Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 1 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Signature Page
Author’s Signature:
_______Justin Skoble____________
Typed Name of Author
__Not Required__________________
Signature of Author
__________
Date Signed
Acceptance by Subcontracting Institution:
__Suzanne Margerum______________
Typed Name of Subcontracting PI
Not Required _____________________
Signature of Subcontracting PI
__________
Date Signed
Acceptance by the University of New Mexico:
________________________________
C. Rick Lyons, MD. PhD
_ Not Required ____________________
Signature
__________
Date Signed
Acceptance by NIAID:
Freyja Lynn_______Accepted on 3/2/2009____________
Typed Name of NIAID Interim Project Officer
Not Required __Heidi Holley Contract Officer accepted 3/3/09
Signature of NIAID Project Officer
Date Signed
Page 1 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 2 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Table of Contents
1
2
3
4
Milestone Summary ...................................................................................................................................................2
Milestone Objectives..................................................................................................................................................2
Methods, Critical Reagents and SOPs........................................................................................................................2
Salient Original Data, Results, Interpretation, Quality Control .................................................................................4
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location annotations) .................4
4.2
Interpretation ............................................................................................................................................ 13
4.3
Quality Control ......................................................................................................................................... 13
5 Deliverables Completed ........................................................................................................................................... 14
6 Appendices ............................................................................................................................................................... 15
6.1
Appendix 1: Original Data Tables and Figures ....................................................................................... 15
6.2
Appendix 2: Quality Assessment of Milestone Completion and Report ................................................. 17
6.3
Appendix 3: Additional Data/Figures not included in the Text of the Milestone Completion Report
(Section 4) .............................................................................................................................................. 20
1
Milestone Summary
Nucleotide excision repair (NER)-deficient strains of Ft novicida were constructed by UTSA and
transferred to Cerus to determine whether these mutations result in a significant attenuation
allowing the strains to be used as killed but metabolically active (KBMA) vaccine platform strains.
We determined that uvrB and uvrA single and the uvrA uvrB double mutant strains grow at
the same rate as the wild-type U112 strain in Chamberlain’s defined medium (CDM), in J774
macrophages, and in lungs, livers and spleens of Balb/c mice following intravenous (IV) injection.
NER-deficient strains were all highly virulent when administered IP, but when delivered IV or SC,
all Ft novicida NER mutants were approximately 1 log reduced in virulence compared to U112.
This correlated with a slight decrease in the ability of the uvrB mutant to disseminate to the lung
following SC administration. The conclusion of these studies is that NER abrogation does not
result in significant virulence attenuation of Ft novicida and that other attenuating mutations would
be required to ensure the safety of vaccines based on type A F. tularensis strains.
2
Milestone Objectives
Phenotyping of Ft novicida nucleotide excision repair mutants; Measure degree of attenuation of
uvr mutants in macrophages and in mice.
3
Methods, Critical Reagents and SOPs
Bacterial media. Cultivation of Ft novicida strains was performed at 37oC using Cystine Heart
Agar (Difco) supplemented with 10% bovine hemoglobin (Becton, Dickinson) as solid medium
(CHAH) for colony enumeration (Media preparation PRP01.002 and INS 00313.v21.0). For
liquid cultivation of Ft novicida strains, individual colonies were inoculated into Chamberlain’s
Page 2 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 3 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
defined medium (CDM) and incubated at 37oC with shaking at ~200 RPM. For preparation of
CDM liquid media, 24.2 grams premixed formulation (Teknova cat# C0715) is added to water, the
pH is adjusted to 6.26 and sterile filtered through 0.22 m filters (Prep and QC bacterial culture
media PRP 01.ver002),
Intracellular growth of Ft novicida.
Intracellular growth assays were performed as previously described (Portnoy, D.A., et. al. Journal
Experimental Medicine 1988; 167:1459-311471). Briefly,1 ml of overnight CDM Ft novicida
culture was harvested by centrifugation and washed once with 1.5 ml cell culture growth media
(DMEM with 10% FBS). The bacteria were diluted 1:50 into cell culture growth media (~2 x 109
cfu/mL) and added to J774A1 macrophage cells (ATCC) seeded at 0.25 x 106/well in 24 well
plates the day before, (the J774A1 macrophage cell density is ~ 0.5E6/well for the MOI
calculation). 250 ul/well of bacterial suspension was added to cells (thus delivering ~5 x 107 cfu
for a MOI of ~10) and incubated at 37oC for 1h in 5% CO2. Infected monolayers were washed
twice with PBS, and fresh cell culture growth media was added and incubated for another 1.5h at
37oC after which media was aspirated and replaced with 1 ml/well cell culture growth media
containing 50ug/ml gentamicin and incubated at 37oC. Gentamicin was added to kill residual
extracellular bacteria. At each time point, the infected J774A1 cells to be lysed were washed once
with PBS and then 1 ml/well sterile distilled water was added. Cells were incubated at RT for 5
min pipetted up and down a few times and the cell lysate was collected into eppendorf tubes,
vortexed vigorously for 30 sec. 1:10 serial dilutions were made for titration and 100 ul /plate from
each dilution tube was spread on CHAH plates incubate 24-48 hours at 37oC for bacterial colony
enumeration (Bacterial colony count QCP-01.003),. Three wells per timepoint were plated
individually and the cfu were reported as means and standard deviations.
