Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
Contract No.
ADB Contract No.
HHSN266200500040-C
N01-AI-50040
Contract Title:
Tularemia Vaccine Development Team
Performance Period: 4/1/06 to 9/30/06
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: 10/15/2006
Annual Meeting:
Presentations from the 9/29/2006 Annual meeting
are the sources for the contents of this semi-annual
report (PowerPoint slides and meeting recording)
1 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal
models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by Milestone
Active milestones: 2, 3, Working Group, 5, 12 (UNM &LBERI), 26, 33, 40, 41, 43, 46, 49,
50
Milestones completed with this report: 25, 32, 39
Previously Completed milestones: 1, 16
Inactive milestones: 4, 6-11, 13-24, 27-31, 34-38, 42, 44-45, 47-48, 51-54
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. NIAID is working on the IAA with USAMRIID and a legal and financial liability review is
pending.
b. Dr. Bob Rubin, President/CEO of LRRI, is discussing access to the LVS vaccinations with
Dr. Ed Nuzum, who is the Chief of the Product Development Section in the Office of
Biodefense Research Affairs at NIAID. Dr. Rubin wants the LVS vaccine for the LRRI
scientists working on tularemia. LRRI wants best protection for their team and is willing
to waive the indemnification issue for their scientists.
c. Dr. Chuck Hobbs, of LRRI, is providing a cost analysis, based on travel, EOH pre and
post health screening costs provided by Barbara Griffith. Currently, Barbara estimates a
per person cost of $3,970.92, which includes two trips to USAMRIID, and the prehealth
and post vaccination health screenings. This does not include labor costs or the costs of
the vaccinations from USAMRIID.
d. Dr. Hobbs and Dr. Lyons have contacted Bev Fogtman requesting the cost of the LVS
vaccinations at USAMRIID. In December 2005, UTSA paid $47,065.00 for 5 participants.
4. Significant decisions made or pending
a. UNM and NIAID continue to wait for a change in the status of the IAA between NIAID
and USAMRIID.
b. UNM and LBERI will use their biobubbles as additional physical protective equipment, but
LBERI does not want to begin aersolizations with SCHU S4, until their staff are
vaccinated with LVS.
c. NIAID will need to provide UNM access to human cells from other LVS vaccinated
individuals which are needed to develop in vitro immunoassays. For possibly another
year, UNM will not have access to a local source of human cells from LVS vaccinated
individuals
2 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
d. UNM EOHS has obtained many of the laboratory documents
i. Documents pending
1. Radiology Facility Accreditation Certificate
5. Problems or concerns and strategies to address
a. UNM will need an external source of human cells from LVS vaccinated individuals, in order to
develop the immunoassays in humans.
b. LBERI does not want to begin SCHU S4 aerosols until after their staff receive the LVS
vaccinations; progress on milestones would be impacted.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
16%- no change relative to 9/15/06 report
9. Work plan for upcoming 6 months
a. Ross Kelley will continue to monitor the progress of whether Martin Crumrine's IAA between
NIAID and USAMRIID will inform UNM when and whether the TVD Contractors can be
vaccinated under this IAA.
b. LRRI and UNM are researching possible collaboration with the TRUE Foundation
c. Barbara Griffith is contacting J. Stringfield, UNM Legal Counsel, regarding UNM’s position on
the medical/legal liability for the potential LVS vaccinees employed by UNM.
10. Anticipated travel
Travel could occur in September 2006 to September 2007, depending on the completion of the
IAA.
11. Upcoming Contract Authorization (COA) for subcontractors
UNM may request a COA to allow 1-2 UNM EOHS nurses to travel to USAMRIID for training on
LVS site vaccination evaluations. The timing of the COA request depends on the achievement of
the IAA.
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

LBERI has been optimizing the growth media for LVS (see Working Group below) and
optimizing the bioaerosol setup.
 Plotted the OD(600) vs. Log10 CFU/ml of LVS grown in Chamberlains for 48 hr and
for 72 hrs (LBERI Sherwood ppt; slide 16). Though the curves differ slightly, the OD
difference impacts the actual sprays to optimize the bioaersol technique.
 LBERI described their 2 bioaerosol rooms, bioaerosol setup, and bioaerosol
determinations (LBERI Sherwood ppt; slides 17-19). The hepa filtered air transfers
the spray from the Collison nebulizer to the head only exposure chamber. An air
sample is recovered in the AGI impinger. Both the spray solution and the sample
from the impinger are serially diluted, grown on CHAB plates, and the CFU/ml are
determined to characterize the aerosol.

Lyophilized LVS vs. frozen LVS vs. vegetative cultures of LVS are being compared
to determine their relative spray factors. Lyophilized LVS had a spray factor of zero
3 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble






indicating that lyophilized LVS does not survive the Collison nebulizer. The
lyophilized LVS didn’t survive the sheer forces (~20psi through the port) of the
Collison nebulizer. (LBERI Sherwood ppt; slide 20).
QUESTION: how measure sheer forces?: Tae Kim/Ed Barr discussion- Trying to
compare sheer forces in a fermentor at Cerus to those in the Collison at LBERI.
Collison is a small glass vial, which is constantly receiving high pressure air into the
base causing a bubbling up and falling back down of the fluid. It may be hard to
compare the spray factors of these two different systems.
Frozen LVS with the Collison nebulizer had spray factors ranging from 1x10 -6 to 4x106 which is very good.
Compared Biosampler to AGI and the AGI recoveries were better. (LBERI Sherwood
ppt; slide21)
Spray factor definition: (concentration in CFU/liter of the agent in the spray air as
measured by the impinger) ÷ (CFU/liter of the agent in the generator solution). Very
high efficiencies are in the 10-6 range and 10-8 is poor. 10-7 is range for most sprays
and matches literature. Henderson established the spray factor in the literature for
comparing sprays.
Since the doses for LD50 are expected to be low, like single digits, in nonhuman
primates, LBERI needed to establish the lowest dose in the spray that gave
reproducible results. The lowest LVS spray concentration is ~5x10 4 CFU/ml which
gave 10 CFU/L of air. (LBERI Sherwood ppt; slide 22-23). 10 CFU is standard
microbiological level minimal level.
Data files are found in \\Saturn\ABSL3\Study Data (2005-2006)\Francisella tularensis\FY06078 (Tul-03)\Bioaerosol Data
4. Significant decisions made or pending
a. Cannot aerosolize lyophilized LVS using a Collison nebulizer
b. Frozen LVS provides good spray factors
c. Lowest LVS spray concentration is ~ 5x104 (10 CFU/Liter air)
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
27%
9. Work plan for upcoming 6 months



Perform bioaerosol experiments on vegetative LVS with Collison generator to compare with
frozen and lyophilized LVS
 Repeat of studies performed on thawed LVS, but now with vegetative. The studies
will include measurement of spray factors, reproducibility, high and low doses.
 Plan to grow LVS in CB
 Plan to quantitate LVS on CHAB
Perform initial aerosols with additional generators
 Sparging generator
 Micropump generator
 LDI Sonik generator
 Down select in discussions with NIAID Project Officers
Repeat Collison studies (frozen and fresh LVS)
4 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble


