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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 1 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
Signature Page
Author’s Signature:
_Justin Skoble_____________________
Typed Name of Author
Acceptance by Subcontracting Institution:
_Suzanne Margerum_______________
Typed Name of Subcontracting PI
Acceptance by the University of New Mexico:
Rick Lyons_______________________
C. Rick Lyons, MD. PhD
Acceptance by NIAID:
_Freyja Lynn______________________
Typed Name of NIAID Project Officer
_Heidi Holley______________________
Typed Name of NIAID Contract Officer
___10/1/2009_
Date Accepted
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 2 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
Table of Contents
1
2
3
4
Milestone Summary ...................................................................................................................................................2
Milestone Objectives..................................................................................................................................................2
Methods, Critical Reagents and SOPs........................................................................................................................3
Salient Original Data, Results, Interpretation, Quality Control .................................................................................5
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location annotations) .................5
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) .............................................................................................................................................. 19
1
Milestone Summary
The goal of this milestone was to develop conditions for production of killed but metabolically
active (KBMA) Francisella tularensis (Ft) subspecies novicida vaccines. KBMA vaccines are
based on nucleotide excision repair (NER)-deficient organisms that are photochemically
inactivated with a synthetic psoralen (S-59) and ultraviolet light (UVA) that crosslink DNA.
Replication of NER-deficient organisms can be prevented with as little as a single DNA crosslink
which allows them to maintain a high degree of metabolic activity and therefore secrete antigens
and induce a robust immune response.
In order to select a NER-deficient background for further development of KBMA tularemia
vaccines, we characterized three Ft novicida mutants constructed by UTSA that should all have
been NER deficient. We determined the level of sensitivity of NER mutants to photochemical
inactivation and measured the degree of metabolic activity of each strain after inactivation. We
optimized photochemical inactivation conditions at a 3.5 mL scale and a 400mL scale and
produced a lot of KBMA uvrB Ft. novicida for potency testing in MS42. We demonstrated that
KBMA Ft. novicida was highly attenuated for virulence. Frozen formulated KBMA Ft. novicida
vaccine lots maintained metabolic activity at –80oC for at least 2 months.
Ft novicida uvrB and uvrA single, and the uvrA uvrB double mutant strains were equally
sensitive to photochemical inactivation, but the conditions required were only slightly less
stringent than those required for inactivation of wild-type Ft. novicida. This lack of dramatically
increased sensitivity resulted in inactivated NER-mutant strains that were not more metabolically
active than photochemically inactivated wild-type Ft. novicida (typically a hallmark of KBMA
vaccines). These results are strikingly different from what has been demonstrated with NER
mutant strains of Listeria monocytogenes or Bacillus anthracis. This lack of sensitivity to
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 3 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
photochemical inactivation was also true for other methods of induced DNA damage. These data
suggested that Francisella tularensis may maintain a redundant DNA repair mechanism that
limits the sensitivity of the NER mutants to DNA damage and thereby limit the metabolic activity
and potency of KBMA Ft novicida strains. Overall these data suggest that Francisella species
may not be suitable as platforms for KBMA vaccines. This milestone was terminated prior to
completion due to the strategic decision to change the vaccine platform from a Francisella-based
tularemia vaccine to a Listeria-based vaccine platform expressing Francisella antigens.
2
Milestone Objectives
Cerus will optimize photochemical treatment conditions and characterize KBMA Ft novicida:
Cerus will determine the amount of S-59 and UVA required to photochemically inactivate various
NER mutants (∆uvrA and/or ∆uvrB), will determine the extent of metabolic activity of NER
mutants after S-59 and UVA inactivation, and will determine the level of virulence attenuation of
KBMA NER mutant strains in mice.
Cerus halted pending work on Milestone 41which was 85% complete. Cerus optimized the
photochemical treatment regimen for Ftn NER mutants and demonstrated that ∆uvrA and ∆uvrB
mutants of Ftn are not substantially more sensitive to photochemical inactivation than wild-type
Ftn.