Mouse studies. 6-12 week-old-female BALB/c mice were obtained from Charles River
Laboratories (Wilmington, MA). The mice were housed in individually HEPA-filtered cages with
autoclaved bedding. Food and water were supplied to the mice ad libitum (Animal procedure FAP01.001 and FAP-01.003). Animals were allowed to acclimate to their surroundings for at least 1
week prior to use. Animals were injected IP (Animal procedure FAP-01.028), IV via the tail vein
(Animal procedure FAP-01.026), or subcutaneously (Animal procedure FAP-01.027), with 100200ul diluted bacterial suspension as described previously (Brockstedt, D.G. et al., Proc Natl Acad
Sci USA. 2004; 101(38):13832-7). LD50s were calculated using the method of Reed and Muench
(Reed, L. J., and H. Muench. 1938. Amer Journal Hygiene 27:493) (Toxicity studies LD50 in
mice-PRP 01-004, appendix 1&2) and (Anesthesia FAP-01.016). The level of infection in each
mouse was determined at the indicated time after Listeria challenge by enumerating bacteria in
liver and spleen organ homogenates (Auerbuch, V. et al., Infect Immun. 2001; 69(9):5953-7). All
animals were treated according to National Institutes of Health guidelines, and protocols requiring
animal experimentation received prior approval from the Cerus Animal Care and Use Committee.
List of Critical Reagents
Cystine Heart Agar (Difco)
Page 3 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 4 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Bovine hemoglobin (Becton, Dickinson)
Chamberlains Defined Medium (Teknova cat# C0715)
J774A1 macrophage cells (American Type Culture Collection; ATCC)
BALB/c mice (Charles River Laboratories. Wilmington, MA)
uvrA F novicida (UTSA KKF72 strain; MS#39)
uvrB F novicida (UTSA KKF71 strain; MS#39)
uvrA, uvrB F novicida double mutant (UTSA KKF100 strain; MS#39)
U112 wildtype F novicida
SOP Number1
PRP01 ver.002
INS00313 v 21.0
FAP-01.001
FAP-01.003
FAP-01.026
FAP-01.027
FAP-01.028
FAP-01.029
FAP-01.016
PRP-01-004, appendix 1&2
QCP-01-003
SOP Title
Preparation and Quality Control of Bacterial Culture Media
Media Preparation Using the S8000 Auto preparer and APS300
Pourer Stacker
Animal Facility Gowning
Animal Handling and Restraint
Intravenous (IV) Injections
Subcutaneous (SC) Injections
Intraperitoneal (IP) Injections
Intramuscular (IM) Injections
Isofluorane Anesthesia
Determination of Murine Toxicity (LD50) of Viruses and Bacteria
Determination of Viable Bacterial Count of a Bacterial Culture
1
Individual Standard Operating Procedures will be reviewed separately and accepted by the Subcontracting PI and UNM
PI
4
Salient Original Data, Results, Interpretation, Quality Control
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location
annotations)
Rationale. Our ultimate goal is to produce a Killed But Metabolically Active (KBMA)
Francisella tularensis (Ft) vaccine. Because Francisella tularensis is highly infectious, one
concern about using a KBMA Ft vaccine is that a single organism that escapes inactivation could
potentially cause fatal disease. Thus it is essential to construct a KBMA vaccine platform strain
Page 4 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 5 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
that is attenuated for virulence. Because type A strains of Ft are the most likely to be used as a
bioweapon, protecting against these strains is the highest priority. Unfortunately Ft subspecies
tularensis strains are much more difficult to manipulate genetically than Ft subspecies novicida.
For this reason, proof-of-concept studies were performed using Ft novicida which are extremely
virulent in mice (but not in humans) to evaluate the feasibility of constructing safe and effective
KBMA Ft vaccines. In order to construct KBMA vaccines, the bacteria need to be nucleotide
excision repair (NER)-deficient to allow for photochemical inactivation with minimal crosslinking
of DNA. In some organisms (e.g. Mycobacterium tuberculosis) it has been demonstrated that
mutations in NER genes can result in severe attenuation of virulence. We therefore wanted to
determine whether the NER-deficient mutants of Ft novicida are attenuated for growth or virulence
in vitro, in macrophages, and in mice. In our previous studies with KBMA Listeria
monocytogenes and Bacillus anthracis, two genes in the NER-pathway were deleted (uvrAB)
because they were adjacent to each other on the chromosome. In Ft the uvrA and uvrB genes are in
separate chromosomal locations, thus we evaluated 3 strains uvrA, uvrB, single mutants and a
uvrA uvrB double mutant.
