp) were wild type LVS as the negative controls. Lane3 (about 600bp) and lane8(about 1500bp) were
the positive controls. The colony6,12 and 36(lane4-6 and lane9-11) had the same size bands as the
positive controls (see the detailed explanation in last month technical report).The colony6 was designated
KKF343 and frozen away.
h
Ping Chu got her access to BSL-3 lab in the end of Feb, and she started to work there
since then. The wild type SCHUS4 was inoculated from the frozen stock onto
TSA++/Ampicillin(100ug/ml) plate and incubated at 37C for 1-2 days. The wild type
SCHUS4 from the starter culture was cultivated onto the fresh TSA++/Ampicillin plate
and incubated at 37C for overnight for cryotransformation.
i
The plasmid pKEK1186 (at 720/721 retarget site) was transformed into wild type
SCHUS4 using cryotransformation. After incubation in Chamberlain’s liquid media at
30C for at least 4 hours, the transformed cells were plated onto
TSA++/Kanamycin(30ug/ml) agar media and incubated at 30C for at least 4 days. The
transformation was just done recently and the colonies have not been able to be
observed.
Data recorded on UTSA TVDC notebook #6, page11-14 for figure1 and 2.
4
Significant decisions made or pending
None
5
Problems or concerns and strategies to address
None
6
Deliverables completed
KKF343 (RecA mutant LVS)
7
Quality of performance
Good
8
Percentage completed
Approximate 18% of scientific work completed on the milestone
9
Work plan for upcoming month
i Screen the transformants for transformation of pKEK1186 into wild type SCHUS4.
ii Select the pure RecA mutant SCHUS4 without being mixed with wild type SCHUS4.
iii Remove the plasmid from the SCHUS4 mutant strain.
10 Anticipated travel
Page 45 of 46
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2008 to 2/29/2008
Due Date: 3/20/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Karl Klose, Bernard Arulanandam
Ping Chu will participate in the Tularensis workshop in Albany, NW from 31st Mar to 1st Apr ,2008.
11 Upcoming Contract Authorization (COA) for subcontractors
None.
Page 46 of 46