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 following PRP01.002. For liquid cultivation of Ft novicida
strains, individual colonies were inoculated into Chamberlain’s 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
3.5mL Scale Photochemical Inactivation. 50mL of CDM were inoculated with 1mL of cell
bank material and grown overnight at 37oC. 3.15 mL of CDM containing various concentrations of
S-59 were then added to each well of two 6 well dishes in parallel (plate A and B). 0.35 mL of
overnight culture was added to each well and incubated at 37oC. Plate A was used to monitor the
replication of the bacteria and when the growth rate slowed to ½ the maximal rate (early stationary
phase) and plate B was illuminated with 6.5 J/cm2 of UVA light. Bacteria from plate B were then
distributed on CHAH plates for viable cfu determination using diluted and undiluted culture.
Bacteria that had not been illuminated (from plate A) were used to determine the number of viable
bacteria that had been inactivated.
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 4 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
MTS Metabolic Activity Assay. The metabolic activity of vaccine stocks was measured using the
Cell Titer 96 Aqueous Assay (Promega) according to the manufacturer’s instructions. This assay
measures the reduction of the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) in the presence of the
electron coupling reagent phenazine methosulfate (PMS) into a formazan product. The rate of
formazan production corresponds to both the number of cells and the metabolic activity of the cells
(Leverone, M. R., T. C. Owen, F. S. Tieder, S. G.J., and D. V. Lim. 1996. Resting-cell
dehydrogenase assay measuring a novel water-soluble formazan detects catabolic differences
among cells. Journal of Microbiological Methods 25:49-55). Serial dilutions of KBMA and live
bacteria were prepared using CDM in 96 well plates and 20µl MTS/PMS solution was added. The
absorbance of the formazan was measured at 490nm, every 15 minutes at 37oC in a SPECTRAmax
plus 384 plate reader (Molecular Devices).
400mL Photochemical Inactivation, 100mL of CDM were inoculated with 1mL of cell bank
material and grown overnight at 37oC. The following day, 40 mL of overnight culture was used to
inoculate a 400mL culture containing CDM with 40µM S-59. The culture was incubated until
early stationary phase, then transferred to a PL2410 container and illuminated with 7J/cm2 UVA
light. Illuminated bacteria were transferred to a centrifugation bottle, concentrated by
centrifugation, washed twice with buffer, and resuspended in cryopreservation solution at a
concentration of greater than 1x1010 particles/mL and frozen in aliquots at -80oC. KBMA bacteria
were thawed and distributed on CHAH plates for viable cfu determination using undiluted culture.
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. Animals were allowed
to acclimate to their surroundings for at least 1 week prior to use. Animals were injected IP, IV via
the tail vein, or subcutaneously with 100-200ul 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 Journ Hygiene
27:493). 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. Depending on the study design, 5 to 10 mice may be used per treatment
group.
Minimum inhibitory concentration (MIC) assay. 15 mL of CDM were inoculated with 1mL of
cell bank material and grown overnight at 37oC. In the morning, cultures were diluted 1:100 in
fresh CDM. Serial dilutions of DNA damaging agents were made in 96 well plates and 50 ul/well
were transferred to duplicate 96 well plates to which 100 ul/well of diluted overnight cultures of
U112 or uvrB were added. The OD600 nm was read at Time 0 and again after incubation at 37oC
for 16h without shaking.
List of Critical Reagents
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 5 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
Amotosalen HCl (S-59) (Cerus)
Cystine Heart Agar (Difco)
Bovine Hemoglobin (Becton, Dickinson)
Chamberlains Defined Medium (Teknova cat# C0715)
BALB/c mice (Charles River Laboratories. Wilmington, MA)
∆uvrA Ft novicida (UTSA KKF72 strain; MS#39)
∆uvrB Ft novicida (UTSA KKF71 strain; MS#39)
∆uvrA,∆uvrB Ft novicida double mutant (UTSA KKF100 strain; MS#39)
U112 wild type Ft novicida (UTSA)
Cell Titer 96 Aqueous Assay (Promega)
SOP Number1
Cerus_Ft_protocol_005
Cerus_Ft_protocol_006
Cerus_Ft_protocol_010
SOP Title
Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
MTS Metabolic ActivityAssay
400mL Scale Photochemical Inactivation of Ft novicida ΔuvrB
1
Individual Standard Operating Procedures will be reviewed separately and accepted by the Subcontracting PI and UNM
PI. Ft_protocol _005 was submitted to NIAID in association with the MS 42 MSCR.