Optimization of cultivation of Ft novicida. Prior to initiation of the phenotypic characterization
of Ft novicida mutants, in vitro growth medias were evaluated and optimal liquid and solid culture
medias were selected. We determined that Chamberlain’s media supports robust growth of Ft.
novicida with a doubling time of 1h. This rate of growth is equal to or greater than Modified TSB
and Modified MHB and final OD was approximately 3 fold higher than with complex medias.
Chamberlain’s media has numerous advantages: it is optically clear, chemically defined, and
animal-product free. We contracted with a custom media company (Teknova) to prepare 1 Kg of a
dry premixed formulation of Chamberlain’s media to facilitate the ease of preparation and reduce
the batch-to-batch variability of weighing out the many components in this chemically defined
medium. This premixed formulation supported robust growth of Ft novicida. For solid Media:
Evaluation criteria were: maximal cfu, growth rate, ease of colony enumeration, ease of media
formulation and cost. Cystine heart agar plates supplemented with hemoglobin (CHAH) prepared
in-house performed as well as commercially available CHAB (Remel) and were significantly less
expensive and were therefore selected for use for cultivation of Ft novicida strains. The method
for preparing CHAH plates (PRP01 ver.002) is included in the SOP section.
Comparison of growth rates of Ft novicida NER mutants in broth. In order to evaluate
whether the NER mutant strains provided by UTSA were attenuated for virulence it was critical to
evaluate whether the deletions introduced into strains affect growth in general or growth
specifically in animals. As each strain was received from UTSA its growth rate in CDM was
compared to the wild-type strain U112. The uvrA, uvrB, and uvrA uvrB double mutant were
all found to replicate with identical kinetics when compared to U112 (F-1, F-2). These data
demonstrate that deletion of the nucleotide excision repair machinery does not have negative
impact on Ft novicida replication rates in vitro.
Page 5 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 6 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Growth Curve in Chambalain Media
Growth Curve in Chambalain Media
F-1
10
U112
U112
U112
uvrA
U112 uvrA dl
U112 uvrA dl
U112 uvrB dl
uvrB
U112 uvrB dl
1
600
OD600
OD600
OD
10
1
0.1
0.1
0.01
0.01
0
5
0
10
5
15
10
15
hours
Time
hours(h)
20
25
20
25
NB920-p103
F-1. The single mutant strains Ft novicida uvrA and Ft novicida uvrB grow at the same
rate and to the same density as the parental U112 strain in CDM at 37oC. Cultures were
grown overnight in CDM and then diluted 1:100 into 350 mL fresh CDM and incubated at 37oC
with shaking at 300 RPM.
Page 6 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 7 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
F-2
Ft novicida Growth in Chamberlain's Media
1.E+10
Ft novicida Intracellular Growth in J774 Cells
10
1.E+09
cfu/ml
1.E+07
1
OD600
1.E+08
U112
uvrAB
0.1
1.E+06
1.E+05
0.01
1.E+04
0
1.E+03
0
5
10
10
20
Time (h)
15
20
25
30
Time (h)30
NB920-p120
F-2. The double mutant strain of Ft novicida uvrAuvrB grows at the same rate and to the
same density as the parental U112 strain in CDM at 37oC. Cultures were prepared as described
in F-1.
Comparison of growth rates of Ft novicida NER mutants in macrophages. Ft novicida is a
facultative intracellular pathogen that has the ability to replicate in the cytosol of macrophages.
Macrophages have the ability to kill pathogens, and one mechanism by which macrophages are
bacteriacidal is by the induction of damage to nucleic acids by reactive oxygen and nitrogen
species. In order to determine whether the NER-mutant bacteria were more susceptible to
macrophage-mediated killing, we compared the ability of each strain to replicate in the J774
murine macrophage cell line. The strains were found to replicate with identical kinetics when
compared to U112 in the presence or absence of 50g/mL gentamicin which prevents extracellular
replication (F-3, F-6). These data demonstrate that the NER-mutant strains were not compromised
in their ability to replicate intracellularly.
Page 7 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 8 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Ft novicida Intracellular Growth in J774 Cells
F-3
1.E+10
1.E+09
1.E+08
U112
cfu/ml
1.E+07
uvrAB
1.E+06
1.E+05
1.E+04
1.E+03
0
10
20
Time (h)
30
NB920-p121
F-3. The double mutant strain of Ft novicida uvrAuvrB grows at the same rate as the
parental U112 strain in J774 macrophages. J774 macrophages were infected at an MOI of 100
for 1h, washed, and media was replaced with medium containing 50g/mL gentamicin. At
indicated time points, monolayers were lysed by hypotonic shock, diluted, and plated on CHAH
for CFU enumeration.
