Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Contract No.
ADB Contract No.
HHSN266200500040-C
N01-AI-50040
Contract Title:
Tularemia Vaccine Development Team
Performance Period: 10/1/07 to 3/31/08
Contractor Name:
University of New Mexico Health Science Center
Contractor Address: Controller’s Office MSC-09-5220
1 University of New Mexico
Albuquerque, New Mexico 87131-0001
Authors:
C. Rick Lyons, MD, PhD and Barbara B. Griffith, MS
Date of submission: 4/15/2008
Semi-Annual
Technical Report:
Based on Contributions from ASU, Cerus, LBERI,
UNM and UTSA
Page 1 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, 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, 26, 27, 28,
35(ASU/UNM), 49, 50, 52
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
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. LBERI has 33 staff and scientists vaccinated as of 4/1/2008.
b. First group of 4 UNM participants received the LVS vaccination on 3/18/2008
c. 1 UNM participant and 1 LBERI participant declined the vaccination. 1 UNM participant
is not yet medically cleared.
d. 1 LBERI staff was vaccinated on 4/1/08.
e. 1 UNM participant is scheduled for LVS vaccination on 4/29/08
f. UNM and USAMRIID are actively using the LVS vaccine web database to track Risk
Assessment form submission and acceptance, Informed Consent submission and
acceptance, Health screening appointments, planned dates for receipt of LVS
vaccinations, dates of medical clearance, and travel arrangements
g. LBERI LVS vaccinees have received an invitation to donate blood for immunoassay
development on the TVDC at UNM. The volunteers will be consented prior to scheduling
blood draws at UNM
4. Significant decisions made or pending
a. UNM and LBERI are using their biobubbles as additional physical protective equipment
b. Dr. Lyons received UNM IRB approval for blood draws on the vaccinated LBERI and
UNM scientists after their LVS vaccinations. The LBERI and UNM scientists and staff are
being offered the opportunity to volunteer to donate bloods for the development of
immunoassays, approximately 2 months after receiving the LVS vaccination.
c. UNM (4) and LBERI (33) are offering the LVS vaccinations up to 9 more scientists to total
46; USAMRIID will continue to provide the LVS vaccinations until 4/29/08 and then
Page 2 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
USAMRIID is pausing the LVS vaccinations until a new protocol is approved in
approximately late summer 2008
d. The CRDA with USAMRIID is valid for 2 years, ending June 2009.
5. Problems or concerns and strategies to address
Starting in April 2008, UNM will have access to the blood of LBERI scientists who have been
vaccinated with LVS at USAMRIID. UNM’s IRB has been approved.
6. Deliverables completed
33 LBERI and 4 UNM scientists and staff have received the LVS vaccination between 9/11/07
and 4/1//08.
7. Quality of performance
Excellent
8. Percentage completed
65%
9. Work plan for the next month
a. Complete health screenings for the next group of 1 UNM participant and 1 LBERI
participant.
b. Make travel arrangements for medically eligible participants to enter LVS Vaccination
program at USAMRIID on 4/29/08 – possible second group of UNM participants.
Participants will be at USAMRIID for 2 days following the vaccinations.
c. Maintain excellent communications with UNM EOHS, LBERI and USAMRIID
10. Anticipated travel
1 UNM LVS vaccination participant will be traveling to USAMRIID on 4/28/08..
11. Upcoming Contract Authorization (COA) for subcontractors
UNM received a signed COA letter for COA 15 on 9/11/07.
Milestone 3
Milestone description: Bioaerosol technique selected and optimized
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08
a. Bioaerosol testing with LVS and SCHU S4 was completed
i.
Based on cumulative data using LVS, the optimal bioaerosol approach was
down-selected to the following criteria:
i. Brain-heart infusion broth (BHIB) as the aerosol generation and
collection substrate.
ii. Use of an all-glass impinger (AGI-4) for the collection of the bioaerosol.
iii. Maintenance of 60-90% humidity in the exposure line during bioaerosol
runs.
iv. Bacterial cultures performed on BCGA. This was modified from the
previously used CHAB that proved to be inconsistent and problematic.
v. Use of the Collison and Aeromist nebulizers for bacterial generation
(discussed below).
Page 3 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
ii.
Cumulative results indicated that LVS (when tested under similar conditions) was
not predictive for SCHU S4 (i.e., the two F. tularensis strains behaved differently
as bioaerosols). In general, the trends were the same, but SCHU S4 was
influenced more by external factors (i.e., it proved to be more fragile). This
pointed towards the need to optimize bioaerosol stability as discussed below.
iii.
Cumulative data indicated that the Aeromist was more efficacious versus the
Collison for F. tularensis bioaerosol generation for the following reasons:
i. It was easier to handle using the current LBERI ABSL-3 exposure
laboratory setup
ii. It was more cost effective
iii. Increased LVS and SCHU S4 bioaerosol viability (i.e., better spray
factors) were observed. This was an important consideration for animal
exposures in that a greater percentage of viable bacteria could
potentially be delivered using the Aeromist.
iv.
Cumulative data indicated that fresh cultures were optimal for bioaerosol studies.
i. Frozen cultures demonstrated poor spray factors regardless of the
generator used.
ii. SCHU S4 bioaerosols prepared from 48- and 72-hour cultures grown on
BCGA (solid medium) and in Chamberlains liquid broth medium,
respectively, were compared. Based on spray factor results, bacteria
harvested from the BCGA were more stable as a bioaerosol. For
consistency across the TVDC study groups, however, it was decided that
all future bioaerosols, including animal exposures, were to be performed
using 48h Chamberlains broth cultures.
b. A new working stock of SCHU S4 was created on 28FEB08. The titer of this stock was
determined to be 2.3x109 CFU/mL. Because of consistently observed drops in bacterial
titer, LBERI creates a new working stock every six months.
c. Cumulative LVS data from Milestone 3 were presented in the poster entitled,
“Optimization of Bioaerosol Generation Techniques for Francisella tularensis” at the
Annual ASM Biodefense Meeting in February 2008.
Monthly-March 2008:
a. No Francisella tularensis (LVS or SCHU S4) bioaerosol technique optimization
experiments were conducted in March 2008.
4. Significant decisions made or pending
The final decision concerning which generator (Aeromist or Collison) to use for all future
bioaerosol exposures is pending. This will ultimately depend on SCHU S4 lung deposition testing
in mice using the two generators. This is currently scheduled to take place on 7APR08.
5. Problems or concerns and strategies to address
It is unclear as to specifically how the Aeromist and Collison nebulizers affect SCHU S4
bioaerosols with respect to viability upon animal inhalation and lung deposition. This is currently
being addressed in MS4.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
98%
Page 4 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
9. Work plan for next month and next six months
a. 1 month
i. Complete Milestone Completion Report Draft
ii. Complete and submit SOPs used on MS3
b. 6 months
iii. Complete necessary revisions and finalize Milestone Completion Report
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
Semi-Annual: 10/1/07 to 3/31/08
a. Prior to NHP aerosol challenge, LVS and SCHU S4 virulence was verified in mice:
i.
The first LVS and SCHU S4 bioaerosol virulence study in Swiss Webster mice
using the Collison nebulizer was completed in November 2007. Results
demonstrated that the two LVS and SCHU S4 strains grown at LBERI were
virulent when delivered as an aerosol.
i. Mice were challenged with LVS harvested from a fresh, 48h culture
grown in Chamberlains broth. Material for SCHU S4-challenged mice
was directly diluted from frozen stock because of growth problems in the
Chamberlains broth. On several occasions SCHU S4 has failed to grow
in Chamberlains broth. The reason for this has not yet been clearly
ascertained, but is thought to be due to low inoculum volume and/or
media preparation. To address the first issue, the inoculum volume has
since been increased by 10; concerning the media, though routine
quality control measurements are in place at LBERI, the true expiration
date for Chamberlains has not yet been ascertained.
ii. 100% mortality was observed in the SCHU S4-challenged mice by five
days post-exposure. The calculated challenge dose was 1,580 CFU
delivered per mouse.
iii. Approximately 50% mortality was observed in the LVS-challenged mice
by 11 days post-exposure. The calculated challenge dose was 10,200
CFU delivered per mouse.
iv. Data clearly indicated that the LBERI SCHU S4 working stock was
significantly more virulent in a Swiss Webster mouse model when
compared to LVS.
b. The initial mouse virulence study led the way for the NHP virulence study conducted in
December 2007:
i.
Two naïve cynomolgous macaques were exposed to a high aerosol dose of
SCHU S4 and monitored for up to 14 days post challenge:
Page 5 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
ii.
Animals were challenged with bacteria harvested from a fresh, 72h BCGA
culture. This approach was taken based on the aerosol stability testing
mentioned under Milestone 3. The decision to use only 48h Chamberlains broth
cultures for exposures had not yet been made.
iii.
Calculated delivered doses were 2,670 and 5,030 CFU.
iv.
One animal succumbed to infection on Study Day 13 whereas the other was
euthanized on Study Day 14.
v.
Primary clinical signs over the duration of the infection included general lethargy,
lack of eating, hunched posture, and coughing.
vi.
Blood and select tissues were analyzed upon necropsy. The absence of F.
tularensis in the blood and the low numbers present in the spleen, liver, and
TBLN of one of the animals was suggestive of a primarily pneumonic disease.
The high tissue concentrations of SCHU S4 observed in the remaining animal
indicated that septicemia likely did not occur until end of life.
vii.
The pathology report was submitted in March 2008 (see monthly update below).
c. Following the NHP study, LBERI questioned the virulence of the SCHU S4 working stock
used for challenge. Based on the initial mouse study and the available literature, LBERI
believed that the two NHPs should have succumbed to infection within one week. It was
hypothesized that growth on solid medium (here, BCGA) may have decreased the
bacterial virulence in some unknown fashion; this was based on previous Y. pestis
studies conducted at LBERI. In order to test this, another mouse virulence study was
conducted:
i.
44 naïve BALB/c mice were challenged with SCHU S4 harvested from either a
48h Chamberlains broth culture or a 72h BCGA culture.
ii.
Target doses were 10 and 1000 CFU for each cohort.
iii.
One mouse from each group was sacrificed approximately 10 minutes following
aerosol challenge in order to assess bacterial lung deposition.
iv.
Bioaerosol and morbidity/mortality data demonstrated correct challenge doses
and virtually no difference in virulence between the two growth methods.
v.
Lung cultures demonstrated very low deposition rates though a high degree of
contamination was noted. Deposition samples had not been cultured on
selective plates, but will be in future studies.
d. Based on the second mouse virulence study, it was officially decided to use 48h
Chamberlains broth cultures for all future SCHU S4 bioaerosol exposures. Aerosol
delivery of SCHU S4 to the mice, however, resulted in a lower lung deposition than
expected (approximately 5% expected, 0.2 to 0.5% observed). It was hypothesized that
the bacteria were potentially damaged by the Collison nebulizer. Results observed in
Milestone 3 indicated that the Aeromist nebulizer was gentler. A direct comparison of
Collison and Aeromist bioaerosol delivery to an additional 20 mice is planned for the
week of April 7. This follow-on study will focus on lung deposition as influenced by the
two generators.
Monthly-March 2008:
a. The pathology report for the December 2007 NHP SCHU S4 exposures was completed.
Results are summarized below (NOTE: the full pathology report contains additional
details not included here):
i.
The gross and histologic lesions in A04344 were compatible with primary
pneumonic tularemia, with equivocal pathologic evidence of systemic
dissemination. The gross and histologic lesions in A04339 were compatible with
primary pneumonic tularemia, with unequivocal pathologic evidence of systemic
dissemination. The lung lesions in A04339 were more extensive and more
Page 6 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
necrotizing than for A04344. In addition, A04339 had evidence of possible
primary nasopharyngeal tularemia (rhinitis) with subsequent infection of the
lymph nodes which drain the head, neck and gastrointestinal tract
(retropharyngeal, submandibular, and mesenteric lymphadenitis) and secondary
bacterial rhinitis (Staphylococci). Lung lesions from both animals are
represented in Figure 1, and tracheobronchial lymph node, spleen, and nasal
cavity lesions from A04339 are represented in Figure 2.
Figure 1. Pulmonary histopathology from cynomolgus macaques exposed to aerosols of F. tularensis
SCHU S4. A, C, and E are from A04339, and B, D, and F are from A04344. Arrowheads (A, B) indicate
primary foci of pyogranulomatous to necrotizing bronchopneumonia. Small arrows (C) indicate smaller
foci of pyogranulmatous to necrotizing embolic pneumonia detected only in A04339. Large arrow (E)
indicates a colony of small coccobacilli.(report filed in Saturn\\ABSL3\Agent and Study Specific
Data\Study Specific Data\FY07-083 and -089 (TUL04)\14DEC07 NHP Exposure\07-083 path report.doc)
Page 7 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 2. Histopathology of the tracheobronchial lymph node, spleen and lung from a cynomolgus
macaque (A04339) exposed to aerosols of F. tularensis. Arrowheads in the lymph node (A) and spleen
(B) indicate foci of pyogranulomatous to necrotizing inflammation. Small arrows indicate foci of nasal
turbinate ulceration with fibrinosuppurative rhinitis in the nasal cavity (C).