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 was to produce a Killed But Metabolically Active (KBMA)
Francisella tularensis (Ft) vaccine. KBMA vaccines are produced by a process of photochemical
inactivation of organisms using a synthetic psoralen (S-59) and ultraviolet light (UVA). The
ultraviolet light activates the S-59 to crosslink nucleic acids and prevents the replication of the
bacterial chromosome. Photochemical inactivation is a process used commercially by Cerus for
pathogen inactivation in plasma and platelet products for transfusion. Nucleotide excision repair
(NER) is the primary mechanism of repair of crosslinked DNA. This NER process is initiated by
the exinuclease complex composed of the products of the uvrA, uvrB, and uvrC genes. While a
broad range of wild-type pathogens can be killed by photochemical inactivation, bacteria that are
engineered to be NER deficient are exquisitely sensitive to the process, and replication can
theoretically be prevented with a single crosslink. Photochemical inactivation with randomly
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 6 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
distributed infrequent crosslinks allows the NER-deficient bacteria to maintain a high degree of
metabolic activity while preventing replication leading to a state we refer to as KBMA. This
KBMA vaccine concept has been demonstrated with uvrAB mutants of Listeria monocytogenes,
Bacillus anthracis, and Salmonella typhimurium. With all three species, the NER- mutant bacteria
were significantly more sensitive to photochemical inactivation, maintained a higher degree of
metabolic activity, and a broader expression profile when compared to wild type organisms.
Genetic manipulation of LVS and Ft tularensis strains is difficult and slow, the aim of this
milestone was to demonstrate proof-of-concept that KBMA Francisella vaccines could be
produced using Ft novicida as a model. One difference between Ft novicida and the other
organisms that have been turned into successful KBMA vaccines is that the uvrA and uvrB genes
are in separate loci on the Ft chromosome as opposed to being located within an operon. Thus,
separate uvrA and uvrB gene deletions were produced in the laboratory of Karl Klose at UTSA.
Ultimately a double mutant, containing both ∆uvrA and ∆uvrB alleles, was made. The rationale
was that the mutants would be evaluated to determine the level of sensitivity to photochemical
inactivation and the degree of metabolic activity after inactivation, and then one genotype would
be selected for formulation as a KBMA vaccine and used to demonstrate proof of concept in the
mouse model. The selected genotype would then be recapitulated in LVS or perhaps an attenuated
type A Ft tularensis strain for formulation as a KBMA human vaccine in later milestones.
Development of small-scale photochemical inactivation conditions for Ft novicida. In
Milestone 40 we determined that chamberlains defined medium (CDM) was an excellent medium
for cultivation of Ft, and was used as the medium in which photochemical inactivation would be
performed. We developed a small-scale photochemical inactivation process using 6 well dishes
containing 3.5ml of culture in each well in order to evaluate the sensitivity of the wild-type and
mutant strains to photochemical inactivation (Cerus_Ft_protocol_005). As the mutants were
delivered sequentially to Cerus by UTSA, Cerus optimized the assay with the ∆uvrB mutant and
then repeated the assays with all three mutants once we were in possession of them.
To determine whether the NER-deficient strains were more susceptible than the parental U112
strain to photochemical inactivation, two independent experiments were performed in which
increasing concentrations of S-59 were added to each well of 6 well dishes containing one bacterial
strain. The dishes were illuminated with 4J/cm2 of UVA light and 1x1010 bacteria were plated onto
5 CHAH plates and incubated for 2-3 days to allow for colonies to form. Cfu were enumerated
and the data were averaged and are presented below (F-1). In both experiments the wild type
strain U112 was more resistant to S-59 and UVA killing than all of the NER mutants. The
minimum S-59 concentration required to inactivate ~1 x 1010 cfu of U112 was 40M while 20 M
S-59 was required for inactivation of uvrA, uvrB, and uvrAuvrB mutant strains of Ft.