NB 920 p.75
CFU/ml
F-6
Intracellular Growth of Ft novicida in J774 Macrophages
in the Presence or Absence of Gentamicin
1.E+10
1.E+09
uvrB - Gent
1.E+08
U112 - Gent
1.E+07
1.E+06
uvrB + Gent
1.E+05
U112 + Gent
1.E+04
1.E+03
0.0
10.0
20.0
30.0
40.0
hours post infection
F-6. The uvrB mutant strain of Ft novicida grows at the same rate as the parental U112
strain in J774 macrophages in the presence or absence of gentamicin.
Page 8 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 9 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Virulence determination of Ft novicida NER mutants in mice. We next wanted to determine
whether the nucleotide excision repair mutants were attenuated for virulence in mice. Virulence of
Ft has been demonstrated to be highly route dependent, suggesting that certain tissues represent
barriers to systemic dissemination of the organisms. Thus we compared the virulence of the NERmutant bacteria by three routes of administration: intraperitoneal (IP), intravenous (IV), and
subcutaneous (SC), in decreasing order of virulence.
IP LD50 determination. Since mice are exquisitely sensitive to Ft novicida infection when
challenged by the IP route, we determined the median lethal dose of U112, uvrA, uvrB, and
uvrA uvrB strains in Balb/c mice. Five 5-fold serial dilutions of seed stocks were prepared with
calculated doses expected to be from 50 to 0.08 cfu and was administered in 100ul into the
peritoneal cavity in groups of 4 animals per dose group. CFU were determined empirically by
plating 100ul of the first and second serial dilutions on CHAH plates. The median lethal dose was
then calculated based on the actual cfu enumerated (and were calculated to be from 52 CFU to
0.0016 CFU). For U112, uvrA, uvrB, and uvrAuvrB strains the LD50 was calculated to be
less than 1 CFU (T1). Because it is difficult to ensure that any given animal was actually injected
with an organism when the concentration of bacteria is so low, it does not appear that there are any
significant differences in the median lethal dose of the strains. Furthermore, all animal deaths were
reported within 3 or 4 days after injection of each strain, suggesting that the pathogenic capacity of
each mutant administered IP is the same in mice.
T-1 Median lethal dose of Ft novicida NER-mutant strains by route of administration
IP LD50 (cfu)
IV LD50 (cfu)
SC LD50 (cfu)
SC LD50 (cfu)
SC LD50 (cfu)
U112
0.70
0.95
1.17 x 103
2.10 x 105
5.26 x 103
uvrA
0.82
27
ND
2.43 x 105
3.25 x 104
uvrB
0.20
8.1
1.28 x 105
1.17 x 105
1.25 x 104
uvrA uvrB
.33
38
ND
3.48 x 104
2.46 x 104
Study #*
AS06-090
AS06-112
AS07-014
AS07-025
AS07-039
Serial dilutions of seed stocks were prepared and administered by the indicated route. Median
lethal dose was calculated by the method of Reed and Muench.
*The Study# “AS” is also the location of the primary data in the “Animal Study” folders at Anza/Cerus.
IV LD50 determination. Serial dilutions of frozen seed stocks were prepared for each strain as
described above, and dose verification was performed by CFU analysis. All Ft novicida strains
were highly virulent when administered by the IV route, but LD50 values ranged from 1 for wild
type to 38 cfu for the uvrA uvrB double mutant demonstrating that the NER double mutant may
be a log attenuated compared to the parental strain (T-1). While the wild-type Ft novicida strain
administered IV had an LD50 that was again less than 1 CFU, the animals died between 4 and 7
days after infection, which is longer survival than seen with comparable doses administered IP.
This delay in mortality suggests that IV administration represents a modest barrier to uncontrolled
Page 9 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 10 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
replication of the organisms. While both the uvrA, and uvrA uvrB mutants appear to be more
than 20 fold attenuated compared to the wild type strain when administered IV it is not clear
whether these differences are meaningful, because the numbers of CFU are so low that accurate
determination of virulence is difficult. Furthermore, despite demonstrating modest attenuation,
these mutants are still considered highly virulent in mice.
SC LD50 determination. SC is the route of administration by which LVS is the least virulent
(Green et. al, Vaccine. 2005: 23(20):2680-6); thus, this is the route by which subtle differences in
virulence may best be evaluated. Unfortunately, the LD50 determination with the wild type strain
was somewhat variable with a nearly 200-fold range, from 1.17 x 103 to 2.10 x 105 (T1) but we
believe that the one high value is an outlier because in this experiment one animal per group over a
4 log range (made by 10x serial dilutions of the same stock) died. Thus the animals in this study
did not respond in a dose-dependant manner. One possible explanation is that SC injections may
provide inconsistent virulence based on stochastic factors such as the proximity of the injection site
to a blood vessel. As demonstrated above, if a single wild type bacterium gets into the blood
stream the mouse will die, thus while the virulence should be dependent on the dose of bacteria, it
may also depend on their ability to disseminate based on proximity to the blood vessels or other
factors of which we have less contol. The third LD50 determination (AS07-039) 10-fold serial
dilutions ranging from 1x107 to 10 cfu were administered to 3 animals per group. In all cases the
animals responded in a dose-dependent manner. The LD50 of U112 was 5.26 x 103 which is similar
to the first LD50 determination (1.17 x103) and suggests that the second LD50 data were indeed
spurious. Each of the NER mutants had similar virulence ranging from 1.25 x104 to 3.25 x104.