Report is stored electronically:
<File://\\citron\jhutt\FY07-083>\citron\file://\\citron\jhutt\FY07-083
Slides are stored in Julie Hutt’s office:
LBERI Building 14, room 126A
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
As discussed previously, aerosol delivery of SCHU S4 to mice resulted in a lower lung deposition
than expected. The SCHU S4 may be damaged by the Collison nebulizer. A mouse bioaerosol
study to address this has been scheduled for the week of 4/7/08 with results expected by 4/12/08.
These results will influence the decision on which generator to use for upcoming NHP challenges.
6. Deliverables completed
Page 8 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
None
7. Quality of performance
Good
8. Percentage completed
30%
9. \Work plan for next month and next six months
a. 1 month:
i.
Complete mouse lung SCHU S4 deposition study comparing the Aeromist and
Collison nebulizers.
ii.
Decide upon choice of generator for all future NHP studies.
iii.
Plan ABSL-3 move-in and challenge of additional two NHP. Tentative exposure
date is set for end of April.
b. 6 months:
i.
Complete additional NHP virulence study
ii.
Complete Microbiology reports for initial and follow-on NHP studies.
iii.
Initiate Milestone Completion report
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 5 - UNM
Milestone description: Small species tested for sensitivity to LVS & generation of immunity
against a pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
Fischer 344 rats
a. We determined the kinetics of SCHU S4 proliferation, dissemination and clearance in
naïve and s.c. LVS-vaccinated Fischer 34 rats after i.t. challenge (Fig. 1)
i. In naïve rats, SCHU S4 grew uncontrollably until the bacterial burden in the
lungs, spleen and liver reached 109/tissue when the rats died 4 days after
challenge.
ii. The amount of SCHU S4 in the lungs of LVS vaccinated rats was initially
very similar to that of naïve rats. However, vaccinated rats were able to gain
control over bacterial growth 3 days after challenge. The number of SCHU
S4 then hovered between 107 and 108 cfu for an undetermined period of time,
but by day 42, 4 of 5 rats had cleared SCHU S4 from the lungs.
iii. The bacterial burden in the spleen and liver of vaccinated rats tracked
closely. SCHU S4 grew exponentially in the first 3 days, reaching and
staying at a plateau of ~ 105 cfu, and then was cleared by day 21. It is
interesting that although vaccinated and naïve rats had similar lung burdens
in the first 3 days of challenge, the vaccinated rats always had lower spleen
Page 9 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
and liver burdens than the naïve rats (especially d2 and d3). This suggests
that vaccinated rats may be more effective at 1) limiting systemic
dissemination from the lungs or 2) controlling systemic bacterial growth
Figure 1. Kinetics of SCHU S4 growth, dissemination and clearance in naïve and LVSvaccinated Fischer 344 rats
b. We continued to test quantum dots as a tracker for pulmonary infection
i. The best angle for imaging the rats depends on the location of the quantum
dots. In Fig 2, the quantum dots were located in the left lung lobes and
therefore the best image was obtained from the left side
ii. We found a discrepancy between the Xenogen image and the actual lung
deposition. For example, when the Xenogen image indicated that all of the
quantum dots are located in the lungs, we would only recover 10% of the
inoculum from the lungs. It is possible that the lung homogenate is toxic to
the bacteria because we consistently find lung deposition of < 30% even
without quantum dots. It is also possible that the remaining inoculum is
deposited elsewhere in the rat. We are performing experiments to test these
two possibilities
iii. We also found that a small amount of bacteria in the lungs after esophageal
delivery
Figure 2. Xenogen images taken of a single rat from 4 different
perspectives after intratracheal inoculation with a mixture of
SCHU S4 and quantum dots
Page 10 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Monthly-March 2008
a. 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
b. Rat model data was presented at the Tularemia Workshop in New York (3/30 to
4/1/08)
4. Significant decisions made or pending
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c. Rat model completed
7. Quality of performance
NA
8. Percentage completed
69%
9. Work plan for upcoming month
For the next one month
a. None
For the next six months
a. Milestone completion reports and associated SOP for the mouse, guinea pig and rat
will be written within the next 6 months
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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:
Semi-Annual: 10/1/07 to 3/31/08
This section is not applicable as the start date for this milestone was 2/25/08.
Page 11 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Monthly-March 2008:
SCHU S4 LD50 determination in cynomolgus macaques is in planning stages. The objective of
the study is to determine the actual 50% effective dose (ED50) in cynomolgus macaques
following inhalation exposure to Francisella tularensis SCHU S4. ED50 will be used instead of
LD50 because of IACUC and USDA concerns associated with the LD50.
We will plan three rounds of exposure. The goal will be to subsequently narrow down the ED50
value based on morbidity/mortality observations in the primates. Exposures will be performed as
follows:
1. 3 groups of 4 animals (n=12 total). Target doses will be below, at, and above the
achieved values obtained for the December 2007 NHP virulence study
2. 2 groups of 4 animals (n=8 total). Target doses will be below and above the estimated
ED50 value observed in the first round.
3. 2 groups of 4 animals (n=8 total). Target doses will be below and above the estimated
ED50 value observed in the first and second rounds.
The endpoints for each set of exposures will be clinical observations, temperature monitoring,
body weight records, gross necropsy, and viable bacterial blood/tissue cultures.
Tentative start dates are as follows:
1. Phase I: 5/8/08
2. Phase II: 6/6/08
3. Phase III: 7/11/08
4. Significant decisions made or pending
Confirmation of firm start dates pending. Specific procedures and endpoints need to be detailed
in protocol format.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Fair
8. Percentage completed
3%
9. Work plan for next month and next six months:
a. 1 month:
i.
Confirm dosing scheme
ii.
Confirm endpoints and schedule appropriate personnel
iii.
Confirm bioaerosol method
a. Dependent upon mouse deposition study described under MS 4
iv.
Initiate ABSL-3 move-in and challenge dates
b. 6 months:
i.
Complete the three rounds of SCHU S4 bioaerosol challenges
ii.
Determine ED50 value under the conditions tested
iii.
Initiate Milestone Completion report
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 12 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 11 - UNM
Milestone description: In vivo GLP model efficacy SOPS developed in one small species
and primate and efficacy testing of vaccine candidates
Institution: UNM
1. Date started: 1/16/2008
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. We found that serum from LVS vaccinated rats was sufficient to protect naïve rats
from a lethal i.t. SCHU S4 challenge (Table 1;Ptran 1 experiment). The mechanism
of protection may be different from that generated by active s.c. LVS vaccination
because the passively immunized rats appeared to clear SCHU S4 with a slower
kinetics. Surprisingly, naïve serum also protected 2 of 6 naïve rats, but the
significance of this result is not yet clear
Table 1. Passive immunization protected naïve rats from respiratory SCHU S4
Groups
Vaccination status
Treatment2
Survival ratio (No.
live/total)3
1
1
s.c. LVS vaccinated
None
6/6
2
Naïve
None
0/6
3
Naïve
Normal rat serum
2/6
4
Naive
Immune rat serum
6/6
1
Rats vaccinated s.c. with 5 x 107 LVS
Sera collected 36 d after vaccination and 3 ml transferred i.p.
3
Rats challenged i.t. with ≤ 72 SCHU S4 1 d after serum transfer
2
b. The observation that immune rat serum protected naïve rats from a lethal i.t. SCHU
S4 challenge prompted us to test whether immune rat serum would also protect
naïve BALB/c mice against a lethal i.n. SCHU S4 challenge. Unfortunately, we could
not interpret the results from the first experiment because the LVS vaccinated mice
that we used as positive control did not survive the challenge.
c. We would like to determine the importance of CD4 and CD8 T cells in protecting LVS
vaccinated rats against i.t. SCHU S4 challenge. To this end, we generated ascites
fluids from W3/25 (mouse anti rat CD4), OX-8 (mouse anti rat CD8) and TS2/18.1.1
(isotype control; anti-human LFA-2) hybridoma for in vivo depletion. This experiment
will be underway soon
Monthly-March 2008
a. Experiment Ptran2 (Notebook 112, page 68-75)
i. The purpose of this experiment is to repeat Experiment Ptran 1 which
showed that immune rat serum was sufficient to protect naïve rats from a
lethal i.t. SCHU S4 challenge. The experimental design is exactly the same
as Ptran1. The rats have already been passively immunized and challenged
i.t. with SCHU S4. We will present the results in the next monthly tech report
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Page 13 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
6%
9. Work plan for upcoming month
For the next one month
a. Titrate amount of vaccinated rat serum required for protection
b. Increase the SCHU S4 challenge dose to determine the level of protection conferred
by passive immunization
c. Determine the impact of CD4 and CD8 T cell depletion on the protection induced by
LVS vaccination
For the next six months
a. Compare active and passive immunization
i. Bacterial growth, dissemination and growth
ii. Histopathology
b. Determine whether immune rat serum is sufficient to protect naïve mice against i.n.
SCHU S4 challenge
c. Determine whether vaccinated human serum is sufficient to protect rats against
SCHU S4. This experiment will be performed as soon as sera from vaccinated
human volunteers become available
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. We optimized the IFN Elispot assay for measuring antigen-specific IFN production
by Fischer 344 rat splenocytes. The best balance of sensitivity and background was
obtained with 2 x 105 splenocytes/well and 8 x105 heat-killed or formalin-fixed LVS.
We could not increase the assay sensitivity by adding more splenocytes per well
because that caused non-specific IFN production or by boosting the LVS vaccinated
mice with live or heat-killed LVS
Monthly-March 2008
a. Experiment MFT3.6 (ABD notebook 1 and ABD flow binder 2 section 6) (electronic
file location on Dr. Dubois’ PC in BMSB roomG34: D:\My
Documents\Balbc&C57black6\Mouse flow\MFT3.6)
Page 14 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
i. The purpose of this experiment was to compare the quality of the CD4+ T
cell response in the spleen, lungs, and lung associated lymph node (LALN) in
naïve, LVS vaccinated, and LVS vaccinated BALB/c mice 4 days after i.n.
challenge with 125 cfu of SCHU S4.
ii. When compared to naïve animals, mice vaccinated i.n. with 500 cfu of LVS
showed an increased frequency of CD4+ cells that were simultaneously
secreting IFNγ, TNFα, and IL-2 (multifunctional T cells) in the spleen but not
the LALN when the cells were stimulated with heat-killed LVS (HK-LVS) (Fig
3).
iii. The percentage of multifunctional T cells was reduced in the spleen and
increased in the LALN after vaccinated mice were challenged with SCHU S4,
potentially indicating that the multifunctional cells were migrating from the
spleen to the lymph nodes draining the site of primary infection (Fig 3)
iv. In the spleen of vaccinated mice, the multifunctional (+++) cells express
higher levels of each cytokine than the double (++) or single (+) positive
cells. After challenge, the (+++) T cells appear to produce less IFN and
TNF than before challenge (Fig 4).
v. In the LALN of vaccinated mice, the amount of each cytokine produced by
the multifunctional cells increased dramatically after SCHU S4 challenge.
The TNF/IFN (++) cells produced the largest amount of TNF per cell, with
the multifunctional T cell population producing only slightly less (Fig. 5)
vi. Optimization of the multifunctional T cell assay using lung cells is ongoing.
Figure 3. Percentage of CD4+/TNFα+/IL-2+/IFNγ+ cells (multifunctional T cells) in each organ in naïve,
LVS vaccinated, and LVS vaccinated then i.n. SCHU S4 challenged BALB/c mice. Organs were collected
and processed to single cell suspension followed by treatment in vitro with media alone (none), α-CD28
(CD28), or α-CD28 and HK-LVS (LVS).
Page 15 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 4. Cytokine production by multifunctional cells, double, and single positive cells in the spleen
following stimulation with α-CD28 and HK-LVS.
Figure 5. Cytokine production by multifunctional cells, double, and single positive cells in the lung
draining lymph node following stimulation with α-CD28 and HK-LVS.
Page 16 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
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
For the next one month
a. Optimize the multifunctional T cell assay for use with lung cells.
b. Quantify multifunctional cells in the lungs, lung draining lymph nodes, and spleens
from BALB/c mice vaccinated with increasing doses of LVS
c. Determine whether LVS vaccinated mice with an active SCHU S4 infection could be
used to increase the sensitivity of the IFN Elispot assay
For the next 6 months
a. Quantify multifunctional cells in lungs, lung draining lymph nodes, and spleens after
s.c. and i.n. LVS vaccination in BALB/c mice.
b. Develop similar assay for multifunctional cells for the Fischer 344 rats
c. Help Julie Wilder develop the multifunctional T cell assay for NHP
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and compared to those in other species.