novicida. These data demonstrate that that the NER mutants are more sensitive than the parental
strain of Ft novicida (albeit modestly), and that the phenotype of each mutant is identical, therefore
these is no advantage of having a double mutant. However, it is important to note that the
concentration of S-59 required for inactivation is 400 and 200 times higher than required for
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 7 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
inactivation of B. anthracis or L. monocytogenes NER mutants (requiring 50nM and 100nM
respectively). Another difference between Ft novicida and these other bacteria is that the
difference between S-59 concentrations required to inactivate wild type and mutant was only 2fold with Ft novicida but was 20-fold with both B. anthracis and L. monocytogenes. These data
suggest that while the F. novicida NER mutants were indeed slightly more sensitive to
photochemical inactivation than the wild-type strain, the degree of sensitivity was not on the scale
typically achieved for KBMA vaccines in other bacteria.
F-1
Photochemical Inactivation of Ft novicida Strains
1.E+10
1.E+08
cfu/ml
U112
uvrA
1.E+06
uvrB
1.E+04
uvrAuvrB
1.E+02
1.E+00
0
10
20
[S-59] uM
30
40
F-1. Titration of S-59 required to kill 1x1010 cfu Ft novicida strains. Strains were grown in 6
well dishes containing 3.5mL CDM and increasing concentrations of S-59. Dishes were then
illuminated with 4J/cm2 of UVA and plated for cfu enumeration on CHAH plates. NB963-010
Measurement of Ft novicida metabolic activity after photochemical inactivation. After
establishing the conditions under which Ft novicida strains were unable to replicate, we next
compared the metabolic activity of each strain after photochemical inactivation. For these assays
the 3.5 mL scale inactivation process was performed using twice the minimum concentration of S59 required for complete inactivation (this increase is typically done for production processes) and
4J/cm2 UVA. The bacteria were either photochemically treated (KBMA) or untreated (live) and
serially diluted in 96 well microtiter plates and (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent was added
(Cerus_Ft_protocol_006). Plates were incubated at 37oC and the ability to reduce the MTS reagent
to a formazan was used as a measure of metabolic activity every 15 minutes for 12 hours (F-2).
Each strain had less metabolic activity when it was inactivated when compared to untreated. All 4
inactivated strains had similar metabolic activity that persisted for at least 12 hours. The
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 8 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
inactivated wild-type strain U112 (which was treated with 80uM S-59) had slightly less activity
than the NER mutants that were inactivated with 40uM S-59. The KBMA uvrB strain had
slightly more metabolic activity than uvrA, and uvrA uvrB strains. These data demonstrate
that there is no advantage to having both uvrA and uvrB genes knocked out. After producing these
data, we selected the uvrB strain for further evaluation.
It is important to note that the difference in metabolic activity between the inactivated wild-type
strain and the NER mutants was not nearly as extensive as was observed with L. monocytogenes or
B. anthracis. This lack of increased metabolic activity with the NER mutants could have been
attributable to 1) an inactive NER pathway, thus allowing all 4 strains to have the same degree of
metabolic activity, or 2) a redundant repair mechanism requiring a high degree of crosslinking to
overwhelm the repair systems whether the NER genes were intact or not. If scenario 1 was the
case then a KBMA Ft novicida vaccine should perform well compared to a heat killed vaccine, if
scenario 2 was the case then a KBMA Ft novicida would not be expected to perform well. Thus we
opted to continue with the plan of increasing the scale of the inactivation process in order to
produce a vaccine lot of suitable size for testing in the mouse model in Milestone 42.
F- 2
Metabolic Activity of KBMA vs. Live Ft novicida Strains
2.0
1.8
1.6
Ftn wt Live
Ftn uvrA Live
Ftn uvrB Live
Ftn uvrAB Live
Ftn wt KBMA (80uM S59, 4J)
Ftn uvrA KBMA (40uM S59, 4J)
Ftn uvrB KBMA (40uM S59, 4J)
Ftn uvrAB KBMA (40uM S59, 4J)
OD490
1.4
1.2
1.0
0.8
0.6
0.4
0.2
NB 963-054
0.0
0
2
4
6
Time (h)
8
10
12
F-2 Metabolic activity of photochemically inactivated Ft novicida strains. Photochemically
treated or untreated Ft novicida strains were incubated with MTS reagent at 30oC and the ability to
reduce the reagent to a formazan was monitored in a spectrophotometer at 490 nm every 15
minutes for 12 hours. NB 963-054.
