Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, 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, 7, 11, 12/13(UNM/LBERI), 14, 17, 19, 21(UNM/LBERI), 26, 27,
28, 35(ASU/UNM), 49, 50, 52, 55
Completed milestones: 1, 25, 32, 33, 34 (UNM/ASU), 16, 39, 40, 43 (UTSA), 48, 51
Milestones terminated after initiation: 41, 42, 44, 46, (MSCR will be written)
Milestones terminated before initiated: 43 (Cerus), 45, 47 (MSCR will not be written)
Inactive milestones: 6, 8, 9, 10, 15, 18, 20, 22, 23, 24, 29, 30, 36, 37, 38, 53, 54, 56,
57, 58, 59
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/2005
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. 13 LVS vaccinees have volunteered to donate blood for immunoassay development
under the TVDC.
b. UNM EOHS is clarifying the USAMRIID annual health screening requirements
4. Significant decisions made or pending
a. Dr. Lyons received UNM IRB approval to allow blood draws on the vaccinated LBERI and
UNM scientists after their LVS vaccinations. The LVS vaccinated LBERI and UNM
scientists and staff are being offered the opportunity to volunteer to donate bloods for the
development of immunoassays, approximately 2 months after receiving the LVS
vaccination.
b. USAMRIID has temporarily halted offering the LVS vaccine as of 4/29/08, until a new
study protocol is activated, possibly by late summer 2008
c. UNM (4) and LBERI (33) are vaccinated; UNM and LBERI could offer the LVS
vaccinations up to 9 more scientists to total up to 46. The CRDA with USAMRIID is valid
for 2 years, ending June 2009.
5. Problems or concerns and strategies to address
a. One UNM and one LBERI scientist are medically pending. One UNM scientist may be
rescheduled for LVS vaccination. The next LVS vaccinations will not occur until
USAMRIID’s new protocol is activated.
b. The UNM TVDC Project Manager has requested pricing for the annual health screening
to be performed at the UNM Employee Occupational Health Services for the LBERI and
UNM LVS vaccinees.
Page 1 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
c. The 37 LVS vaccinees will not begin annual health screenings until approximately
9/11/2008.
d.
6. Deliverables completed
A total of 37 participants (33 LBERI and 4 UNM participants)have received the LVS vaccination
since 9/11/07.
7. Quality of performance
Excellent
8. Percentage completed
66% of the scientific work is complete
9. Work plan for the next month
a. Obtain UNM EOHS pricing for the annual health screening required by USAMRIID and
being performed at UNM for the LBERI and UNM LVS vaccinees.
b. UNM will be obtaining blood donations from LVS vaccinees for immunoassay
development and reimbursing participants $40/ donation.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
UNM will be requesting a modification to COA#15, once the annual health screening cost is
established.
Milestone 3
Milestone description: Bioaerosol technique selected and optimized
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. Work on the milestone completion report was continued. It will be completed once the
SCHU S4 growth procedures have been completed (discussed below in section c).
b. The Aeromist nebulizer is no longer being manufactured. Because of this change a
similar nebulizer, the Hospitak, was tested with Bacillus globigii (BG) spores and
compared to the Aeromist nebulizer. The data reported in Figure 1 is based on 3
Aeromist runs and 6 Hospitak runs.
Page 2 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Spray Factors vs CFU/L: Freshly Reconstituted Bacillus globigii Spore
Testing with the Aeromist and Hospitak Nebulizers
6.0
CFU/L (log10)
5.0
4.0
3.0
2.0
1.0
0.0
-4.5
-5.0
-5.5
-6.0
-6.5
-7.0
-7.5
-8.0
Spray Factor (log10)
Aeromist, 10 psi
Hospitak, 10 psi
Figure 1: Spray Factors versus CFU/L of freshly reconstituted Bacillus globiggi spore testing with
the Aeromist and Hospitak nebulizers.
c.
The mean spray factor was 1.30 X 10 -6 ± 1.61 X 10-7 for the Hospitak which is similar to
the Aeromist. Hospitak can hold a working volume up to 20 mL where as the Aeromist
can only hold up to 10 mL. This increase in volume will allow for longer exposure times.
These data are located in the following folder: \\Saturn\absl3\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\\FY06\FY06-078 TUL03\TUL-03 data files\BSL-2 (BG) testing
SCHU S4 growth curves were prepared due to issues with the preparation of challenge
material for the 14MAY08 challenge of two primates (refer to Milestone 4 for further
detail). Two working stocks were tested- 6SEP07 and 28FEB08. The bacteria were
cultured onto BCGA plates and then one colony was picked, normalized to an OD600 of
approximately 0.05 and inoculated into 100 mL Chamberlain’s broth in 500 mL baffled
flask and incubated in the dark with shaking (200 RPM) for up to 48 hours. Two flasks
were independently initiated from 2 separate colonies on the BCGA plates as biologic
replicates, so the graph shows 2 OD and 2 CFU growth curves from the 6SEP07 working
stock and 2 OD and 2 CFU growth curves from the 28FEB08 working stock. At defined
times during the incubation (hourly beginning at 12 h post-inoculation) a sample was
removed, analyzed at 600 nm, and cultured to determine viable CFU/mL. The graph
below outlines the growth curve data for the two stocks. These data are located in the
following folder: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY
SPECIFIC
DATA\\FY06\FY06-078
TUL-03\Schu4
Growth
characterization.
Page 3 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Time (h)
10
1.50E+10
9
1.30E+10
8
9.00E+09
6
5
7.00E+09
4
CFU/mL
Normalized OD600
1.10E+10
7
5.00E+09
3
3.00E+09
2
1.00E+09
1
0
-1.00E+09
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
OD600, 6SEP07 WS, Flask 1
OD600, 6SEP07 WS, Flask 2
OD600, 28FEB08 WS, Flask 1
OD600, 28FEB08 WS, Flask 2
CFU/mL, 6SEP07 WS, Flask 1
CFU/mL, 6SEP07 WS, Flask 2
CFU/mL, 28FEB08 WS, Flask 1
CFU/mL, 28FEB08 WS, Flask 2
Figure 2: SCHU S4 growth curves prepared with 6SEP07 and 28FEB08 working stocks.
These data show that the log phase for SCHU S4 initiated at approximately 18 hours
post-inoculation and the stationary phase was reached approximately 32 hours postinoculation. These data will be confirmed with a third set of growth curves using the
28FEB08 stock. Once the data is confirmed, two bioaerosol sprays with bacteria grown
for 24 hours will be compared to two bioaerosol sprays with bacteria grown for 48 hours
to verify that bacteria harvested at 24 hours is viable and stable under the aerosol
conditions compared to bacteria harvested at 48 hours.
Data indicate that the CFU counts for the 6SEP07 cultures were significantly lower than
the 28FEB08 flasks. The reasons for this are currently unknown, but it may be
hypothesized that the low and/or missing values were due to halting of the growth curve
analysis at the early log phase (22h post-inoculation) thus excluding the majority of the
measurable colony counts. Cultures were performed again at 36 and 38h postinoculation and indicated a decrease in titer similar to what was observed at 48h postinoculation for the 28FEB08 working stock. It is possible that the 6SEP07 stock reached
stationary/death phase significantly sooner than 28FEB08 stock though further testing
would be needed to confirm this. However, based on the age of the former stock (greater
than 6 months), it will not be used in future bioaerosols. Virulence of the two working
Page 4 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
stocks should be similar since they both originate from the same seed stock shown to be
highly virulent using our bioaerosol methods in a mouse model.
4. Significant decisions made or pending
a. The challenge material preparation will be redefined upon completion of the growth curve
studies.
b. Based on the growth curve data obtained to date, bacteria will be harvested for
bioaerosols during the mid-log to late-log phase (approximately 26-30 hours).
5. Problems or concerns and strategies to address
a. The Aeromist nebulizer is no longer manufactured. Preliminary data shows that the
Hospitak nebulizer is similar to the Aeromist nebulizer. If it becomes necessary to switch
nebulizers, the Hospitak will be tested with SCHU S4 to demonstrate similarity between
the two nebulizers.
b. The pre- and post-spray and AGI suspensions from the NHP challenge were negative for
bacterial growth and the growth curve had a poor R 2 value and a conflicting concentration
compared to the original suspension (from the 6SEP07 working stock) cultured on a
BCGA plate (refer to Milestone 4 section for further details). Based on the hypothesis
that the bacteria may have been dying due to depletion of nutrients or the stationary
stage they were in at the time of the challenge, multiple growth curves will be performed
to determine when the bacteria reach lag, log, and stationary phase of growth.
Additionally, the volume of broth will be increased to 100 mL and the speed increased to
200 rpm. Two of these curves have been prepared and are outlined above in section 3c.
Preparation of a third set of growth curves is pending. Aerosols with bacteria grown for
24 hours and 48 hours will be performed upon completion of the growth curve
characterization to demonstrate viability and stability of the bacteria under the aerosol
conditions.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
98.5% of the scientific work is complete
9. Work plan for next month
a. Complete Milestone Completion Draft Report
b. Complete and submit SOP drafts used on MS3
c. Confirm the results of the growth curves with a third curve with the 28FEB08 stock. Once
the results are confirmed, perform bioaerosol sprays to demonstrate predictable sprays.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Page 5 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions:
a. A bioaerosol challenge with two additional primates (A05254 and A05262) was
performed to confirm that aerosolized Schu S4 freshly grown in Chamberlain’s broth is
virulent in NHPs. Before challenge the primates were screened for background titers
(data are reported in Milestone 12/13 section). The primates were then challenged on
May 14, 2008. The targeted challenge delivered dose was 25,000 CFU SCHU S4
(working stock used was 6SEP07), which was approximately 1 log higher than December
2007 NHP challenge dose with the goal of exposing the NHP to a definitely lethal dose of
SCHU S4. Pre- and post- spray and AGI suspensions were cultured onto BCGA in
triplicate at three dilutions. There was little to no growth on all of the plates. The original
bacterial suspension (pre-spray) had growth at a concentration equivalent to 1 X 109
CFU/mL. This concentration was at least 2 logs lower than what was expected based on
the previous growth curve (2 X 1011 CFU/mL). Of note, the growth curve had a poor R2
value (0.24) and there was significant clumping of bacteria and relocation to the side of
the flasks. The challenge dose could not be properly calculated though it can be
assumed that the animals were delivered a dose equal to or less than the dose delivered
in December 2007 (2500-5000 CFU)
Although the cause of these issues is unknown, it is hypothesized that the bacteria may
have been dying and/or in a weakened state (i.e. post-stationary growth phase)
unsuitable for bioaerosols. It was noted that 50 mL of broth may not be enough volume
to support the number of bacteria present and so the nutrients are depleted rapidly.
Additionally shaking at 150 rpm may be too low and lead to the clumping and relocation
of bacteria to the sides of the flasks. These issues will be addressed in additional studies
(discussed in Milestone 3).
d. After challenge, both of the primates (A05254 and A05262) presented typical signs of
tularemia infection: coughing, sneezing, decrease in appetite, piloerection, hypoactivity,
and hunched posture. On study day 9, A05254 was euthanized due to clinical signs of
disease. Culture data confirmed the presence of F. tularensis in the spleen, liver,
tracheobronchiolar lymph node, and lung of A5254 (Table 1). Gross pathology
demonstrated bilateral, necrotizing bronchopneumonia. Based on the culture data,
A05254’s bacterial load in tissue and blood culture was less than A04339 (challenged
December 2007) who died on Study Day 13 but greater than or similar to A04244
(challenged in December 2007) who was euthanized at the completion of in-life (Table 1).
These data are located in the following folder: \\Saturn\absl3\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY07\FY07-083 and -089
(TUL-04)\14MAY08 NHP exposure.
Page 6 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Table 1: Blood and tissue culture data for animal A05254, a naïve Cynomolgus Macaque exposed
to an aerosol challenge of Francisella tularensis.