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08
a. We determined that both the Cerus and CTL freeze/thaw protocols compared favorably
with the Lyons protocol as regards the recovery of total number of PBMCs after thawing as
well as the recovery of the proliferative response
i. We chose to continue using the Cerus protocol due to its relative ease compared
to the CTL protocol
b. Historical data suggested that PBMCs from NHPs vaccinated with LVS via the SC route
produced more IFNγ than those isolated from NHPs vaccinated via the ID route; however,
Page 17 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
side by side comparison of PBMCs from such NHPs on the same day did not confirm this
difference
c. Although we cannot detect IgA anti-LVS in the sera of LVS-vaccinated NHPs, we
confirmed that our reagents detect human IgA and thus should be able to detect monkey
IgA if it were present
d. PBMCs from non-LVS vaccinated NHPs have consistently been shown to respond to
LVS
i. The highest responses in non-LVS vaccinated NHPs is to FF LVS, especially when
measuring IFNγ by ELISPOT
ii. The IFNγ response to FF LVS is less when fewer cells are plated/well (1.0 x 10 6/ml vs.
1.33 x 106/ml) and appears to dilute out in a non-linear fashion suggesting that a small
percentage of PBMCs in non-LVS vaccinated NHPs may be secreting large amounts of
IFNγ
iv.
IFNγ production by PBMCs from LVS-vaccinated NHPs decreases in a linear
fashion when fewer cells are plated/well
v.
Non-LVS vaccinated NHPs rarely have IgG anti-LVS titers above background
e. Unstimulated PBMCs from both non-LVS vaccinated and LVS-vaccinated NHPs
sometimes show background responses in the proliferation and IFNγ ELISPOT assays
i. These responses are not associated with individual NHPs; the same NHP can
show low or high background responses upon repeated testing
ii. Background responses in the IFNγ ELISPOT assay are often due to high RBC
content
1. We now continue lysing RBCs until the RBC contamination is less than
2%
2. High RBC content does not lead to high backgrounds in the proliferation
assay
iii. High background in one assay (IFNγ or proliferation) does not predict high
background responses in the opposite assay when tested on the same day
iv. High background generally does not prohibit observation of an LVS-specific
response in the proliferation assay but high background responses in the IFNγ
ELISPOT assay, particularly due to high RBC content, can obscure LVS-specific
responses
Monthly-March 2008:
a. Occasionally we observe responses to LVS in PBMCs prepared from non-LVS vaccinated
NHPs in both the IFNγ ELISPOT and proliferation assays
i. We were unsure whether these background responses were consistent (i.e. always
associated with particular NHPs) because we had rarely tested the PBMCs from such
NHPs on more than one occasion
ii. We set such an experiment up on 3/27 with 3 non-LVS vaccinated NHPs that had
been tested before
a. As we usually see this response to ff LVS, and not often to hk LVS, we tested
an even higher dose of hk LVS and various doses of ff LVS
b. We also tested various doses of hk and ff SCHU S4 for the first time
iii. Figure 1 shows the results of the IFNγ ELISPOT assays; Figure 2 shows the results
of the proliferation assay
Page 18 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Cell Mean for IFNg Spots
200
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
A. A04260
160
120
80
40
NT
0
TUL26
Cell Mean for IFNg Spots
350
TUL31
B. A04168
300
250
200
150
100
NT
50
NT
0
TUL21
TUL23
TUL31
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 19 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Cell Mean for IFNg Spots
450
Media
LVS hk Hi
C. A05477
400
LVS hk Mid
350
LVS ff Hi
300
LVS ff Mid
250
LVS ff Lo
200
LVS hk Super
150
SCHUS4 hk Super
NT
SCHUS4 hk Hi
100
50
SCHUS4 hk Mid
NT
SCHUS4 ff Super
0
TUL21
TUL23
SCHUS4 ff Hi
TUL31
Figure 1: IFNγ production by PBMCs from non-LVS vaccinated NHPs to LVS and SCHU S4 antigens. A:
A04260; TUL 26 PBMCs plated at 1.33 x 106/ml; TUL31 PBMCs plated at 1 x 106/ml; B. A04168 and C:
A05477, all PBMCs plated at 1.33 x 106/ml.
Cell Mean for RLU small
1800000
1500000
1200000
900000
Media
Con A
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
A. A04168
600000
NT
300000
0
TUL21
Cell Mean for RLU small
1800000
TUL23
B. A05477
1500000
1200000
900000
Media
Con A
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
NT
600000
300000
0
TUL21
TUL23
Page 20 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 2: Proliferation of PBMCs from non-LVS vaccinated NHPs to LVS and SCHU S4 antigens. A:
A04168; B. A05477, all PBMCs plated at 1 x 106/ml.
b. Data Interpretation
i. We observed some consistent responses when we compared the responses of
non-LVS vaccinated NHPs tested on separate days (ex. Figure 1, A04260 (A)
and Figure 2 (A: A04168 and B: A05477)
ii. We observed some divergent responses when we compared the responses of
the same NHPs on separate days (ex. Figure 1, A04168 (B) and A05477 (C))
iii. We are suspicious that LVS hk Hi and LVS ff Hi stimuli were reversed in TUL21;
if so, the responses of A05477 and A04168 would look more consistent in the
IFNγ ELISPOT assay
iv. Proliferative responses (Figure 2) are uniformly more consistent than are IFNγ
ELISPOT responses (Figure 1)
v. We tested a higher dose of HK LVS (LVS HK super; 4 x 105/ml) in anticipation of
testing it on LVS-vaccinated NHPs in an effort to increase the IFNγ ELISPOT
response; however, it stimulates PBMCs from non-LVS vaccinated NHPs to a
greater extent than lower doses of LVS
vi. A04168 did not respond to SCHU S4 antigens in the IFNγ ELISPOT assay
(Figure 1B) but A05477 did (Figure 1C)
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay040608.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 040608.svd; TVDC 1 bound notebook
(8628): TUL 21 (pps. 135 – 140), and TUL 23 (146 – 151); and TVDC 2 bound notebook (8935): TUL26
(1 -2) and TUL 31 (pps. 32 – 38).
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Concern that IFN ELISPOT assay needs to be optimized further, specifically regarding HK and FF
LVS and SCHU S4 antigen concentrations and potential mitogenic activity of the FF LVS
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
96% of scientific work has been completed
9. Work plan for upcoming month and next 6 months
a. One Month
1. Continue to test PBMCs from LVS-vaccinated and non-vaccinated NHPs in the IFN ELISPOT
assay to determine the effect of HK and FF LVS at different concentrations.
2. Screen the 26 new non-LVS vaccinated NHPs in the proliferation, IFNγ ELISPOT and IgG
anti-LVS ELISA assays.
3. Continue to test the effect of the Cerus freeze-thaw protocol on the performance of the
PBMCs in the immunoassays.
Page 21 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
b. Six Months
1. Continue to screen the non-LVS vaccinated NHPs in the proliferation, IFNγ ELISPOT and IgG
anti-LVS ELISA assays for their potential use as future LVS vaccinees or controls in SCHU S4
challenge experiments.
2. Write milestone completion report.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. At risk laboratory workers at UNM and LBERI have been undergoing LVS vaccination
under the Special Immunization Program at USAMRIID
b. Protocol for collecting blood from vaccinees received IRB approval
c. LBERI vaccinees have been offered the opportunity to donate blood for
immunoassay development at UNM.
Monthly-March 2008
a. same as above
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
For the next one month
a. Experiments will be performed from the 6-month plan as blood from vaccinees
become available
b. LVS vaccinee volunteers will be consented to donate blood for the immunoassay
development
c. LVS vaccinee volunteers will be scheduled for blood draws
For the next six months
a. Develop SOP for isolating and stimulating PBMC from the peripheral blood of control
and vaccinees
b. Optimize the sensitivity of the IFN Elispot and proliferation assays
Page 22 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
c.
i. Number of cells
ii. Concentration of bacteria
iii. Length of incubation
Develop SOP for quantifying multifunctional T cell cells in control and 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
1. Date started: 2/29/2008
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. At risk laboratory workers at UNM and LEBRI have been undergoing LVS vaccination
under the Special Immunization Program at USAMRIID
b. Protocol for collecting blood from vaccinees received IRB approval
Monthly-March 2008
a. same as above
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
For the next one month
d. Experiments will be performed from the 6-month plan as blood from vaccinees
become available
For the next six months
a. Develop SOP for isolating monocytes from the peripheral blood of control and
vaccinees and 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
Page 23 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 19-UNM
Milestone description: Interaction between human alveolar macrophages and F. tularensis
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. We made very little progress on this milestone due to limited availability of human
alveolar macrophages, though two human donors are scheduled in April 2008
Monthly-March 2008
a. Experiment Ftc36 study 8 (Notebook 115, pages 75-78)
i. The purpose of this experiment was to determine the optimal MOI for
infecting human alveolar macrophages with LVS and SCHU S4 and to show
that recombinant IFN can suppress bacterial growth
ii. Human alveolar macrophages were infected with MOI of 1:1, 10:1 and 100:1
(bacterial to macrophages) and then either left untreated or treated with 15
ng/ml recombinant human IFN. The bacterial load in the macrophage
culture was determined after 3 days
iii. The results showed that the optimal MOI is 10:1 for LVS and 1:1 for SCHU
S4 and that human alveolar macrophages can be activated with recombinant
IFN to suppress F. tularensis growth
Figure 6. Determining the optimal MOI for infecting human alveolar macrophages T-0
is day zero, T-3 is day 3 and T-3 IFN is day 3 with IFNγ treatment. ND = below
detection level
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
Page 24 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
7. Quality of performance
Needs improvement
8. Percentage completed
10%
9. Work plan for upcoming month
For the next one month
e. Experiments will be performed from the 6-month plan as human alveolar
macrophages become available
For the next six month
a. Compare the growth kinetics of LVS and SCHU S4 in alveolar macrophages cultures
from mice, rats, and human
b. Determine the growth kinetics of LVS and SCHU S4 in human monocyte derived
macrophages after F. tularensis infection with and without recombinant IFN
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. We have been trying to develop a functional assay for effective vaccination, looking
specifically for the ability of mouse T cells from vaccinated individuals to induce
macrophage killing of intracellular SCHU S4. We have shown that macrophage
cultures with vaccinated splenocytes have fewer SCHU S4 than macrophage cultures
with naïve splenocytes (Fig 7). However, this has been extremely difficult to
reproduce on a consistent basis. We have also been working on a similar assay
using rat macrophages
Page 25 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 7. Titration of MOI for macrophage killing assay with SCHU
S4. BMM infected with SCHU S4 were cultured with naïve and
vaccinated splenocytes for 3 days. The data show the mean of
triplicates ± SD
Monthly-March 2008
a. Experiment Ftc30.17 (Notebook 109, pages 69-72), Ftc30.18 (Notebook 109, pages
81-84)
i. As indicated previously, it has been extremely difficult to reproduce the
macrophage killing assay on a consistent basis
ii. These two experiments were part of our effort to troubleshoot this problem
iii. Murine bone-marrow derived macrophages were infected with LVS at MOI =
1:20 (bacteria:macrophages) or with SCHU S4 at MOI = 1:100 and then
either left untreated or treated with 50ng/ml recombinant murine IFN
iv. As shown in Fig 8, both LVS and SCHU S4 grew exponentially over a 3 day
period. Recombinant IFN activated the macrophages to completely
eliminate all intracellular LVS and reduced SCHU S4.
v. We will repeat these experiments a few more time to make sure it is
reproducible before we add T cells from vaccinated mice
Figure 8. Murine macrophage killing assay with LVS and SCHU S4. ND = below detection
b. Experiments Ftc 61.3d (Notebook 109, pages 73-76), Ftc61.4 (Notebook 109, pages
77-80), and Ftc61.4b (Notebook 109, pages 89-92)
i. Our goal for these experiments was to develop the macrophage killing assay
with rat bone marrow derived macrophages in a way that would allow us to
get consistent and reproducible results
Page 26 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
ii. Bone marrow-derived macrophages from Fischer 344 rats were infected with
SCHU S4 at MOI form 1:1 to 1:80 (bacteria:macrophages) and bacterial load
was measured after 3 days.
iii. The macrophage monolayer stayed intact and remained healthy at all the
MOI tested (Fig 9). Since we would like to use the lowest MOI possible, MOI
of 1:40 or 1:20 may be ideal for infecting rat macrophages.
iv. We will test the effect of recombinant rat IFN next
Figure 9. Titrating the MOI for infecting rat macrophages with SCHU S4
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Needs improvement
8. Percentage completed
27%
9. Work plan for upcoming month
For the next one month
a. Test splenocytes from naïve and LVS vaccinated mice on Ft-infected murine bone
marrow derived macrophages. This will be repeated several times for consistency
For the next six months
b. Determine whether recombinant rat IFN can suppress Ft growth in rat bone marrow
derived macrophages
c. Test splenocytes from naïve and LVS vaccinated rats on Ft-infected rat bone marrow
derived macrophages
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 27 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
Semi-Annual: 10/01/2007 to 3/31/2008 – SUMMARY/HIGHLIGHTS ONLY
a. We have developed and optimized HTP PCR production of modular linear expression
elements (LEEs). Depending on the need, LEEs can be assembled for prokaryotic or
eukaryotic expression, and can generate a polypeptide fused to a tag such as His or HA
on either or both ends, or fused C-terminally to a thioredoxin peptide (thio).
b. All details of IVT protocols have been optimized. We replaced Proteomaster with
HiGrow, optimized amount of template, feeding conditions, length of the incubation and
the shaking rate of the reaction, and analyzed effect of chaperon addition. We found the
optimized protocol sufficiently reproducible and robust for HTP applications. For most
tested templates prokaryotic IVT expression yield exceeded 25ug protein per reaction.
c. Since use of total E. coli IVT extract leads to reactivity against LVS-immune splenocytes,
direct use IVT reaction for T-cell stimulation is not considered possible. We have
extensively explored the possibility of IVT depletion as an alternative to affinity
purification of IVT made proteins. Sulfate ammonium and acetone precipitations were
found inefficient in removing non-specifically reactivity (background). Size exclusion by
filtration through 100kDa filter units was found to reduce background but was associated
with unacceptably high IVT product losses.
d. We investigated effect of chaperones and thioredoxin fusion on the efficiency of Ni-bead
protein purification and decided against their addition because we didn’t find them to be
universally useful.
e. We investigated efficiency of His tag based purification of IVT made proteins using Ni
beads. Different bead kinds, binding, washing and elution conditions have been tested.