Development of 400mL scale inactivation conditions for Ft. novicida. In order to produce a lot
of KBMA Ft novicida of suitable titer for stability and potency testing in mice, we increased the
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 9 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
scale of the inactivation process from 3.5 mL to 400mL. This required alteration of the UVtransparent vessel from a 6 well dish to a PL2410 bag used for inactivation of pathogens in plasma.
We initially attempted to use 2 and 4 times the minimum S-59 (40M and 80M) concentration
required in the small scale process for inactivation of Ftn uvrB. Neither of these lots were
completely inactivated after illumination with 4J/cm2 UVA (T-1, lots 948-136 Arm-1 and Arm-2).
Since neither lot was sterile, and the concentration of S-59 was higher than used for the small-scale
process, we next optimized the scaled-up inactivation process by titrating the UVA dose required
to achieve complete inactivation. Ftn uvrB was grown in CDM in shaker flasks to early
stationary phase in the presence of 40 or 120M S-59. 400mL of culture was transferred to a UVtransparent container and illuminated with 2, 4, 6, 8 J/cm2 of UVA. After each UVA exposure
1mL of bacterial suspension was plated for cfu on CHAH plates. The results are presented in T-1.
Complete inactivation of the uvrB strain was achieved after illumination with 6 J/cm2 UVA at
both S-59 concentrations. This UVA dose is comparable to what is used for making lots of KBMA
L. monocytogenes and B. anthracis. These results demonstrated that complete inactivation of up to
1x109 cfu/mL of the uvrB strain can be achieved with 40M S-59 and 6 J/cm2 at the 400ml scale.
These conditions were then used to produce a 400ml lot of KBMA Ft novicida uvrB . The
bacteria were then concentrated by centrifugation and formulated in cryopreservation solution for
storage at -80oC
T-1 Optimization of photochemical inactivation conditions at the 400mL scale
400ml scale photochemical inactivation results
Lot number
[S-59]
UVA
Particles/mL
948-136 Arm-1
4J/CM2 4.8 x1010
40M
948-136 Arm-2
4J/CM2 4.84 x1010
80M
948-155 Arm-1
2J/CM2 1 x109
40M
948-155 Arm-1
4J/CM2 1 x109
40M
948-155 Arm-1
6J/CM2 1 x109
40M
948-155 Arm-1
8J/CM2 1 x109
40M
948-155 Arm-2
2J/CM2 1 x109
120M
948-155 Arm-2
4J/CM2 1 x109
120M
948-155 Arm-2
6J/CM2 1 x109
120M
948-155 Arm-2
8J/CM2 1 x109
120M
948-164
6J/cm2 3.9 x 1010
40M
948-202 arm-1
6J/cm2 5.4 x 1010
40M
948-202 arm-2
7J/cm2 5.12 x 1010
40M
cfu/mL
3
13
TMTC
18
0
0
TMTC
1
0
0
1
91
0
Production of 400mL scale KBMA lot of KBMA Ft novicida uvrB for potency testing in
Milestone 42. (NB 948 pages 136, 155, 164, 202) We then produced a 400mL scale lot of KBMA
Ft novicida uvrB using the conditions identified above. Briefly, overnight starter cultures were
diluted 1:10 in fresh CDM media containing 40µM S-59. The bacteria were incubated at 37oC in
disposable shaker flasks until they reached early stationary phase. 400mL of culture was
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 10 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
transferred to a PL2410 bag and illuminated with 6J/cm2 UVA and then washed twice and
suspended in cryopreservation solution at a final OD600 corresponding to approximately 5x1010
cfu/mL. The KBMA lot was then tested for sterility by plating 1mL on 5 CHAH plates. However,
we found incomplete inactivation using these conditions. This is likely due to the fact that the
concentration of bacteria was increased by approximately 15-fold for freezing and storage, and the
limits of detection were increased concomitantly. After 2 lots were found to be unsterile using
these conditions, a third lot (948-202 arm 2) was produced using 7J/cm2 UVA and was found to be
free of live cfu (T-1). The metabolic activity of the sterile lot (948-202 arm-2) was compared to
the unsterile lot 948-164 (which was produced with 6 J/cm2 UVA and as a result had 1 cfu per 3.9
x 1010 particles) (F-3). These lots had similar degrees of metabolic activity, thus this change in
UVA dose from 6J/cm2 to 7J/cm2 did not adversely affect metabolic activity. The final conditions
for photochemical inactivation of Ft novicida uvrB at a 400mL scale were thus established at
40uM S-59 and 7J/cm2 UVA and are included in the final protocol (Cerus_Ft_protocol_010).