FY07-083 Naïve Cynomolgus Macaque Francisella tularensis SCHU S4 Bioaerosol Challenge Data
Tissue Culturec
Animal
ID
Challenge
Date
Challenge
Dose
(CFU)a
Nx Dateb
A05254
14-May-08
Unknown
23-May-08
1.88E+04
Mes
LN
BLD
1.48E+05
5.70E+04
8.20E+04
Blood
Spleen
Liver
TBLNd
BLD
3.12E+03
1.28E+02
A04339
14-Dec-07
2670
27-Dec-07
1.70E+06
1.20E+06
3.50E+04
7.20E+06
NAe
A04344
14-Dec-07
5030
28-Dec-07
BLD
2.60E+02
2.20E+02
6.70E+02
NA
Lung
a
Dose is based on viable bacteria collected into an all-glass impinger following achievement of 3.5L inhaled
bAnimal A05254 was euthanized on Study Day 9 and A04339 was euthanized on Study Day 13.
c
Blood data presented as CFU/mL; tissue data presented as CFU/g; BLD, below limit of detection
d
In addition to F. tularensis, one contaminant was noted in the TBLN of A05254; currently being characterized
Not applicable
e
3A
3B
Figure 3: Pneumonia in animal A05254 euthanized on Study Day 9. 3A-Dorsal lung;
3B- Ventral lung.
4. Significant decisions made or pending
Due to the issues with the two animal challenges (Dec 2007 and May 2008), an additional
challenge will be performed once the growth curve data are obtained and the challenge material
preparation defined. Additionally, aerosols with bacteria grown for 24 hours and 48 hours will be
performed upon completion of the growth curve characterization to demonstrate viability and
stability of the bacteria under the aerosol conditions. The additional challenge will not be
performed until two growth curves are performed in duplicate with similar/same results and two
bioaerosol sprays are performed in duplicate from two separate growth preps with similar/same
results.
Page 7 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
5. Problems or concerns and strategies to address
The pre- and post-spray and AGI suspensions from the NHP challenge were negative for
bacterial growth and the growth curve had a poor R 2 value and a conflicting concentration
compared to the original suspension (from the 6SEP07 working stock) cultured on a BCGA plate.
Based on the hypothesis that the bacteria may have been dying due to depletion of nutrients or
the late growth phase they were in at the time of the challenge, multiple growth curves will be
performed to determine when the bacteria reach lag, log, and stationary phase of growth.
Additionally the volume of broth will be increased to 100 mL and the speed increased to 200 rpm.
Refer to Milestone 3 for details on these additional studies
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
50% of the scientific work is complete
9. Work plan for next month
a. Complete the NHP challenge study that initiated on May 14, 2008. If animal A05262
does not succumb to infection or is not euthanized due to illness, terminal sacrifice is
scheduled on June 11, 2008, study day 28. Post mortem will be similar to NHP study of
December 2007 but will also include also include Mesenteric lymph nodes, nasal cavity,
gastrointestinal tract.
b. Perform bioaerosol challenge with two additional primates to confirm that aerosolized
SCHU S4 freshly grown in Chamberlain’s broth is virulent in NHPs. Before challenge the
primates, the chosen primates will have been screened for background titer. The
challenge delivered dose will be 25,000 CFU of viable SCHU S4, which is approximately
1 log higher than December 2007 NHP challenge dose. The Aeromist generator will be
used. The primates will be observed daily for up to 28 days for clinical signs of illness.
NHP will be offered enriched vegetable/fruit food, as a clinical measure of health status.
NHP will not be euthanized unless moribund. Moribund has been defined for this study
as animals who are demonstrating seizures or coma; respiratory distress or severe
dyspnea; persistent recumbency and weakness; unresponsiveness to touch or external
stimuli; or a combination of these along with other parameters such as appearance,
appetite, body weight, temperature, pulse, and interaction with staff. Post mortem will be
identical to the NHP study performed in May 2008.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 5 - UNM
Milestone description: Small species tested for sensitivity to LVS & generation of immunity
against a pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Page 8 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
a. Experiment Ftc70 study 1 (Notebook 115, pages 98-100)
i. We have repeatedly observed occasional survivors in SCHU S4 challenge
experiments when the other rats in the group challenged with the same dose
died. We do not know if this is due to individual variation in susceptibility or
to technical errors in delivering the inoculum; we have been using a nonsurgical inoculation method that involves inserting a flexible catheter down
the trachea and have occasionally seen the catheter end up in the
esophagus instead.
ii. To address the possible technical problem, we plan to do a side-by-side
comparison between the non-surgical inoculation method and a surgical
inoculation that the laboratory has been using for years to infect mice. The
advantage of the surgical method is that we can see the needle being
inserted into the trachea. In several surgical attempts with the rats, we have
had contamination problems that we had not encountered before with the
non-surgical i.t. We are now troubleshooting this problem by using longer
needles and refining our techniques.
iii. To address the possibility that Fischer 344 rats may have individual variation
in susceptibility to SCHU S4 infection, we are consulting with a biostatistician
at UNM, Dr. Ron Schrader, to determine the significance of the data we have
collected so far and the design of future experiments
b. Characterization of the Fischer 344 rat model is currently being done under Milestone
11, as the efforts on the Fischer 344 rat model are shifting toward GLP model
efficacy
4. Significant decisions made or pending
Rat model is under development on MS 11.
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
72%
9. Work plan for upcoming month
a. Complete sub-milestone completion reports for the mouse, rat, and guinea pigs
b. Continue development of surgical i.t. for rats
Continue consultation with GCRC (General Clinical Research Center) biostatistician
at UNM
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 9 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 7
Milestone description: SCHU S4 LD50 in primates determined from selection of challenge
dosing
Institution: LBERI
1. Date started: 2/25/08
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions:
a. A draft protocol was written and submitted to the IACUC. IACUC review is complete and
the comments are being addressed.
4. Significant decisions made or pending
Confirmation of firm start dates pending completion of Milestone 4.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Fair
8. Percentage completed
8% of the scientific work is complete
9. Work plan for next month
a. Schedule appropriate personnel.
b. Initiate ABSL-3 move-in and challenge dates.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 11
Milestone description: In vivo GLP model efficacy SOPS developed in one small species
and primate and efficacy testing of vaccine candidates
Institution: UNM
1. Date started: 1/16/2008
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Cdep1.5 (Notebook 122, page 14-27, 33-36)
i. The purpose of this experiment was to determine the role of CD4 and CD8 T
cells in protecting LVS vaccinated rats against i.t. SCHU S4 challenge
ii. We purchased the OX-8 (anti-CD8) and W3/25 (anti-CD4) hybridomas from
ECACC and the isotype control TS2/18.1.1 hybridoma from ATCC and
contracted with Taconic to produce ascites fluids in mice.
iii. OX-8 is a depleting antibody and W3/25 is a non-depleting, inactivating
antibody that has the same effect as a depleting antibody in a rat model of
Pneumoncystis pneumonia [TD Thullen et al. Infect Immun (2003) 71:62926297]
Page 10 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
iv. To determine the effect of depleting CD4 or CD8 T cells, rats that had been
vaccinated 3 months earlier were treated i.p. with 1 mg of ascites fluid and
challenged with a target dose 105 SCHU S4 (due to a technical problem, we
could not determine the actual lung deposition).
v. In this experiment, we also incorporated self-illuminating quantum dots into
the inoculum in order to track intratracheal delivery using the Xenogen IVIS
imaging system. Figure 1 shows the three potential outcomes. (Left) Image
from the left side of infected rats showed that the inoculum was in the lungs.
(Middle) Image from the right side but not the left side showed that the
inoculum was in the lungs. (Right) Neither image from the left or the right
side showed that the inoculum was in the lungs. This rat was excluded from
analysis.
vi. Although SCHU S4 killed all 5 unvaccinated rats, it also killed 2 of 5 LVS
vaccinated rats and 2 of 4 vaccinated rats treated with isotype control
antibody (Fig. 2). This reduced our ability to determine the effects of CD4
and CD8 T cell depletion and therefore no conclusion could be drawn from
this experiment
vii. It is possible that the protection generated by LVS vaccination wanes over
time in rats like it does in mice. Therefore, we will repeat this experiment
earlier after vaccination
Figure 1. Tracking localization of inoculum in the lungs with self illuminating quantum dots. After
an inoculum containing SCHU S4, self illuminating quantum dots and substrate was inoculated
into rats, the rats were imaged using the Xenogen IVIS Imagine system for 30 to 60 sec.
Page 11 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 2. Effect of CD4 and CD8 T cell depletion on the survival of LVS vaccinated rats after
i.t. SCHU S4 challenge. LVS vaccinated rats were challenged with a target dose of 105
SCHU S4 and monitored for survival. Each group contains 3 to 5 animals, based on
localization of quantum dots in the lungs.
b. Experiment Ptran4 (Notebook 122, page 28-31 and 54-55)
i. The purpose of this experiment was to determine whether immune serum
from LVS vaccinated Fischer 344 rats is able to protect BALB/c mice from
intranasal SCHU S4 challenge. This is a repeat of Experiment Ptran3
ii. BALB/c mice were injected i.p. with 250 l of normal or immune rat serum
and 1 day later challenged i.n. with 2.74 x 104 LVS or ~100 SCHU S4
iii. As seen previously in Experiment Ptran3, immune serum protected mice
against an intranasal challenge with LVS but not SCHU S4 (Fig. 3).
Page 12 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 3. Immune serum from LVS vaccinated Fischer 344 rats protected BALB/c mice
against LVS but not SCHU S4. BALB/c mice (n = 5) were injected i.p. with 250 l of normal
or immune sera and 1 day later challenged with 2.74 x 104 LVS or ~100 SCHU S4
c.
Experiment Ptran5 (Notebook 122, page 36-47 and 51-55)
i. The purpose of this experiment was to determine the maximum volume of
normal rat serum that can be transferred into naïve rats without inducing nonspecific protection.
ii. We observed previously that 2 to 2.5 ml normal rat serum protected 4 of 6
rats against i.t. SCHU S4 challenge in one experiment and 2 of 6 rats in a
second experiment. This really affected our ability to interpret the results
with the immune serum
iii. In this experiment, naïve Fischer 344 rats were injected i.p. with 0.25, 0.5, 1
and 2.5 ml of normal serum collected from Fischer 344 rats from NCI and
then challenged with an i.t. target dose of 500 SCHU S4. The inocula also
included quantum dots and substrate to track lung delivery using the
Xenogen IVIS imaging system
iv. It is important to point out that the rats used in this experiment were from
Harlan instead of NCI, which usually supplies the rats that we use. We
initially ordered the Fischer 344 rats from Harlan to test the robustness of the
rat model. However, it was decided in subsequent discussions that the
passive immunization experiment should have a higher priority and therefore
the rats were diverted to this experiment
v. Unlike the two previous experiments, there was not protection by normal
serum at any volume (Fig. 4)
vi. This may be attributed to genetic differences between Fischer 344 rats from
NCI and Harlan. Ann Sutton suggested that if the Harlan rats are in deed
less susceptible to the effects of serum transfer, we may be able to use this
to our advantage in examining passive immunization
Page 13 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam

Figure 4. Titration of normal rat serum that would confer no protection to naïve rats to i.t.
SCHU S4 challenge. Fischer 344 rats were injected i.p. with the indicated volume of normal
serum and challenged 1 day later with a target dose of 500 SCHU S4. Each group contained
2 to 5 rats and rats without clear indication of lung inoculation were excluded from analysis.
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
15%
9. Work plan for upcoming month
a. Titrate amount of immune rat serum required to protect rats against i.t. SCHU S4
challenge using Fischer 344 rats from NCI for both donor and recipient
b. Repeat experiment to determine the role of CD4 and CD8 T cells in protecting LVS
vaccinated rats
c. Expand the OX-38 (CD4 T cell depleting antibody) and 55-6 (isotype control antibody
for OX-38) hybridomas and send them to Taconic for production of ascites fluid
d. Titrate the amount of F. tularensis-specific antibodies in immune sera
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 14 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 12/13
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. No new work done this month.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse proliferation assay, IFN and IL-2 Elispot, anti-Ft antibody titration
Rat IFN Elispot, anti-Ft antibody titration
Guinea pig anti-Ft antibody titration 
7. Quality of performance
Good
8. Percentage completed
60%
9. Work plan for upcoming month
a. Determine whether LVS vaccinated mice with an active SCHU S4 infection could be
used to increase the sensitivity of the IFN Elispot assay
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and compared to those in other species.