These data suggest that the abrogation of the NER machinery causes approximately a one log
virulence attenuation when the bacteria are administered SC.
Determination of in vivo growth rates after IV administration. In order to determine whether
the Ft novicida NER mutants were attenuated for growth in vivo, we evaluated the growth rates of
the uvrA, uvrB, and uvrA uvrB mutant strains compared to wild-type Ft novicida U112 in
spleen, liver, and lungs of Balb/c mice that were infected IV. Mice were injected with
approximately 100 cfu (which represents approximately 10-100 LD50). Cohorts of 3 mice were
sacrificed at 4, 24, 48, and 72 hours post infection. At each time point, spleen, liver, and lungs
were harvested and homogenized in HBSS. 100ul of each homogenate were plated directly on
CHAH plates and a series of tenfold dilutions were plated to calculate the number of CFU per
organ (F4). Each symbol represents the mean of three organs and the bars represent the standard
error. All strains were able to replicate by 4-5 logs in livers and spleens within 24 hours, whereas
increases in bacterial burden in lungs primarily increased between 2 and 3 days. While there were
some significant differences in the number of cfu at day 3, all Ft novicida NER mutant strains
replicated at the same rate or more rapidly than the wild-type strain. These data suggest that the
NER machinery is not required for growth in lungs livers or spleens following IV administration,
and that the mutants are not attenuated for grown in vivo.
Page 10 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 11 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
AS07-034
Spleen
F-4
1.0×10
AS07-034
8
F.t.n.
F.t.n.
F.t.n.
F.t.n.
CFU/Organ
1.0×10 7
1.0×10 6
1.0×10 5
U112
uvrA
uvrB
uvrAuvrB
1.0×10 4
1.0×10 3
1.0×10 2
LOD
1.0×10 1
1.0×10 0
0 (4hr)
1
2
3
Day
Lungs
Liver
F.t.n.
F.t.n.
F.t.n.
F.t.n.
CFU/Organ
1.0×10 7
1.0×10 6
1.0×10 5
1.0×10 4
1.0×10
3
1.0×10 2
LOD
1.0×10 1
U112
uvrA
uvrB
uvrAuvrB
F.t.n.
F.t.n.
F.t.n.
F.t.n.
1.0×10 5
CFU/Organ
1.0×10
1.0×10 6
8
1.0×10 4
1.0×10 3
1.0×10 2
LOD
1.0×10 1
1.0×10 0
1.0×10 0
0 (4hr)
1
2
Day
3
0 (4hr)
1
2
3
Day
F-4. In vivo growth rate of NER mutant Ft novicida strains administered IV is similar to wild
type. 100 CFU of U112, uvrA, uvrB, and uvrAuvrB were administered to Balb/c mice via
the tail vein. Animals were euthanized and organs were harvested at 4h, 1d, 2d, and 3d post
infection. Tissues were homogenized, serially diluted, and plated on CHAH plates at 37oC for
CFU enumeration. Determination of in vivo growth rates after SC administration. We also
monitored the growth of Ft novicida uvrB and U112 in lungs, livers, and spleens following SC
infection in order to determine whether the nucleotide excision repair machinery is required for
growth or dissemination to specific organs (F-4). 5x105 CFU of U112 and uvrB were
administered SC (representing ~100x LD50 of U112 and ~10xLD50 of uvrB). Cohorts of 3 animals
were sacrificed at 4, 24, 48 and 72 hours post-infection, and lungs, livers and spleens were
harvested, homogenized and serial dilutions were plated onto CHAH plates for enumeration of
CFU. Both U112 and the uvrB mutant were able to disseminate from the SC injection site near
the base of the tail to the liver within 4 hours and then replicate logarithmically for 48 hours.
Surprisingly, the bacteria were cleared to below the limit of detection in livers and spleens by 72
hours. In both groups one animal was found dead at day 3, whereas the other animals were found
to have no detectable CFU in livers and spleens, and decreased burden in the lungs. This suggests
Page 11 of 20
U112
uvrA
uvrB
uvrAuvrB
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 12 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
that perhaps these animals represent examples of the kinetics of clearance of animals that would
survive lethal challenge. The peak bacterial burden in livers and spleen was indistinguishable
between the wild type and the uvrB mutant, but there appears to be a significant difference in the
ability of the uvrB mutant to disseminate toAS07-045
the lung: no cfu were detectable at 24hours postinfection, and the peak CFU at 48 hours post-infections was approximately one log lower than with
U112. These data suggest that perhaps the reason for the decrease in virulence of the NER mutants
is due to a decreased capacity to disseminate to the lungs following SC injection.