F-3
400mL KBMA F. tularensis novicida delta uvrB (Nominal 1.5e8 particle/mL)
Lot 948-164 (40uM S-59, 6J/cm2 UVA) vs. Lot 948-202 Arm-2 (40uM S-59, 7J/cm2 UVA).
1.40
Lot 948-164, T=2 Month, Run date: 02-23-07
OD (490nm)
1.20
Lot 948-202 Arm-2, T=1 Month, Run date: 02-23-07"
1.00
0.80
0.60
0.40
0.20
0.00
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (hours)
F-3 Comparison of KBMA vaccine lots of Ft novicida uvrB produced with 6 or 7 J/cm2
UVA. The MTS assay was performed with lot 948-164 which was inactivated with 6J/cm2 UVA
and lot 948-202 Arm-2 which was inactivated with 7J/cm2 UVA. These assays were performed at
the same time and thus used material that was either 2 months or 1 month old, respectively. The
curves appear very similar in shape suggesting that the increase in UVA dose does not negatively
impact the metabolic activity. NB968-050.
Stability of KBMA Ft novicida uvrB vaccine after storage at -80oC. We next wanted to
determine whether KBMA Ft novicida vaccine lots maintained their metabolic activity during
storage at –80o C in cryopreservation solution containing 10% sucrose. This solution was found to
maintain the viability of frozen stocks of LVS at -80oC in Milestone 46. To measure stability of
KBMA vaccines, we monitored the metabolic activity of the vaccine lots using the MTS assay.
The metabolic activity of Lots 948-164 and 948-202 were measured over a 2 month period. The
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 11 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
stability data for KBMA Ft novicida uvrB lot#948-202 arm-2 is presented in F-4 and for 948164 is presented in appendix 3 (section 6.3.1). For both lots, metabolic activity after 2 months of
storage was essentially equivalent to the initial metabolic activity immediately after storage. For
lot#948-202 arm-2 there appeared to be a slight drop in metabolic activity after the first month of
storage, but this was likely due to experimental variability as the 2 month curve was essentially
overlapping with the 2 week curve. For lot 948-164 the metabolic activity curve after 1 month of
storage was overlapping with curve generated prior to freezing. These data suggest that 10%
sucrose is an appropriate cryopreservation agent and that KBMA Ft novicida vaccine stocks can be
stably stored for 2 months without loss in metabolic activity.
F-4
OD (490nm)
Lot 948-202 Arm-2 (Nominal 1.93e8 cfu/mL)
1.90
1.70
1.50
1.30
1.10
0.90
0.70
0.50
0.30
0.10
-0.10
T=2 weeks
T=1 Month
T=2 Months
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (hours)
F-4 Stability of metabolic activity of KBMA lot 948-202 Arm-2 over time. The metabolic
activity of lot 948-202 was determined using the MTS assay after storage for 2 weeks (blue), 1
month (pink), or 2 months (yellow) in 10% sucrose cryo-preservation media at -80oC . The assays
were performed on serially diluted samples of bacteria and the dilution corresponding to 1.93x108
cfu/mL are presented. Results from samples run at different times were then plotted on the same
graph. NB968-072.