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. We have continued to screen non-LVS vaccinated NHPs in both the IFNγ ELISPOT and
proliferation assays in order to avoid choosing any high responders to serve as LVSnaïve controls
Page 15 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
ii. In the past month, we have screened 6 naïve NHPs for their responsiveness to LVS
and SCHU S4, both heat-killed (HK) and formalin-fixed (FF) preparations (these are in
addition to the 12 NHPs we presented in the April 2008 report).
iii. Figure 4 shows the results of the proliferation assay for all monkeys screened in the
last two months (from the March 2008 shipment from Covance). Figure 5 shows the
results of the IFNγ ELISPOT assays for all monkeys screened to date (in the last two
months (from the March 2008 shipment from Covance). Figure 6 shows the IgG anti-LVS
ELISA titer for all the monkeys screened to date in the last two months (from the March
2008 shipment from Covance).
4A
Cell Mean for RLU small
250000
200000
150000
100000
50000
0
A02314
A04169
A04308
A05254
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
4B
Media
LVS hk Hi
250000
Cell Mean for RLU small
LVS hk Mid
LVS ff Hi
200000
LVS ff Mid
LVS ff Lo
150000
LVS hk Super
SCHUS4 hk Super
100000
SCHUS4 hk Hi
50000
SCHUS4 hk Mid
SCHUS4 ff Super
0
A05987
A06199
A06589
A06592
SCHUS4 ff Hi
SCHUS4 ff Mid
Page 16 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
4C
Media
LVS hk Hi
LVS hk Mid
Cell Mean for RLU small
400000
350000
LVS ff Hi
300000
LVS ff Mid
250000
LVS ff Lo
200000
LVS hk Super
150000
SCHUS4 hk Super
SCHUS4 hk Hi
100000
SCHUS4 hk Mid
50000
SCHUS4 ff Super
0
A04643
A04645
A04713
SCHUS4 ff Hi
SCHUS4 ff Mid
4D
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
Cell Mean for RLU small
450000
400000
350000
LVS ff Mid
300000
LVS ff Lo
250000
LVS hk Super
200000
SCHUS4 hk Super
150000
SCHUS4 hk Hi
100000
SCHUS4 hk Mid
50000
SCHUS4 ff Super
0
A04994
A05262
A06587
SCHUS4 ff Hi
SCHUS4 ff Mid
Page 17 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
4E
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
Cell Mean for RLU small
250000
200000
LVS ff Lo
150000
LVS hk Super
SCHUS4 hk Super
100000
SCHUS4 hk Hi
SCHUS4 hk Mid
50000
SCHUS4 ff Super
0
A04999
A05403
A05988
A05997
SCHUS4 ff Hi
SCHUS4 ff Mid
Figure 4: Proliferation of PBMCs from non-LVS vaccinated NHPs to LVS and SCHU S4 antigens (ff
= formalin fixed; hk = heat-killed; Super = 4 x 105/ml, Hi = 1 x 105/ml; Mid = 025 x 105/ml; Lo =
0.0625 x 105/ml). All PBMCs were plated at 1 x 106/ml. Panels A and B show NHPs that did not
respond to any stimuli, whereas those NHPs in panels C – E demonstrated responsiveness to at
least one stimuli.
5A
Cell Mean for IFNg Spots
225
200
175
150
125
100
75
50
25
0
A04994
A05987
A05997
A06199
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
Page 18 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
5B
Media
LVS hk Hi
Cell Mean for IFNg Spots
140
LVS hk Mid
120
LVS ff Hi
LVS ff Mid
100
LVS ff Lo
80
LVS hk Super
60
SCHUS4 hk Super
40
SCHUS4 hk Hi
SCHUS4 hk Mid
20
SCHUS4 ff Super
0
A02314
A04169
A04643
SCHUS4 ff Hi
SCHUS4 ff Mid
Cell Mean for IFNg Spots
5C
Media
LVS hk Hi
LVS hk Mid
250
225
200
LVS ff Hi
LVS ff Mid
175
150
125
100
75
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
50
25
0
SCHUS4 hk Mid
SCHUS4 ff Super
A04713
A05254
A06592
SCHUS4 ff Hi
SCHUS4 ff Mid
Page 19 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
5D
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
Cell Mean for IFNg Spots
400
350
300
LVS ff Mid
250
LVS ff Lo
200
LVS hk Super
SCHUS4 hk Super
150
SCHUS4 hk Hi
100
SCHUS4 hk Mid
50
SCHUS4 ff Super
0
A04308
A04999
SCHUS4 ff Hi
A06587
SCHUS4 ff Mid
5E
Cell Mean for IFNg Spots
250
200
150
100
50
0
A04645
A05262
A05403
A05988
A06589
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
Figure 5: IFNγ production by PBMCs from non-LVS vaccinated NHPs to LVS and SCHU S4
antigens (ff = formalin fixed; hk = heat-killed; Super = 4 x 105/ml, Hi = 1 x 105/ml; Mid = 025 x
105/ml; Lo = 0.0625 x 105/ml). Panels A - C show NHPs that either did not respond to any stimuli
(A04994) or responded only to LVS ff; panel D shows NHPs that responded to more than one
stimuli, whereas those NHPs in panel E had background responses high enough to make
interpretation of the response to stimuli difficult to interpret. All PBMCs were plated at 1.33 x
106/ml. Missing bars in panels 5A, 5C and 5E result from too few PBMCs being available to test
those stimuli.
Page 20 of 61
Tularemia Vaccine Development Contract: Technical Report
1000000
100000
10000
1000
100
10
1
A02314
A04169
A04308
A04643
A04645
A04713
A04994
A04999
A05254
A05262
A05403
A05987
A05988
A05997
A06199
A06587
A06589
A06592
Cell Mean for IgG anti-LVS Titer
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 6: Serum from non-LVS vaccinated NHPs was tested for IgG anti-HK LVS reactivity by
ELISA. Titers are shown and are defined as the inverse of the highest dilution of serum showing
OD405 values above the assay blank (all components added to the well except serum). The titer of
the positive control run in these two assays was 125,000 (A00868, day 28 post SC-LVS
vaccination).
Data Interpretation
i. In the proliferation assay, 8 animals out of 18 had little to no response to any
stimuli (Figures 4A and 4B). From this group A02314 had a high IgG anti-LVS
titer, however, so we do not anticipate using this animal for any future study on
this contract.
ii. In the proliferation assay, 10 animals out of 18 had a proliferative response to at
least one stimuli (Figures 4C, 4D, and 4E).
iii. In the IFNγ ELISPOT assay, 1 animal (A04994) had no response to any stimulus
(Figure 5A).
iv. In the IFNγ ELISPOT assay, 9 animals responded only to FF-LVS and the
responses diminished as the concentration of the stimulus was decreased
(Figure 5A, 5B, and 5C).
v. In the IFNγ ELISPOT assay, 3 animals responded to FF-LVS and various other
stimuli, including SCHU S4 (Figure 5D).
vi. In the IFNγ ELISPOT assay, 5 animals had a high background (spots in
unstimulated wells) making interpretation difficult (Figure 5E).
vii. In the IgG anti-LVS ELISA, one animal (A02314) had a titer as high as an LVSvaccinated NHP, but the other 17 had titers ranging from 200 - 5000.
viii. Based on these data, it is clear that there is a continuum of responsiveness in the
non-LVS vaccinated NHPs to LVS and SCHU S4 antigens. As studies require
their use, we will attempt to select NHPs with the lowest cumulative
responsiveness in the IFN, proliferation and IgG anti-LVS assays. These NHPs
will be used first. As we order more NHPs, we will continue to screen them so
that they will also be available for use. Responsiveness to FF LVS Hi appears so
often that we suspect that this dose of antigen is behaving like a mitogen.
Page 21 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Therefore, responsiveness to FF LVS Hi is not an exclusion criterion. The only
absolute exclusion criteria will be if a non-LVS vaccinated NHP exhibits an IgG
anti-LVS titer greater than 5000.
ix. A sample table of selection criterion is presented below using the NHPs shown in
this report. The NHPs were ranked based on their responsiveness in the various
assays with a lower score representing less of a response to LVS and SCHU S4
antigens. When equivalent responses were observed, the same score was
assigned.
ID
Proliferation
IFNg
IgG antiLVS
Sum
Average Score
Note
5254
1
2
1
4
1.33 Used for SCHU S4 Challenge, May '08
6592
2
2
3
7
2.33 Will retest IFNγ due to high background
6589
3
2
3
8
2.67 Will retest IFNγ due to high background
4169
3
5
1
9
3
4308
1
6
2
9
3
4994
5
1
3
9
3
6199
4
3
2
9
3
5262
6
2
2
10
3.33 Used for SCHU S4 Challenge, May '08; IFNγ response uncl
4713
5
5
1
11
3.67
5987
4
5
3
12
4
5988
8
2
2
12
4 Will retest IFNγ due to high background
4645
10
2
1
13
4.33 Will use for SCHU S4 challenge, June '08; would have liked
5403
11
2
2
15
5 Will retest IFNγ due to high background
4643
9
5
2
16
5.33 Will use for SCHU S4 challenge, June '08
4999
7
8
3
18
6
5997
11
4
3
18
6
6587
12
7
2
21
7
Note: NHPs A04643 and A04645 were selected for use in the upcoming SCHU S4 study prior to
screening and analyzing the data from some of the other NHPs.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay060508.svd; TVDC (2)
bound notebook (8935): TUL 33 (pps.48 to 55); TUL 34 (56 to 63); TUL 35 (66 to 72); LVS ELISA (64 –
65); TUL 37 (82 – 87), TUL 38 (88 – 92); TVDC (3) bound notebook (9225): LVS ELISA (p. 1).
4. Significant decisions made or pending
No non-LVS vaccinated NHPs which exhibit an IgG anti-LVS titer greater than 5000 will be used in
future studies on this contract.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
97.5% of the scientific work is complete
9. Work plan for upcoming month
Page 22 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
a. Continue to test PBMCs from LVS-vaccinated and non-vaccinated NHPs in the IFN
ELISPOT assay to determine the effect of HK and FF LVS at different concentrations.
b. Continue to test the effect of the Cerus freeze-thaw protocol on the performance of the
PBMCs in the immunoassays.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 14
Milestone description: Assays in vaccinated humans validated (sensitivity)
Institution: UNM/LBERI
1. Date started: 2/29/2008
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
a. We have obtained materials and reagents necessary for blood collection
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
1%
9. Work plan for upcoming month
a. Work with the Decode project group in the Lyons lab to develop SOP for isolating
PBMCs from the peripheral blood of control and vaccinees
b. Develop the IFN Elispot and proliferation assays
c. Develop SOP for quantifying multifunctional T cell cells in control and human LVS
vaccinees
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 17
Milestone description: In vitro assay for analysis of cellular and humoral elements of the
immune response in vaccinated human and animal’s response to T. tularensis established
Institution: UNM
Page 23 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
1. Date started: 2/29/2008
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
b. We have obtained materials and reagents necessary for blood collection
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
N
8. Percentage completed
1%
9. Work plan for upcoming month
a. Work with the Decode project group in the Lyons lab to develop SOP for inducing
differentiation of monocytes to macrophages
b. Develop SOP for infecting human monocyte-derived macrophages with LVS and
SCHU S4
c. Determine whether PBMC from vaccinees can induce infected monocyte-derived
macrophages to kill intracellular bacteria
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 19
Milestone description: Interaction between human alveolar macrophages and F. tularensis
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc36 study 10 (Notebook 115, pages 125-127 and 133)
i. The purpose of this experiment was to determine the cytokine profile of
human alveolar macrophages infected with LVS and SCHU S4
ii. Two batches of human alveolar macrophages were collected: there were 1.7
x 107 cells for the 5/13/08 collection and 1.9 x 107 cells for the 5/14/08
collection
iii. Macrophages were plated into 24 well plates at a concentration of 10 6/ml/well
and then allowed to adhere overnight. The next day, the wells were slightly
Page 24 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
more than confluent and the medium was yellow, but no contamination was
seen under a microscope with 40X objective. Since there was a possibility of
low level contamination, the wells were gently washed with warm PBS.