No universally successful protocol has been developed so far. In HTP format only 20%
of IVT made proteins were successfully purified.
f. To assist UNM with developing immunodetection protocols we generated a test set
constructs consisting of eight FTU products (full size groES and Tul4, subfragments of
groEL, IglC, katG and two non-FTU antigen OVA and CalM3. The set was generated in
multiple formats. Namely: unpurified E. coli and rabbit retic IVTs; Ni-purified IVT
products; Ni-beads bound IVT products; E. coli expressed purified proteins.
Page 28 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Monthly-March 2008:
A. Select and test ORF expression constructs
1. Ten eukaryotic in vitro expression cassettes for five complete FTU (Francisella tularensis)
genes (groES, groEL, IglC, katG, Tul4) and two non-FTU antigen (OVA and CalM3) have
been generated and used in rabbit retic IVT system.
B. Select and test IVT Protocols
1. The IVT protocols developed for E. coli based IVT systems have been successfully
adapted for rabbit retic IVT system. The observed protein yield of 2-20 ng/reaction is
within the manufacturer suggested range.
C. Select and test protocols for protein purification
1. Based on the results of T-cell stimulation we dismissed IVT depletion approaches and
concentrated on protein purification. Previously we reported that proper protein
conformation seems to be critical for efficient Ni binding. Two approaches have been
tested to improve folding of the IVT made proteins;one, addition of chaperons to the
reaction and another, use of a thioredoxin fusion. Two chaperone proteins have been
tested: GroE and DnaK. Supplementing IVT with either one does not appear to
significantly influence the reaction (fig. 1).
Fig.1 Effect of chaperones on IVT. Odds – Roche, evens – Invitrogen IVT system.
File location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP
IVT 35S gels\IVT chaperones Roche vs. Invit test 3-4-08 02.jpg
Addition of GroE had no effect on any of the three tested proteins. DnaK appears to
have improved folding of FTU0721a, but had no effect on the other two (fig. 2).
Page 29 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Fig. 2 Effect of chaperones on purification of sub-protein polypeptides.
Files location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP
IVT 35S gels\Chaperone supplement purification\Chaperone sup purification of
FTU1696a 03-21-08.jpg, Chaperone sup purification of FTU0721a 03-13-08.jpg,
Chaperone sup purification of FTU901 03-21-08.jpg
We didn’t find effect of chaperone addition to be universal and consistent enough to
justify their use in HTP format.
2. Effect of thioredoxin fusion has been tested on two model proteins CPV172 and GFP,
previously tested without fusion. Autoradiography of PAGE analysis with different
purification fractions is shown on fig. 3.
Fig. 3. Ni-beads purification of IVT made thio-fusions.
File location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT
35S gels\Thio fusion and GFP purification 03-10-08.jpg
The Ni purification recovery rate of these two fusions (~10%) is similar to that for the
stand alone proteins (data shown previously), but if more consistently generated for
numerous FTU proteins, would be advantageous.
Page 30 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
4. Significant decisions made or pending
We plan to focus our efforts on developing a magnetic Ni-bead loading protocol for the IVT
generated polypeptides, which has shown to be both free of cross-reactive E. coli lysate
components, and more efficient in stimulating T cells than protein-antigen in solution (Dennis
Casper lab, unpublished results).
5. Problems or concerns and strategies to address
We are concerned about being able to universally deliver sufficient quantities of sufficiently
pure FTU polypeptides from IVT generated samples so as to stimulate FTU-immune T cells
above background. Toward the goal of efficiently purifying IVT products we are testing the
utility of adding the Ni mag beads directly to the IVT reactions. We envision this approach as
being universally applicable, thus permitting Ni access to the nascent His tag. This should
ensure tag binding. If we use washed beads directly in T cell assays, Ni release will not be
necessary.
6. Deliverables completed
None
7. Quality of performance
Very good
8. Percentage completed
99%
9. Work plan for upcoming month
Currently we are working on generating several sets of test proteins, free and bound to
magnetic Ni and protein G beads for evaluation in T-cell assay. Samples will be shipped to
UNM this week.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will use
ASU’s protein fragments in lymph node proliferation assays to define vaccine candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. We have been helping ASU troubleshoot the problem that the E. coli in vitro
translation (ivt) system they have been using to produce F. tularensis proteins causes
non-specific IFN production. LVS vaccination of BALB/c mice, Fischer 344 rats, and
possibly cynomolgus macaque appears to induce T cells specific for epitopes in the
E. coli ivt system but not in wheat germ or rabbit reticulocyte ivt systems. We have
tried various strategies to eliminate this non-specific reactivity such as 1) binding the
ivt proteins to magnetic beads and then washing extensively, 2) adding ATP to the ivt
reactions to remove chaperones, 3) size exclusion, and 4) differential acetone
precipitation. The only strategy that appeared to work consistently was size
exclusion through filter with 100 kDa or less MW cutoff.
Page 31 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
b. We also determined the sensitivity of the IFN and IL-2 Elispot assays. Since we are
not aware of any F. tularensis protein that consistently and strongly stimulates T cells
from LVS vaccinated mice, we turned to the DO11.10 T cell receptor (TCR)
transgenic mice. Approximately 20% of the CD4 T cells from these mice express a
TCR that is specific for OVA323-339 presented in the context of MHC class II molecule
I-Ad. By stimulating the DO11.10 T cells with OVA protein and peptides, we found:
i. The assay sensitivity increases with increasing antigen concentration used
for restimulation.
ii. Restimulation with peptide is more sensitive than with protein, presumably
because peptides do not need to be processed to be presented
iii. IL-2 ELispot is more sensitive than IFN Elispot, presumably because all
activated T cells produce IL-2 but only a subset of them secrete IFN
iv. The assay sensitivity is low. The sensitivity of the IL-2 Elispot with 5 M
protein is about 0.2% of total splenocytes; it is 1% according to figure 5, but
only 20% of the DO11.10 splenocytes are OVA specific. The sensitivity of
the IFN Elispot with 5 M protein is about 1% of total splenocytes
Figure 10. Titration of DO11.10 splenocytes in IFNγ Elispot assay to determine assay
sensitivity
Monthly-March 2008
No new work done

4. Significant decisions made or pending
NA
Page 32 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
18%
9. Work plan for upcoming month
For the next one month
a. Determine whether we can increase the T cell frequency by taking splenocytes from
vaccinated mice with active SCHU S4 infection
For the next six months
a. We will test all ivt proteins from ASU as they devise new strategies to eliminate the
crossreactivity problem.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of polypeptide libraries (Optimize IVT proteinfragment production, Develop IVT protocol for high-throughput production, Validate
immunogenecity of protein-fragments, Full scale production of protein-fragment library,
Purification of protein-fragment library, Array protein-fragment into overlapping pools, Ship
to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome (active)
 Generate complete protein-fragment library (inactive)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)
Institution: ASU-Sykes
3. Date started: 03-01-2007
4. Date completed: Pending
5. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/01/2007 to 3/31/2008 – SUMMARY/HIGHLIGHTS ONLY
a. We have all PCR protocols, templates and primers readily available for HTP production
of IVT templates. They all have been tested in HTP format and showed over 90%
success rate.
b. HTP test of IVT efficiency showed ~70% reaction success rate on first pass, with average
yields of ~25ug of IVT made protein per reaction.
Page 33 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Monthly-March 2008:
A. Build ORF expression library corresponding to proteome
In anticipation that milestone 26 is nearly complete, we have pooled PCR primers ORF
library production. We will proceed following decisions on expression system, yield
needs, delivery format, pooling capacity, and purification requirements.
4. Significant decisions made or pending.
The decision to complete the polypeptide purification/optimizations of milestone 26 are pending
but near.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
30%
9. Work plan for upcoming month
Wait for MS 26 to be completed.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 33
Milestone description: Microarrays constructed and confirmed; First printing of arrays,
Testing with DNA from Ft, Arrays GDPs validated at ASU.
Institution: ASU-Johnston
1.
2.
3.
Date started: 08-01-2006
Date completed: 10-31-2007
Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. This milestone was completed shortly after the beginning of this 6 month period. We
had previously established the microarray platform and established that the in house
poly-L-lysine coating for the substrate was superior to Corning Ultragaps slides.
These arrays were tested with unamplified RNA from SCHU S4 and LVS strains of
F. tularensis.
b. The final series of experiments in this milestone were to validate the GDPs by
performing reconstitution experiments using normal mouse lung RNA spiked with
known amounts of SCHU S4 RNA before performing the LAPT procedure. We had
performed multiple spiking experiments and the last one reported during this period
Page 34 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
validated that we could dilute SCHU S4 RNA in 10 micrograms of total normal mouse
lung RNA to 0.0001 μg and still have significant signal detection.
4.
Significant decisions made or pending
a.
5.
This milestone was completed with the repeat validation of the GDP’s with the
reconstitution samples.
Problems or concerns and strategies to address
None
6.
Deliverables completed
This milestone was completed shortly after the beginning of this 6 month period. We established
that we had a good quality microarray for 1804 genes of SCHU S4. We validated the GDPs and
LAPT process by the amplification of SCHU S4 RNA in the presence of mouse lung RNA down to
0.0001 μg.
7.
Quality of performance
Good
8.
Percentage completed
100%
9.
Work plan for upcoming month and next 6 months
Will write the MS33 Completion report
Will finalize the 10 SOPs supporting the transcriptome approach
10. Anticipated travel
Describe request
11. Upcoming Contract Authorization (COA) for subcontractors
Describe request
Page 35 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 34-UNM
Milestone description: Pilot Studies for the optimization of RNA isolation and hybridization
conditions
Institution: UNM
1.
2.
3.
Date started: 03/01/2006
Date completed: 1/31/2008
Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. We provided ASU with RNA isolated from the lungs of mice infected i.n. with 10 1 to
107 CFU SCHU S4. This was used to determine the sensitivity of the F. tularensis
expression microarray developed at ASU
Monthly-March 2008
a. This milestone was completed by UNM and ASU recently and UNM is now providing
infected mouse organ RNAs to ASU under Milestone 35
4. Significant decisions made or pending
This milestone has been completed.
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming month
UNM and ASU need to write a MS completion report for MS 34.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 34-ASU
Milestone description: Pilot studies for optimization of RNA isolation & hybridization
conditions done.
Institution: ASU-Johnston
1. Date started: 03-01-2007
2. Date completed: 12-31-2007
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
Page 36 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
a. We had previously established that infected tissue RNA isolated by Tri-Reagent
procedure needed further purification via the Qiagen RNAeasy cleanup columns
before processing in the LAPT process.
b. We tested various hybridization conditions to include MAUI wave system versus the
static Array-IT slide chamber system. The Array-IT system consistently gave the
best hybridization results.
c. We compared hybridization efficiency of ASU prepared arrays to the TIGR provided
arrays. We concluded that the ASU arrays performed with superior hybridization
characteristics. We verified this result by hybridization of 10 defined but randomly
chosen PCR products to the ASU array. Two genes did not find an ASU probe on
the array. After bioinformatic analyses were completed, these two PCR products that
failed to hybridize to the ASU array did not amplify the section of a gene to which the
probe was designed to hybridize. Thus, the failed detection was the result of
randomly picking 10 genes and not a failure of the probe or amplification process.
4. Significant decisions made or pending
All RNA for LAPT amplification needs to be highly purified. Qiagen RNAeasy columns are a
preferred method for final cleanup. The ASU arrays perform well and the Array-IT static
hybridization chambers provide economical and highly efficient hybridization conditions.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
This milestone was completed shortly during this 6 month period. We have a standardized
RNA extraction process and validated that the ASU prepared microarray provided consistent
results.