Liver
F-5.
AS07-045
10000000
F.t. n U112
F.t.n. urvB
CFU/organ
1000000
100000
10000
1000
100
limit of dection
10
1
0 (4hr)
1
2
3
Day
* One animal from each group w as found dead on Day 3.
Lungs
Spleen
1000000
10000000
F.t. n U112
F.t.n. urvB
1000000
CFU/organ
CFU/organ
100000
10000
1000
100
limit of dection
10000
1000
100
limit of dection
10
10
.
F.t.n. U112
F.t.n. uvrB
100000
1
1
0 (4hr)
1
2
3
0 (4hr)
1
2
3
Day
Day
* One animal from each group w as found dead on Day 3.
* One animal from each group w as found dead on Day 3.
F-5. In vivo growth rate of Ft novicida uvrB mutant delivered SC is similar to wild type. 5
x 105 CFU of U112 and the uvrB mutant strain were administered to Balb/c mice SC. Animals
were euthanized and organs were harvested at 4h, 1d, 2d, and 3d post infection. Tissues were
homogenized, serially diluted, and plated on CHAH plates at 37oC for CFU enumeration
Page 12 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 13 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
4.2
Interpretation
Together the data presented here suggest that abrogation of the NER-machinery by deletion
of uvrA, uvrB or both uvrA and uvrB genes, does not result in a dramatic virulence
attenuation of Ft novicida strains. Thus, in constructing a KBMA vaccine based on highly
virulent type A strains it would be required to have a secondary attenuation mutation to add a
degree of safety if any bacteria escaped photochemical inactivation.
4.3
Quality Control
Each experiment was documented in a laboratory notebook that was read and understood and
countersigned by an individual who did not participate in the experiment. In all experiments,
the wild type F. novicida strain (U112) was used as a comparative control. After noting that
CDM changes color over time, a new 5 Kg lot (# c071516F601) of CDM was ordered from
Teknova. When the two independent lots of CDM were evaluated they both sustained
equivalent growth of LVS which is more sensitive than F. novicida to growth media (F-7).
Growth of LVS was identical to original lot (#c071110D601). This data suggests that
although changes in CDM occur, they do not alter growth characteristics of LVS growth.
F-7
LVS Growth in CDM
10
OD600
1
Old lot
New lot
0.1
0.01
0
5
10
15
20
Time (h)
NB837-p14
F-7. Two lots of CDM support equivalent growth of U112.
In vitro growth experiments were performed multiple times with single mutants, but were not
repeated with double mutant because the data demonstrated no differences when compared to
wild type. For LD50 experiments 3 or 4 mice per group were used, depending on the
availability of animals and the number of groups used.
Page 13 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 14 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
5
Accepted Date:1/30/09
Deliverables Completed
The deliverable for MS#40 is information and experimental results rather than tangible
reagents or bacterial strains. The deliverable is the MS#40 Milestone completion report
containing the assessment of the degree of attenuation of the Ftn uvrB strain through inv
vivo testing in mice, as compared to the wildtype F novicida U112 strain, including LD50
and difference in time to death.
Deliverable Reagents
Bacterial
Strain
# of
vials
Vial
Concentra
-tion
Storage
media
Date Stored
or Date
Transferred*
Storage location*
Date Stored
or Date
Transferred*
Storage location*
(Institution, room, shelf, etc)
NA
Tissue
identifier
# of
blocks
Tissue
Type
~ Mass
per
block
(Institution, room, shelf, etc)
NA
RNA/DNA
# of
vials
Vial
Concentra
-tion
Storage
media
Date Stored
or Date
Transferred*
Storage location*
#
Concentra
-tion
Storage
media
Date Stored
or Date
Transferred*
Storage location*
(Institution, room, shelf, etc)
NA
Polypeptide
(Institution, room, shelf, etc)
NA
*The storage location should allow a future researcher to specifically find the stored reagent.
When the “storage location” is equal to the creator’s location, enter the “Date Stored” in the “Date
Stored or Date Transferred” column. When the “storage location” indicates that the reagent has
been transferred to another institution, enter the “Date Transferred” in the “Date Stored or Date
Transferred” column.
Page 14 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 15 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
6
Accepted Date:1/30/09
Appendices
6.1
Appendix 1: Original Data Tables and Figures
Table/
Figure1
Title
Notebook
Location2
Electronic Location2
(Full Path & File Name)
(Notebook # and
page numbers)
F-1
The single mutant strains Ft novicida
uvrA and Ft novicida uvrB grow at the
same rate and to the same density as the
parental U112 strain in CDM at 37oC.