Unfortunately, many of the cells were removed during this process.
iv. Nevertheless, the macrophages were infected with LVS and SCHU S4 at
MOI of 1:1, and 10:1 (bacteria to macrophages based on the number of
macrophages initially plated)
v. Supernatants were collected 1 day after infection for cytokine analysis.
vi. Results of the analysis will be reported in the next technical report
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
12%
9. Work plan for upcoming month
a. Repeat the cytokine analysis of human alveolar macrophages infected with LVS and
SCHU S4. To minimize the loss of macrophages grown in 24 well plates, we will try
to grow macrophages in suspension. The macrophages should adhere to each other
instead of the plate to form spheres but we will be able to centrifuge the
macrophages when we wash them, if necessary.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc30.19a (Notebook 109, pages 110-112)
i. The results of several macrophage killing assays with mouse cells and
SCHU S4 show large variations among replicates within a single experiment
and across several experiments.
ii. The purpose of this experiment is to improve the consistency of this assay by
1. lncreasing the amount of time allowed for the bone marrow derived
macrophages to phagocytose SCHU S4
Page 25 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
2. Plating a larger volume of cell lysate using the autoplater 100 from
Sprial Biotech
3. Lysing the infected macrophages with the detergent deoxycholate
instead of water
iii. The results (Table 1) showed after 48 h culture that
1. Increasing the phagocytosis incubation time from 1 h to 4 h improved
the consistency across replicates
2. Plating a larger volume (50 l) of cell lysates with the autoplater
improved the consistency across replicates than plating a smaller
volume (10 l) by the microdot technique
3. There was no difference between cell lysis with deoxycholate and
water
Table 1. Optimization of murine macrophage killing assay for SCHU S4
Total CFU recovered (Each cell shows a replicate sample)
Autoplater (50 l)
Microdot (10 l)
Water
Deoxycholate
Water
Deoxycholate
1h
3.9x103 8.2x104 7.6x104 6.4x103 7.29x104 7.15x103
1.0x105 8.0x103 8.0x104 4.4x103 1.0x105 4.9x103
4h
ND
ND
ND
ND
ND
ND
3.1x106 1.7x106 1.8x106 2.5x106 1.2x106 2.0x106
ND = not determined because the culture was not diluted enough when plating
b. Experiment Ftc61.15 (Notebook 109, pages 113-115)
i. The purpose of this experiment was to determine whether recombinant IFN
can suppress LVS growth in rat bone marrow-derived macrophages (BMM).
This will serve as a positive control when we add T cells from LVS
vaccinated rats
ii. BMM were infected with LVS at MOI of 1:10 and 1:20 (bacteria:macrophages)
and then incubated with 50 or 500 ng/ml recombinant rat IFN
iii. The results showed clearly that IFN reduced the number of LVS recovered,
suggesting that it induced macrophage activation (Table 2). Moreover, 500
ng/ml of IFN may produce more dramatic inhibition
Table 2. Effect of recombinant IFN on killing of LVS by rat BMM1
Total CFU recovered (each cell shows a replicate sample)
rIFN (ng/ml)
0
3.6 x 106
4.5 x 106
6.2 x 106
50
2.4 x 103
1.0 x 103
1.1 x 105
500
> 20
> 20
> 20
1
Results show MOI of 1:10
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 work
8. Percentage completed
29%
9. Work plan for upcoming month
Page 26 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
a. Repeat the macrophage killing assay with SCHU S4 with more replicates per
experiment
b. Determine the effect of immune T cells on LVS growth in rat macrophages
c. Determine the MOI for infecting rat macrophages with SCHU S4
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21
Milestone description: Correlates of protection: in vitro assay or other readout of effector
function of Ft developed for multiple species.
.
Institution: LBERI
1. Date started: 4/8/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. Intracellular cytokine staining was performed using 3 LVS-vaccinated NHPs and data is
currently being analyzed.An aliquot of PBMCs from each of the same animals were
tested for IFNγ ELISPOT activity (Figure 7). A00659 and A00868 are 523 days post SCLVS vaccination. A00908 is 532 days post ID-LVS vaccination.
Cell Mean for IFNg Spots
350
300
250
200
150
100
50
0
A00659
A00868
A00908
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
Figure 7: IFNγ production by PBMCs from LVS vaccinated NHPs to LVS and SCHU S4 antigens (ff
= formalin fixed; hk = heat-killed; Super = 4 x 105/ml, Hi = 1 x 105/ml; Mid = 025 x 105/ml; Lo =
0.0625 x 105/ml). All PBMCs plated at 1.33 x 106/ml.
Page 27 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Data Interpretation
i. The two S.C. vaccinated NHPs (A00659 and A00868) appear to still have a
response to LVS FF (at different doses), as well as a small, but detectable
response to LVS HK (A00659 > A00868) and high doses of SCHU S4 FF.
ii. A00908 (I.D. vaccinated with LVS) appears to respond primarily to LVS FF
antigen and to SCHU S4 FF antigens. However, the slightly higher background
makes the interpretation difficult.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay060508.svd; TVDC (2)
bound notebook (8935): TUL 36 (pps. 73 – 81).
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
1.5% of the scientific work is complete
9. Work plan for upcoming month
Analyze the results of the completed staining. Set up another intracellular cytokine assay once
the current results are analyzed.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated.
Milestone 26
Milestone description: Confirmation of gene and protein expression (develop HTP SOPs
for ORF library production, protein library production, and protein purification)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Select and test ORF expression constructs
1. Proper conformation appears to be critical for efficient separation of cross-reacting
molecules from polypeptides by:
Page 28 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
i. size filtration
ii. acetone precipitation
iii. Affinity (Ni-binding) purification
2. Decision was made to evaluate methods to improve folding or alternative methods of
purification that are folding-independent.
3. We placed thioredoxin (trx) into our LEE IVT construct designs. IVT templates and
recombinant bacterial expression plasmids were used to generate two FTU proteins,
one that was folding properly without the thio and another FTU polypeptide and
another that was behaving as a misfolded protein.
4. These samples have been sent to UNM.
B. Select and test IVT Protocols
C. Select and test protocols for protein purification
1. Current Status:
• Adding the Ni beads during the IVT reactions significantly improves polypeptide
“capture” without urea, thereby improving purification.
• Pre-clearing the IVT lysates of bead-sticky proteins prior to IVT, does not reduce
polypeptide yield. This is done by adding beads to lysate and then removing this
sample. This is referred to as “depletion” in the figure.”
• Such IVT made OVA products specifically stimulate immune T cells.
• In our last trial we found no cross-reacting spots with our depleted lysates.
However, the positive controls were not included in this run.
• We noticed that the number of spots stimulated by the killed LVS varied
significantly between UNM experiments.
2. Based on these data, a new set of IFN
same antigen samples were tested on two sets of LVS-immune T cells: one
harvested 3 months following immunization and the other harvested 2 months after
immunization.
Page 29 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Samples tested on splenocytes harvested 3 month post LVS vaccination (UNM
expt Ftc study 19
Data Location: (Notebook 115, pages 118-124)
OVA
LVS
3 mos. after
vaccination
A
B
C
D
E
F
G
H
1
No
template
No
template
No
template
No
template
Without depletion
2
3
OVA
Ftu 1695
OVA
Ftu 1695
OVA
Ftu 1695
OVA
Ftu 1695
4
Ftu 901
Ftu 901
5
No
template
No
template
No
template
No
template
With depletion
6
7
OVA
Ftu 1695
OVA
Ftu 1695
OVA
Ftu 1695
OVA
Ftu 1695
8
Ftu 901
Ftu 901
9
Sigma
OVA
OVA
peptide
Sigma
OVA
OVA
peptide
Controls
10
11
FF LVS HK LVS
neat
neat
FF LVS HK LVS
1:10
1:10
FF LVS HK LVS
neat
neat
FF LVS HK LVS
1:10
1:10
12
No Ag
Ni beads
50% naïve +
50%
Protein G
DO11.10
beads
No Ag
Ni beads
LVS
Protein G vaccinated
beads
Page 30 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Samples tested on splenocytes harvested 2 month post LVS vaccination (UNM
expt Ftc study 19
Data Location: (Notebook 115, pages 118-124)
OVA
LVS
2 mos. after
vaccination
A
B
C
D
E
F
G
H
1
No
template
No
template
No
template
No
template
Without depletion
2
3
OVA
Ftu 1695
OVA
Ftu 1695
OVA
Ftu 1695
OVA
Ftu 1695
4
Ftu 901
Ftu 901
5
No
template
No
template
No
template
No
template
With depletion
6
7
OVA
Ftu 1695
OVA
Ftu 1695
OVA
Ftu 1695
OVA
Ftu 1695
8
Ftu 901
Ftu 901
9
Sigma
OVA
OVA
peptide
Sigma
OVA
OVA
peptide
Controls
10
11
FF LVS HK LVS
neat
neat
FF LVS HK LVS
1:10
1:10
FF LVS HK LVS
neat
neat
FF LVS HK LVS
1:10
1:10
12
No Ag
Ni beads
50% naïve +
50%
Protein G
DO11.10
beads
No Ag
Ni beads
LVS
Protein G vaccinated
beads
3. In red circled wells, no spots are expected. In green circled wells, spots are
expected. In both the 3 month and 2 month data, the OVA peptide and ASU-made
protein stimulated the OVA transgenic T cells, and the FF LVS positive controls
antigen stimulated IFN reactivity in the LVS-immunized T cells. , However, the
number of spots were significantly different between the two T cell samples.
4. From column 9 we note that even with the highly reactive 2 month samples, the
Sigma purchased ova protein did not stimulate the transgenic T cells. This is an
assay sensitivity concern.
5. From column 2 we note that the ASU IVT-made ova stimulated more ova-immune T
cells when presented on G beads than when presented on Ni beads.
6. We cannot draw any conclusions about the FTU samples since the reactivities are
too high without polypeptide.
7. We found that the length of time since immunization influenced the number of
reactive T cells. Shorter timeframes generated stronger responses. Samples
harvested 2 months post vaccination show stronger overall reactivity than samples
harvested 3 months post vaccination. Rick agrees that this is very possible.
Page 31 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
8. This explains the last data set, and we have not removed the cross-reacting antigen
by the bead depletion step using either with the Ni or protein G beads.
9. Based on these data, we pursued several approaches to preparing antigen:
a. Use thio fusion constructs as second tag opportunity. Affinity purify via thio
onto anti-thio antibody, attached to protein G sepharose beads. The Ni
beads are stickier than the G beads, and we have shown higher assay
sensivity with the G beads. The thio fusion is also likely to facilitate proper
folding.
b. Deplete IVT reaction post synthesis with anti IVT or anti-bead antibodies.
These samples are being generated.
c. Other approaches include:
i. Wash polypeptide bound Ni beads more stringently with higher
imidazole concentrations
ii. Pre-clear (or depleting) IVT lysates with anti-LVS antisera, prior to
use in IVT reactions
iii. Pre-clear IVT products (post-synthesis) with anti-E. coli antibodies
iv. Use rabbit retic IVT products, with enhanced sensitivity by
attachment to protein G beads.
10. We have previously considered that ribosomal proteins are highly conserved, and a
major component of the IVT lysates.
11. We BLASTed the amino acid sequence of E. coli and F. tularensis. The proteins
displaying the highest level of sequence identity (85%) were ribosomal components
and other translation factors.
12. As a pilot, we will test whether we can deplete E. coli proteins with a commercial antiE. coli antisera post IVT reaction to remove cross-reacting antigen. If this indicates
that the antigen can be removed with the antisera then we will immunize our own
mice with the IVT lysate itself and make lots of our own antisera, The IgG could then
be used to clean up the sample post-reaction.