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming month and next 6 months
Will write the MS34 Completion report
Will finalize the 10 SOPs supporting the transcriptome approach
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35 - UNM
Milestone description: Array hybridization with mouse RNA from virulent SCHU S4
infection and RT PCR confirmation of candidates
Institution: UNM
1. Date started:
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. We have been providing SCHU S4 RNA and DNA as needed to ASU
Page 37 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
March 2008
a. Ftc64 (Notebook 115, pages 79-84)
i. The purpose of this experiment purpose of this experiment was to determine
the SCHU S4 gene expression pattern at early time points (1, 3, 5, 7, and 24
h) after intranasal infection with SCHU S4 and to compare that with in vitro
SCHU S4 gene expression pattern in culture. By comparing the genes
expressed in vivo in mouse lungs (stressful conditions) and in vitro in liquid
Chamberlain’s culture (unstressful conditions), it may be possible to
determine the pathways that get turned on in response to the stresses of in
vivo exposure.
ii. We infected mice i.n. with 103 SCHU S4 and prepared RNA from lungs
collected after 1, 3, 5, 7 and 24 h). The RNA was sent to ASU
iii. We also prepared RNA from SCHU S4 grown in culture, but because of the
low initial inoculum, we did not isolate enough RNA. We are repeating the
cultures with higher initial inoculum.
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
4%
9. Work plan for upcoming month
For the next one month
a. Isolate RNA from SCHU S4 grown in Chamberlain’s broth
For the next six months
a. We will provide RNA and DNA when needed by ASU, as the experiments are
planned by ASU and UNM
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35-ASU
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
Page 38 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
a. Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
i. We have received two independent biological replicate RNA samples of a dose
response challenge in mice from UNM. Mice were challenged with doses of
SCHU S4 F. tularensis ranging from 10 up to 107 organisms per animal. Lungs
were harvested at 4 hours post infection for total RNA isolation.
ii. The consistently detectable signal required 103 organisms per challenge.
Comparison of the dose response range across two amplifications of each of the
two experimental sets revealed 50 - 70% of the genes being reproducibly
detected.
b. Monthly-March 2008:
i. From the two dose-response challenge experiments, we bioinformatically
averaged the individual doses across two amplifications per sample Figure 1.
The top 300 expressing genes were identified in each of the two different
experiments and the intersection determined. We obtained between 56 to 64%
overlap between the two amplifications. We then bioinformatically averaged all
of the array data for the two independent experiments and determined the
overlap among and between all the doses (representative intersections are
shown in Figure 2).
Figure 1. The top 300 genes were identified in
two different biological experiments. The
number of genes identified in both
experiments are in the yellow intersection.
Figure 2. Examples of averaging all of the
amplification data (4 array experiments per
dose).
ii. The intersection of all of these cross comparisons lead to identification of 253
genes being routinely detected across the dose response at 4 hours post
challenge. We bioinformatically subtracted genes that could also be detected in
uninfected normal mouse lung (NML) (Figure 3). This revealed a list of 65 genes
and the expression patterns were then mapped back onto the average of the
array data for each of the key doses (Figure 4, Left Panel). We used the pattern
mapping algorithm in GeneSpring GX to identify genes consistently highly
expressed across all doses revealing a list of 20 genes (Figure 4, Right Panel
and Table 1). DNA topoisomerase 1, FTT906c was also detected as a possible
vaccine candidate in the PPG expression library immunization project.
Page 39 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 3. Bioinformatic subtraction of genes
that are amplified in uninfected normal mouse
lung (NML)
Figure 4. Pattern analysis of the top 65 genes
identified as shown in Figure 3 (left panel). Pattern
mapping analyses to locate the high expressing
patterns across the dose responses revealed a list of
20 genes (right panel).
Table 1. Twenty genes induced at 4 hours after challenge.
Page 40 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam

Notebook/File locations …, Notebook 514, LAPT 20, pages 143-166 LAPT 22, pages 174183; Notebook 661, LAPT 23,, pages 1-12.
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-20 (Pooled NM Samples).
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-22 (second run of NM
Samples)
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-23 (Comparison of NM EXP
I vs. EXP II Samples)
4. Significant decisions made or pending
Each sample will be amplified twice for experimental averaging to determine the
reproducibility of gene identification via the LAPT process. Genomic normalization should
provide a method to enhance the ability to compare multiple experiment results.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
We have a set of 65 genes reproducibly detected across the SCHU S4 dose response
challenge experiments utilizing the data from mice challenged with 103-107 CFU per mouse.
A subset of 20 genes was identified by pattern mapping of with high responses across the
dose response. One of these genes, FTT0906c DNA Topoisomerase 1, was also identified
as a possible protective component using Expression Library Immunization, efforts funded
under a separate grant.
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month and next 6 months
a. For Next one month
i. We have received samples from a time course experiment where mice were
challenged with 103 CFU SCHU S4 and lungs harvested at 0, 1, 3, 5, 7 and 24
hours post infection. These samples have been amplified and hybridizations are
planned.
ii. Establish the Q-RT-PCR process for gene expression validation.
iii. Assess gene expression levels of known potential antigens (TUL4, KatG, IglC,
GroEl) in the current data set.
b. For Next 6 months
i. Design continuing experiments to include challenge of immune and non-immune
animals to determine differences in microbial gene expression under
immunological attack.
ii. Determine gene expression changes of infected animals in different tissues, i.e.
compare gene expression in infected lungs, liver, and spleen.
iii. Compare in vivo gene expression patterns to bacteria grown in vitro.
iv. Compare in vivo gene expression in the rat and mouse model of tularensis.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 41 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Milestone 41
Milestone description: Optimization of photochemical inactivation and characterization of
KBMA Ft. novicida; determine the amount of S-59 and UVA required to inactivate uvr mutants;
determine extent of metabolic activity of uvr mutants after S-59 and UVA inactivation; determine
the level of virulence attenuation of KBMA uvr strains in mice
Institution: Cerus
1. Date started: 3/2/06
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: We have determined that all the NER-deficient strains of Ft. novicida are only
slightly more sensitive to photochemical inactivation than wild type Ft. novicida. We have
optimized photochemical inactivation conditions at a 3.5 mL scale and a 400mL scale and
produced a lot of KBMA uvrB Ft. novicida for potency testing in MS42. We have
demonstrated that KBMA Ft. novicida are highly attenuated for virulence. Frozen KBMA
uvrB Ft. novicida maintain metabolic activity at –80oC for at least 3 months. Inactivated
NER-deficient strains have a similar degree of metabolic activity as the wild-type Ft. novicida
strain (which is different than has been seen with L. monocytogenes or B. anthracis), and we
have demonstrated that this lack of sensitivity to DNA damage is universal to numerous DNA
damaging agents.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
Nov 16, the vaccines research program was “spun out” of Cerus Corporation. All of the
personnel, intellectual property and other assets related to immunotherapy were transferred
from Cerus to Anza Therapeutics Inc. Cerus and Anza are working to establish a
professional service agreement that will allow the work on the TVDC to proceed using Anza
personnel and that is acceptable to UNM and NIAID. Numerous discussions have been held
between Cerus and Anza representatives and with UNM to discuss possible mechanisms for
continuing with the TVDC contract going forward. Cerus, Anza, and UNM have agreed in
principle to establish a service agreement between Cerus and Anza. However, until this
relationship is approved by NIAID there will be no direct costs charged by Anza to Cerus for
the TVDC. Anza is currently in the process of getting an IACUC established in order to
obtain our OLAW assurance and Anza has submitted APHIS documents to the USDA for
transport of Francisella tularensis strains (application # 07361052).
4. Significant decisions made or pending
All NER mutants (uvrA, uvrB, and uvrA uvrB) of Ft. novicida were equally sensitive to S-59
and had comparable metabolic activity after inactivation. We have chosen to use the uvrB
single mutant for further experimentation. We have selected 40M S-59 and 7J/cm 2 as the
conditions for making 400ml-scale KBMA lots, and have produced a lot of KBMA uvrB Ft
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
Page 42 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Francisella species as a platform. A new concern is that Cerus may no longer have enough
human resources to complete this milestone in a timely manner.
6. Deliverables completed
400mL-sacle photochemical inactivation process defined
7. Quality of performance
fair progress
8. Percentage completed
85% of scientific work completed on the milestone
9. Work plan for upcoming month
 Cerus has generated a modified set of milestones that are scientifically appropriate and
achievable. Cerus is awaiting NIAID contract officer approval of the modifications to the
Cerus subcontract, which were requested on 4/2/2008
 This milestone has been terminated due to the Cerus to Anza transition and a milestone
completion report will be written
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 42
Milestone description: Determine whether KBMA F.t. novicida vaccine protects against wildtype F.t. novicida challenge in mice: Vaccination route and regimen optimization, measure
durability of protection
Institution: Cerus
1. Date started: 2/1/07
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: KBMA Ft novicida uvrB vaccine stocks produced in MS41 have been tested in
mice for virulence and protection against a 100 x IP LD50 challenge of Wild-type Ft novicida.
KBMA Ft novicida uvrB were 100% protective when a single dose was administered at or
near the LD50 of the KBMA vaccine (1 x 109 IP, 1 x 108 IV). 100% protection was also
achieved by administration of 1 x 107 KBMA particles IV when the vaccine was given twice
separated by 3 weeks. Depletion of CD4+ T cells prior to the challenge decreased the
survival rate to 80%, depletion of C8+ T cells had no effect, and depletion of both cell
populations resulted in 90% survival. Together, these data demonstrated that CD4 T cells
contribute to a protective immune response in a non-CD8 T cell-dependent manner. These
data suggest that the CD4 T cells may be boosting humoral immunity by stimulating B cells.
This interpretation was supported by an adoptive transfer experiment in which only the hightiter serum from CD8-depleted animals provided any protection against a lethal U112
challenge. Together these data demonstrate that the protection we see after vaccination with
KBMA Ft novicida uvrB correlates with humoral immune responses and explains why the
KBMA vaccine does not perform better than heat killed vaccine. This also makes it nearly
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 ovalbumin epitope tag and are awaiting the
construction of this strain from UTSA.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
Page 43 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
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
 Cerus has generated a modified set of milestones that are scientifically appropriate and
achievable. Cerus is awaiting NIAID contract officer approval of the modifications to the
Cerus subcontract, which were requested on 4/2/2008
 This milestone has been terminated due to the Cerus to Anza transition and a milestone
completion report will be written
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 44
Milestone description: Formulation and evaluation of KBMA LVS: establish photochemical
inactivation regimen of selected uvr mutant of LVS and measure metabolic activity and virulence
of KBMA LVS.
Institution: Cerus
1. Date started: 6/18/2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary: using a small-scale inactivation procedure we have determined that the S-59 psoralen
concentration required to inactivate uvrB LVS is 5uM. This is the same concentration at which
we have been able to inactivate WT LVS. The uvrB LVS was also not more sensitive to DNA
damaging agents compared to WT. This suggests that there may be redundant DNA repair
mechanisms in LVS that may be functioning to repair photochemically induced crosslinks.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
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
Page 44 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
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
 Cerus has generated a modified set of milestones that are scientifically appropriate and
achievable. Cerus is awaiting NIAID contract officer approval of the modifications to the
Cerus subcontract, which were requested on 4/2/2008
 This milestone has been terminated due to the Cerus to Anza transition and a milestone
completion report will be written
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale LVS
culture conditions, Establish 3L culture scale purification conditions, Optimize 3L scale
photochemical inactivation process, Verify protective immunogenicity of vaccine candidates
produced by optimized large-scale process
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: we have demonstrated that LVS grows robustly in Chamberlains Defined Media
(CDM) and have prepared expanded DVC lot 16 LVS cultures grown in CDM for 36 hours,
and stored at -80oC. We have determined that the minimum concentration of S-59 required
for complete inactivation of DVC lot 16 LVS is 5µM and that photochemically inactivated LVS
maintain metabolic activity for at least 12 hours. We produced a 3L lot of LVS in our
fermentor using .001% Sigma antifoam A in CDM and have demonstrated stability for 4
months at -80o in 2 cryopreservation medias. We have found that the LVS provided by DVC
is greatly attenuated for virulence in mice when administered IP compared to literature
reports. We have demonstrated that LVS replicate rapidly in livers and spleens of mice
immediately following IV injection; however, it appears that there is a lag that specifically
affects growth in the lungs. We have also demonstrated that LVS is nearly avirulent when
administered by the SC route.