High dose spray factors
Low dose spray factors
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Working Group
Milestone description: Determine appropriate solid and liquid media for growth of tularemia for
project team
Institution: LBERI/UNM
1. Date started: 2/23/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions:
a. LBERI tested the growth of LVS on 9 solid agar media. Based on quantitative recovery,
colony morphology and colony color, the best agars were BCGA, SBCGA, and CHAB.
(LBERI Sherwood ppt; slides 1-12) for consistent colony size, blue color, and recovery.
b. LBERI tested growth of LVS in 6 different liquid media . Chamberlains media
outperformed all other media as measured by growth. (LBERI Sherwood ppt; slides 1315). The LVS growth curve in Chamberlains showed that late lag is in the 48 to 72 hr
period.
c. UNM tested the virulence in BALB/c mice of LVS directly reconstituted from the
lyophilized lot#16 vs. LVS grown in Chamberlains broth media vs. LVS grown on CHAB
plates. The three sources of LVS were used to infect mice intranasally and to determine
the LD50. The LVS grown in Chamberlains and LVS grown on CHAB were at least as
virulent as the LVS directly reconstituted from the lyophilized vial with calculated LD50 of
1x104 to 6x104 CFU/ml. (Working Group TWu final report 10/3/06)
d. UNM, NRC and DSTL are growing LVS in Chamberlains broth with one vaccine vial of
LVS (DVC lot#16) resuspended in 250ul water, inoculated 240ul into 60mls and grown
in a 125 ml flask at 370C for 48 hrs at 150 rpm. UNM and NRC will be testing for
consistent LVS broth growth under these conditions from DSTL. (Working Group Tech
Call Minutes 092006)
4. Significant decisions made or pending
a. A meeting of the working group examined blue/grey colony morphology data produced by
DSTL and determined that Chamberlain’s broth and CHAB agar outperformed the other
media tested and recommended that these media be used in growth of LVS stocks for
the TVDC.
b. A lyophilized vaccine LVS vial from DVC lot#16 can be grown once in Chamberlains
media to generate working stocks that can be frozen.
c. A final decision on the LVS growth conditions is pending.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Determined liquid and solid media for LVS growth
b. The virulence of LVS after growth in liquid or solid media, is at least as good as the
virulence of LVS reconstituted from the lyophilized lot#16 vial.
7. Quality of performance
Excellent
5 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
8. Percentage completed
100% for LBERI and 95% for UNM
9. Work plan for upcoming 6 months
UNM is performing experiments to examine the consistent growth of LVS in Chamberlains under
DSTL specific culture conditions.
10. Anticipated travel
COA #11: authorizes travel for 7 UNM TVDC scientists to the 5th International Tularemia
Conference in Woods Hole, MA
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 5
Milestone description: Species tested for sensitivity to LVS & generation of immunity against a
pulmonary challenge of Schu4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. Objective: to test five different animal species as potential models for vaccine
development, including NIH-Swiss out bred mice, Fischer 344 rats, guinea pigs,
hamsters and rabbits.
b. Model selection criteria include sensitivity to respiratory challenge with SCHU S4,
tolerance to LVS vaccination, and protection against respiratory SCHU S4 challenge.
For each animal model, UNM will test different LVS vaccination doses and will test
multiple routes of infection including subcutaneous, intradermal and respiratory
c. Murine Model:
i. Experiments Ftc6 (notebook 81 pages 36-45), Ftc6B (notebook 81 pages 4853 and 64-67), Ftc7 (notebook 81 pages 54-58 and 68-75), Ftc7B (notebook
81 pages 76-79 and 95 and notebook 85 pages 9-12), and Ftc17 (notebook
85 pages 49-54 and 78-82).
ii. BALB/c is the best model so far; mice can be vaccinated with LVS and get
protection against respiratory challenge with SCHU S4. Mouse model could
be improved as only get protection with intranasal/aerosol vaccine delivery
instead of conventional subcutaneous or intradermal routes. UNM also
doesn’t know the mechanism of protection through the intranasal route. Also
the window of protection is narrow, as vaccinated mice tolerate 200-300 CFU
of SCHU S4. The mouse model shows “all or nothing” protection, rather
than partial protection against SCHU S4 challenge. The protection is
relatively short lived, as the mice lose protection in 2-3 months.
iii. NIH Swiss mice were tested with the hope of a better model in an out bred
strain. Sensitivity of naïve NIH Swiss mice to intranasal challenge of SCHU
S4 was determined and they are sensitivity to as few as 1 CFU of SCHU S4.
(UNM Wu ppt; slide 4) NIH Swiss mice are as sensitive as BALB/c mice.
UNM has compared tolerance to LVS vaccination in BALB/c and NIH Swiss
mice, with intranasal, subcutaneous and intradermal routes. BALB/c and
NIH Swiss handle intranasal vaccination similarly (UNM Wu ppt; slide5). Five
to 10 mice were used per group. For intradermal and subcutaneous
vaccinations, the highest possible dose of LVS was used ( 5 to 8 x10 6) and
6 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
both strains of mice survived completely. By all 3 routes of vaccination,
BALB/c and NIH Swiss mice are similar. (UNM Wu ppt; slide 6).
iv. UNM followed the clearance of LVS from lung, spleen and liver 5 weeks after
immunization by intranasal, subcutaneous or intradermal vaccination in both
strains. With prior LVS isolates, UNM saw clearance in 3 weeks but with
DVC lot#16, UNM saw complete clearance at 7 weeks. (UNM Wu ppt; slide
7). Then LVS intranasally vaccinated mice were challenged intranasally with
SCHU S4 at multiple doses at 8 weeks. With the two lower doses, the
BALB/c and NIH Swiss mice behave similarly. The higher dose in BALB/c
was lost at the initial challenge. (UNM Wu ppt; slide 8) The intradermal and
subcutaneously vaccinated mice were challenged intranasally with 3 doses
of SCHU S4. The SCHU S4 challenge doses were five fold lower than the
SCHU S4 doses used with the intranasally vaccinated mice. With 40 CFU,
get over 50 % killing in both strains. So the intranasal LVS vaccination route
provides better protection and the intradermal and subcutaneous vaccination
routes provide about 5 fold less protection. (UNM Wu ppt; slide 9)
v. Summary of mouse models: NIH Swiss out bred mice are not advantageous
over BALB/c in bred mice as measured by sensitivity to intranasal,
intradermal and subcutaneous LVS vaccination, clearance of the LVS from
organs, sensitivity to intranasal SCHU S4 challenge and vaccine induced
protection against intranasal SCHU S4 challenge. In both strains, intranasal
vaccination is better than intradermal or subcutaneous vaccination.
d. Rat Model
i. Experiments Ftc12 (notebook 85 pages 4-8), Ftc12 study 4 (notebook 85,
page 13), Ftc12 study 5 (notebook 85 pages14-15), Ftc12 study 6 (notebook
85 pages 16-17), Ftc12 study 7 (notebook 85 pages 18-19), Ftc12 study 8
(notebook 85 pages 20-24), Ftc12 study 9 (notebook 85 page 29), Ftc12
study 10 (notebook 85 pages 39-41), Ftc16 (notebook 85 pages 55-56)
ii. Literature: Jemski paper from 1982 showed that Fischer 344 rats may be a
good model as they were protected from SCHU S4 after any route of LVS
vaccination. The paper didn’t discuss the lung deposition or the LD50 or the
level of protection. Jemski’s work has not been repeated to date.
iii. To develop the rat model, UNM is evaluating the method of anesthesia, the
doses, the reproducibility of the method, the lung deposition, the clinical
symptoms etc.
iv. Anesthesia: UNM compared isoflurane to ketamine/xyazine and found that
ketamine/xyazine caused excess constriction of the trachea which made
intratracheal infection difficult. Isoflurane was chosen, but has a short time
duration. (UNM Wu ppt; slide 13)
v. Depositions: UNM developed homogenization method using larger bead
beater tubes in conjunction with DNase I addition, with alternating cooling
and homogenization, to prevent heat build up and bacterial killing. (UNM Wu
ppt; slide 15). Two minutes of homogenization was sufficient. Without
DNase I, the homogenate became viscous. The DNase I for 1 hour had no
effect on bacterial viability (UNM Wu ppt; slide 16).
vi. Pulmonary delivery method: They tested the intranasal delivery volume with
and without a wash, by placing the inoculum at the tip of the nares and
allowed the rats to inhale it. The volume with the most reproducible and best
dose delivery to the lung was 400ul. (UNM Wu ppt; slide 17). However, UNM
had to balance the short duration of the isoflurane anesthesia with the
volume delivered. With too little anesthesia, the rats awoke and blew out the
400ul and with too much anesthesia, the rats drowned with 400ul.
7 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
vii. Intratracheal delivery: This method bypassed the nares problems. UNM
tried a microsprayer to deliver a fine mist in 50ul increments, which had
worked well with mice. (UNM Wu ppt; slides 19-21). UNM found that the
microsprayer caused a 5 fold loss in the bacteria being delivered to the
lungs, probably due to the sheer forces of the microsprayer. The
microsprayer also posed a contamination problem because of the way its has
to be loaded would have been unsuitable with an agent like SCHU S4.
viii. QUESTION: Alex- would it be possible to go directly past the ribcage and
into the lungs? Rick and Terry said “no” as it would collapse the lungs.
ix. Intratracheal delivery using i.v. catheter needle: The flexible needle is
associated with a disposable syringe, avoiding frequent reloading of agent.
Terry inserts the plastic sheath into the trachea and tests placement by
pumping air, sensing the vacuum if it hits the esophagus, and feeling the
rales if it hits the trachea/lungs. (UNM Wu ppt; slide 24). After the tubing is
placed correctly, the needle is used to delivery the inoculum.
x. Sensitivity of Fisher 344 rats to pulmonary SCHU S4 challenge: In all the
animals with rales, the rats were infected and the lung deposition was
approximately 50% of the inoculum dose. At 106 CFU SCHU S4, the rats’
mean time to death was 6.5 days. At lower doses down to 102, the rats all
survived. (UNM Wu ppt; slide 25). These doses were similar to Jemski’s.
xi. Summary of Rat model: have developed the standard operating procedure
for pulmonary infection of Fischer 344 rats including using isoflurane
anesthesia, delivery using a laryngoscope and i.v. catheter, immediate
confirmation of deposition by rales, and a lung deposition of 50 %. The naïve
rat is sensitive to 106 CFU of SCHU S4.
xii. QUESTION: Justin- is a dose of 106 CFU rather high? Terry said rats are
naturally resistant to Biovar A strain, so it is a high dose but this is an
important question. Rick added that we need to generate as much data on
each of the animal models as we can so each can be evaluated. Mice die at
rather low doses of LVS, which isn’t good for a model either. Vicki and Rick
added that rabbits are fairly resistant to LVS. Karl remembers that rats are
very resistant to novicida infections. Rick commented on the Sprague
Dawley rat that is extraordinarily resistant to SCHU S4 – you can’t even kill it
with SCHU S4. The rat physiology is different. Karl thinks one of the reasons
for that is the macrophages – Rick affirmed that rat macrophages are
extraordinarily good at regulating their immune response, much better than
mice. The question is whether LVS will provide protection to the rats.
4. Significant decisions made or pending
1. For the mouse model, NIH Swiss mice offer no advantage over the BALB/c mice.
2. For the rat model, we will use isoflurane anesthesia and use an iv catheter for the
intratracheal delivery of inoculations. The Fischer 344 rat is sensitive to 106 CFU of
SCHU S4.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
The NIH Swiss mouse and the BALB/c mouse models are completed
7. Quality of performance
Good
8. Percentage completed
25%- mouse model completed
9. Work plan for upcoming 6 months
8 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
a. Fischer 344 Rats:
i. Determine the resistance of s.c. and i.d vaccinated rats to i.t. SCHU S4
challenge
ii. Determine the i.t. LD50 for SCHU S4
iii. Compare the effects of LVS vaccination by i.n., s.c., and i.d and whether it
will protect them from a pulmonary challenge.
b. Guinea pigs:
i. Identify method of pulmonary infection
ii. Start training on handling, anesthetizing, and infecting guinea pigs
10. Anticipated travel
10/30 to 11/4/06: Rick Lyons, Terry Wu and Barbara Griffith to attend Collaborative
meeting with DVC and NIAID and to attend 5th International Tularemia Conference in
Woods Hole MA
11. Upcoming Contract Authorization (COA) for subcontractors
COA #11: authorizes travel for 7 UNM TVDC scientists to the 5th International Tularemia
Conference in Woods Hole, MA
QUESTION: Kathy – is there an end-date for completing one of these models? Is there some criteria you
have set so at some point you can decide this is a good model and then you can move on to the next
animal? Rick – if they were extraordinarily sensitive to LVS then you could vaccinate them and move on
to the next model. Terry said we may have to run through all 5 models and then decide at the end which
is the best. Vicki affirmed that we need all the preliminary data as so much of the data in the literature is
anecdotal so until we repeat the experiments to compare, we won’t know which is the best model. Rick
added that no model is likely to be perfect and the chosen model is likely to be a compromise.
COMMENT: Vicki – if this team and Dynport both look at some of the same models, if we come to the
same conclusions then we can be pretty confident we’ve got the right answer. Rick said then the data
could be shown to the FDA and a working group could be requested. Vicki spoke with Karen Elkins about
the likelihood of a working group – Karen doesn’t see this as a possibility until we have more animal data.
We can’t have a working group with data on only one model.
COMMENT: Rick/Vicki – we must remember that you will need some immunological tools and many of
these animal models don’t have available immunological tools. Rick compared to plague for which there
are antibody tests but with cell mediated immunity, we don’t have the reagents. Vicki mentioned a rabbit
study that spawned a working group which developed a website for rabbit models. This improved the
networking communication with other researchers.
Milestone 12-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: UNM
1. Date started: 7/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Challenge: efficacy testing in humans will be difficult, if not impossible and a high risk
population for pulmonary tularemia is not readily available. We will be extrapolating
from animals to humans to develop correlates of protection.
9 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
b. Example: In the UK, there was a horrible disaster with an antibody to stimulate T
cells for a disease. The antibody had gone through mass tox studies and even
possibly some primate studies; it appeared fine. Then 5 young men were given the
antibody and all suffered severely with septic shock. They all almost died. The
report vaguely referred to some signs in the primate studies that this could happen.
So we really need to understand the differences between the animals so we know
what to expect with our vaccine candidate development
c. This TVDC project will assist other future emerging infection assay needs by
providing a better understanding between animals and humans, and providing more
information for orphan diseases.
d. Goal: to identify and develop an immunoassay that will predict whether an individual
has developed a protective immune response following vaccination.
i. This has never been done for a bacteria for which cell mediated immunity
has been required.
ii. Protection often result of a cascades of events, rather than the activity of one
cell or protein. For example, in TB, no one has shown that a macrophage
alone can kill cells infected with TB.
iii. The final mechanism of protection may be undetermined. Even if the
mechanism is understood in animal model, final effector mechanism in
human cells would still unknown. The defense system is very different
between humans and mice.
iv. Sensitivity of detection may be limiting and need to detect in blood, rather
than lung lavages.
v. How to develop surrogates when the protection is due to a complex series of
events that requires the interaction among multiple cell populations?
e. Historically, we have had strict humoral measures and you can show for plague that
a certain level of antibody leads to protection. With cell mediated immunity as
measure by cell proliferation, cells are sensitized but doesn’t address their ability to
protect. A typical DTH response with a positive PPD does not guarantee protection.
f. T cell proliferation following LVS intranasal vaccination mice vs. protection against
virulent F tularensis.
i. Two strains of mice (BALB/c and C57BL/6) received a sub-lethal dose of
LVS down the lungs. The C57BL/6 lung cells have a better proliferative
response than the BALB/c but only the vaccinated BALB/c are actually
protected (UNM Lyons ppt; slide 5). The cytokine profiles between these two
strains of mice look extraordinarily similar. (data not provided)
g. Example of TB community: Developing an in vitro System to evaluate the
antimycobacterial activity in whole blood as a surrogate marker of immunity after
vaccination against Tuberculosis.
i. They measured growth of luciferase reporter gene-tagged BCG in blood
from pre, and post-vaccinated infants
ii. Significant Growth Inhibition was seen for population of vaccinated infants.
h. UNM’s approach: Will work for all animal species as it doesn’t depend on
immunological reagents. Will use a whole blood or PBMC approach to collect cells
from LVS vaccinated individuals and determine whether the cells can kill or inhibit Ft
infected cells or not.
i. Want to use a luciferase bacteria that gives off light and so that killing is
measured by a reduction of light. Karl developed the Luc LVS for UNM but
the quantity of light is low
ii. So UNM is using a microdot assay, in which the whole blood cellular assays
are set up in microtiter plates, then do serial dilutions, and if you see killing or
inhibition as a drop by a log or two, then the assay is working. This is an
accurate, reproducible assay that requires few bacterial plates. (UNM Lyons
10 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
ppt; slide 8). UNM has vaccinated mice and using spleens as a surrogate for
whole blood, since a mouse has so little blood volume. UNM is currently
working out the cell numbers etc with LVS. Since LVS is easily killed, UNM
may have to use SCHU S4 for the assay to show that cells from vaccinated
mice or primates can actually inhibit SCHU S4.
iii. (UNM Lyons ppt; slide 9; Experiment Ftc18 study 4 pages 83-87):
Evaluation of bacterial inactivation methods on ability to stimulate
splenocytes from LVS vaccinated BALB/c mice- determination which antigen
works best. Tested heat killed, uv inactivated, and formalin fixed LVS as
antigens. All 3 types of LVS antigen worked well. Historically, most use
formalin fixed bacteria as antigens so we can discuss the choice at Woods
Hole meeting since these 3 types are comparable. UV lamp efficiencies may
be more variable than formalin fixation or heat killing.
i.
General Schema for evaluating and comparing lymphocyte- macrophage interactions
(UNM Lyons ppt; slide 10). UNM hopes to identify the different populations of cells
involved in the inhibition assays, and then gradually isolate cell populations to look at
the roles of specific T cells, macrophages, specific cytokines or antibodies.
Antibodies may or may not play a role, but this could be studied. We could create an
ex vivo in vitro system.
j.
U19 Contract (value added to TVDC) : Looking at LVS and Biovar A bacteria on
human dendritic cell function. UNM has expertise in working with primary human
dendritic cells, both MDC and PDC. This assay looks at the update of bacteria by
labeling the bacteria with Alex 488 and then put them in trypan blue. IF the bacteria
sits on the outside of the cell, the flow cytometer can’t detect whether the bacteria is
inside or outside of the cell. However, if you treat with trypan blue, then only the
signal from the bacteria inside the cell is detected. The trypan blue quenches A488
signal from outside the cell, so you can quickly determine whether the LVS has been
taken up by the cells. (UNM Lyons ppt; slide 12-13). UNM incubated A488 LVS with
MDDC or MDM and looked at the uptake of the A488 labeled LVS. The MDM took
up more LVS than the MDDC and LVS was taken up better than FTNMA1, which is a
Biovar A isolated from a person in Mexico. UNM measured the percentage of
apoptotic MDDC after F tularensis treatment and found LVS with a 100x MOI gave
very high apoptosis (50%). UNM looked at the maturation of MDDCs, using CD86+
as a marker, after treatment with LVS or Biovar A for 24 hrs, and both LVS and
Biovar A were able to induce some MDDC maturation. This is not true for mice.
Interestingly, when the human MDDC cells are treated with LPS, the cells become
primed to respond to the LVS. This LPS priming and enhancement is not seen with
Biovar A. This may lead to an understanding of why humans make a better immune
response to LVS than to Biovar A. (UNM Lyons ppt; slides 16-18) and explain why
LVS makes a better vaccine than Biovar A. UNM also looked at cytokines produced
by mDC.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
Currently, UNM is replacing the LVS luciferase assay with the microdot assay for cellular
inhibition of LVS.
6. Deliverables completed
None
7. Quality of performance
11 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
Good
8. Percentage completed
8%
9. Work plan for upcoming 6 months
a. Determine the best antigen type and the lowest antigen concentration required to
stimulate proliferation of T cells from vaccinated BALB/c mice
b. Optimize macrophage killing of intracellular SCHU S4 by microdot analysis
c. Vaccinate BALB/c mice for peptide screen
10. Anticipated travel
a. 10/30 to 11/4/06: Rick Lyons, Terry Wu and Barbara Griffith to attend Collaborative
meeting with DVC and NIAID and to attend 5th International Tularemia Conference in
Woods Hole MA
11. Upcoming Contract Authorization (COA) for subcontractors
COA# 10: authorized travel to the CMI Workshop in Rockville MD.
QUESTION: Karl- asked about the LPS and LVS enhancement and the impact on a wild
type B strain. Rick does not have any wild type B strains in his lab, but Karl asks a good
question. Rick could get it from CDC or from Karl’s lab. The defining mutations between LVS
and type B strains have been observed so this enhancement may be able to be pinpointed
down.
Milestone 12-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
a. Goal: Develop assays for detecting relevant (anti- Francisella tularensis) immune
responses in vaccinated and challenged animals and in vaccinated humans.
b. Task 1: Tested 3 protocols for preparation of cynomolgous macaque PBMC and found that
LymphoPrep separation procedure from the Purdue Cytometry Mailing List gave the best
yield of cells, lowest RBC contamination, best percentage of viable mononuclear cells, good
proliferation in culture to mitogenic stimuli and ease of procedure. The T cells and monocytes
in the blood will be key to the assays. The Ficoll-Hypaque Plus method was too labor
intensive and the 90% Ficoll-Hypaque didn’t result in any white cells in the band, but put all
the wbc at the bottom of the tube. (LBERI Wilder ppt; slides 3-5)
c. Full LymphoPrep protocol can be found in TVDC Binder 1 (Wilder Lab)
ii. Raw data:
 TVDC Binder 1 (Wilder Lab), TUL-3 (8/14/06; repeat of Purdue Cytometry
Nycomed Lymphoprep method for purification of cynomolgous macaque
PBMCs);
 C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
Contract\tul3 elispot .xls
 C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
Contract\Tul 3 prolif .xls
iii. Summary data:
12 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble



C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
Contract\TUL3 summary.xls
C:\Documents Settings\jwilder.LOBOS\My Documents\Tularemia Contract\tul3
elispot 081606 incl summary.xls
C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
Contract\PBMC assays.svv
d. Table below shows that the results of 5 PBMC isolations using the LymphoPrep procedure
with whole blood from naïve cynomolgous macaques, from a breeding colony at LRRI. The
colony is supported by a Pharmaceutical company that allows LRRI to use the animals for
studies. Animal 63909 had a high PBMC yield due to being sick and the animal was
sacrificed after the blood draw. The blood from Animal 74863 was clotted leading to higher
RBC contamination. The PBMC yields varied. The concentration in a normal animal is
approximately 7x106/ml to 19x106/ml. The percent viability is percent viable white blood
cells. The percent RBC contamination is acceptable and it hasn’t affected the
immunoassays. (LBERI Wilder ppt; slide 6)
(C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia Contract\tul 1-3
summary tables 090606)
Tul 1-3 summary tables 092706.doc
Yield and Purity of PBMCs: LymphoPrep Procedure
Date
Animal
Yield
Concentration % Viable
7/12/06
F2519
8/14/06
63909
9/11/06
74863
9/15/06
0307B
9/18/06
0307B
* Animal sick
19.4 x
106/4.2
ml
94.75 x
106/4.5
ml
2.6 x
106/2.4
ml
2.05 x
106
/3.1ml
21.1 x
106/8.65
ml
%
RBCs
4.62 x 106/ml
74%
10.7%
21.06 x
106/ml*
82.8%
7.6%
1.08 x 106/ml**
48.8%
46.5%
0.66 x 106/ml
70.69%
28.45%
2.44 x 106/ml
84.5%
13.5%
** Clotted blood
e. For the cell proliferation assay, LBERI is using a BRDU Elisa method to quantitate the
response to mitogenic stimuli, ConA and anti-CD3. Figure below shows the responses in light
units. Three controls were assayed (see figure footnotes). When BRDU is incorporated into
13 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
the DNA, the relative light units increase. (LBERI Wilder ppt; slide7)(Cell Proliferation ELISA,
BrdU (chemiluminescent), Roche Applied Science, Cat. # 11 669 915 001)
f. Results for proliferation assay with mitogenic stimuli are in the figure below. They
observed good proliferative capacity to Con A, but not anti-CD3, at three different cell
concentrations (0.5 x 106/ml, 1 x 106/ml and 1.5 x 106/ml). Anti-CD3 was expected to be a
good stimulus for B cells but was actually a poor mitogen in this assay. Though the data is
not shown, PHA was also a good mitogen.
QUESTION: Was this a bound CD3? Julie responded that this is not a bound CD3 assay. In
the Elispot kit, the CD3 is just added and is not bound, which might impact the proliferative
response.
Proliferation data shown below and also in TVDC Binder 1, Tul 3
a. Raw data: C:\Documents and Settings\jwilder.LOBOS\My
Documents\Tularemia Contract\ Tul 3 prolif 081806.xls
b. Summary data: C:\Documents and Settings\
jwilder.LOBOS\My Documents\Tularemia Contract\ TUL3
summary.xls
TUL3 Summary.xls
Cyno PBMC Proliferation: Tul3
900000
800000
700000
RLU
600000
0.5 x 106/ml
1 x 106/ml
1.5 x 106/ml
500000
400000
300000
200000
100000
0
2255
15599
Nothing
Media only
Cells only
Cells + ConA
Cells + anti-CD3
Key to Graph:
-Nothing: No cells in wells; no addition of BRDU or anti-BRDU or substrate to those wells
-Media only: Media in wells for 4 days; addition of BRDU, anti-BRDU and substrate
-Cells only: Unstimulated cells in wells; addition of all reagents to detect proliferation
g. Cells from the sick animal also were set up in a IFN ELISPOT assay to test their response to
mitogenic stimuli (see figure below) (LBERI Wilder ppt; slide 8). The x axis is the number of cells
per well. In ConA stimulated cells, need to use less than 104 cells per well. Cells are counted by
automated counting system. The anti-CD3 stimulated a little with the highest concentration of
14 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
cells per well. They saw good stimulation of
anti-CD3
IFN production after Con A stimulation, but not
Data locations:
i. Raw data:
C:\Documents Settings\jwilder.LOBOS\MyDocuments\Tularemia Contract\ tul3 elispot
081606.xls
C:\Documents and Settings\jwilder.LOBOS\MyDocuments\Tularemia Contract\PBMC
assays.svd
ii.Summary data:
C:\Documents and Settings\ jwilder.LOBOS\My Documents\Tularemia Contract\tul3
elispot 081606 incl summary.xls
C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia Contract\PBMC
assays.svv
TUL-3: ELISPOT Results
Cell Mean for IFNg Spots
300
Media
Con A
Anti-CD3
250
200
150
100
50
0
14063
28125
56250 112500 225000
Number of Cells Plated/Well
h. Using flow cytometric methods, LBERI is looking at the cells in the PBMC population relative to
the whole blood. The data in the table below represents a short survey of the markers used. In
whole blood, about 37% of the lymphocyte- monocyte gate are CD4+ . Almost all of the CD4+
cells are also CD3+ and they are enriched in the PBMC population. The CD8+ cells are not as
enriched in the PBMC population. Interestingly, in 3 assays, the B cells which are CD20+ appear
to be depleted in the PBMC preparation. Perhaps this is a marker issue? There is no inherent
reason why B cells should not behave similar to T cells in this PBMC preparation. The number of
15 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
NK cells is low. The monocytes are also being purified by the preparation method. (LBERI Wilder
ppt; slide 9)
Cellular Characterization of NHP PBMCs
Cell Type
Whole Blood
PBMCs
% of Lymph
gate
% of Total
cells
% of Lymph
gate
% of Total
cells
CD4+
37
16.75
47.46
11.55
CD8+
35.85
16.05
36.56
8.56
CD3+
61.6
27.58
77.2
18.8
CD4+CD3+
37.5
16.99
49.06
11.95
CD8+CD3+
23.84
10.67
28.48
6.67
CD20+
25.7
11.63
4.59
1.09
CD56+
3.07
1.4
2.22
0.51
4. Significant decisions made or pending
1. LBERI will use the Purdue Cytometry Mailing List (PCML) protocol employing Nycomed
Lymphoprep separation media for the preparation of PBMCs from cynomolgous macaques
2. LBERI will use flow cytometry to examine cell populations in the PBMC preparations
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
80% of scientific work has been completed on the NHP section of this Milestone
9. Work plan for upcoming 6 months
1. Further analyze flow cytometry data to determine if we are truly losing B cells in the PBMC
prep (I.e. perhaps CD19 staining will pick them up, rather than CD20). Will use flow
cytometric methods rather than differential count for detection of lymphocytes, monocytes,
contaminating neutrophils, eosinophils etc. Flow cytometry allows detection of the
granulocyte gate and can stain with markers for neutrophils so neutrophil contamination can
be detected. There are few eosinophils in these preps. Differential counts are more variable
than the flow cytometric profile. Flow cytometry methods also have the advantage of
requiring few cells.
2. Develop intracellular cytokine staining with mitogen-stimulated PBMCs; start with IFN
16 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
3. Investigate freezing protocols for PBMCs and begin some pilot studies designed to test
their recovery, viability and function in proliferation and IFN ELISPOT assays
4. Write IACUC protocol and begin to vaccinate cynomolgous macaques with LVS and begin
to examine antigen specific responses; these experiments may fall under Milestone 13. They
will use healthy cynomolgous macaques for the LVS vaccination using scarification, not from
the LBERI colony. They will track response to the vaccination using the flow cytometry, the
IFNElispot, and the BRDU proliferation in response to a killed antigen, to be decided upon
by the group.
10. Anticipated travel
Julie Wilder attended the CMI Workshop by DMID on September 20-21, 2006, but does not
anticipate other travel at this time.
11. Upcoming Contract Authorization (COA) for subcontractors
None for this milestone.
QUESTION: SUBCUTANEOUS VS SCARIFICATION: Vicki asked a question regarding scarification for
monkeys – are you following the clinical program for humans? Julie responded: Yes. Vicki asked: Do
you have plans to include a subcutaneous arm in the protocol or might you do this later? Rick
commented that the number of animals should be considered. UNM could use information from NIAID
and Vicki commented this would be good because a subcutaneous arm would mimic the phase 1 trial.
The FDA will want to know if scarification is quantifiable method for vaccination; subcutaneous
vaccination is a more quantifiable, clinical method. Julie has 3 animals reserved now and they could
order more for a subcutaneous arm, depending on budgetary issues.
Action: Vicki thinks she can get access to the Phase I trial, just finished yesterday (9/26) and so is now
capable of being un-blinded. Vicki will check GDP regulations to see if she can share the protocol.
Hopefully, they can share volume type details.
COMMENT: FREEZING MACAQUE PBMC
Julie commented that it would be a good idea to perfect our freezing methods so that cells can be frozen
as a reference standard for each assay being developed. This may be a priority for assay validation.
Rick commented that freezing down macaque cells is not well standardized.
Action: Vicki hopes to share information from other contractors regarding freezing macaque cells–
they’ve been through the same problems UNM/LBERI is now facing. Vicki is researching the
nondisclosure agreements that NIAID has with the other contractor prior to sharing the freezing protocol
for macaque cells.
QUESTION: Kathy Sykes asked whether other pure antigens, like peptides or non-protein antigens, might
be used in the immunoassays. Rick responded that right now there are no plans for non-protein antigen,
but individual proteins could be used. Kathy clarified that what UNM is doing with T-cells matches what
LBERI is doing with monkeys.
QUESTION: Do we have plans to move from protein to an overlapping peptide library? Keith was afraid
we’d see a lot of CD4 and not much CD8 but Mitch didn’t fully agree. Rick thinks we’ll still get good
antigen processes. Kathy clarified that UNM will have the comparison of peptides vs. proteins from the
UNM pilot study.
Milestone 25
Milestone description: Design protein-fragment library based on SCHU S4 sequence
Institution: ASU-Sykes
17 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
1. Date started: 3/02/2006
2. Date completed: 10/7/2006
3. Work performed and progress including data and preliminary conclusions
a. Genomic-scale protein synthesis for determination of all tularensis T cell immunogens
(immune-ome). Immune-ome is a suggested new term. All of the proteins will be built in
vitro transcription/translation systems by Cheryl Selinsky. Which proteins are stimulating
the T cells? If we use that as a filtration step, then we will be able to test all
immunostimulatory antigens as subunit vaccine candidates.
b. Synthesize a complete library of all SCHU S4 coding sequences as 3,484 gene
fragments (ORFs), and then manipulate them into in vitro translation constructs.
c. Approach: take Genbank sequences for SCHU S4, will make all of the ORFs, then
translate all the ORFs into in vitro into protein fragments. These are not peptides and not
proteins, but are protein fragments. 200AA is nice size for folding shape and eliminating
potential technical or functional problems associated with full length proteins. The
protein fragments will be arrayed in 3 overlapping pools so each protein fragment will be
in 3 different pools. Those will be tested in T cell immunoassays to see which stimulate
the T cells. The identified T cell immunogen will tested as vaccines in animal models.
d. Kathy and Rick have a separate collaboration to do subunit vaccine development with no
filtration and this provides value added to the TVDC. It has taught ASU to build all of the
genes and proteins of Tularensis.
e. First aim: make all the ORFs. Changed the design after the contract was awarded. This
was not simple due to the AT rich genome, it doesn’t clone well, and it is hard to get
robust reproducible invitro products in microtiter plates, due to primary and secondary
issues in the genome. The approach is Primer Design Plus which Alex and Preston
wrote to fix and rebuild the genome to a Tm matched fashion. This software is very
complex, user friendly and transparent so you can see each parameter.
f. QUESTION: Joe- is the software publicly available? Kathy said it is almost ready, it will
be made public, but it is not ready yet. It will be on a website in the future.
g. QUESTION: Julie- What is fixing the genome? Kathy said it is fixing the 3rd positions to
make the sequence work better in PCR. Fixing the genome is using codon optimization.
This also normalizes to give the same level of expression in vitro. Technical advantage is
to decrease pcr failures, due primary and secondary considerations being removed.
h. (ASU Sykes ppt; slide 8)- demonstrates that predicted ORFs worked for 7 protein
fragments. A company in Houston can build the oligos using microfluidic”chips” which is
10 times cheaper than by standard column based synthesis. They supply 1 mix of 4000
oligos, which can be then co-assembled into ORFs and amplified with specific primers.
The gene building is a 2 step PCR, with a ligase chain reaction, which gives stronger
yields, better success rates and higher fidelity gene sequence. Each ORF is 250bp and
is made from a pool of 1200 oligos. (ASU Sykes ppt; slide 9) ASU showed that there is
no bias in the relative efficiencies of the block amplifications of the individual ORFs. The
yield of the oligos is approximately 5 fmoles of each oligo which is enough to assemble a
full ORF.
i. (ASU Sykes ppt; slide 10-14) Gene assembling by overlapping blocks- all work and the
yield is good and specific. More than 58% of ORFs are perfect, when a prior standard
protocol would have given maybe 5% perfect. The protocol is in place for high fidelity and
high production.
The list of 500 synthetic peptides covering MHC Class I and Class II epitopes have been
reviewed by Drs. Sykes, Johnston, Lyons, and Breen. The order has been placed but has not yet
been received.
Files are stored at R:\GeneVac\FTU\Contract\Proteome\Milestones\26\MHC\ftu_mhc_output.xls
18 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
4. Significant decisions made or pending
A protocol is in place for high throughput, high efficiency assembly of high-fidelity ORFs and gene
specific oligos have been ordered.
5. Problems or concerns and strategies to address
None.
6. Deliverables completed
A protocol is in place for high throughput, high efficiency assembly of high-fidelity ORFs and gene
specific oligos have been ordered.
7. Quality of performance
Good
8. Percentage complete
100%
9. Work plan for upcoming 6 months
a. Receive, dilute, and array pools of peptides for sending to UNM for in vitro testing.
b. Write final milestone report
10. Anticipated travel
10/30 to 11/4/06: Kathy Sykes will attend the collaborative meeting with DVC and NIAID and to
attend 5th International Tularemia Conference in Woods Hole MA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 26
Milestone description: Confirmation of gene expression (Design HTP SOPs, Test HTP SOPs,
ORF library production, confirm gene expression)
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
 Aim 2 (ASU Sykes ppt; slide 16): In vitro translate the coding sequence library, generating a