13. These samples have been sent to UNM:
Page 32 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Sample
1
2
3
4
5
Depletion of E coli lysate by LVS immunized sera
E coli lysate as is without depletion
E coli lysate depleted with 10ul protein G-IgG beads
E coli lysate depleted with 20ul protein G-IgG beads
E coli lysate depleted with 50ul protein G-IgG beads
E coli lysate depleted with 100ul protein G-IgG beads
Depletion of E-coli lysate by E coli antibody after IVT
Sample reaction for Ova
1
NEB IVT as is
2
NEB IVT depleted with 10ul proteinG-IgG beads
3
NEB IVT depleted with 50ul proteinG-IgG beads
4
NEB IVT depleted with 100ul proteinG-IgG beads
5
Invitrogen IVT as is
6
Invitrogen IVT depleted with 10ul proteinG-IgG beads
7
Invitrogen IVT depleted with 50ul proteinG-IgG beads
8
Invitrogen IVT depleted with 100ul proteinG-IgG beads
Sample
IVT of Ova, FTU 901, FTU1695 by Rabbit Ret system
1
Ova IVT as is
2
FTU 901 IVT as is
3
FTU 1695 IVT as is
4
Ova IVT bound on protein G beads
5
FTU 901 IVT bound on protein G beads
6
FTU 1695 IVT bound on protein G beads
Thio fusion protein samples for
UNM shipped 06-02-2008
Sample
1
2
3
4
Sample
5
6
7
Sample
8
9
10
IVT proteins bound on G beads via α-Thio
antibody
No template
Ova
FTU 901
FTU 1695
IVT proteins bound on G beads via α-His
antibody
Ova
FTU 901
FTU 1695
IVT proteins denatured with 6M urea and
bound on Ni magnetic beads
Ova
FTU 901
FTU 1695
Page 33 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
14. To establish the composition of the proteins sticking to the beads and generating a
cross-reacting response by the LVS immune T cells, we performed a mass spec
analysis of IVT lysate (without template reaction) bound beads .
15. This list confirmed our hypothesis that ribosomal proteins are sticking to the beads,
even after a high concentration imidazole wash (500mM)
Elongation factor Tu (EF-Tu) (P-43) - Escherichia coli
Tryptophanase (EC 4.1.99.1) (L-tryptophan indole-lyase) (TNase) - Escherichia coli
Cold-shock DEAD box protein A (EC 3.6.1.-) (ATP-dependent RNA helicase deaD) Escherichia coli
30S ribosomal protein S3 - Escherichia coli
Malate dehydrogenase (EC 1.1.1.37) - Escherichia coli
50S ribosomal protein L9 - Escherichia coli
50S ribosomal protein L2 - Escherichia coli
50S ribosomal protein L7/L12 (L8) - Escherichia coli
DNA-binding protein HU-alpha (NS2) (HU-2) - Escherichia coli
50S ribosomal protein L6 - Escherichia coli
Acetate operon repressor - Escherichia coli
Alkyl hydroperoxide reductase subunit C (EC 1.11.1.15) (Peroxiredoxin) (Thioredoxin
peroxidase) (Alkyl hydroperoxide reductase protein C22) (SCRP-23) (Sulfate starvationinduced protein 8) (SSI8) - Escherichia coli
50S ribosomal protein L15 - Escherichia coli
50S ribosomal protein L4 - Escherichia coli
30S ribosomal protein S13 - Escherichia coli
50S ribosomal protein L17 - Escherichia coli
30S ribosomal protein S5 - Escherichia coli
Chaperone protein skp precursor (Seventeen kilodalton protein) (Histone-like protein HLP-1)
(DNA-binding 17 kDa protein) - Escherichia coli
Catabolite gene activator (cAMP receptor protein) (cAMP-regulatory protein) - Escherichia coli
50S ribosomal protein L24 - Escherichia coli
30S ribosomal protein S4 - Escherichia coli
50S ribosomal protein L22 - Escherichia coli
50S ribosomal protein L10 (50S ribosomal protein L8) - Escherichia coli
6-phosphogluconate dehydrogenase, decarboxylating (EC 1.1.1.44) - Escherichia coli
30S ribosomal protein S7 - Escherichia coli
Succinyl-CoA synthetase beta chain (EC 6.2.1.5) (SCS-beta) - Escherichia coli
30S ribosomal protein S18 - Escherichia coli
Thiol peroxidase (EC 1.11.1.-) (Scavengase P20) - Escherichia coli
30S ribosomal protein S20 - Escherichia coli
30S ribosomal protein S15 - Escherichia coli
50S ribosomal protein L28 - Escherichia coli
Phosphocarrier protein HPr (Histidine-containing protein) - Escherichia coli
50S ribosomal protein L13 - Escherichia coli
50S ribosomal protein L21 - Escherichia coli
Enolase (EC 4.2.1.11) (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase)
- Escherichia coli
10 kDa chaperonin (Protein Cpn10) (groES protein) - Escherichia coli
Page 34 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Translation initiation factor IF-3 - Escherichia coli
30S ribosomal protein S1 - Escherichia coli
Acyl carrier protein (ACP) (Cytosolic-activating factor) (CAF) (Fatty acid synthase acyl carrier
protein) - Escherichia coli
Isocitrate dehydrogenase [NADP] (EC 1.1.1.42) (Oxalosuccinate decarboxylase) (IDH)
(NADP(+)-specific ICDH) (IDP) - Escherichia coli
50S ribosomal protein L20 - Escherichia coli
30S ribosomal protein S21 - Escherichia coli
50S ribosomal protein L3 - Escherichia coli
SsrA-binding protein (Small protein B) - Escherichia coli
Cold shock-like protein cspE (CSP-E) - Escherichia coli
30S ribosomal protein S19 - Escherichia coli
Trigger factor (TF) - Escherichia coli
Elongation factor Ts (EF-Ts) - Escherichia coli
Data location: \\peptide\Research\CIM\GeneVac\FTU\Contract\Proteome filename: MS results 052308.xls
16. These data suggest that anti-lysate antibodies may be able to clear the lysates postreaction.
17. We have immunized mice with a lysate sample, and also a preparation of lysatebound beads.
4. Significant decisions made or pending
We will perform the ELISpots with T cells from a non-murine source. We will evaluate both rat
and primate LVS-immune T cells.
5. Problems or concerns and strategies to address
See above. Although the form of the antigen delivered to animal T cells at UNM is not
solidified, nor has the type of T cells to use been established. It is clear that sufficient
quantities of synthetically proteins can be made robustly to specifically stimulate small animal
splenocytes in an ELISpot assay. This has been repeatedly demonstrated with the ova
positive control samples. We will address the cross-reactivity between FTU and E. coli by
removing IVT proteins post reaction and by evaluating non-murine sources of T cells
(monkey and rat).
6. Deliverables completed
None
7. Quality of performance
Very good
8. Percentage completed
99.8%
9. Work plan for upcoming month
Currently we are:
 Generating test proteins, attached to protein G beads via the thio tag, with and
without anti-lysate depletion for evaluation in UNM’sT-cell assay.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 35 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 27
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
UNM has been supporting ASU’s effort to troubleshoot the crossreactivity problem with
the in vitro translated proteins. Details and results of the UNM ELIspot work performed
are described by ASU under MS26. Records of the testing done at UNM are located in:
Ftc59 study 18 (Notebook 115, pages 109-112 and 116-117)
Ftc59 study 19 (Notebook 115, pages 118-124)
Ftc59 study 20 (Notebook 115, pages 128-132)

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
22%
9. Work plan for upcoming month
Perform IFN ELISpot assays for ASU as the need arises
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of polypeptide libraries (Optimize IVT proteinfragment production, Develop IVT protocol for high-throughput production, Validate
immunogenicity of protein-fragments, Full scale production of protein-fragment library,
Purification of protein-fragment library, Array protein-fragment into overlapping pools, Ship
to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome (active)
 Generate complete protein-fragment library (inactive)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)
Page 36 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Institution: ASU-Sykes
3. Date started: 03-01-2007
4. Date completed: Pending
5. Work performed and progress including data and preliminary conclusions
A. Build ORF expression library corresponding to proteome
PCR primers are ready for ORF library production. We will proceed following decisions on
expression system, yield needs, delivery format, pooling capacity, and purification
requirements.
B.
C.
Generate polypeptide library
Array polypeptide library
4. Significant decisions made or pending.
The decision to complete the polypeptide purification/optimizations of milestone 26 will be made
this month
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
37%
9. Work plan for upcoming month
Prepare for initiating library production.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35
Milestone description: Array hybridizations with mouse RNAs from virulent Schu 4
infection & RT PCR confirmation of candidates.
Institution: UNM/ASU-Johnston
1. Date started: 08-01-2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

Previous Results: From the two initial dose-response challenge experiments at a single 4 hr
timepoint, two different amplifications were performed for each of the two experiments. Using all
Page 37 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
the data for the challenge doses between 103 to 107 CFU, the data were averaged and we
identified 141 genes consistently elevated among the dose responses at 4 hours post challenge.
We received lung RNA samples from UNM of a time course after a 10 3 CFU challenge. Samples
were collected at 1, 3, 5, 7, and 24 hours after infection. Samples labeled T0 were from
uninfected animals. We were able to establish that there were patterns that increased,
decreased, or maintained expression levels over time.

We received samples of SCHU S4 RNA of microbial cultures in vitro at the same time points of
incubation as listed above (0, 1, 3, 5, 7, and 24 hours). The RNA was checked by bioanalyzer
and purified by Qiagen’s RNAeasy technique. We amplified a starting amount of 1.0 micrograms
of input RNA through the LAPT- process. After purification we obtained an average of 20
microgram yield (Table 1).
Table 1. Amplification yields of 1 microgram SCHU S4 RNA from in vitro cultures after the LAPT process.
Notebook 711, LAPT 26, pages 6.


Notebook/File locations …, Notebook 711, LAPT 26, pages 1-10.
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-26
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We have hired several new personnel due to turnover and these new people are in training to
perform the LAPT and gene expression analyses.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
30%
9. Work plan for upcoming month and next 6 months


Finish the labeling and hybridization of amplified RNA of F. tularensis grown in culture
medium and compare the in vitro gene expression results to those identified in vivo gene
expression patterns.
Establish the Q-RT-PCR process for gene expression validation.
Page 38 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam

Assess gene expression levels of known potential antigens (tul4, katG, iglC, groEl) in the
current RNA from the initial dose response and time course experimental sample set.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35
Milestone description: Array hybridization with mouse RNA from virulent SCHU S4
infection and RT PCR confirmation of candidates
Institution: UNM
1. Date started:
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
No new work done
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
6%
9. Work plan for upcoming month
a. We will provide RNA and DNA when needed by ASU
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
Page 39 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
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 milestone is currently paused pending approval of modification #2 to subaward
agreement. NIAID contract officer approved the Cerus subcontract modification 4R2 on
5/19/08. UNM is waiting for Cerus to sign and return the subcontract modification before
6/15/08.
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-scale 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
This milestone has been paused. Following approval of Modification #2 to subaward agreement,
this milestone will be halted and a milestone completion report will be written comprising the
progress to date.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors

Modification 4R2 to subaward agreement received from NIAID on 5/19/08. UNM is
waiting for full execution by Cerus before 6/15/08.
Page 40 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
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 LD 50 of
the KBMA vaccine (1 x 109 IP, 1 x 108 IV). 100% protection was also achieved by administration
of 1 x 107 KBMA particles IV when the vaccine was given twice separated by 3 weeks. Depletion
of CD4+ T cells prior to the challenge decreased the survival rate to 80%, depletion of C8+ T cells
had no effect, and depletion of both cell populations resulted in 90% survival. Together, these
data demonstrated that CD4 T cells contribute to a protective immune response in a non-CD8 T
cell-dependent manner. These data suggest that the CD4 T cells may be boosting humoral
immunity by stimulating B cells. This interpretation was supported by an adoptive transfer
experiment in which only the high-titer 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 milestone is currently paused pending approval of modification #2 to subaward
agreement. NIAID contract officer approved the Cerus subcontract modification 4R2 on 5/19/08.
UNM is waiting for Cerus to sign and return the subcontract modification before 6/15/08.
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
Page 41 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
This milestone has been paused. Following approval of Modification #r2 to subaward agreement,
this milestone will be halted and a milestone completion report will be written comprising the
progress to date.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
Modification 4R2 to subaward agreement received from NIAID on 5/19/08. UNM is waiting for full
execution by Cerus before 6/15/08.