We have produced a 400mL lot of KBMA wild-type LVS using 10 uM S-59 and 6 J/cm 2 UVA
for initial proof of concept studies, and for later comparison with NER-deficient uvrB LVS
and we have demonstrated that the metabolic activity of this lot is stable for 3 months. We
have demonstrated that KBMA WT LVS IV LD50 is 6.8x108, which represents a 4-5 log
attenuation compared with live LVS. We have demonstrated that doses of KBMA WT LVS as
low as 1 x107 provide protection against 100 x IP LD50 challenge of live LVS. However, none
of the mice vaccinated with the equivalent doses of HK LVS died either. This is consistent
with protection against an LVS challenge being largely humoral. b We recently attempted to
measure the T-cell response to a CD4 Tul4 epitope in mice vaccinated with live or KBMA
LVS by intracellular interferon-gamma (IFN-) cytokine staining (ICS) or ELISpot assay, but
Page 45 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
were unable to detect an induced response to this epitope. This may be because this epitope
does not bind the MHC molecule with high affinity, or the T cell response elicited by LVS may
actively suppress T cell responses. We recently demonstrated that LVS does not induce IL-6
or MCP-1which are critical hallmarks of a protective inflammatory response. Furthermore,
co-vaccination with LVS decreased the innate inflammatory response to Lm. Administration
of LVS decreased the ability of the elicited T cells to produce the cytokine IL-2. Terry Wu at
UNM completed a protection study with KBMA WT LVS in which neither a (IV or IN) prime
nor a prime and boost (separated by 3 weeks) vaccination regimen with KBMA WT LVS
protected against a lethal SchuS4 challenge in mice. KBMA WT LVS vaccine appears to be
less potent than live attenuated LVS.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
4. Significant decisions made or pending
Because wt Ft novicida is inactivated with S-59 concentrations that are only slightly higher than
uvrB mutant we have been investigating the efficacy of a wild-type KBMA LVS vaccine. Now
that we have received the uvrB mutant we will focus on producing a lot of KBMA uvrB LVS
5. Problems or concerns and strategies to address
The protection seen with the KBMA WT LVS against a lethal LVS challenge is independent of
metabolic activity. This suggests that comparison of various routes, regimens, or formulations will
be difficult to optimize by protective efficacy. The SchuS4 challenge model in mice is more
stringent, but KBMA LVS failed to protect after two doses. It is possible that the rat model may
allow a higher degree of sensitivity. The suppression of the innate inflammatory response and
the suppression of CD4 T cell cytokine production may potentially indicate that LVS is not a
potent inducer of protective T cell responses. We would like to screen for T-cell responses using
the peptides generated by ASU as an alternative method for optimization of vaccine potency or
construct an overlapping peptide library for IglC.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
53% of scientific work completed on the milestone
9. Work plan for upcoming months
 Cerus has generated a modified set of milestones that are scientifically appropriate and
achievable. Cerus is awaiting NIAID contract officer approval of the modifications to the
Cerus subcontract, which were requested on 4/2/2008
 This milestone has been terminated due to the Cerus to Anza transition and a milestone
completion report will be written
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 46 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
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
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a.
Cloning of an igLC plasmid was completed (KEK1160) and was used to generated a
SCHU S4 igLC mutant.
i.
This mutant was verified by PCR experiments using igLC intron specific and
igLC gene specific oligos.
ii.
Protein isolations were prepared from this igLC mutant for a western blot
experiment using anti-rabbit igLC serum which indicated that no wild type igLC protein was
produced in this mutant.
iii.
Genomic DNA was isolated from this mutant and digested with various
enzymes (EcoRI, HindIII, and BamHI) to prepare a Southern blot using a probe of 400 bp
DNA specific to the intron portion of this construct. This was done to verify that the igLC
“intron” integrated at the expected locations in the SCHU S4 chromosome. These results
are discussed in the next section of this report.
b.
Started construction of the pdpD plasmid to use to create a SCHU S4 pathogenicity
island mutant. The deletion construct was prepared in pwsK30 initially (KEK1188) this was
then used to move the pdpD Flp::Kan deletion sequence into pUC118. The pwsk30 cloning
vector offers more unique restriction sites to use during cloning than does the pUC118 vector.
The pdpD construct was in process in our lab and was to be used with the F.novicida strain.
Transformation experiments done in the past using low copy plasmid pwsk30 (KEK906) did
not yield any deletion (igLC) integrations. The pUC118 is a high copy plasmid and has been
shown to yield a desired integration. The first deletion made to ultimately create this FPI
deletion mutant (pdpA) was made using a pUC118 cloning vector.
c.
Generated vgrG plasmids (KEK1161 and KEK1162, targetron plasmids targeted to
vgrG) which were used in a transformation experiments with SCHU S4. These experiments
generated thousands of colonies and we are screening for correct clones. Screening of
these clones gave rise to potential vgrG mutant strain in Schu S4
d.
Generated igLD plasmids (igLD30a and igLD255a) which were used in
transformation experiments with SCHU S4. These experiments generated thousands of
colonies. The initial screening for each of the igLD insertion sites 30/31a or 255/256s groups
revealed that the igLD255a clones contained at least one insertion in all of the original ten
clones screened. We proceeded with this set to screen for a correct clone.
Monthly-March 2008:
a. Continued with additional vgrG screening by PCR of potential vgrG mutants in Schu4. Clone
1 (of last month) was taken and streaked for single colonies on a fresh TSA+++Kan plate at
30 degrees Celsius.
Isolated colonies were patched on to a TSA+++Kan plate.
Chromosomal preps were performed on 10 of the patched clones. This genomic DNA served
as a template for PCR with a primers specific to FTT1346 (FTT1346 fwd NdeI) and a primer
specific to the intron (EBS Universal). Remember, a primer specific to vgrG would prime past
Page 47 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
the intron insertion due to the intron inserting at base pair 3 of vgrG. Wild type vgrG should
yield a PCR product of ~500 bp whereas a mutant should be shifted to ~1600 bp. Of the 10
clones screened, clone 10 had the most visible shift in PCR product size (figure 1). The
majority of clones had a shift which is difficult to see in the digital image. However, clone 10
will be streaked again for isolated colonies in hopes of driving the population to total insertion
(shifted product only), not partial (wt size and shifted). Data in TVD UTSA Notebook 1, p27.
Figure 1.
1.
2.
3.
4.
5.
6.
ladder
Schu4 wt
clone 1
clone 2
clone 3
clone 4
7.
8.
9.
10.
11.
12.
clone 5
clone 6
clone 7
clone 8
clone 9
clone 10
b. Continued to screen for an igLD SCHU S4 clone which has both igLD genes disrupted by the
insertion of the igLD group II intron (igLD255a). Four potential igLD clones in SCHU S4 were
grown in Chamberlain’s media and passaged for four days. These cultures were then diluted
and plated on TSA +++ Kan plates to generate single colonies. Some of these colonies were
patched on another TSA+++Kan plate. Ten single candidates from each of the original clone
groups (2D1, 2D2, 2D3 and 2D4, respectively) were selected to isolate genomic DNA for
further screening. Using oligos specific to the intron (EBS Universal) and to the igLD gene
(IgLD Nco For) first verified that the insertion was in the chromosome (Figure 2).
Subsequently, using oligos specific to the 5’ and 3’ ends of the igLD gene (Figure 3) we
determined if both gene copies were interrupted. This profile should illustrate only one
product which will be ≈800 bp larger (≈1900 bp mutant) than the wild type SCHU S4 profile
(≈1100 bp). Although 40 clones were screened only two sets (2D3 and 2D4) are illustrated in
the figures below. Furthermore, all screen clones did contain an insertion of igLD255a intron
but only one appeared to be correct (2D3B).
Figure 2.
1 Kb
Legend
1 2 14 15 15 17 18 19 20 21 22 23 24
1. 1 Kb Ladder 14. Ori 2D4 clone
2. KKT1 Δβlac2 15. 2D4A clone
2.0
3. Ori 2D3 clone 16. 2D4B clone
0.5
B
2.0
A
0.5
4. 2D3A clone
17. 2D4C clone
5. 2D3B clone
18. 2D4D clone
6. 2D3C clone
19. 2D4E clone
7. 2D3D clone
20. 2D4F clone
8. 2D3E clone
21. 2D4G clone
9. 2D3F clone
22. 2D4H clone
10. 2D3G clone
23. 2D4I clone
11. 2D3H clone 24. 2D4J clone
1 2 3 4 5 6 7 8 9 10 11 12 13
12. 2D3I clone
13. 2D3J clone
Figure 2 represents PCR products when using 255a igLD tulatron transformants’ genomic preparations as
templates in respective reactions (lanes 3-24). The 255a igLD tulatron transformants are designated as 2D
followed by their respective original clone number. The letter designation represents a single colony from
this respective original clone (e.g. 2D3A) which resulted from the cell passaging. Both panels represents
the PCR products generated when using EBS Universal (intron specific) with IgLD Nco I for (gene specific)
Page 48 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
oligos. The expected size of ≈400 base pairs (bp) will indicate that the IgLD intron is in the Schu S4
chromosome. Lane 2 represents the Schu S4 wild type and should not yield a product. All of the clones
resulting from the passaged cultures retained the igLD intron. Data located in TVD UTSA Notebook 5, page
112.
Figure 3.
1 Kb
Legend:
1 2 13 14 15 16 17 18 19 20 21 22
1. 1Kb Ladder 13. 2D4A clone
2. KKT1Δβlac2 14. 2D4B clone
3. 2D3A clone 15. 2D4C clone
3.0
A
1.0
4. 2D3B clone 16. 2D4D clone
5. 2D3C clone 17. 2D4E clone
6. 2D3D clone 18. 2D4F clone
3.0
B
1.0
7. 2D3E clone
19. 2D4G clone
8. 2D3F clone
20. 2D4H clone
9. 2D3G clone 21. 2D4I clone
10. 2D3H clone 22. 2D4J clone
11. 2D3I clone
12. 2D3J clone
1 2 3 4 5 6 7 8 9 10 11 12
Figure 3 represents PCR products profiles which were generated when using genomic templates from the
255a igLD tulatron group 2D3 (panel B) and 2D4 (panel A) clones. The oligo set used with these genomic
templates were igLD NdeI for and igLD NcoI rev. Lane 2 is the KKT1 control which will illustrate the
wild type expected size when these oligos are used (≈1100 bp). Panel A represents the 2D4 single clones
A-J, respectively. Panel B represents the 2D3 single clones A-J, respectively. The correct clone should
show a ≈800 bps shift to ≈1900 bps and contain no wild type product size. Lane 4 is clone 2D3B and
appears to be correct. Lane 1 is the 1 Kb ladder from Invitrogen. Data located in TVD UTSA Notebook 5,
page 112.
c.
The tulatron-igLC mutants (KKT5 and KKT6) were prepared for a Southern blot; however, the
BamHI digested DNA did not provide a good separation to differentiate the igLC in the FPI-I
(≈77.5 kb expected size) and FPI-II (38.4 kb expected size). Therefore, prepared more DNA
from these mutants to use in a Southern blot which will illustrate that the tulatron introns
integrated at the expected sites. I used Hind III and EcoRI restriction endonuclease enzymes
in the experiment. The expected band profile for each digestion when using a probe (400 bp)
directed to an intron specific sequence was confirmed. Since the background Schu S4 had a
Δβlac2 (KKT1) intron insertion we expected two bands for each digestion profile on the igLC
mutants to resolve and only one in the KKT1 wild type strain (figure 4). Data located in TVD
UTSA Notebook 5, pages 107, 108, 118-120 and 122-124.
Page 49 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Figure 4.
Hindi III
EcoRI
A
1 2 3 4
B
5
2 3 4 5
1 Kb
Legend:
12.5
1. 1 Kb Ladder
2. Schu S4
7.0
3. KKT5 d igLC
4. KKT6 d igLC
3.0
5. KKT1 d Blac2
2.0
Figure 4 represents results from a Southern blot where various Schu S4 genomic DNAs were
digested with either Hind III (panel A) or EcoRI (panel B) restriction endonucleases, respectively.
Subsequently, the transferred DNA was hybridized with a 400 bp probe directed to the intron
sequence portion of the igLC intron. The background Schu S4 strain used in creating the igLC
mutant was the KKT1 ∆βlac2 mutant; this mutant will also have an intron portion therefore, the
results should show two bands for the correct igLC construct. The Hind III digestion will yield a
band at 2389 bp for igLC mutants (Lanes 3 and 4); and the KKT1 mutant should yield 7819 bp
(lane 5). The EcoRI digestion will yield a 14.8 kb band for the igLC mutants (lanes 3 and 4) and the
KKT1 mutant will yield 3580 bp band (lane 5). Lane 2 is the wild-type Schu S4 which will not yield
any band. Data located in TVD UTSA Notebook 5, page 123 and 124.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
The milestone 49 will not be completed in the next 6 months however, we hope to have the vgrG
and igLD mutants completed and checked for their potential attenuation in mice. The iglC mutant
is completed in SCHU S4, though UTSA may be able to visualize the two igLC genes separated
by doing a pulse field electrophoresis experiment to further verify this mutant. Also, we probably
should do a complementation for this mutant to verify the phenotypes are due to the igLC gene
being disrupted and not other downstream genes.
7. Quality of performance
Good
8. Percentage completed
63.9%
9. Work plan for upcoming month and next 6 months
a. Will continue the screening for potential igLD mutants in KKT1 SCHU S4 strain. Need to do a
western blot on any potential correct igLD intron double insertion clones. Once confirmed we
need to remove the plasmid from the strain by growing at 37°C and subsequently screen for
kanamycin sensitivity.
b. Will continue the screening for potential vgrG mutants in KKT1 SCHU S4 strain by PCR. Once
potential correct vgrG intron double insertion clones are identified we will remove the plasmid
from the strain by growing at 37°C and subsequently screen for kanamycin sensitivity.
c. Will continue with the cloning to move the pdpD flp::Erythromycin deletion construct into
pUC118.
Next 6 months:
Page 50 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
a. Will finish the vgrG Schu S4 mutant and verify by PCR and subsequent sequencing.
b. Will characterize the vgrG Schu S4 mutant by checking intra-macrophage survival and
potential attenuation in mice.
c. Will finish the igLD Schu S4 mutant and verify by PCR and subsequent sequencing.
d. Will characterize the igLD Schu S4 mutant by checking intra-macrophage survival and
potential attenuation in mice.
e. Will finish the pdpD flp::erythromycin pUC118 vector construct and generate a complete
FPI mutant where a complete FPI will be missing from this Schu S4 mutant.