complete proteomic library of ~200 amino acid F. tularensis protein-fragments. A schematic
of the in vitro template shows a linear template and nothing is done in E coli. This is a
synthetic ORF with tags like ubiquitin which facilitates translation and refolding after
synthesis.
(ASU Sykes ppt; slide 18-20) Kathy showed the Ft. linear in vitro constructs and the proteins
that they produced in invitro translation reactions. ASU uses genebuilt ORFs for more
uniform levels and higher yields of products.
QUESTION: What do you mean by 58% perfect? It means that 58% had 200/200 or 100%
perfect sequence and the other 42% had perhaps 1 substitution but the other 199/200 were
perfect.
Now there is a company making peptides by microfluidics cheaper so we have ordered 500
potentially immunogenic peptides for MHC I or MHC II of SCHU S4 for testing the high
throughput method. We took only 1 epitope from 1 protein so these are 500 different
peptides for 500 different proteins. (ASU Sykes ppt; slides 21-23)
Aim 3: Affinity purify IVT reactions and then strategically array protein-fragments/peptides into
150 overlapping pools of 70 components each (ASU Sykes ppt; slides 24-29). These will be
made on a large scale with a biotin attached for purification, a his tag for quantification and
ubiquitin for folding. ASU will send the antigen pools to UNM to stimulate T cells from LVS
immunized/ SCHU S4 challenged mice, looking for T cell proliferative response to the protein
fragments which then would become the potential vaccine candidates.
19 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
4. Significant decisions made or pending
Seven protein fragments are being prepared as per IVT protocols and these will be sent to UNM
as material to initiate development of Tcell stimulation assays.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None.
7. Quality of performance
Good
8. Percentage completed
42.5%
9. Work plan for upcoming 6 months
Comparatively evaluate IVT constructs in several reaction mixes. Select the optimal construct and
IVT reactions based on ease of purification, yield and specificity of products and robustness of
the protocol.
10. Anticipated travel
10/30 to 11/4/06: Kathy Sykes will attend the collaborative meeting with DVC and NIAID and to
attend 5th International Tularemia Conference in Woods Hole MA
11. Upcoming Contract Authorization (COA) for subcontractors
None
QUESTION: Julie – is it important that the
T cells come from SCHU S4 challenged rather than just
LVS immunized mice? Rick responded yes. There are some genes missing from LVS that are in SCHU
S4.
COMMENT: Rick- Is a little concerned that the linear ivt vector may have too many tags and that they
may interfere. Alex/ Cheryl: ASU is making the modular constructs with and without the various tags, so
they can remove tags if necessary.
QUESTION: Karl-asked about obtaining protein chips from ASU (non-contract related). Kathy said
they’re not spotting peptides. It’s not really chip, it’s actually a micro-fluid device. They’re soluble. Joe
recommended contacting Phil Felgner to satisfy Karl’s request.
COMMENT: Vicki commented that she wants to promote collaboration but we first need to speak to Ross
about ownership of the reagents.
Milestone 32
Milestone description: Oligos selected for microarray production; Oligos list refined, 70mer
oligos procured, GDP oligos defined, Based on SCHU S4 sequence.
Institution: ASU-Johnston
1. Date started: 3/02/2006
2. Date completed: 08/28/2006
3. Work performed and progress including data and preliminary conclusions

Previous design was based on 2003 annotations of SCHU S4 genome and reanalysis
with 2005 annotations detected a number of misalignments. A complete reanalysis of a
20 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble







70 mer sequence was performed by Jonathan Lawson. (ASU Johnston/Magee ppt; slide
3)
1804 coding sequences of which 201 were pseudogenes. They designed a new probe
for each one of the genes.
75% of the probes are targeted for the 3’ half of the transcript to insure full read through
and less than 7% were in the first quarter of the gene. (ASU Johnston/Magee ppt; slide 4)
A Tm profile shows an average of 910 C and a good distribution around this average
(ASU Johnston/Magee ppt; slide 5).
ASU has received all of the probes and are working with a small subset to establish
hybridization conditions. UNM has provided LVS and SCHU S4 RNA. Mitch has printed
a 100 gene chip to check hybridization conditions.
Mitch has compared gene expression profile with LVS RNA and SCHU S4 RNA. ((ASU
Johnston/Magee ppt; slide 6). Preliminary hybridization studies show expression
differences between SCHU S4 and LVS. In the analysis, Mitch limited to genes that are
1.5 fold up in LVS vs. SCHU S4 or 1.5 fold up in SCHU S4 vs. LVS. The genes had to
have detectable expression in both LVS and in SCHU S4 in the analysis so it is not a set
of genes that are expressed in one bacterial strain but are not expressed at all in the
other bacterial strain. Genome directed primers were not used in the cDNA preparations,
but standard methods were used with 10ug of pure RNA.
QUESTION: Karl- when you designed the oligos, did you match the LVS and SCHU S4,
as they are very similar but are not identical? Mitch responded that the primers were only
designed based on SCHU S4 genome. ASU could easily return and do the analysis to
check this and has room on the chip to add other probes, given the resources. This is a
small gene chip with only 1804 genes so replicates will be printed per microarray slide.
ASU could add a full other set of genes.
The GDP (Genome Directed Primer) program was used to create a set of 183, 7mer
primers for amplification of the SCHU S4 genome in the context of murine tissue. The
region of design for the GDPs was restricted to the ensure encompassing of the
microarray probe. Blast analysis revealed no significant homology with the mouse
genome. (ASU Johnston/Magee ppt; slide 7) These GDP will selectively amplify the
SCHU S4 transcripts in the background of the mouse transcripts so ASU can selectively
amplify the bacterial RNA within the constraints of the probe sequence and analyze the
bacterial gene expression. The 183 GDP have been received but haven’t used them in
amplifications yet.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
 A set of 1804, 70mer probes designed to cover entire transcriptome is available
 Master printing plates are made and full-scale microarray production is ongoing
 GDP primers have been predicted and obtained.
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming 6 months
Write completed milestone report
10. Anticipated travel
None
21 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
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. Date started: 08-01-2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
 We received 19, 96 well plates containing a minimum of 65 microliters of an 80 µM stock
solution of each of the 1,824, 70mer oligonucleotides and controls. These have been rearrayed into master and working 384 well plates for printing. The test plate has been used to
print microarray slides which will be used for test hybridizations with LVS and/or SCHU S4
genomic DNA
4. Significant decisions made or pending


Test spotting buffers, as assessed by spot morphology and hybridization
Test printing on in house PLL slides versus Corning UltraGAPS, as assessed by spot
morphology and hybridization
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
30%
9. Work plan for upcoming 6 months





Start on the full production of the arrays including some substrate comparisons
Testing PLL coated slides versus Corning UltraGAPS slides, as the UltraGAPS are much
more expensive than the in house PLL coated slides
Philip Stafford wants Mitch to run 5 replicates per sample.
Will start with GDPs with purified RNA first
Within 6 months, will begin gene expression pilot studies from RNAs isolated from infected
mouse tissues provided by UNM to ASU.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
QUESTION: Joe- what types of studies will be done with the mouse tissue RNAs? Mitch
described the dose response curves, the detection of the lower limit of pure LVS RNA and will
also be done in the context of excess mouse RNA. Phil will perform the linear analysis
statistically.
22 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
QUESTION: Karl- Are you aware of the pathogen array slides from the NIH resources for SCHU
S4? Mitch is aware but has not compared the NIH slides to the ASU slides.
QUESTION TO Phil: Rick- Concerning best statistics – I think in many ways, more than
replicates, if you can do more mice of the same condition and less slides, that’s more powerful
than doing more replicates of the same sample. Phil responded that the technical replicates are
mostly useful in the development phase of the microarray experiments. Once the minimal
detectable fold change is, it should always be the same. Then you can start by doing pooling and
individual biological replicates. The five replicates are only during the development phase.
COMMENT: Joe- TIGR has been making pathogen arrays, but Joe is unsure how effective they
are or what is being printed, LVS or SCHU S4 or both. Karl knew that NIH had SCHU S4 and
they are free or at a minimal cost for approximately 150/year. Rick commented that ASU had to
design their own gene probes around the genome directed primers to assure detection of the
bacterial genes in the background of the host RNA. The GDP are designed to eliminate any
cross reaction with mouse RNA, and yet pick up all transcripts of F. tularensis. Rick said that this
strategy has worked with the Mtb studies in the past.
Milestone 39
Milestone description: Create uvrA or uvrB mutant F. tularensis subsp. novicida
Institution: UTSA
1. Date started:4/3/2006
2. Date completed: 10/7/2006
3. Work performed and progress
a. Goal: To generate and characterize live, attenuated F. tularensis susp. Novicida strains
for their vaccine potential. The uvrA and uvrB genes are involved in DNA repair following
UV damage and their inactivation facilitates KBMA technique of Cerus
b. Strains to be constructed:
a. F. tularensis subsp. novicida DuvrA::ermC
b. F. tularensis subsp. novicida DuvrB::ermC (9/1/06)
c. F. tularensis subsp. novicida DuvrA&DuvrB::ermC- made also
c. “3 step PCR “ strategy for creating gene mutations in F. novicida (UTSA Klose ppt; slide
6)
a. Doesn’t use groELpromoter. This promoter came up in a screen in the
University of Washington where they did saturation mutagenesis of novicida
genome and identified specific promoters that gave very high levels of
antibiotic resistance. They gave Karl the sequence of the promoter and it is an
excellent driver of antibiotic resistance in novicida and works well in other
subspecies too. Karl doesn’t know the gene associated with the promoter.
b. Overlapping PCR technique, amplifying upstream and downstream regions and
the ermC marker gene, then amplify the entire fragment with the marker gene
in the middle, put the PCR fragment directly into F novicida and get double
recombination in one step.
c. This method works easily in F novicida but does not work at all in LVS or
SCHU S4.
d.
d. Status of strain creation:
a. DuvrB::ermC strain (KKF71) sent to Cerus on 5/31/06
b. uvrA::ermC strain (KKF72) sent to Cerus on 7/28/06
c. uvrA::KanR construct made by same techniques
d. uvrA::KanR transformed into KKF71, KanR transformants screened, correct
mutant identified: uvrA::KanR, uvrB::ermC double Mutant
23 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
e. uvrA::KanR, uvrB::ermC mutant sent to Cerus
f. Milestone complete
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
No problems so far.
6. Deliverables completed
a. DuvrB::ermC strain (KKF71) sent to Cerus on 5/31/06
b. uvrA::ermC strain (KKF72) sent to Cerus on 7/28/06
c. uvrA::KanR, uvrB::ermC mutant sent to Cerus ~9/25/06
7. Quality of performance
Complete.
8. Percentage completed
100% of scientific work completed on the milestone
9. Work plan for upcoming 6 months
Write a completed milestone report for MS 16 and 39
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None.
Milestone 40
Milestone description: Phenotyping of Ft novicida mutants; Measure degree of attenuation of
uvr mutants in macrophages and in mice
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions







KBMA vaccine background: potency of live vaccines because they are metabolically
active and the safety profile of killed vaccines because the bacteria can’t replicate. UVA
light causes the S-59 psoralen to cause crosslink DNA in bacteria. In the parental strain
with functional nucleotide excision repair intact, the expression profile is diminished. In
the uvrAB mutants without nucleotide excision repair mechanism, the expression profile
is preserved. (Cerus Skoble ppt; slides 2-3)
Justin’s results are similar to Bernard Arulanandam’s results.
Cerus chose Cystine Heart agar with hemoglobin for plate cultivation and chose
Chamberlain’s media for liquid broth culture. (Cerus Skoble ppt; slides 4-5)
Question: Karl-Can others purchase the Chamberlains from Teknova? Yes, per Justin
and the two lots tested at Cerus have been comparable in CFU and growth rates.
Probably is best to order a fresh lot rather than one large long term lot because some
components are labile. Media should be sterile filtered. Action: Barbara will send ordering
information to Karl (completed by 9/29/2006)
Ft. novicida uvrA and uvrB mutants each have no growth defect in broth culture; they
grow very similarly to U112 wild type. (Cerus Skoble ppt: slide 8)
Ft. novicida uvrA and uvrB mutants each have no growth defect in J774 macrophages;
they grow very similarly to U112 wild type (Cerus Skoble ppt: slide 9)
Cerus has received the uvrA+uvrB double mutant in Ft novicida from Karl.
24 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
4. Significant decisions made or pending
a. We have selected Chamberlain’s Defined Medium (CDM) and Cystine Heart Agar with
Hemoglobin (CHAH) as liquid and plate medias for cultivation and enumeration of Ft novicida
5. Problems or concerns and strategies to address
Abrogation of the nucleotide excision repair pathway through uvrA or uvrB deletions does
not appear to result in a macrophage growth defect. If this trend persists in animals, it
suggests that a secondary attenuating mutation would be required if the SchuS4 strain were
to be used as the vaccine background (since LVS is already attenuated in humans it does not
require a secondary attenuating mutation). We will be screening attenuated Ft novicida
mutants that also have uvr mutations for immunogenicity in milestone 43, with the ultimate
goal of selecting a defined attenuating mutation to construct in SchuS4. We will also test a
double mutant uvrA +uvrB just received from UTSA.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
35% (updated 10/8/06)
9. Work plan for upcoming 6 months
 Characterize the uvrA+uvrB double mutant for growth in broth and in J774 cells
 Compare the in vivo rate of growth of U112, uvrA, uvrB, and uvrA+uvrB in mice.

however Cerus may reconsider this goal of working with just uvrA or uvrB single mutants
but just do double mutants, in light of the data presented by Bernard Arulanandam at the
annual meeting.
COMMENT: Karl pointed out that Bernard only did i.n. infection and Justin would do IV,
SC, IP infections. Also, Karl mentioned that all the virulence protein in this strain is
functional even though the uvrA gene is knocked out.
Enumerate CFU from lung, liver, spleen
Determine the LD50 of U112, uvrA, uvrB, and uvrA+uvrB in mice IV, SC, IP


10. Anticipated travel
10/30 to 11/4/06: Justin Skoble will attend the collaborative meeting with DVC and NIAID and
attend 5th International Tularemia Conference in Woods Hole, MA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 41
Milestone description: Optimization of psoralen treatment and characterization of KBMA F.
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
 For the KBMA approach it is critical that Cerus assures that the bacteria cannot
replicate and yet are still metabolically active. It is important to assure that the
25 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble








KBMA vaccine is not simply a low dose of a live vaccine. Cerus is titrating the S-59
dose and UVA light dose to optimize for KBMA.
S-59 titration: uvrB is more sensitive to S-59 (5uM) and UVA photochemical
inactivation than U112 (20uM)(Cerus Skoble ppt; slide 12). uvrB is not as
dramatically sensitive as uvrAB mutants of Listeria or B. anthracis
S-59 titration: uvrA (15uM) is less sensitive than uvrB to photochemical inactivation
(Cerus Skoble ppt; slide 13). The different S-59 sensitivities suggest that something
different is happening with these two mutations. Maybe there is a functional
redundancy of the urvA and uvrB genes and that a double mutant may be
synergistic.
UVA titration: 4 joules/cm2 is the dose that achieved reproducible killing of all 3
strains (uvrA, uvrB and U112 Ft novicida). (Cerus Skoble ppt; slide 14). For
Listeria and Anthrax, Cerus uses 6.5 joules/cm2.
Metabolic activity: measured using a MTS assay that relies on the bacteria’s ability to
convert a dye into a formizam as a demonstration of metabolic activity. Must
remember that the live bacteria curves also include increasing numbers of bacteria
due to replication. The KBMA bacteria are not replicating, so at 6 hrs when 3
doublings of the live bacteria have occurred the curves of the live bacteria and KBMA
bacteria diverge more. The KBMA bacteria maintain some degree of metabolic
activity for the 12 hours followed. So if the bacteria get into cells, they may be
perceived by the host as metabolically active for at least 12 hrs and this may be
sufficient time for the host to mount an immune response (Cerus Skoble ppt; slide
15).
QUESTION: Karl- question about relative activity at 12 hrs with Anthrax. Tae
responded that with Anthrax the KBMA is about 30% as active as live and that F
novicida is about 40-50% as active as live. Justin added that it is hard to compare
metabolic activity of Anthrax and Ft novicida at 12 hrs because the two bacteria have
different doubling rates.
Metabolic activity: Found no dramatic difference in metabolic activity between U112
wild-type novicida and uvrB mutant. Maybe the U112 strain is very sensitive to
photochemical inactivation, rather than having a redundant repair mechanism that
hasn’t been knocked out. (Cerus Skoble ppt; slide 16)
Both KBMA wild-type U112 and uvrB mutants have high degree of metabolic
activity (Cerus Skoble ppt; slide 17)
KBMA uvrA Ft novicida maintains metabolic activity and the heat killed shows no
metabolic activity. (Cerus Skoble ppt; slide 18) over 12 hrs.
4. Significant decisions made or pending
The minimum S-59 dose required for inactivation of U112 is 20 µM, the minimum S-59
concentration required for inactivation of Ftn uvrB is 5 µM and for Ftn uvrA is 15 µM. The
minimum UVA dose required to achieve complete inactivation is 4 J/Cm 2.
5. Problems or concerns and strategies to address
We previously have determined the concentration of S-59 required to inactivate Ftn uvrB is
only ¼ the concentration required to inactive the wild-type U112 strain. Currently, we have
determined that the concentration of S-59 required to inactivate Ftn uvrA is ¾ the
concentration required to inactive the wild type U112 strain. We have also determined that
the level of metabolic activity between Ftn U112 and uvrB or uvrA mutant strains is
26 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
indistinguishable. These differences are less than we have observed for other organisms. It
is still possible that the Ftn uvrA+uvrB strain will be more sensitive to photochemical
inactivation, and hence may display a higher degree of metabolic activity. The uvrA+ uvrB
double mutant will be evaluated next.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
30% of scientific work completed on the milestone (updated 10/8/06)
9. Work plan for upcoming 6 months
 Optimize photochemical inactivation conditions for Ft novicida uvrA+uvrB mutant
o S-59 and UVA doses will be optimized
 Compare S-59 sensitivity with uvrA, uvrB, and U112
 Compare metabolic activity with uvrA, uvrB, and U112
 Begin to scale up the inactivation process.
 Finalize photochemical inactivation regimen for all uvr mutants
 Determine the LD50 of KBMA F.t. novicida in mice
 Develop a storage formulation for KBMA strains.
10. Anticipated travel
10/30 to 11/4/06: Justin Skoble will attend the collaborative meeting with DVC and NIAID and to
attend 5th International Tularemia Conference in Woods Hole MA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 43
Milestone description: Create uvrA or uvrB mutants in LVS
Institution: UTSA
1. Date started: 5/01/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. Goal: To generate and characterize live, attenuated F. tularensis susp. Holarctica (LVS)
strains for their vaccine potential. LVS is a type B strain. The uvrA and uvrB genes are
involved in DNA repair following UV damage and their inactivation facilitates KBMA
technique of Cerus
b. Strains to be constructed:
a. F. tularensis subsp. holarctica LVS DuvrA::ermC
b. F. tularensis subsp. holarctica LVS DuvrB::ermC (6/1/07)
c. Mutation strategy
 For homologous recombination in LVS, larger segments of flanking homology
necessary
 1.5 kb uvrB upstream fragment from LVS PCR-amplified,1.5 kb uvrB downstream
fragment from LVS PCR-amplified cloned into pUC19. The plasmid is critical
because Francisella is an extremely AT rich genome and tends not to be stable
in E. coli.
27 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble

QUESTION: Joe-what do you mean by unstable? Karl said deletions are
incurred, but seemingly randomly and spontaneously. Can’t get around working
in E. coli but Karl is moving into lower copy number vectors for E. coli.
 This creates uvrB (LVS) with large flanking homology
 We are inserting FpKan into the deletion to create uvrB::Kan, this will be used
to transform LVS and obtain mutant.
 Same is being performed for uvrA::Kan LVS mutant
d. Homologous recombination technique
a. Overview (UTSA Klose ppt; slide 9)
b. We’ve cloned uvrB into pUC-ori plasmid, are cloning FpKanR into deletion
c. Cryotransformation followed by selection for KanR will result in first recombination.
Second recombination occurs at high rate naturally in novicida following cycling in liquid
culture Also in LVS (?), screen for loss of pUC ori by PCR . This is the only reported
origin to work.
d. We will add sacB for counterselection if cycling doesn’t lead to second recombination,
then plate on sucrose. The second recombination is required to finalize the mutant.
All the data were documented in page 58-61, TVD UTSA notebook #2.
4.Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
42% scientific work completed
9. Work plan for upcoming 6 months
a. Finish construction of uvrB::Kan in LVS
b. Finish construction of uvrA::Kan in LVS
10. Anticipated travel
Karl Klose will travel to Cerus in December 2006 to share knowledge of the genetics of Francisella with
Justin Skoble’s laboratory.
11. Upcoming Contract Authorization (COA) for subcontractors
None- No COA required for domestic travel between UNM and subcontractors.
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale
Francisella tularensis 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
28 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble






MS 46 is a 3-year milestone; presented by Tae Kim.
Cultivation of LVS (Cerus’ old strain) in Chamberlains for 26 hrs, taking time points every 2
hours to determine OD (600nm) and CFU/ml. Detected a biphasic curve with maximum OD of
4.6. They found the CFU/ml to be more variable than the OD which is more consistent
measure of growth.(Cerus Skoble/Kim ppt; slide 21)
Cultivation of LVS lot#16 from DVC in shaker for 11 hrs, taking time points hourly, measuring
OD and CFU/ml. See steady doubling time of 2 hrs and in log phase for the full 11 hrs, when
it reaches OD 1 and 1x1010 CFU/ml. When the OD of 1 was reached, they made frozen
stocks by replacing with fresh media plus DMSO and stored frozen. (Cerus Skoble/Kim ppt. ;
slide 22)
QUESTION: Bob asked about the freezing media. Novicida is fine in glycerol but LVS does
not survive well in glycerol. The NRC and DSTL are using 10% sucrose for freezing. Vicki
commented that sucrose is good for human administration and good for lyopholization. Rick
mentioned that DMSO is used clinically in bone marrow transplant and remembers the
patients smelling like DMSO.
S-59 Titration with LVS Lot#16 DVC: At 6.5 joules/cm 2, the LVS are completely inactivated at
5uM S-29 . (Cerus Skoble/Kim ppt. ; slide 23)
Fermentation system: First experiment failed because they used glycerol cell bank and LVS
doesn’t survive glycerol. In second experiment, grew overnight in shaker flask to OD of 1 and
then transferred into 2 liters of fresh chamberlains media and then transferred to the
fermentor. Just like DVC, they have no pH control and pH will go from 6 to high 7 over the
fermentation time. Use low air-flow rate of about 0.5 vvm (volume of air per volume of liquid
per minute). Cerus had a problem with the last fermentor culture and it is probably a
contaminant picked up from the media in the fermentor; contaminant maybe from an enzyme
detergent, which hasn’t impacted Listeria but maybe effecting LVS. Cerus is switching
cleaning procedures for the fermentor to an acid wash type. They are also looked at oxygen
poisioning but that wasn’t the problem. Cerus considered the possible degradation of
Chamberlains media as powder. Cerus hasn’t seen a problem with the liquid media.
4. Significant decisions made or pending
We have selected Chamberlain’s Defined Medium (CDM) and Cystine Heart Agar with
Hemoglobin (CHAH) as liquid and plate medias for cultivation and enumeration of LVS. We have
switched from glycerol to DMSO for cryopreservation of stocks. We have determined the
minimum concentration of S-59 psoralen required for complete inactivation is 5uM.
We believe it is worthwhile investigating whether LVS maintains metabolic activity and
immunogenicity when photochemically inactivated (prior to receiving a uvr mutant from UTSA)
5. Problems or concerns and strategies to address
Cerus is testing whether a contaminant from the cleaned fermentor is inhibiting LVS growth in the
fermentor.
6. Deliverables completed
None
7. Quality of performance
fair progress
8. Percentage completed
15% of scientific work completed on the milestone (updated 10/8/06)
9. Work plan for upcoming 6 months
a.
b.
c.
d.
e.
f.
Optimize 3L fermentor conditions
Increase scale of UVA inactivation to 400ml
Repeat S-59 dose titration
Perform UVA dose titration
Determine the extent of metabolic activity of KBMA LVS
Determine the LD50 of live LVS
29 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
10. Anticipated travel
10/30 to 11/4/06: Justin Skoble will attend the collaborative meeting with DVC and NIAID and to
attend 5th International Tularemia Conference in Woods Hole MA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Plans for next 6 months on milestones to begin: MS 42, 43, 44
•
•
•
Milestone 42: Protection experiments with KBMA F.t. novicida
» Vaccinate mice with KBMA F.t. novicida strain and challenge with U112
» Begin optimizing vaccination route and regimen
Milestone 44: Formulation and evaluation of KBMA LVS
» Optimize PCT regimen for non-uvr mutant of LVS
» Measure degree of attenuation of KBMA LVS
Milestone 45: Protection experiments with KBMA LVS
» Initiate protection studies with non-uvr mutant of LVS
QUESTIONS Rick- do you expect that using the higher level of psoralen will affect wild type by changing
the transcriptome significantly from the uvrA or uvrB mutants or is it a function more of the UVA dose
used? Justin said the difference in psoralen dose is around 5 fold and it is possible that it accounts for
the difference in the number of crosslinks that are around, but there is evidence from anthrax data that
there could be a difference in the transcriptome between the strains.
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
c.
Goal: To generate and characterize live, attenuated F. tularensis susp. tularensis strains
for their vaccine potential. The pdpD, IglC, IglD, IglA, IglB, genes are the virulence
genes located in the pathogenecity island and have a high potential to attenuate the
strain. The recA gene facilitates genetic recombination and its inactivation will stabilize
potential vaccine strain.
d. Strains to be constructed:
a. F. tularensis subsp. tularensis DiglC (3/1/07)
b. F. tularensis subsp. tularensis DiglD
c. F. tularensis subsp. tularensis DpdpD (3/1/08)
d. F. tularensis subsp. tularensis DiglA
e. F. tularensis subsp. tularensis DiglB (3/1/09)
f. F. tularensis subsp. tularensis DrecA
g. F. tularensis subsp. tularensis DrecA DiglC (3/1/10)
30 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
e. Construction of IglC F. tularensis subspecies tularensis strain (SCHU S4)
• This involves genetic manipulation of SCHU S4, requires BSL-3 which increases
inherent difficulty.
• We have tried and continue to try many different techniques to facilitate targeted
genetic recombination in SCHU S4
• Some of the things we have learned:
• Use of Ft promoter to drive antibiotic resistance is essential (both ermC
and KanR work well)
• Conjugation is not essential, we achieve recombination with both
electroporation and cryotransformation
• Amount of flanking homology is likely critical (>=1 kbp) for recombination
• QUESTION: Joe- is it a matter of efficiency? Karl- good question. With
shorter flanking homologies we don’t see co-integrants so I would say it
is inefficient, but not unfaithful homologous recombination. We just don’t
get much recombination.
• Second recombination (loss of plasmid) does not occur at high enough
frequency without counterselection. This was a surprise because F.
novicida looses the plasmid so easily.
•
Things we have tried (that did not work):
• Linear DNA with large flanking homology
• Genomic DNA with marked mutation
• Conjugation with marked mutation (Ftn DNA, 500 bp homology),Sucrose
counterselection
• Transformation with deletion, pUC-plasmid carrier Ft-expressing
antibiotic marker, followed by cycling for loss of plasmid (second
recombination). Plasmid went in, but would not recombine out.
• This last attempt was closest to succeeding, we are now adding sacB
(driven by Ftp) for sucrose counterselection. Allows to select for strains
that have lost the plasmid.
• We have cloned iglC fragment (Schuh4 DNA, 1.5kb flanking homology)
into low-copy plasmid .
• We have tried to move construct into mating plasmid constructed
specifically for Schuh4 work (pUC ori) which only resulted in deletions.
The DNA went in, but wasn’t stable.
• We have tried to move construct into plasmid used to create mutants in
novicida and LVS (pUC ori), which only resulted in deletions. For some
reason, the DNA around the IglC gene hasn’t worked.
• We are now moving construct into low-copy mating plasmid used
extensively by my lab for allelic replacement in multiple bacterial species.
This plasmid has conditional ori (R6K), tra genes, and sacB for
counterselection. We need to add FpKan before trying in Schuh4. since
SCHU S4 is naturally amp resistant. This conditional origin needs the
helper gene.
• We are attempting additional genetic techniques in lab that may impact:
e.g. Targetron system, removal of 1 copy of FPI, etc. This is
independent of the host. The advantage of this approach is that it is
independent of the recombination system of the host bacteria.
Everything needed for targeting is on the plasmid so it is almost host
independent.
• There are two copies of the pathogenecity island in Ft in the virulent
strains and this Sigma system targets both copies of the gene
simultaneously.
31 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
•
Karl’s lab is working on eliminating one copy of the pathogenecity island
as well.
e. Data located in TVD UTSA Notebook 3, page 31-36.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None that weren’t anticipated, because there has been so little genetic manipulation on type A
strains. We knew that genetic manipulation of Schuh4 and LVS would be challenging, we feel
confident that we are making significant progress and should soon have relatively straightforward
means for knocking out genes in Schuh4. (see above too)
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
20%
9. Work plan for upcoming 6 months
a. Prepare more mini plasmid preparations from the transformation resulting from
KEK229+iglC deletion sequence in section I.h. above. We are hopeful that this set will
yield the correct construct containing the longer iglC deletion to use in making the Schu4
igLC deletion.
b. Once we verify the correct construct, I will try cryotransformation and
electrotransformation to attempt to generate the IglC deletion in Schu4.
c. Will work also in cloning in the SacB gene in KEK1023 to check if this would be helpful in
pushing the second recombination event needed to generate a mutation in Schu4.
d. Order more supplies as needed
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
QUESTION: Phillip- what is the genomic identity across these strains? 97-99% identity.
QUESTION: Alex- Is there something unusual about the pUC origin? Karl thinks it is not totally inert and
may have some functionality.
QUESTION: How much is known about the recombinant system in Francisella? Essentially nothing. It’s
never really been tested . …There’s something toxic about IglD that for some reason is very toxic in E.
coli. We could use vectors with strong transcriptional terminators…really the issue is not that the DNA
itself is bad for the cell, I think that the products you’re trying to clone are toxic to the cell, so we can
improve the selection for those by putting in terminators. Karl has some of these vectors in the lab.
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,
32 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
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
a. Phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB
i. Intramacrophage survival of F.novicida uvrB (UTSA Arul ppt; slide3)
1. In J774 cells, the uvrB mutant of F novicida replicates nicely in 24 hrs
with an MOI of 10 or 100. It is the same as wildtype F novicida
ii. Mouse survival with F. novicida uvrB intranasal challenge with a dose range of
15 to 1250 (UTSA Arul ppt; slide4)
1. Isoflurane anesthesia, using BALB/c mice, similar to Terry Wu’s
procedure, all the mice die in 8 days
2. uvrB F novicida is not attenuated and is similar to wild type F novicida
iii. Bacterial burden of after intranasal infection with F novicida uvrB in lung, liver
and spleen over 24,48, and 72 hrs (UTSA Arul ppt; slide5)
1. Lung, liver and spleen are the 3 major organs for bacterial replication
2. The F novicida uvrB are replicating well in all 3 organs and are first
detected in lung
3. QUESTION: Vicki- you got really tight error bars in all the organs except
spleen at 48 hrs. did something unusual happen? This is 3-4 animals
per group and at that time point was a large error. We did it again and
the error bars did tighten up a little.
iv. Intramacrophage survival of F.novicida uvrA (UTSA Arul ppt; slide 6)
1. In J774 cells, the uvrA mutant of F novicida replicates nicely in 24 hrs
with an MOI of 10 or 100. It is the same as wildtype F novicida
v. Mouse survival with F. novicida uvrA intranasal challenge with a dose range of
10 to 1250 (UTSA Arul ppt; slide 7)
1. uvrA F novicida is not attenuated and is similar to wild type F novicida
vi. Bacterial burden of F novicida uvrA in lung, liver and spleen over 24,48, and 72
hrs(UTSA Arul ppt; slide 8)
1. The F novicida uvrA are replicating well in all 3 organs and are first
detected in lung
b. Baseline immune correlates with LVS strain Lot#16 from DVC
i. LD50 with intranasal challenge of 1x103, 2x103, 4x103 CFU (UTSA Arul ppt; slide
10)
1. Baseline for later comparison with LVS mutants
2. LD50 of 2000 to 2500, which is similar to Terry Wu’s data
ii. Cytokine recall responses after vaccination with LVS lot 16 from DVC (UTSA Arul
ppt; slide 11)
1. Challenge with sublethal dose of LVS intranasally and then 14 days later,
we make suspensions of spleen cells and challenge with UV inactivated
LVS in vitro for 3 days and take supernatant to detect IFN gamma and IL
4
2. See IFN gamma response but little IL4 or IL5
iii. Serum antibody profiles after vaccination with LVS lot 16- day 42 (UTSA Arul ppt;
slide 12)
1. Total antibody and IgG2a are high and low IgG1 is low
2. Whole cell elisa assay
33 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
iv. Respiratory cellular infiltration after pulmonary challenge (UTSA Arul ppt; slide
13)
1. Baseline study; when do cells show up after challenge
2. C57Bl 6 mice intranasally challenged with either LVS or F novicida and
harvested lungs and spleen 24,48 and 72 hrs after challenge
3. Looked at phenotypic markers in cell populations in lungs and spleen by
flow cytometry
4. At 24 hrs, LVS and F novicida elicit similar responses in the lungs
5. At 48 and 72 hrs, more cells are infiltrating the lungs relative to the PBS
control
6. Primary cells at 48 and 72 hrs in lungs are macrophages, dendritic cells,
and neutrophils.
7. With a gram negative bacteria, you would see a more robust response
earlier on.
8. Innate responses to Francisella which may be dampening or inhibiting
the response.
9. Chart shows the % positive cells for the marker.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Characterization of uvrA and uvrB mutants of F novicida
7. Quality of performance
Good”
8. Percentage completed
17% of scientific work completed on the milestone
9. Work plan for upcoming 6 months
LVS uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
COMMENT: Rick commented that the mice die with a huge burden in their spleens – seems like a much
higher burden than it should be, you’d think mice would be dead by that point. Tularensis is very stealthy.
The structure of the spleen histologically doesn’t look that bad and the lungs don’t look that bad either.
QUESTION: Kathy pointed out that LVS is indeed a vaccine because it stimulates some response that is
sufficient for it to act like a vaccine in the right context. Something is happening that works – though it’s
only marginal. Rick commented that people tolerate Biovar A for a while and they don’t go into septic
shock, like they do with plague. People have been sick with tularensis for weeks before they arrive at the
hospital. Julie commented that for some of these assays, we see great proliferation but it doesn’t
necessarily correlate with protection.
QUESTION: Karl- Is there an experiment where a co-infection is added along with SCHU S4 to see if an
inflammatory infection could…stimulate? Rick – yes, others have tried that with an immunoadjuvant and
got a roaring inflammatory infection.
34 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
QUESTION: Rick-What is a good intracellular infection? We’re too used to thinking about vaccines. Rick
doesn’t think we can prevent the infection process; focus should be on reducing morbidity.
COMMENT: Karl – IglC mutant persists for a long time in the mouse, perhaps is a component for inducing
the immune response.
Administrative Presentations at the Annual Meeting
1. Marlene Hammer: Differences between NIH Contracts and Grants- (NIH Hammer ppt)
Ross Kelley reviewed Marlene’s slides, listened to the presentation remotely and supported the contents
of the presentation. Contracts are very different than grants, as in contracts the government is more
involved in the technical, reporting and financial oversight.
2. Barbara Griffith; Biotracker- a Laboratory Information Management System (LIMS)
(UNM Griffith ppt). UNM is working with Ocimum Biosolutions to customize the software for Rick’s lab
and this will be available via web access, to view and possibly enter data, for the TVDC subcontractors.
QUESTION: Phil- how much work is required of the administrator on a daily basis? Barbara- we are at the
end of the beta testing and will know more in the future.
QUESTION: Alex- ASU has existing databases and wondered if theirs and ours would be compatible.
UNM could have Ocimum contact ASU to discuss. Data can always be saved as a blob or pdf file, or
parsed into the database. In the future, subcontractors should be able to view UNM’s data
3. Mindy Tyson: New TVDC Website (UNM Tyson ppt)
Mindy welcomed the subcontractors to visit the TVDC website, using the temporary group login and
password and to provide suggestions to Mindy for website improvements. The TVDC website is
designed to simplify and enhance communications and be a common repository for reporting forms and
reports.
Discussion Session at the Annual Meeting
1. Working Group and LVS culture
QUESTION: Barbara- Growth in Chamberlains and freezing the LVS is being developed by the Working
Group. What is our UNM TVDC consensus? Rick provided the Working Group background as the LVS
growth at UNM was not as consistent as is needed. Growth or no growth depended on the initial