Milestone 44
Milestone description: Formulation and evaluation of KBMA LVS: establish photochemical
inactivation regimen of selected uvr mutant of LVS and measure metabolic activity and virulence
of KBMA LVS.
Institution: Cerus
1. Date started: 6/18/2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary: using a small-scale inactivation procedure we have determined that the S-59 psoralen
concentration required to inactivate uvrB LVS is 5uM. This is the same concentration at which
we have been able to inactivate WT LVS. The uvrB LVS was also not more sensitive to DNA
damaging agents compared to WT. This suggests that there may be redundant DNA repair
mechanisms in LVS that may be functioning to repair photochemically induced crosslinks.
1) This milestone is currently paused pending approval of modification #2 to subaward
agreement. NIAID contract officer approved the Cerus subcontract modification 4R2 on 5/19/08.
UNM is waiting for Cerus to sign and return the subcontract modification before 6/15/08.
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
This milestone has been paused. Following approval of Modification #r2 to subaward agreement,
this milestone will be halted and a milestone completion report will be written comprising the
progress to date.
10. Anticipated travel
none
11. Upcoming Contract Authorization (COA) for subcontractors
Page 42 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Modification 4R2 to subaward agreement received from NIAID on 5/19/08. UNM is waiting for
full execution by Cerus before 6/15/08
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale LVS
culture conditions, Establish 3L culture scale purification conditions, Optimize 3L scale
photochemical inactivation process, Verify protective immunogenicity of vaccine candidates
produced by optimized large-scale process
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: we have demonstrated that LVS grows robustly in Chamberlains Defined Media
(CDM) and have prepared expanded DVC lot 16 LVS cultures grown in CDM for 36 hours, and
stored at -80oC. We have determined that the minimum concentration of S-59 required for
complete inactivation of DVC lot 16 LVS is 5µM and that photochemically inactivated LVS
maintain metabolic activity for at least 12 hours. We produced a 3L lot of LVS in our fermentor
using .001% Sigma antifoam A in CDM and have demonstrated stability for 4 months at -80o in 2
cryopreservation medias. We have found that the LVS provided by DVC is greatly attenuated for
virulence in mice when administered IP compared to literature reports. We have demonstrated
that LVS replicate rapidly in livers and spleens of mice immediately following IV injection;
however, it appears that there is a lag that specifically affects growth in the lungs. We have also
demonstrated that LVS is nearly avirulent when administered by the SC route.
We have produced a 400mL lot of KBMA wild-type LVS using 10 uM S-59 and 6 J/cm 2 UVA for
initial proof of concept studies, and for later comparison with NER-deficient uvrB LVS and we
have demonstrated that the metabolic activity of this lot is stable for 3 months. We have
demonstrated that KBMA WT LVS IV LD50 is 6.8x108, which represents a 4-5 log attenuation
compared with live LVS. We have demonstrated that doses of KBMA WT LVS as low as 1 x107
provide protection against 100 x IP LD50 challenge of live LVS. However, none of the mice
vaccinated with the equivalent doses of HK LVS died either. This is consistent with protection
against an LVS challenge being largely humoral. b We recently attempted to measure the T-cell
response to a CD4 Tul4 epitope in mice vaccinated with live or KBMA 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 milestone is currently paused pending approval of modification #r2 to subaward
agreement. Modification 4R2 to subaward agreement received from NIAID on 5/19/08. UNM is
waiting for full execution by Cerus before 6/15/08.
4. Significant decisions made or pending
Page 43 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
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
This milestone has been paused. Following approval of Modification #2 to subaward agreement,
this milestone will be halted and a milestone completion report will be written comprising the
progress to date. NIAID contract officer approved the Cerus subcontract modification 4R2 on
5/19/08. UNM is waiting for Cerus to sign and return the subcontract modification before 6/15/08.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
Modification 4R2 to subaward agreement received from NIAID on 5/19/08. UNM is waiting for
full execution by Cerus before 6/15/08
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis (SCHU S4)
(iglC, pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F. tularensis
subsp. tularensis (SCHU S4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F. tularensis
subsp. tularensis (SCHU S4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHU S4 we need to develop tools to generate successful deletions.
Therefore, our focus is two fold, one is cloning experiments to get our target deletions into vectors
that we can use in creating these deletions and experiments with SCHU S4 itself using constructs
that we believe will allow us to make deletions into SCHU S4.
Page 44 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
I.
Cloning:
In order to delete an entire pathogenicity island (FPI) from Schu S4 we need to move the
pdpD deletion containing the flip recombinase recognition sites (Flp) from the pwsK30
plasmid into pUC118 vector. In the April’s report we had possible pUC+pdpD::Flp::ErmC
clones based on the PCR screen and the EcoRI digestion which linearize the plasmid
constructs. However, further enzyme restriction analysis with Bgl II enzyme indicated
that these clones were actually the original pwsk30 construct. Therefore, will begin the
cloning again by preparing new pUC118 and generating more pdpD::Flp::ErmC fragment
via PCR using the pKEK1188 as the template. Bgl II will be used in the screening of the
new clones. Data located in UTSA TVD Notebook 5, page 132.
II.
Experiments to generate mutants in Schu4:
a. Continued with additional vgrG screening by PCR of potential vgrG mutants in Schu4.
Since clone 10’s single colonies resulted in all wild type genotypes we decided to go back
to our original clones (C1-C10) and selected 10 additional (C11-C20) clones and do
colony PCR using a primer specific FTT1346 (FTT1346 fwd NdeI) and a primer specific
to the vgrG stop codon. Wildtype vgrG plus FTT1352 should yield a PCR product of
~900 bp whereas a mutant should be shifted to ~1600 bp. Of the 20 clones screened,
none showed a shift in sized compared to wildtype, lane 2 (figure 1).
Our initial
screening of the vgrG transformants yielded a clone that showed both the wild type and
the mutant’s shifted band, we seem to have lost this plasmid’s integration from these
clones therefore we’ve decided to begin again.
That is, we will do another
cryotransformation experiment with freshly prepared plasmid from KEK1162 bacteria
strain. This is the strain that has the VgrG deletion construct (plasmid).
Figure 1.
Figure 1 represents PCR products generated when colony lifts from the original vgrG
transformants (C1-20) were used with a FFT1346 specific and vgrG specific oligo set. Lane 2
represents the wildtype PCR product size expected at ≈900 bp and the expected vgrG mutant
PCR product is expected to be ≈1600 bp. Lanes 3-22 are colony PCR products resulting from
the original vgrG transformants. None of these appear to be correct.
Data located in UTSA
TVD Notebook 1, page 28.
b.
Continued with evaluation of the SchuS4 igLD mutant candidates via western blotting
experiment. Based on the PCR profiles from April’s report, we have several potential
igLD mutants. By using igLD specific antibody (mouse anti-IgLD) we can determine by
Page 45 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
the absence of igLD protein in the immunoblot whether both copies of the igLD gene has
been disrupted. Therefore, first we grew liquid (Chamberlains) cultures of the necessary
clones and added 2XSDS treatment buffer to each harvested pellet, respectively. Then
two 10% acrylamide gels were prepared. Once the treated clones were loaded in wells
the gels were electrophoresed until the blue dye reached the bottom of the gels. One gel
was used for staining in coomaisse blue dye and the other was used to transfer the
protein onto a nitrocellulose membrane. This membrane was then incubated in blocking
reagent overnight at 4 °C; washed 3 times in 1XTBS buffer; incubated in mouse anti-IgLD
serum (1:250) overnight at 4 °C; washed again with 1x TBS buffer; incubated in
peroxidase labeled anti-mouse antibody (1:8,000 Amersham NIF825) for 4 hours at room
temperature. This membrane was washed three times in 1xTBS then exposed to
autoradiograph film (figure 2). We will continue processing some of the correct clones
by generating third set of single colonies which will be grown at 37 C to remove the
tulatron igLD plasmid.
Figure 2.
Figure 2 represents results of an immunoblot experiment using mouse anti-igLD serum
(1:250) with a peroxidase labeled (HRP conjugated) antibody using the ECL Western
Analysis Kit (Amersham #RPN2108). All second single clones were used to test for loss
of protein expression (igLD). Lane 2 is the positive control KKT1 wild type expression of
igLD is shown at 47 KDa (¤). Lanes 3-11 are potential SchuS4 igLD clones and all did
not react with the igLD antibody. Lane 9 and 10 protein samples (*) were prepared on
the same day the others were prepared at an earlier date. Eight individual clones appear
to be correct. Data located in UTSA TVD Notebook 5, page 135.
c.
Prepared chromosomal isolations from some of the second and third single isolates
generated from the passaging of the 255a tulatron igLD mutant candidates. The third
single isolates were grown at 37 °C to cure the plasmid. The third single isolates (e.g.
2D1g2.26) illustrated on figure 3 were determined to be kanamycin sensitive. Oligos
specific to igLD gene (igLD NdeI for and igLD NcoI rev) with the various genomic
templates were used in PCR analysis to determine possible igLD SchuS4 mutants (figure
3)
Page 46 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 3.
Figure 3 represents PCR products generated when using SchuS4 genomic DNA templates with
igLD specific oligos (igLD NdeI For; igLD NcoI Rev). Lane 2 is the wild type control product from
KKT1 strain (≈1100 bp). Lanes 3 and 6 are first singles igLD mutants this illustrate both the wild
type and mutant’s product (≈1900 bp). Lane 4 and 7 are examples of a second single igLD
mutants (these look correct) and lanes 5 and 8-13 are third igLD single mutants. Clones
2D1g2.26, 2D1J3.23, 2D1J3.36, and 2D1J3.39 appear to be correct in lanes 5, 9, 10 and 11.
Data located in UTSA TVD Notebook 5, page 136.
d.
e.
Two of the potential correct igLD mutants (2D1J3.39 –KKT9 and 2D1g2.26 – KKT10)
were frozen and placed in the -85 freezer for long storage while we wait for confirmation
sequence results of various PCR products that will be generated and sent for sequencing
in this coming month.
Did some ordering for enzymes and general supplies for ongoing experiments.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
69%
9. Work plan for upcoming month
b. Will continue the screening for potential igLD mutants in KKT1 SCHU S4 strain. The clones
which are cured of the plasmid will be used in western blot analysis. In additional, various PCR
products will be generated with various oligo sets to further verify that these clones are correct
(KKT9 and KKT10).
c. Once the immunoblot is repeated with plasmid negative clones and illustrates no igLD protein, we
will infect mice to check for attenuation of the igLD mutant
d. Will continue experiments to generate the VgrG mutant in KKT1 SCHU S4 strain. Which involve
doing another cryotransformation of the KKT1 SchuS4 strain.
Page 47 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
e. If time allows will continue with cloning the pUC pdpD::Flp::ErmC fragment into the pUC118
plasmid. This should allow a complete pathogenicity island to be removed if we can generate the
pdpD deletion in these mutants using this resulting construct (pUC pdpD::Flp::ErmC).
10. Anticipated travel
None
11.Upcoming Contract Authorization (COA) for subcontractors
UTSA has signed subcontract modification 6R2.
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 05/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
50A: (1) Evaluate the protective efficacy of intragastric F. novicida iglB vaccination (prime
and one boost) against SCHU S4 intranasal challenge in C57BL mice. (Note book #4, page
153-155). Mice were given intragastrically a single or a second dose of iglB (103 CFU) and
challenged intranasally with either 50 or 200 CFU of SCHU S4 three weeks after the final
immunization. Single i.g. vaccination with iglB did not protect C57BL/6 mice against i.n.
SCHU S4 challenge (50 CFU), however, survival rate has increased to 40% when mice
received a booster dose (Fig. 1). All mock control mice were succumbed to SCHU S4
infection by day 6. C57BL/6 mice received a single or two iglB immunizations were not
protective against the higher dose (200 CFU) of SCHU S4 challenge (data not shown).
Page 48 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
100
Moc k
Prime
Prime/ Boos t
% Survival
80
60
40
20
0
0
3
6
9
12
15
18
21
Days after challenge
Fig. 1. Protective efficacy of F. novicida ΔiglB immunization against F. tularensis
infection. C57BL/6 mice (10 per group) were intragastrically (i.g.) primed or
primed and boosted with 103 CFU of ΔiglB and challenged intranasally with F.
tularensis SCHU S4 strain (50 CFU) three weeks after the final immunization.