10. Anticipated travel
None
11.Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 04/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
(50A) We evaluated the protective efficacy of two Ft subsp. novicida mutants (uvrBiglA and
uvrBpdpD) against pulmonary Ft novicida challenge using a murine tularemia model.
Additionally, we also evaluated the protective efficacy of the Ft novicida iglB mutant and Ft
tularensis (SCHU S4 strain) iglC mutant against SCHU S4 challenge.
a. uvrBiglA double mutant of F. novicida
i. This mutant is highly attenuated in mice with 50% lethal dose (LD 50) greater than 107
CFU (bacterial colony forming unit) when infected intranasally. The LD 50 of wild type F.
novicida (U112 strain) is less than 10 CFU.
ii. Mice vaccinated intranasally (i.n.) with uvrBiglA produced significant amounts of
antigen-specific antibody.
iii. Mice vaccinated intranasally (i.n.) with this mutant (as low as10 5 CFU) were protected
from subsequent lethal pulmonary challenge with F. novicida (1000 CFU, 100LD50).
b. ΔuvrBpdpD double mutant of F. novicida
i. LD50 of this mutant is between 105 and 106 CFU.
ii. uvrBpdpD replicated in the lungs and disseminated to the liver and spleen after i.n.
challenge. Bacteria were still detectable in the lungs, liver, spleen and lymph nodes 2
weeks after i.n. challenge. This replication pattern may account for the robust priming
of the immune system, with this attenuated vaccine candidate.
iii. Mice vaccinated with uvrBpdpD produced significant amounts of antigen- specific
antibody.
iv. Mice immunized with uvrBpdpD (1000 CFU, i.n.) were protected from subsequent
lethal pulmonary challenge with F. novicida (1000 CFU, 100LD50).
Page 51 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
c. ΔiglB mutant of F. novicida
i. Mice immunized with iglBC intragastically (i.g. 103 CFU) produced significant amount
of antigen-specific antibody.
ii. Vaccination with ΔiglB (i.g.) protected mice against lethal pulmonary SCHU S4
challenge (20 CFU, 20 LD50) with a 67% survival rate 3 weeks after challenge.
d. ΔiglC mutant of Ft. tularensis (SCHU S4)
i. ΔiglC unlike its parental SCHU S4 strain replicated poorly in macrophages.
ii. Mice immunized with iglC by either intradermal (i.d.) or i.g. route produced significant
amount of antigen-specific antibody.
iii. Mice immunized with iglC (103 CFU, i.d.) were partially protected from both i.d. and
i.n. SCHU S4 challenge (100 CFU) with survival rates of 17% and 33%, median time to
death (MTD) of 5.5 days and 11 days, respectively, when compared to no survival and
MTD of 4 days for the PBS/mock vaccinated mice.
iv. Mice i.g. immunized with iglC (103 CFU, i.g.) and i.d. challenged with SCHU S4 (100
CFU) exhibited a prolonged MTD of 6 days, compared to 4 days of the PBS/mock
vaccinated mice.
(50B) We have been evaluating the intragastric route of immunization as an alternative
immunization strategy for F. holarctica LVS against F. tularensis SCHU S4 challenge in a
murine model of tularemia.
a. Protective Efficacy of LVS Intragastric Immunization Against SCHU S4 Challenge
i. Intragastric immunization with LVS (i.g. 10 3 CFU) was highly protective (80-100%
survival) against subsequent SCHU S4 challenge (100 and 500 CFU) at 3 weeks after
vaccination.
b. Cellular and Humoral Responses to LVS Intragastric Immunization
i. Mice immunized i.g. with LVS (103 CFU) produced significant amounts of antigenspecific IFN- at two weeks after vaccination compared to mock (PBS) vaccinated
mice. IFN- was also significantly produced at four weeks after vaccination, although in
waning amounts.
ii. Mice immunized i.g. with LVS (103 CFU) produced high levels of antigen specific total
antibody, IgG1 and IgG2a in the serum at three weeks after vaccination. They also
produced high levels of antigen specific total antibody, IgG1, IgG2a, IgA and IgM in the
bronchoalveolar lavage fluid (BAL) and significant levels of IgA in the G.I tract.
iii. Mice immunized i.g. with LVS (103 CFU) continued to produce high levels of antigen
specific total antibody in the serum at eight weeks after vaccination. Mice that were
given a second booster dose of LVS i.g. (103 CFU) after eight weeks had somewhat
higher total serum antibody titers three weeks after the booster dose.
c. Bacterial Dissemination After LVS Intragastric Immunization
i. LVS disseminated to the lungs 2 days after i.g. immunization with 10 3 CFU, and to the
liver and spleen by 5 days after immunization. After 5 days, bacterial numbers started
to decrease and there were no detectable bacteria in any of the organs by 21 days
after vaccination.
Monthly-March 2007:
50A: (1) Continue monitoring the survival of the iglC-immunized/SCHU S4-challenged
mice in the ongoing experiment (Note book #1 pages 146-147). Results of this experiment
are summarized in Table 1. BALB/c mice intradermally immunized with SCHU S4 iglC
survived longer than PBS/mock immunized mice when challenged with wild type SCHU S4
both intradermally and intranasally. The median survival time for i.g. iglC- immunized
mice/i.d.-SCHU-challenged mice was also significantly longer than the PBS treated mice.
Page 52 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Table1:
Route of
Vaccination
Route of
Challenge
iglC mutant against homologous wild type challenge
Challenge
% Survival
Median survival
Kaplan-Meier
Dose (CFU)
(at day 28)
(days)
Survival analysis
i.n.
20
100
20
100
20
100
20
100
20
100
20
100
i.d.
i.d.
i.n.
i.g.
i.d.
i.n.
Mock
i.d.
0
16.7
16.7
33.3
0
0
0
0
0
0
0
0
6.5
5.5
8
11
5
4
5.5
6
5
4
4
4
*
**
**
**
**
**
* p<0.05 compared to mock vaccinated group
** p<0.01 compared to mock vaccinated group
(2) Evaluate the protective efficacy of intragastric F. novicida iglB vaccination against SCHU
S4 intranasal and intradermal challenge in C57BL mice. (Note book #1 pages 150-152 and
153-155): Groups of C57BL mice (female, 4-6 weeks) were immunized 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 immunized with iglB induced significant
amount of serum antibody as shown in Fig. 1. 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. 2.).
5000
Titer
4000
3000
2000
1000
0
Total Ab
IgG1
IgG2a
Fig.1. Humoral responses induced by F. novicida iglB immunization. Mice
were vaccinated with 1000 CFU of the iglB-null mutant by the intragastric
route. Sera were prepared 3-week after immunization, and assayed for antiiglB specific antibody.
Page 53 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
1.00
1.0
A405
0.80
0.8
0.60
0.6
0.4
0.40
0.2
0.20
0
0.00
IgA
IgM
IgA
 iglB
IgM
Mock
Fig.2. Mucosal immune responses induced by F. novicida iglB
intragastric immunization. Mice were vaccinated with 103 CFU
of the iglB-null mutant and fecal pellets were collected 3-week
after immunization, and assayed for anti-iglB specific
antibody. Mice received PBS are used as mock control.
These mice were challenged i.n. or i.d. with 20 CFU of SCHU S4 3-week after vaccination.
Animals were monitored for survival. The iglB-immunized and i.n.-SCHU-challenged mice
had a 66.7% survival rate (Table 2), while only 16.7% PBS/mock immunized mice survived
by day 21 post-infection.
Table 2. Protective efficacy of F. novicidaiglB mutant (103 CFU, i.g.) against SCHU
S4 (20 CFU) challenge
Antigen
iglB
PBS/mock
Route of Challenge
% Survival (@ day 21)
Median survival (days)
i.n.
66.7 %
21
i.d.
16.7 %
6.5
i.n.
16.7 %
5
i.d.
16.7%
5.5
50B: Measure the presence of LVS in various tissues at early time points after i.g.
immunization by PCR. (Notebook #8, pages 68-74) We have previously looked at bacterial
dissemination after i.g. LVS vaccination by plating of whole tissue homogenates. By this
method we found that bacteria were present at 2 days after immunization in the lungs, but
were not detectable in the liver or spleen until 3 days later. In order to see if there are
indeed bacteria present in these two organs at earlier time points, we are using PCR as a
more sensitive detection method. Groups of BALB/c mice (3 per time point) were
immunized i.g. with 103 CFU of LVS. At 24, 48 and 72 hours after vaccination, mice were
sacrificed and the lungs, livers and spleens were removed and stored at –80 degrees C.
Tissues were also collected from naïve mice as negative controls, and from mice that had
an established lethal LVS infection as positive controls. We have optimized our protocol for
Page 54 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
extracting genomic DNA from the tissues and have found that our primer sets work
correctly by using our control tissues. We are now ready to run the PCR reactions on all of
our sample tissues to look for presence or absence of bacteria at these early time points
with data to follow shortly.
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
65% of scientific work completed on milestone 50A (original plans) NORMALIZED TO 32.5%
42% of scientific work completed on milestone 50B (intragastric plan)NORMALIZED TO 21%
9. Work plan for upcoming month and next 6 months
a. For Next one month
50A: I Evaluate the protective efficacy of intragastric F. novicida iglB vaccination
(prime and one boost) against SCHU S4 intranasal challenge in C57BL and
BALB/c mice.
50B: I Examine by PCR the presence or absence of bacteria in the lungs, livers
and spleens for the first three days following i.g. vaccination.
II Analyze the complete antibody profiles for serum and fecal samples taken
at both eight and twelve weeks after LVS i.g. immunization either with or
without a second booster dose of LVS i.g.
b. For Next 6 months
50A: I Further evaluate F. novicida iglB as vaccine candidate.
II Evaluate Ft tularensis (SCHU S4) vgrG and/or iglD mutant as vaccine
candidate when mutants are generated and confirmed.
50B: I Evaluate long term protective immunity to SCHU S4 challenge following
i.g. LVS immunization
II Examine the immune correlates of protection conferred by i.g. LVS
immunization by using neutralizing antibodies against several cell types.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 52
Milestone description: Create RecA mutants in F. tularensis subsp. tularensis(Schu S4)
Institution: UTSA
1. Date started: 9/15/2007
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
To inactivate RecA in Schu S4, we constructed a Targetron vector for targeting and inactivating the
RecA gene. The Targetron vector was constructed with the intron expression vector pKEK1140 for
the backbone, and a 350bp PCR product to retarget the intron RNA for insertion mutagenesis of
RecA.
Page 55 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Semi-Annual: 10/1/07 to 3/31/08-SUMMARY/HIGHLIGHTS ONLY
a. Constructed a tulatron vector for insertion mutagenesis of recA.
I. Selected the plasmid pKEK1140 to serve as the backbone of the tulatron vector. The intron
expression plasmid pKEK1140 (cloned and modified previously) was sensitive to
temperature, and contained Kanamycin marker for selection and FtgroELP to drive
expression of intron in Ft.
II. Introduced a 350bp PCR fragment into pKEK1140 to retarget intron RNA. This plasmid was
named pKEK1186 (at 720/721bp target site) or pKEK1187 (at 840/841bp target site).
b. Creation of RecA mutant LVS
I. Transformed pKEK1187 into wild type LVS using electroporation to insert the mutated intron
RNA into RecA. The RecA mutants were chosen by selection of Kanamycin resistance.
II. Screened the potential RecA mutants for insertion using colony PCR with the RecA specific
primers flanking the insertion site, and it indicated the insertion was in RecA compared to wild
type LVS.
III.
Screened the same mutants using colony
PCR with the primer in intron RNA insertion and the primer out the insertion. The PCR
confirmed the insertion. Sent the gel purified PCR DNA for sequencing and the result
confirmed the insertion was at 840/841bp in RecA.
III.
Isolated the RecA mutant LVS with wild type LVS on TSA++
agar medium with 30ug/ml Kanamycin at 30C. Screened the single colonies using colony
PCR with the same primers as Section b.ii. and the colonies without wild type LVS being
mixed were selected for the procedure in Section v.
IV.
Removed the plasmid from the RecA mutant LVS.
1. The recA mutant LVS with the plasmid was streaked onto TSA++ agar medium with
100ug/ml Ampicillin and incubated at 37C for removal of the plasmid since the backbone
of the plasmid was sensitive to temperature and change of temperature from 30C to
37C could accomplish the loss of the plasmid.
2. Patched the single colonies onto both TSA++/Kan and TSA++/Amp plates and incubated
at 37C to select the colonies sensitive to Kanamycin.
3. Screened the Kanamycin sensitive colonies using colony PCR with the primers described
on Section b.ii. and iii. to make sure they were the RecA mutant LVS. This RecA mutant
LVS was designated KKF343.
c. Creation of RecA mutant SchuS4
I. Transformed the plasmid pKEK1186 (at 720/721bp target site) into wild type SchuS4 using
cryotransformation.
II. Screened the transformants using colony PCR with the primers flanking the insertion. The PCR
indicated the insertion was in recA.