NB 920 p.105
Electronic files are all
stored on Jskoble
C:\Documents and
Settings\jskoble\My
Documents\francisella
papers\Data File name:
TULARENSIS GROWTH
CURVE 090606
F-2
The double mutant strain of Ft novicida
uvrA uvrB grows at the same rate and to
the same density as the parental U112
strain in CDM at 37oC
NB 920 p.120
File name :
uvrABJ77410_17_06
F-3
The double mutant strain of Ft novicida
uvrAuvrB grows at the same rate as the
parental U112 strain in J774 macrophages
NB 920 p.121
File name :
uvrABJ77410_17_06
T-1
Median lethal dose of Ft novicida NERmutant strains by route of administration
AS06-090,
AS06-112,
AS07-014,
AS07-025,
AS07-039
File names: AS06-090
Tularemia IP LD50
Summary TG1, Repeat
AS06-112Tularemia IV
LD50 Summary TG,
AS07-014Tularemia SC
LD50 Summary TG1
AS07-025 Tularemia SC
LD50 Summary
Repeat AS07-039
Tularemia SC LD50
Summary TG1
F-4
In vivo growth rate of NER mutant Ft
novicida strains is similar to wild type
AS07-034
File name:
AS07-034
F-5
In vivo growth rate of Ft novicida uvrB
AS07-045
AS07-045 SC
Page 15 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 16 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Table/
Figure1
Title
Notebook
Location2
Accepted Date:1/30/09
Electronic Location2
(Full Path & File Name)
(Notebook # and
page numbers)
biodistribution
mutant delivered SC is similar to wild
type.
T-2
Ft novicida growth on solid media
(See Appendix section 6.3)
NB 937 pp.510
Testing the Suitability of
Different Agar Media for
Growth
F-6
The uvrB mutant strain of Ft novicida
grows at the same rate as the parental
U112 strain in J774 macrophages in the
presence or absence of gentamicin.
NB 920 p.75
NA
F-7
Two lots of CDM support equivalent
growth of U112
NB 837 p. 14
C:\Documents and
Settings\jskoble\My
Documents\francisella
papers\Data
CDM lot comparison with
LVS
Use abbreviation “T” for table and “F” for Figure (e.g. T-1 for table 1 or F-3 for figure 3)
If the data location has changed relative to the location reported in the original monthly
technical report, then provide both the previously reported data location and the final data location
1
2
Page 16 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 17 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
6.2
Accepted Date:1/30/09
Appendix 2: Quality Assessment of Milestone Completion and Report
Assessment Criteria for Milestone Completion
Evaluation of Milestone
Completion Report
Yes No N/A Comment
Has the Milestone Completion Report
format been used and all sections
completed, including Milestone Summary,
Milestone Objectives, Methods Reagents
& SOPs, Salient Original Data Results
Interpretation & Quality Control,
Deliverables Completed, and Appendices?
Does the Milestone Summary include the
milestone’s goals, milestone results, an
overall interpretation of the milestone’s
data and conclusion?
Do Methods, Critical Reagents and SOPs
include summarized methods and details
necessary to re-perform critical
experiments? A list of critical reagents?
The completed table of SOPs?
X
Are salient negative and positive original
data included in the Milestone Completion
Report?
Has the Deliverables Table been
completed?
X
Have the Appendices been completed?
Are the specific original data associations
with experiments clearly annotated in the
“Salient Technical Data” section of the
Milestone Completion report?
X
X
Evaluation of Data included
Are the salient original data and results
included in an organized, easily
interpretable format?
Is the rationale included?
Do tables and figures have legends and
original data location annotations?
Is the data interpretation clear?
Is the data storage location listed in
Appendix 1 sufficient for data retrieval in
X
X
Some SOPs are referenced to publications with
full reference citations so a person could find the
publication and full published SOP; 9 SOPs are
provided, but some are missing sections (e.g.
introduction, calculations/formulas). 3/2/09Freyja Lynn accepted the 9 SOPs in Cerus/Anza
format without adding the few missing sections
NA
No physical deliverables BG added text describing
the MS#40 MSCR as the deliverable for MS#40,
per the SOW in the Cerus subcontract
Yes No N/A Comment
X
X
X
X
X
Page 17 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 18 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Assessment Criteria for Milestone Completion
the future? (E.g. notebook numbers and
pages, electronic file locations including
directory paths and file names). Are prior
data locations cross-referenced to final
data locations?
Is the data backed up electronically or in
hardcopy notebooks?
X
Is the data storage location secured either
in a locked fireproof cabinet for hardcopy
or on a server protected by firewall?
Has the data quality been assessed? How
many replicates and how reproducible is
the data? Has statistical analysis been
performed on the data? What quality
control has been utilized by the
subcontractor during the data generation
and assessment?
X
The notebooks are scanned and PDF files are
stored in \\Anzafs01\terra\CaVaxGeneral\ANZA_Legal\Notebooks
X
If a protein or peptide has been
synthesized, how has the protein or
peptide sequence been verified? What
percentage of the sequences has been
randomly verified?