Mice were monitored for survival rate. Mice received PBS i.g. were used as mock
control.
(2) Evaluate the humoral responses after intragastric F. novicida iglB vaccination. (Note
book #4 pages 156-157): Groups of C57BL mice (female, 4-6 weeks) were primed or primed
and boosted with 103 CFU of iglB intragastrically. Sera and fecal pellets were collected at
day 21 after immunization and assayed for anti-iglB specific antibody titers. Mice received
single immunization of iglB induced significant amount of serum antibody as shown in Fig.
2A. Further IgG isotyping analyses of the sera indicated i.g. immunization of iglB resulted in
producing comparable titers of IgG1 and IgG2a. Intragastric immunization also induced
measurable anti-iglB specific secretory IgA in the prepared fecal pellet samples (Fig. 2B.).
Furthermore, the booster dose of iglB induced even higher titer of serum antibodies and
secretory IgA. This stronger humoral response by the boost may contribute (partially) to the
higher survival rate as shown in Fig.1 above.
Page 49 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
(A)
(B)
4000
1.00
Total Ab
IgG1
0.80
IgG2a
A405
Titer
3000
2000
0.60
0.40
1000
0
0.20
0.00
Prime
Prime/ Boos t
Prime
Prime/ Boos t Prime Prime/ Boos t
IgA
IgM
Fig.2. Mucosal immune responses induced by F. novicida iglB intragastric
immunization. Mice were primed or primed/boosted with 103 CFU of iglB. Sera (A)
and fecal pellets (B) were collected 3-week after final immunization, and assayed for
anti-iglB specific antibody.
50B. (1) Analyze the serum and fecal antibody isotypes of mice intragastrically immunized
with F. holarctica LVS at 12 weeks after vaccination. (Note book #8, pages 7, 15, 59, 77-78,
82-84). Mice were vaccinated intragastrically with 103 CFU LVS or mock immunized with PBS
alone. At 12 weeks after inoculation, blood and feces were collected. Some mice received a
second booster dose of 103 CFU LVS I.G. Blood and feces were collected from these mice
three weeks after booster vaccination dose. Specific anti-LVS total antibody titers, as well as
IgG1, IgG2a, and IgA isotypes for serum and Ig (H+L), IgA and IgM isotypes for fecal
samples, 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 or undiluted fecal samples. Goat anti
mouse Ig(H+L), IgG1, IgG2a, IgA and IgM antibody conjugated with horseradish peroxidase
were used as the secondary antibodies to determine antibody isotypes and titers. As shown
in Fig. 1 below, mice immunized with LVS I.G. have decreased total antibody titers at 12
weeks when compared to analyses performed at 8 weeks after vaccination. Mice which
received a second dose of LVS at 12 weeks after initial vaccination had slightly decreased
titers. Mice also retained a predominantly Th1 response similar to serum analyzed at 3 and 8
weeks after vaccination with mice which received a booster vaccination having relatively
lower 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. As shown in Fig. 2
below, mice retain high levels LVS-specific IgA in the G.I. tract at 12 weeks after vaccination.
However, mice which were given a booster vaccination had decreased levels of IgA. Similar
to the 3 and 8 week time points, there were minimal amounts of LVS-specific total antibody
and almost no IgM isotype. Little to no LVS-specific antibody was detected in mice mockvaccinated with PBS. All tested serum samples showed no reactivity to the unrelated HEL
protein
Page 50 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
20000
LVS 12 wk
10000
LVS 12 wk + Boost
Mock (PBS)
50% Binding Titer
LVS/ HEL
Mock (PBS)/ HEL
1000
100
Total Ab
IgG1
IgA
IgG2a
Fig. 1. Humoral responses to intragastric LVS immunization at 12 week
timepoint. Groups of BALB/c mice were vaccinated I.G. with 103 CFU of LVS or
PBS as a control. Sera were collected 12 weeks later and analyzed to determine
titers for anti-LVS specific antibodies. Some mice were given a second boost
dose of LVS I.G. at 12 weeks and sera was collected 3 weeks later.
LVS 12 wk
LVS 12 wk + Boos t
Moc k (PBS)
LVS/HEL
M oc k (PBS)/HEL
O.D. (630 nm)
0.80
Ig (H+L)
2.00
IgA
0.20
0.60
1.50
0.15
0.40
1.00
0.10
0.20
0.50
0.05
0.00
0.00
0.00
IgM
Fig. 2. Humoral responses to intragastric LVS immunization at 12 week timepoint. Groups of
BALB/c mice were vaccinated I.G. with 103 CFU of LVS or PBS as a control. Fecal samples
were collected 3 weeks later and analyzed to determine titers for anti-LVS specific
antibodies. Some mice were given a second boost dose of LVS I.G. at 12 weeks and fecal
samples were collected 3 weeks later.
Page 51 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
(2) Perform histological analyses on tissues at varying time points after LVS intragastric
immunization and subsequent SCHU S4 challenge. (notebook #8, pages 100-101) BALB/c
mice were vaccinated intragastrically with 10 3 CFU LVS or mock immunized with PBS alone.
Four weeks later, mice were challenged intranasally with 100 CFU SCHU S4. At various time
points after challenge (3, 15 and 30 days), mice were sacrificed and lungs were collected.
Briefly, 10% neutral buffered formalin was injected in to the lungs via the trachea wherein
lungs were removed and set overnight in formalin for fixation. Tissues were then imbedded in
paraffin and sliced in 5m sections and placed on slides, 3 sections per slide. Every fourth
slide was stained with hematoxylin and eosin and visualized using light microscopy. As
shown in Fig. 3, at three days post-infection, mock immunized lungs displayed minimal
inflammatory cellular infiltration. In contrast, lungs from LVS vaccinated mice displayed focal
peri-bronchiolar and peri-vascular mononuclear cell infiltration, which appear to be
predominantly composed of lymphocytes. At 15 days after challenge, lungs from vaccinated
mice showed a greater degree of peri-bronchiolar and peri-vascular lymphocytic infiltration,
including the presence of bronchiolar and vascular cuffing by the inflammatory cells. At 30
days after challenge, lung sections from vaccinated mice displayed minimal inflammatory
cellular infiltrates. Mock vaccinated mice succumb to infection by day 5 after challenge.
10X
20X
40X
Mock (PBS)
3 Days Post
Challenge
LVS
3 Days Post
Challenge
Page 52 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
LVS
15 Days Post
Challenge
LVS
30 Days Post
Challenge
Fig. 3. Lung histology after LVS vaccination and SCHU S4 challenge. Groups of BALB/c
mice were vaccinated I.G. with 103 CFU of LVS or PBS as a control. Mice were
challenged 3 weeks later with 100 CFU SCHU S4 i.n. and lungs were collected at
varying time points after challenge. Lung sections were stained with hematoxylin and
eosin.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed (Averages to 64.5% completed)
71% of scientific work completed on milestone 50A (original plans)
58% of scientific work completed on milestone 50B (intragastric plan)
9. Work plan for upcoming month
50A: Measure intramacrophage (J774) replication of Ft subsp. tularensis (SCHU S4) iglD
mutant
50B: (1)Survival after LVS I.G. vaccination and SHU S4 challenge at 8 weeks after
immunization
(2) Histological analyses of lungs of LVS vaccinated mice at 45 and 60 days after
challenge to check for the presence of long-term pathology.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
UTSA has signed subcontract modification 6R2.
Page 53 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 52
Milestone description: Create RecA mutants in F. tularensis subsp. tularensis(Schu S4)
Institution: UTSA
1. Date started: 9/15/2007
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
Creation of Schu S4 recA mutant
3.1 To inactivate RecA in Schu S4, we were in the process of constructing a Targetron vector for
targeting and inactivating the RecA gene. The Targetron vector was designed to be constructed
with the intron expression vector pKEK1140 for the backbone, and a 350bp PCRproduct for the
insertion to mutate intron RNA.
a. In last monthly technical report, we reported that 27 colonies were sensitive to Kanamycin,
which meant that those colonies had lost the plasmid. Colony PCR was performed using
two sets of primers “recA Schus4 for and recA Schus4 rev”, “recA Schus4 for and EBS
Universal” to screen the colonies to confirm recA was mutated in Schu S4.
Figure1:
Lane5 (about 600bp) and lane10 (about 1500bp) were the positive controls (KKF343, LVS recA
mutant). Lane6 and lane11 (about 630bp) were the negative controls. Lane2 to lane4 had the band
about 500bp as expected since the insertion was at 720/721bp in recA of Schus4, whereas the
insertion was at 840/841bp in recA of LVS (KKF343, positive control), so those three colonies were
correct mutants. The recA mutant Schu S4 strain was designated as KKT11, and the frozen stocks
were made.
Data recorded on UTSA TVDC notebook #6, page19-20 for figure1.
Evaluate the attenuation of LVS recA mutant (KKF343) intraperitoneal inoculation and the protective
efficiency against wild type LVS intraperitoneal challenge in BALB/c mice. KKF 343 (1300CFU) was
inoculated into the mice intraperitoneally on March 14th. The mice survived after 30 days. On April 16th (32
days after vaccination with KKF343), the mice were challenged with wild type LVS (about 700CFU) and
monitored for survival for 30 days. The mice survived.
Page 54 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Table1. Attenuation of LVS recA mutant and protective efficacy against wild type challenge
Route of
Inoculation
I.P.
Mock
Inoculation
Dose
(CFU)
1300
Survival Rate Route of
(D30)
Challenge
7/7
I.P.
I.P.
Challenge Survival Rate
Dose
D6
D30
(CFU)
700
7/7
7/7
110
0/5
b. Evaluate attenuation of Schu S4 recA mutant (KKT11) intranasal inoculation in BALB/c
mice. Inoculated the mice with105 CFU Schu S4 recA mutant (KKT11) intranasally on May
6th. The control group was infected with175 CFU wild type Schu S4. The mice inoculated
with Schu S4 recA mutant died on the 4th day.
Table2. Attenuation of Schu S4 recA mutant
Inoculum
KKT11
Wt Schu S4
c.
Route of
Inoculation
I.n.
I.n.
Inoculation
Dose
(CFU)
105
175
Survival Rate
D1
D2
D3
D4
D5
D6
5/5
5/5
5/5
5/5
5/5 0/5
5/5 5/5 4/5 0/5
Since recA mutant Schu S4 was not attenuated with the dose of 105 CFU, we would like to
eliminate the possibility of the inoculum contaminated with wild type Schu S4. 16 colonies
were selected randomly from the plate streaked with the inoculum, and colony PCR was
performed with the primers “recA Schus4 for” and “recA SchuS4 rev”.
Figure 2: on a 1% agarose gel.
Page 55 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Lane13 and lane 23 were wild type Schu S4 with PCR product at about 630bp, but the bands were not
strong because the template for PCR was not ideal. Lane12 and 22 were the positive controls (LVS recA
mutant) at about 1500bp. Lane2-lane11 and lane16 to lane21 were colony1 to colony16. There wasn’t
any band at the size of wt Schu S4 (630bp) presented on each lane of 16 colonies. This PCR confirmed
that the possibility of wild type Schu S4 mixed in the inoculum (Schu S4 recA mutant) was very limited.
Data recorded on UTSA TVDC notebook #6, page 35-36 for Table1, page37-38 for Table2 and page 3839 for Figure2.
Creation of a LUX operon plasmid in Ft
3.2 A new focus of this milestone is to introduce LUX operon (LuxC, LuxD, LuxA, LuxB and LuxE)
into the plasmid pKEK843 containing Ft groELp promoter to allow the generation of the
bioluminescent LVS and U112. Lux operon is a set of genes which encode the luciferinluciferase system in bacterial. LuxCDABE have been identified as active in the emission of
visible light.
a. The plasmid PUTminiTn5kmlux provides a 7 kb ECoRI fragment carrying the LUX operon.
Both PUTminiTn5kmlux and pKEK843 were digested with ECoRI restriction enzyme at
37˚C for overnight.
Figure 3: on 1% agarose gel.