III. The transformants were screened by colony PCR using the primer in intron RNA insertion and
the primer out the insertion. This confirmed the intron RNA was inserted in RecA
IV. Isolate the RecA mutant SchuS4 with wild type SchuS4 In progress
Monthly-March 2008:
a. In the technical report for last month, it was reported that the plasmid pKEK1186 (at 720/721bp target
site) was transformed into wild type Schu S4 and the transformants were selected on TSA++ agar
plate with 30ug/ml Kanamycin. After 4 days incubation at 30C, the potential transformants were
patched onto TSA++/Kanamycin (30ug/ml) agar medium, and incubated at 30C for 1 to 2 days.
b. The colony PCR was performed to screen these transformants with “RecA SchuS4 for” and “ RecA
SchuS4 rev” primers, which were RecA gene specific primers flanking the insertion site in SchuS4.
The PCR reaction was set up as follow:
ddH2O
34.6 ul
10X Buffer
5.0 ul
MgCl2
2.0 ul
Page 56 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
dNTPs
5.0 ul
RecA Schus4 For (25pmol/ul)
1.0 ul
RecA Schus4 Rev (25pmol/ul)
1.0 ul
KOD DNA polymerase
0.4ul
DNA
1.0 ul
At 98C 1minute, 98C 15seconds// 57C 15seconds// 72C 2minutes/ 30 cycles
Figure 1: on 1% agarose gel
Lane11 (about 1500bp) was the positive control (KKF343--(RecA LVS), whereas lane12 (wt
LVS)
and Lane13 (wt SchuS4) were the negative controls (about 630bp). The yield on lane13 was
lower
than lane12 (wt LVS), but still visible. Colony1 to colony9 (lane2-lane10) presented the
same size
band (about 1500bp) as lane11 (KKF343, RecA mutant LVS), which indicated that each of 9
colonies
had the insertion in RecA gene.
c. To further confirm the intron RNA insertion in RecA of SchuS4, colony PCR with “RecA
SchuS4 for” (in RecA gene) and “EBS Universal” (in mutated intron RNA) was performed
with 9 colonies as the templates. The colony PCR was set up the same as Section b.
Figure2: on 1% agarose gel
Since the insertion site was at 720/721bp in RecA for SchuS4 mutants and at 840/841bp for LVS
mutants, the expected PCR product generated was about 515bp for (RecA SchuS4 and about
630bp for (RecA LVS. Lane11 (about 630bp) was KKF343 ((RecA LVS), and lane2-lane10
Page 57 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
(about 500bp as expected) was colony1-colony9 (potential (RecA SchuS4). Lane12 (no band)
was wild type SchuS4 as the negative control. This PCR result confirmed that the mutated intron
RNA was inserted in RecA of SchuS4 for colony1-9. Data recorded on UTSA TVDC notebook
#6, page14-16 for figure1 and 2.
4 Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
pKEK1186 (the tulatron vector at 720/721bp target site)
pKEK1187 (the tulatron vector at 840/841bp target site)
KKF343 (RecA mutant LVS)
7. Quality of performance
Good
8. Percentage completed.
About 20% of scientific work completed.
9. Work plan for upcoming month
a.
For Next one month
i Send PCR product for sequencing to confirm the insertion.
ii Isolate the mutant RecA SchuS4 from wild type SchuS4 at 30C.
iii Remove the plasmid from the mutant RecA SchuS4 at 37C.
b. For Next 6 months
i Finish creation of RecA mutant SchuS4
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None.
Sections IV: Contract Expenditures and Subcontractor Billing
1. % work completed vs. %cumulative costs incurred to date with regard to planned
effort and proposed budget
The active Milestones over the past 6 months have been: 2, 3, 4, 5, 7, 11, 12/13 (UNM &LBERI), 14, 17,
19, 21, 26, 27, 28, 34(UNM/ASU), 35, 41, 42, 43, 44, 46, 48, 49, 50 and 52. None of these milestones
currently are over budget.
To date, NIAID has been billed for scientific activity on Milestones 3, 4, Working Group (covered under
MS3 and 5), 5, 7, 11, 12/13 (UNM &LBERI) ,14, 16, 17, 19, 21, 25, 26, 27, 28, 32, 33, 34(UNM/ASU) ,
35 (UNM/ASU), 39, 40, 41, 42, 43, 44, 46, 48, 49, 50, 51 and 52.
Page 58 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Data associated with most current UNM invoice submitted to NIAID ( parallels Financial Report by
Milestone and lags behind current Technical Report)
Milestone
% invoiced
94.7
0
93.5
36.7
82.8
0
4.3
56.2
88.2
% work
completed
50
65
98
22
69
0
6
60
94
Over
budget?
No
No
No
No
No
No
No
No
No
2-LBERI
2-UNM
3- LBERI
4-LBERI
5-UNM
7-LBERI
11-UNM
12/13 UNM
12/13 LBERI
14-UNM
16-UTSA
0
100
1
100
No
No
17-UNM
19-UNM
21-UNM
25-ASU
26-ASU
27-UNM
28-ASU
0
52.7
23.7
100
93.8
42.8
52.5
1
10
27
100
99
18
29
No
No
No
No
No
No
No
32-ASU
33-ASU
34-UNM
34-ASU
35-UNM
35-ASU
39-UTSA
100
100
33.3
97.9
6.5
11.3
100
100
100
100
100
4
10
100
No
No
No
No
No
No
No
40-Cerus
41-Cerus
42-Cerus
43-UTSA
100
68.5
37.5
100
100
85
25
100
No
No
No
No
43-Cerus
0
0
No
Discrepancy?
Yes; see explanation below #1
See explanation below #2
NA
See explanation below #3
NA
MS just opened on 2/25/08
NA
NA
NA; note- funds from MS4 were
transferred into MS 12/13 budget in Fall
2007
MS just opened 2/29/08
NA: no activity past 6 months. note
unspent funds from MS 16,39, 43,& 48,
were moved into MS 50 to cover the new
gastric plan as of Inv #21 for 11/07
expenses onward.
MS just opened 2/29/08
See explanation below #4
NA
None, no activity past 6 months
NA
See explanation below #5
See explanation below #6- need KS
review
None, no activity past 6 months
NA
See explanation below #7
NA
NA
NA
NA: no activity past 6 months. note
unspent funds from MS 16,39, 43,& 48,
were moved into MS 50 to cover the new
gastric plan as of Inv #21 for 11/07
expenses onward.
NA; no activity past 6 months
See explanation below #8
See explanation below #8
NA: no activity past 6 months. note
unspent funds from MS 16,39, 43,& 48,
were moved into MS 50 to cover the new
gastric plan as of Inv #21 for 11/07
expenses onward
Terminated before initiated: see
explanation below #9
Page 59 of 61
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
Data associated with most current UNM invoice submitted to NIAID ( parallels Financial Report by
Milestone and lags behind current Technical Report)
44-Cerus
45-Cerus
6.2
0
5
0
No
No
46- Cerus
47-Cerus
34.1
0
53
0
No
No
48-UTSA
100
100
No
49-UTSA
50-UTSA
51-UTSA
52-UTSA
54
43.3
93.7
7.3
55.5
64
100
12
No
No
No
No
See explanation below #8
Terminated before initiated: see
explanation below #9
See explanation #8
Terminated before initiated: see
explanation below #9
NA: no activity past 6 months. note
unspent funds from MS 16,39, 43,& 48,
were moved into MS 50 to cover the new
gastric plan as of Inv #21 for 11/07
expenses onward
NA
See explanation below #10
NA
NA
Discrepancy Explanations
1. MS2- LBERI: The discrepancy between the 94.7% invoiced and 50% completed is due to the
vaccination trip labor expenses at the beginning of the milestone and yet the milestone won’t be
completed till the one year annual physical exam is completed. There will be little LBERI labor associated
with the one year annual physical exam since the physical exam will be performed at UNM Employee
Occupational Health and not at USAMRIID.
2. MS 2- UNM- MS 2 (LVS vaccinations for relevant personnel) has no budget. The expenses for MS 2
consist of the UNM administrative oversight of the LVS vaccinations and also the travel and health
screenings for the LVS vaccinees. The administrative costs are not covered under Milestone budgets.
The travel and health screening costs are included under the “Occupational Health” expense category,
which was not included in the Milestone budgets. Through 4/1/08, 37 LBERI and UNM participants have
been vaccinated.
3. MS4- LBERI: The discrepancy between the 36.7% invoiced and 22% completed is due to holding
NHP, which were LVS vaccinated in the Fall of 2006, on this milestone for the purposes of blood draws
for MS 12/13 immunoassay development and for later SCHU S4 challenge on MS 8.
4. MS 19- UNM: UNM is developing assays with human alveolar macrophages from human donors. The
expenses to date, includes the salary for the bronchoscopy nurse, who is recruiting the normal human
donors. The discrepancy between the 52.7% invoiced and 10% work completed is due to the
developmental aspects of the assays as well as the low number of donors recruited to date. Two more
donors are scheduled in April 2008.
5. MS 27- UNM: UNM is developing assays with peptides from ASU. The discrepancy between the
42.8% invoiced and 18% work completed is due to the E coli cross reactivity of IVT products in the
cellular assays at UNM. UNM anticipates that the % work completed will catch up with the % invoiced,
as the in vitro peptide, T cell assays fully developed.
6. MS 28- ASU: The discrepancy between the 52.2% invoiced and the 29% work completed is partially
due to the purchase of in vitro transcription/translation reagents in bulk at the beginning of the milestone.
This advance purchase will save funds in the long run, because ASU was able to negotiate a reduced
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2007 to 3/31/2008
Due Date: 4/15/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Mitch Magee, Stephen Johnston, Justin Skoble, Bob Sherwood, Trevor Brasel, Julie
Wilder, Julie Hutt, Karl Klose, Bernard Arulanandam
price-per-unit quote from Invitrogen. Also, is partially due to troubleshooting that ASU is performing to
make IVT products that do not cross react with E.coli proteins in the cellular assays performed at UNM.
7. MS34- UNM: UNM isolated RNA and DNA from SCHU S4 and LVS, as well as mice infected with
SCHU S4, and provided these reagents to ASU for testing the microarrays. ASU has completed testing
the microarray slide printing and hybridization conditions so MS34 is 100% work completed for UNM and
ASU.
8. MS 41, 42, 44,46-Cerus: For scientific reasons, these 4 milestones have been terminated after being
initiated. Cerus and UNM have developed a new statement of work for new milestones (55-59) and are
awaiting the NIAID Contract Officer review on the Cerus subcontract modifications submitted on 4/2/08.
The unspent budgets from these 4 milestones will be transferred to the new milestones #55-59.
9. MS 43, 45, 47-Cerus: For scientific reasons, these 3 milestones have been terminated before ever
being initiated. Cerus and UNM have developed a new statement of work for new milestones (55-59) and
are awaiting the NIAID Contract Officer review on the Cerus subcontract modifications submitted on
4/2/08. The residual unspent budgets from these 3 milestones will be transferred to the new milestones
#55-59.
10. MS 50- UTSA- The discrepancy between 43.3% invoiced and 64% work completed relates to the
11/1/07 increase in the budget for MS50 to cover the gastric plan and to the lack of normalization of the %
work completed. Up until the Tech report and FRM of 2/08 on January 2008 activity, UTSA was adding
the % work completed for the non-gastric plan to the % work completed for the gastric plan on MS50.
This would have lead to a 200% work completed at the end of MS50. As of the 2/08 FRM, the % work
completed was normalized so that the maximum reached at the end of the milestone would be 100%. So
the 64% reported in this table, should be normalized down to 32% and then there is essentially no real
discrepancy.
To date, administrative costs have been billed to NIAID that are associated with Milestone 2 and with the
management of the scientific milestones 3, 4, 5, 7, 11, 12/13(UNM/LBERI), 14, 17, 19, 21, 26, 27, 28,
35(ASU/UNM), 49, 50, 52, 1, 25, 32, 33, 34 (UNM/ASU), 16, 39, 40, 43 (UTSA), 48, 51, 41, 42, 44, and
46.
2. Estimates of subcontractors expenses from prior month if subcontractor did not
submit a bill. List for each subcontractor. If subcontractors were not working or did not
incur any cost in current or prior month, then include a statement to this effect.
ASU, LBERI, and UTSA electronically submitted invoices to UNM in the prior month. The UNM invoice
#28 to NIAID being submitted on 4/15/08 will include a subcontractor invoice from LBERI only. UNM
payments to ASU and UTSA were delayed due to delays in UNM receiving the hardcopy signed ASU and
UTSA invoices. UNM expects to invoice NIAID for 2 ASU invoices totaling $137,264 and 2 UTSA
invoices totaling $72,127 on UNM invoice #29 to NIAID in May 2008.
Cerus’ last invoice to UNM was paid by UNM on 2/18/08 and invoiced to NIAID on 3/20/08 on UNM
invoice #27 to NIAID. Cerus has performed no new laboratory work since December 2007 due to the
planned shift in Cerus’ statement of work to include new milestones 55-59. The Subcontract modification
for Cerus is under the NIAID Contract Officer review as of 4/2/2008.
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