Demonstration that KBMA Ft novicida is attenuated for virulence To determine whether
KBMA Ft novicida uvrB was attenuated for virulence, doses of 1x109 and 1x108 particles of
KBMA Ft novicida uvrB lot 948-164 were administered IP, IV, and SC to BALB/c mice and
compared to heat-killed Ft novicida uvrB administered IP. The KBMA vaccine was avirulent at
both doses when administered SC. The KBMA vaccine was more virulent than heat killed Ft
novicida when administered IP in that at the 1x109 dose only 40% of the mice survived compared to
100% survival with heat killed bacteria. The KBMA vaccine was most virulent when administered
IV: all the mice in the 1e9 group died and only 40% of the 1x108 group survived. Despite the fact
that lot 948-164 had 1 cfu per 3.9 x1010 (hence each animal would have received less than 1 cfu),
we believe that the toxicity of the KBMA vaccine in these studies is most likely due to the host
response to a high number of non-replicating organisms introduced systemically rather than an
Page 11 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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 12 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
infection caused by a rare residual live organism. This is consistent with the finding that all of the
animal deaths occurred within the first 24 hours after administration and the time to death following
live Ft novicida infection is usually after 3 days. The LD50 for live uvrB ranges from less than 1
cfu when administered IP to 125,000 cfu when administered SC. Thus, using the IP route of
administration the KBMA uvrB bacteria are attenuated by greater than 8 logs. The data generated
here were used to calculate the doses of Lot#948-202 arm-2 that were used in MS 42 to determine
whether KBMA vaccines protect against lethal Ft novicida challenge in mice.
T-2 Reduced virulence of KBMA Ft novicida ∆uvrB
Route
Live uvrB
KBMA uvrB
Heat Killed uvrB
SC
1.25 x 104
>1 x 109
Calculated LD50
IV
IP
8.1 x 100 2.0 x 10-1
<1 x 108
6.8 x 108
>1 x 109
Study #
Reported in MSCR 40
AS07-009
AS07-009
Serial dilutions of KBMA Ft novicida ∆uvrB lot 948-164 were prepared and administered by the
indicated route. Median lethal dose was calculated by the method of Reed and Muench.
Additional investigations into sensitivity to DNA damaging agents: Because the
photochemically inactivated NER-deficient strains had a similar sensitivity to photochemical
inactivation and equivalent metabolic activity as the wild-type Ft. novicida strain after inactivation
(which is different than has been seen with L. monocytogenes or B. anthracis), we initiated a series
of experiments to determine whether this observation was specific to photochemical inactivation
with S-59 and UVA or was generally translatable to other sources of DNA damage. We evaluated
the sensitivity of the uvrB mutant and U112 to 6 alternative DNA damaging agents: S-303 (a
nitrogen mustard crosslinking agent that is not activated with UV-light), mitomycin C, cisplatin,
doxorubicin hydrochloride, benzo[a]pyrene, and 4 nitroquinoline-N-oxide using a 96-well format
minimum inhibitory concentration (MIC) assay. Briefly, U112 and uvrB mutant Ft. novicida
were incubated overnight in 15 ml CDM. In the morning, cultures were diluted 1:100 in fresh
CDM. Serial dilutions of chemicals were made in 96 well plates and 50 ul/well were transferred to
duplicate 96 well plates to which 100 ul/well of diluted overnight cultures of U112 or uvrB were
added. The OD600 nm was read at Time 0 and again after incubation at 37oC for 16h without
shaking. Doxorubicin hydrochloride and benzo[a]pyrene did not inhibit growth of the bacteria
while the other 4 DNA damaging agents inhibited growth of the bacteria (Data not shown). Of the
4 agents that inhibited growth of Ft novicida, only 2 (S-303 and 4 nitroquinoline-N-oxide)
inhibited growth of the uvrB mutant at slightly lower concentrations than the wild type strain.