PUTminiTn5kmlux (14.2kb) was cut into three fragments (~7kb, ~5kb and ~2.2kb on lane3) since three
ECoRI restriction sites existed in this vector, whereas only one ECoRI site in pKEK843.
b. The 7-kb fragment (the first band) from lane3 and the digested pKEK843 (lane5) were
purified using QIAquick Gel Extraction Kit. The digested pKEK843 was treated with CIP
to prevent it from self-ligation. Then two gel purified fragments were ligated together
using T4 DNA ligase at 16°C for overnight. The ligation solution was purified using phenol
and chloroform.
Data recorded on UTSA TVDC notebook #2, page120-123 for figure3.
4 Significant decisions made or pending
None.
Page 56 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
5. Problems or concerns and strategies to address
None
6. Deliverables completed
KKT11 (Schu S4 recA mutant)
7. Quality of performance
Good
8. Percentage completed.
About 25% of scientific work completed.
9. Work plan for upcoming month
i. Inoculate the mice with low dose Schu S4 recA mutant to evaluate attenuation.
ii. Transform the ligated product (lux operon in pKEK843) into E.Coli.
iii. Screen the transformants from Section ii.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
UTSA has signed subcontract modification 6R2.
Milestone 55
Milestone description: Compare Cellular Immunogenicity of Francisella and Listeria-Based
Vaccine Platforms. Measure cellular immunogenicity of live-attenuated vaccine platforms.
Compare immunogenicity of KBMA tularemia vaccine platforms
Institution: Cerus/Anza
1. Date started: 4/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary of objectives: We will construct and prepare live and KBMA Listeria monocytogenes (Lm)
vaccines expressing Ft antigens. To directly compare the cellular immunogenicity of Lm and Ft-based
vaccines, each Lm vaccine candidate will express an antigen fused to a model ovalbumin epitope
SIINFEKL (SL8) and these will be compared to Ft vaccines expressing pepO-SL8 fusions (provided by
UTSA). We will measure the ability of each vaccine to stimulate a CD8 T cell response in vitro using a
B3Z assay. We will measure the cytokine responses elicited by vaccination with each platform in mice,
compare the CD8 T cell response to SL8 after prime and boost vaccinations in mice using intracellular
cytokine staining (ICS) and ELIspot assays and measure the potency the T cells elicited by use of an in
vivo cytotoxicity assay.
1) Modification 4R2 to the Cerus subaward agreement was has been authorized by the NIAID Contract officer and
is awaiting execution by Cerus before 6/15/08.. This modification will allow for changing the scope of work to
include evaluation of Listeria monocytogenes-based tularemia vaccines and will allow Anza Therapeutics Inc to
provide the services necessary for Cerus to achieve these milestones. A draft service agreement was written
between Cerus Corporation and Anza Therapeutics Inc., and is currently being reviewed by Anza, and will be
discussed with UNM PI, Dr. Rick Lyons, immediately after the abbreviated monthly technical call on June 10. An
MTA that will facilitate the transfer of materials between Cerus, Anza, UNM and other TVDC members has been
drafted and revised by Anza and was sent to Nancy Carr at UNM April 24. An MTA that will facilitate the transfer
of materials Between UCLA (and their collaborators), Anza, UNM and LBERI is in process pending review of the
Anza-TVDC MTA.
Page 57 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
2) Cloning of Listeria monocytogenes (Lm) tularemia vaccine strains. The live-attenuated vaccine
strain BH2106 LmactAinlBKatGSL8 has been constructed, (see table #1 below, NB899 p19). BH2106
is a recloned version of BH1226 except that the plasmid used to make this BH2106 strain does not have
any mutations in the katG sequence resulting in amino acid changes. As with all the TVDC antigen
cassettes constructed to date, the plasmid used to introduce the expression cassette is an integration
vector that integrates at the tRNAArg locus and uses the actA promoter for prfA-dependent transcription
and the N-terminal 100 amino acids of ActA to facilitate secretion.
Table 1
Strain
Genetic Background
Antigen Cassette
CRS-100 actAinlB
none
BH137
ActAN100-Ova
actAinlB
BH1222
ActAN100-IglC-SL8
actAinlB
BH1226
ActAN100-KatG-SL8
actAinlB
BH2106
ActAN100-KatG-SL8
actAinlB
BH1228
ActAN100-IglC-SL8
actAinlBuvrAB
BH1398
ActAN100-KatG-SL8
actAinlBuvrAB
BH2094
ActAN100-IglC-SL8
actAinlBuvrABprfAG155S
BH2096
ActAN100-KatG-SL8
actAinlBuvrABprfAG155S
BH2098
ActAN100-IglC-VacQuad-SL8
actAinlB
BH2100
ActAN100-IglC-VacQuad-SL8
actAinlBuvrABprfAG155S
* yellow highlighted strains are new this month
Status
Sequence verified
Sequence verified
Sequence verified
Sequence error
Complete
Sequence verified
Sequence verified
Complete
Does not express Ag
Complete
Complete
We have recently demonstrated enhanced immunogenicity of KBMA vaccines when the platform strain
has a constitutively activated prfA transcriptional regulator LmactAinlBuvrABprfAG155S (Lauer et al.
manuscript has been accepted for publication at Infection and Immunity). In order to produce KBMA
vaccine strains with the highest potency, we have begun the process of introducing the IglCSL8 and the
KatGSL8 expression cassettes into this enhanced KBMA platform strain. The following enhanced KBMA
vaccine strains have been constructed using sequence verified plasmids: BH2094
LmactAinlBuvrABprfAG155SIglCSL8 and BH2096 LmactAinlBuvrABprfAG155SKatGSL8 (NB899
p 11). To improve the performance of in vivo cytotoxicity studies, the iglC antigen has been constructed
as a fusion protein to our “quatrotope tag”. This tag encodes 4 epitopes from vaccinia virus of varying
strengths as well as the SL8 epitope. This range of epitope strengths allows for more dynamic range in
the in vivo cytotoxicity assay. The following quatrotope-tagged strains have been produced: liveattenuated platform stain BH2098 LmactAinlB VacQuad-SL8 and KBMA platform strain
LmactAinlBuvrABprfAG155S VacQuad-SL8 (NB899 p 13).
3) Characterization of Lm tularemia vaccine candidate strains. The ability of Lm vaccine candidates
to deliver proteins to the cytosol of antigen presenting cells was determined using the B3Z assay (Fig 1).
All strains but BH2096 stimulated the B3Z T cell line (that responds to the SL8-MHC complex) to a similar
degree as the ovalbumin positive control (BH137). The signal from BH2096 is of similar magnitude as the
negative control, thus it is likely that this strain does not have the antigen cassette and will be recloned in
the next month.
Page 58 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 1
NB 1003 p77
B3Z 052308
0.8
OD 595
0.6
0.4
0.2
06
B
H
21
96
B
H
20
98
B
H
13
00
B
H
21
98
B
H
20
94
B
B
H
20
22
H
12
H
13
B
C
R
S1
00
7
0.0
Analysis of antigen expression in vivo. C57BL/6 mice were vaccinated IV with 5 x 106 live-attenuated
vaccine strains expressing IglC or KatG and the ability to induce a CD8 T cell response against the SL8
tag was measured by intracellular cytokine staining (ICS) and ELISpot (Fig 2). The antigen specific
response to each fusion protein was evaluated by measuring the number of SL8-reactive CD8 positive T
cells. BH1228 expressing the IglC-SL8 fusion protein was greater than twice as potent as BH1398
expressing the katG-SL8 fusion protein (mean 17.3% vs. 7.4% of the total CD8 positive cells). This is
consistent with the Western blot data demonstrating that IglC is more highly expressed by Lm in the
cytosol of host cells than KatG. As a control, the ability of each strain to induce a CD4 T cell response
against the Listeriolysin O (LLO) protein secreted from Lm was similar (2.9 vs. 1.9 respectively).
Secreted LLO mediates escape from the host cell phagosome and has a well-characterized CD4 epitope
(LLO 190) that is identical in all of our host strains, thus LLO responses are used as a positive control.
The reduced LLO response may be due to the inefficient secretion of KatG and thus may represent a
bottleneck on the secretion system of Lm. The ELISpot data confirm the higher immunogenicity of the
iglC-expressing strain compared with the KatG expressing strain; however, the magnitude of the
difference is less (722 vs. 491 spot-forming cells (SFC) per 250,000 splenocytes, respectively). This
decreased magnitude is likely due to the ELISpot assay being saturated and difficult to get accurate spot
counts over 600.
1
98
H
13
B
H
12
B
98
B
H
13
28
H
12
28
0
0
1000
500
0
800
unstim
LLO 190
600
400
200
0
BH1398
2
SL8
LLO 190 responses
IFN- SFC/2e5 splenocytes
3
unstim
BH1398
4
1500
BH1228
% IFN- CD4+ T cells
10
B
% IFN- CD8+ T cells
20
SL8 responses
IFN- SFC/2e5 splenocytes
5
30
IM08-042 ELISpot (NB2000 p1-5)
BH1228
Figure 2 IM08-042 ICS (NB2000 p1-5)
SL8 responses
LLO 190 responses
Page 59 of 61
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
We next wanted to measure the impact of the vaccinia quatrotpe tag and the uvrAB and prfA* alleles on
the immunogenicity of our live attenuated IglC vaccine strains (Fig 3). C57Bl/6 mice were vaccinated IV
with 5 x 106 live-attenuated vaccine strains expressing -IglC-SL8 and the ability to induce a CD8 T cell
response against the SL8 tag was measured by ICS or ELISpot (IM08-043). As expected, the strain
containing the uvrAB allele (BH1228) stimulated T cells to the same extent as BH1222 in the ICS assay.
Interestingly, the presence of the prfAG155S allele in strain BH2094 significantly increased the % CD8
cells to 24.6% compared with 15.5% and 16.1% for BH1222 and BH1228, respectively. This increase in
immunogenicity is consistent with the observations in Lauer et al. (in press). Addition of the vaccinia
quatrotope tag to the iglC-SL8 decreased immunogenicity to 8.4% in BH2098, and this was not increased
by constitutive induction of prfA in BH2100. Interestingly both strains with the prfA* allele had increases
in LL0190 responses. This suggests that the increase in immunogenicity mediated by prfA* require
efficient secretion and that the quatrotope tag decreases the efficiency of antigen secretion. The ELISpot
data generated from these mice is not shown because the number of spots generated against both the
were over 500 spots and the differences between the strains were compressed.
LLO190 responses
BH2100
BH2098
BH2094
BH1228
BH2100
BH2098
BH2094
BH1228
BH1222
BH1222
0
0
0.3
0.2
0.1
0.0
4. Significant decisions made or pending
NoneBH1222: actAinlB-iglC
BH1228: actAinBuvrAB-iglC
5. Problems
concerns and strategies to address
BH2094: or
actAinlBuvrABprfA*-iglC
NoneBH2098: actAinlB-QuadVac-iglC
BH2100: actAinlBuvrABprfA*-QuadVac-iglC
6. Deliverables
completed
None
7. Quality of performance
Excellent
8. Percentage completed
8%
9. Work plan for upcoming month

We will reclone the strain LmactAinlBuvrABprfAG155SKatGSL8 to replace BH2096

We will clone a bivalent vaccine strain that expresses both IglC and KatG.

We will clone the gain of function mutations into the inlA gene in order to increase the
affinity for murine E-cadherin and this inlA allele will be introduced into vaccine strains.
These strains will be used to evaluate immunogenicity of Lm vaccines by various routes
of administration.
10. Anticipated travel
None
Page 60 of 61
BH2100
2
0.4
BH2098
10
4
BH1222
20
0.5
% IFN- CD8+ T cells
% IFN- CD4+ T cells
30
% IFN- CD8+ T cells
LLO 296 responses
6
BH2094
SL8 responses
BH1228
Figure 3:IM08-043 ICS (NB2000 p6-9)
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2008 to 5/31/2008
Due Date: 6/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
11. Upcoming Contract Authorization (COA) for subcontractors
Modification 4R2 to subaward agreement received from NIAID on 5/19/08. UNM is waiting for full
execution by Cerus before 6/15/08
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