X
If a genetic mutant has been made, how
has the mutation been verified e.g. DNA
sequencing, PCR sequence verification?
How stable is the mutation? How has the
impact of the genetic mutation on the
bacterial growth been assessed? What is
the sensitivity of the assay?
X
If an aerosol delivery system has been
tested, how reproducible is the delivery to
the animal? Have sufficient animal
numbers been tested to determine
reproducibility?
X
If UVA/psoralen treatment kills the
bacteria but leaves them metabolically
active, how is killing assessed? How
sensitive is the assessment of killing?
How is expression of bacterial epitopes
determined?
X
Do UNM and the subcontractor agree that
the data supports the scientific
interpretation of the milestone?
X
Page 18 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 19 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Assessment Criteria for Milestone Completion
Evaluation of Deliverables, as
outlined in the Statement of
Work
Have Standard Operating Protocols have
been written by subcontractor, reviewed
by UNM, revised by subcontractor as
requested, and accepted by UNM? The
milestone completion report will not be
accepted by UNM until all the SOPs are
accepted by UNM.
Has the Milestone Completion Report
been written by subcontractor, reviewed
by UNM, revised by subcontractor as
requested, and accepted by UNM?
Has data from the milestone been
submitted by the subcontractor, reviewed
by UNM, data presentation revised by the
subcontractor as requested for clarity, and
accepted by UNM?
For deliverable reagents, have the
minimum number of vials, the minimum
concentration, the minimum block size
and the minimum weight of tissue been
mutually agreed by UNM and the
subcontractor?
Have bacterial strains and tissues been
banked at the subcontractor’s institution
and backup stocks and aliquots been
received by UNM for long term storage?
A minimum number of vials of -20C /80C bacterial stocks at specified
concentration in glycerol are stored at
both institutions. A minimum size
paraffin block or minimum weight of
cryopreserved frozen tissues are stored at
both institutions.
Yes No N/A Comment
Many research experiments are not performed
using SOPs; Cerus /Anza SOPs were accepted on
3/2/09
X
X
BG reviewed on 9/25/08 for first time and will
request minor edits from Justin. This report is
VERY well written.
Report revised on 10/24/08 by Justin; BG accepted
JS edits on 10/28/08. SOPs/procedures resolved
and the MSCR is done. BG reviewed again on
1/29/09 and inserted SOP references into the
Methods section. Done 3/2/09
Data has been reviewed 9/25/08 by UNM
X
X
Deliverable is the MS#40 MSCR; deliverable are
not tangible reagents
X
F novicida bacterial stocks were from UTSA and
not from Cerus.
Page 19 of 20
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 40
Institution: Cerus Corp.
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:4/30/07
Report Date: 6/1/08
Version: 1.0
Page 20 of 20
Reviewed by : Barbara Griffith, 9/25/08, 10/28/08,
11/04/08, 1/29/09, 3/2/09
Accepted Date:1/30/09
Assessment Criteria for Milestone Completion
Evaluation of SOPs
Do SOPs contain standard sections e.g.
purpose, list of supplies and equipment
required including vendors and model
numbers, reagent preparation, method,
results expected, description of data
interpretation, criteria for accepting or
rejecting results, description of data
storage location, date SOP is in service,
names of people who prepared and
reviewed the SOP?
Can an independent scientist read and
understand the standard operating
procedure?
6.3
Yes No N/A Comment
X
BG reviewed SOP numbered PRP-0.1 ver.002
9/25/08; Done on 11/4/08; ver. 3 resulted and then
edited ver. 4 on 1/29/09. All 9 SOPs are accepted
by NIAID on 3/2/09
X
BG reviewed SOP numbered PRP-0.1 ver.002
on 9/25/08, ver. 3 on 11/04/08 and ver. 4 on
1/29/09.
Appendix 3: Additional Data/Figures not included in the Text of the Milestone
Completion Report (Section 4)
T-2 Ft novicida growth on solid media
Media
Colony size
at 24h
Cystine Heart w/rabbit blood and
<1 to 1 mm
antibiotics (commercial)
Cystine Heart w/hemoglobin (home-made) >1 mm
Chamberlain’s agar
<0.5 mm
Chamberlain’s agar w/ hemoglobin
<1 mm
VPP w/cystine**
1 mm
VPP w/cystine and hemoglobin
>1 mm
Mueller Hinton w/5% sheep blood
No growth
*TTTC = Too tiny to count Notebook 937, p.5-10.
Colony size
at 48h
3 mm
Log Titer
at 24h
10.07
Log Titer
at 48h
Same
3 mm
2 mm
2 mm
2-3 mm
3 mm
1.5 mm
10.10
TTTC*
10.12
10.11
10.17
NA
Same
10.09
Same
Same
Same
9.68
Page 20 of 20