inoculum. Terry and Wayne were to grow it under the same conditions as Gill Hartley and report back to
the Working Group. There was no concern regarding the media. Vicki noted that we thought we had
parameters defined and then we discovered that all participants weren’t really using the same
parameters. Parameters to be defined included the starting inoculum, the media volume, the size of the
flask, the RPM for shaking. Terry shared Gill’s parameters as resuspending 1 lyopholized vial of LVS in
250ul of water, then inoculate 240ul of the resuspended vial into 60ml of Chamberlains at 37C in a 125
ml flask with shaking for 48 hrs at 150rpm. Once these conditions are confirmed experimentally by UNM,
NRC and DSTL, then these will become the Working Groups’ final recommended conditions for growing
LVS.
QUESTION: Justin- do you want us to always work from a lyophilized vial? Rick- no, the idea is to grow
up working stocks identically from the lyophilized vials and then use those frozen stocks for experiments,
rather deplete the lyophilized stocks. By virulence and % blue/grey, UNM and NRC, respectively,
determined that there was no difference between the LVS in the lyophilized vial and the LVS grown for
48hr in Chamberlains media.
35 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
QUESTION: Vicki- When you are freezing the cultured LVS down in 10% sucrose or something, what
would be the volume frozen down? Rick – thinks we need to look more at the concentration than the
volume since the volume would vary depending on the quantity of LVS needed for the in vivo or in vitro
type of experiments being done in each lab.
COMMENT: Vicki- There was some discussion of what the final titer should be in the vials. BARBARAshare the final titer established in the working group
COMMENT: Tae- Cerus has found that there is no lag time and that the cultures took right off. Rick had
not seen this before. Tae said that they snap freeze in liquid nitrogen, quick thaw at 37C and bring them
up in fresh media and shake them at 225 rpm in a volume of media not more than 20% of the flask
volume. Barbara-UNM has been shaking at 225 but DSTL shakes at 150rpm. DSTL gets high titers
without shaking this fast. Tae said Cerus has always shaken at 225rpm for other bacteria.
COMMENT: Karl- UTSA has done multiple growths, freezing and re-growing and tittering of SCHU S4 in
the past. UTSA has frozen SCHU S4 in glycerol. Crystal commented that freezing SCHU 4 did not cause
it to lose much viability and Crystal uses them for growth curves that look pretty much the same.
QUESTION: Vicki asked Justin and Tae- When you look at the 12 hr culture of LVS to see if they are
metabolically active, when you freeze them down and then thaw them out, are they still metabolically
active? Justin – yes. Vicki- then at this point, is Cerus looking at a potential vaccine that is frozen
down? Tae said yes though they are also looking at a vaccine that is dried down as an option.
Action item: Rick said that determining freezing solution and concentration of bacteria in the frozen vial
must get standardized.
2. Website and Communication
QUESTION: Rick- was there any thing that you didn’t see on the website that you want to see? Barbarawas there anything on the website that you want removed? Kathy- how would the data management
interface with the website.? Barbara- Biotracker would be connected through the website. It would be
nice to have a connection to Biotracker from the TVDC website.
QUESTION: Mindy- would you like a chatroom or discussion thread through the website or do you prefer
discussions through email and teleconferences? Karl said we talk with each other a lot already. Kathy QUESTION: Julie asked about the lack of connection between subs – can subs talk to each other without
UNM being present? Vicki stated this is at UNM’s discretion since we’re the ones who have the contract
with the subs. Rick said he’s fine with subs talking amongst themselves. Barbara added that
contractually subs must include UNM when transferring or exchanging reagents, but other topics can be
discussed between the subs. The subs must email UNM when exchanging reagents, but UNM’s contract
with the subcontractors is silent on other discussions between the subs. Kathy- thinks a chat box is not a
good idea because she doesn’t want one technologist chatting with another technologist in a different lab,
suddenly changing protocols, without the PI knowing about the discussion. Confusion could arise.
Currently, one PI directs their technologist to specifically contact other labs.
COMMENT: Philip- if you use Outlook for email you can search by topics, names, and subjects. BarbaraUNM uses Novell/groupwise and doesn’t use Outlook.
3. Optimal Animal Model: what is the criteria?
QUESTION: Rick on Woods Hole discussions: what do others think about the optimal animal model in
the context of this contract? What do we want out of this animal model?
Karl – bottom line, good prediction of efficacy.
Rick’s prediction is that we’ll see vaccinated animals live a week longer than unvaccinated. Say the
guinea pig can tolerate high levels of LVS like humans can and then are challenged with Schu4 and
reproducibly live 10 days longer than non-vaccinated guinea pigs. So is that reproducible extension good
36 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
enough? The guinea pigs still die but get a reproducible extension. It is more like human because it
tolerates LVS but it doesn’t produce a solid protection, which is different from the mouse that doesn’t
tolerate LVS very well, but at least for a window, you can get solid protection. These are the issues
ahead of us.
Vicki- we need to consider how the FDA looks at the data , not necessarily how NIH looks at the data for
the vaccine model efficacy.
Bob – we would hope to find a model where a vaccinated animal survives but we might be less picky
about the breakthrough level on the protection. If you have a model and it doesn’t survive that suggests
that you aren’t activating all the proper mechanisms. I hope we get an animal that does activate the
proper mechanisms. It need not be robust as it may survive an LD50 challenge but maybe not many
times the LD50 challenge, but it should activate all the proper mechanisms. The breakthrough level may
be more in play than the survival vs. non-survival.
Rick- When you consider that the LD50 of SCHU S4 in mice is less than 1, and it survives a dose of 200
of LVS, then that is 200 times the LD50s. If you break it down that way, it is a significant challenge.
Justin- The sensitivity to LVS goes away when you stop using the LVS so the idea of a better vaccine is
that it is not as toxic. So an attenuated LVS and KBMA strains are all going to be less toxic in the mouse.
So surviving multiple LD50’s is the standard to which the animal model should live up to.
Kathy – there are two components – the vaccine and the animal. We have used models for certain
infections that look good when you review pathogenesis but the models are not good when you determine
survival vs. non-survival.
Julie – to clarify, Bob was saying that everybody should be able to survive a limited dose. You will
overcome the vaccine at some dose level.
Bob – pathology is very important to consider, similar dissemination, similar organ titers, similar pathology
are all relevant to the model.
Fred – vaccines have multiple uses – a vaccine that delays death may have a therapeutic use in parallel
to the anthrax vaccine. A vaccine that works in conjunction with antibiotics can eliminate the 60 day
reactivation and have a therapeutic effect.
Karl: once you have a protective response, you can tweak the vaccine by modulating the immune
response or adding antibiotics or cytokines or whatever to make the vaccine better , but it gives you a
place to start. The expectations are already pretty high with a cell mediated immune response.
Rick: another extreme is that you have a great model, but you don’t have the immunological tools, for
example, like the rabbit.
Julie: Rick, in your mind, what makes the mouse model a poor model is that they’re sensitive to LVS and
their protection wanes. Can they be re-vaccinated?
Terry: No, you can’t boost with LVS because it gets cleared too fast.
Rick: the biggest problem with the mouse is the high level of sensitivity to LVS which is unlike monkeys
and unlike humans.
Julie: Also in the mouse, the protection correlates may be very different from the human
37 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
Rick: We know that the phagocytes of mice and rats are not human like. The LVS sensitivity and the
waning at 60 days is bothersome in mice. Maybe this is a continuously active infection of LVS in mice
that is chronically stimulating the response and there is no memory. It is odd that mice should loose
memory completely. Seems like once there’s no more antigen stimulation, there’s no more protection.
Bob – a vaccine company’s dream! Daily doses!
QUESTIONJulie: With the tularemia vaccine for humans is the vaccination a one time dose?
Vicki: a derivative of LVS is still used in endemic areas of the Soviet Union. In anecdotal, private
conversations, in these endemic areas, when they start seeing breakthrough cases of the disease in
areas they’ve vaccinated, they go around and vaccinate again. There is no documentation for this as we
really know very little. With laboratory workers that are vaccinated with LVS, Vicki doesn’t think that they
get revaccinated. Of course in their work environment, the laboratory workers may be getting some
continuous exposure to LVS.
QUESTION: Tae- is there a lower logistical challenge level? It seems like LD50 of less than 1 or less than
10 would be subject to artifacts and errors. Should ideal model include the criteria so that you never have
to dose below a certain low level?
Rick: good point, because at these low doses, when you calculate the LD 50 some mice aren’t dying
because they never got infected with a single bacteria and others are dying because they got more than
the intended dose; this is problematic. I think in some animals, you can reproducibly infect with a few
hundred bacteria and this has to be demonstrated to be a good model. You just want to make it a
reproducible dose.
Tae: Immunological reagent development is not trivial.
Jason: Two quick thoughts on the small animal model – one caveat is to remember that we want to get
something that shows complete protection even at an LD25 as opposed to extended time to death as an
endpoint. Even if we say all these mice lived, maybe they only lived for 3 weeks. Other thing is that time
to death is in a vaccine, still important so I don’t want us to discount that. Maybe surviving a week longer
(as opposed to 48 hours) would be very useful information. So let’s not discount possible small victories
in case we can’t get the optimal response.
QUESTION: Cheryl: How realistic is it to give post-exposure prophylactics? Because that would justify
whether delayed time to death makes for a good vaccine candidate.
Rick: A post exposure treatment, not a vaccine, is BioShield and is out of the scope of this Tularemia
vaccine development contract.
Rick: Bob’s argument is the best because if you don’t get full protection then you’re clearly missing
something in the vaccine process and in stimulating the appropriate immune response. I think we’re not
used to seeing LD50 of 1, for most pathogens that we make vaccines for. Think it’s a change of mindset.
We are accustomed to thinking of survival of a few hundred thousand bacteria.
Karl: The bar is set high if we rule out mice model because they’re sensitive to LVS because LVS is not
the perfect vaccine. We can probably develop an attenuated organism that mice are very resistant to and
that won’t kill the mice. I think the mouse is an excellent model otherwise because of the immunologic
reagents available for analyzing this intracellular pathogen.
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
Rick: Knowing the FDC, one of the reasons for doing this is because you want to provide the FDA with a
package and show them why you chose what you did. Maybe none of the models are perfect, but you put
together a logical package of the pros and cons. FDA will want to know about the rabbit, and other
animal models and why you did or did not select them as the final animal model.
Bernard: The two model rule does want us to present two different species.
Bob: FDA will also say the limited human data that we have indicates the following: this kind of pathology
based on this kind of dose, based on this type of titer and inoculation rounds, these tissues are involved,
it moves from this tissue to this tissue. Our animal model is going to have to follow this as much as
possible.
Vicki:” As much as possible” is a key phrase. You can have a model that faithfully mimics the human
disease condition but you have no reagents to test the mutants. This will not be an ideal model. I think
the FDA is going to be reasonable and they won’t want a model with no immunologic reagents either.
Vicki: thanked everybody for all the work that took place over the last year. We are looking forward to an
explosion of work this next year.
Rick – thanks to everybody for all the hard work, good meeting!
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: 1, 2, 3, Working Group, 5, 12 (UNM &LBERI) ,
16, 25, 26, 32, 33, 34, 39, 40, 41, 43, 46, 49 and 50. None of these milestones currently are over
budget.
To date, NIAID has been billed for scientific activity on Milestones 3, Working Group (covered under MS3
and 5) , 5, 12 (UNM &LBERI) , 16, 25, 26, 32, 33, 34, 39, 40, 41, 43, 46, 49 and 50.
Data associated with most current UNM invoice submitted to NIAID ( parallels Financial Report by
Milestone and lags behind current Technical Report)
Milestone
% invoiced
3- LBERI
5-UNM
12 UNM
12 LBERI
16-UTSA
25-ASU
26-ASU
32-ASU
33-ASU
34-UNM
39-UTSA
23.7
34.1
12.7
61.4
79.8
21.6
3.3
26.9
2.4
10.1
50.5
% work
completed
22
25
8
60
100
75
60
95
20
0.5
85
Over
budget?
No
No
No
No
No
No
No
No
No
No
No
Discrepancy?
NA
NA
NA
NA
NA
See explanation #1 below
See explanation #1 below
See explanation #1 below
See explanation #1 below
See explanation #2 below
NA
39 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
40-Cerus
41-Cerus
43.1-UTSA
46- Cerus
49.1-UTSA
50.1- UTSA
19.8
8.2
20.4
3.9
35.2
42.1
15
20
25
10
14
12
No
No
No
No
No
No
NA
See explanation #3 below
NA
See explanation #3 below
See explanation #4 below
See explanation #4 below
Discrepancy Explanations
1. MS 25,26,32,33- ASU: ASU has consistently reported a higher percentage work completed than
percentage invoiced as of 8/7/06. ASU has only invoiced UNM for labor for one month, May 2006. The
ASU direct labor for June, July, August and September are expected to be charged on the invoice due to
UNM on 10/7/2006. This delayed charging of labor is due to ASU changing the effort allocations of their
scientists funded on the TVDC. The new allocations were entered into the ASU Financial system in June
but take effect months later. We anticipate that the discrepancy between % invoiced and % work
completed will diminish significantly when the labor expenses become included in the % invoiced. We
anticipate no over expenditures on these milestone budgets.
2. MS34- UNM: UNM isolated RNA and DNA from SCHU S4 and LVS and provided these reagents to
ASU for testing the microarrays. Once ASU begins using these reagents for microarray assessment, we
anticipate that the % completed will rise to match the % invoiced. We anticipate no over expenditures on
this milestone’s budget.
3. MS41,46-Cerus: Cerus anticipates that over time, the % invoiced will increase to match the % effort
completed. Cerus invoices’ lag 2 months behind relative to the month in which the % effort is reported.
So an invoice submitted in September is for expenses incurred in July. They also anticipate that in some
months, the % work completed will take major leaps forward with little expense and in other months, the
% invoiced will be greater than the % work completed. We anticipate no over expenditures on these
milestone budgets.
4. MS49.1, 50.1-UTSA- Each of these milestones (49 and 50) have budgets over 3 years; however,
UTSA’s financial system requires them to create a budget per year. Thus the percentage invoiced is
calculated based on the percentage spent in one year of the milestone rather than against the total 3 year
budget of the milestone. In contrast, the percentage work completed is based on the effort expected over
all 3 years. When we calculate % invoiced based on the 3 years budget for the full milestone, the %
invoiced is 10.9% for milestone 49 overall and 10.2% for milestone 50 overall. At these lower invoiced
percentages, there is no real discrepancy between the % invoiced and % work completed for MS 49 and
50. We anticipate no over expenditures on these milestone budgets.
To date, administrative costs have been billed to NIAID that are associated with Milestones 1 and 2 and
with the management of the scientific milestones 3, 5, 12, 25,26, 32, 33, 34, 39, 40, 41, 43, 46, 49 and
50.
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.
40 of 41
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 4/01/2006 to 9/30/2006
Due Date: 10/15/2006 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Ed Barr, Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Bernard Arulanandam,
Justin Skoble
LBERI, Cerus, ASU and UTSA all submitted invoices to UNM in the prior month. The UNM invoice to
NIAID, being submitted on 10/15/06, will include subcontractor invoices from ASU, Cerus, LBERI and
UTSA.
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