As seen in F-4, the growth of U112 and the uvrB mutant Ft novicida strains in the presence of
cisplatin were indistinguishable but there was a minor difference in the ability of the uvrB mutant
to replicate in the presence of S-303. However, the concentration at which growth was prevented
(1,100uM) was identical between the strains. The fact that there were either no or only minor
differences in sensitivity to DNA damage between the uvrB mutant and wild type Ft novicida
demonstrates that this phenomenon is not unique to photochemical inactivation with S-59 and
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 13 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
UVA and suggests that there is a redundant mechanism for repairing or preventing DNA damage
in Ft novicida.
F-5
Ftn sensitivity to DNA damage by S-303
0.7
0.7
0.6
U112 T16h
uvrB T16h
0.3
0.2
uvrB T0
U112 T0
U112 T16h
0.6
OD600 nm
0.5
0.4
0.5
uvrB T16h
0.4
U112 T0
0.3
0.2
0.1
uvrB T0
Cisplatin ug/ml
0.
9
1.
7
3.
4
6.
9
44
0.
0
22
0.
0
11
0.
0
55
.0
27
.5
13
.8
2.
3
4.
6
9.
3
0.1
11
85
.0
59
2.
5
29
6.
3
14
8.
1
74
.1
37
.0
18
.5
OD600 nm
Ftn sensitivity to cisplatin
S-303 uM
F-5 Sensitivity of Ft novicida wild type and ∆uvrB strains to DNA damage induced by S-303
and cisplatin. Serial dilutions of DNA damaging agents were performed in microtiter plates and
Ft novicida strains were added. The OD600 was measured at t0 and after 16 hours of growth at
37oC. NB920-189.
4.2 Interpretation
Conditions were established for small and medium–scale photochemical inactivation of uvrB Ft
novicida and this vaccine had metabolic activity that was stable after freezing for at least two
months. KBMA uvrB were dramatically attenuated for virulence. However, abrogation of the
NER-machinery by deletion of uvrA, uvrB or both uvrA and uvrB genes in Ft novicida resulted
only in a slight increase in sensitivity to photochemical inactivation with S-59 psoralen and UVA.
This modest increase in sensitivity was the same for all three mutant strains, and did not result in
metabolic activity that was higher than photochemically inactivated wild-type Ft novicida. The
NER mutant strains were not significantly more sensitive to other forms of DNA damage either.
Together, these data suggest that Ft may be fundamentally different than other bacterial strains that
have been used to make KBMA vaccines in that they may have a redundant or alternative
mechanism for repair of DNA crosslinks. It is likely that KBMA Ft may not have sufficient
metabolic activity to perform well as a vaccine.
4.3
Quality Control
 Each experiment was documented in a laboratory notebook that was read, understood and
countersigned by an individual who did not participate in the experiment.
 All inactivation comparisons and metabolic activity assays were repeated at least twice.
 Wild type F novicida was used as a control strain in each photochemical inactivation or
metabolic activity experiment
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 14 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
5
Accepted Date:7/7/09
Deliverables Completed
From Statement of Work: Protocol for treating F. novicida uvr A or uvrB to produce optimally
killed but metabolically active bacteria MS 41.
- Cerus_Ft_protocol_010 is provided as an attachment
- The final conditions for photochemical inactivation of Ft novicida uvrB at a 400mL scale
were established at 40uM S-59 and 7J/cm2 UVA and are included in the final protocol
Cerus_Ft_protocol_010
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)
N/A
Tissue
identifier
# of
blocks
Tissue
Type
~ Mass
per
block
(Institution, room, shelf, etc)
N/A
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)
N/A
Polypeptide
(Institution, room, shelf, etc)
N/A
*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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 15 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
6
Accepted Date:7/7/09
Appendices
6.1
Appendix 1: Original Data Tables and Figures (those included in the above
sections 3 and 4)
Table/
Figure1
Title
Notebook
Location2
Electronic Location2 (Full Path & File
Name)
(Notebook # and
page numbers)
F-1
Titration of S-59 required to kill
1x1010 cfu Ft novicida strains
NB963 p.10
File name: 963-010 data. C:\Documents
and Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\francisella papers\Data
C drive is located on Justin Skoble laptop
PC. All data are backed up on to a
second hard drive.
F-2
Metabolic activity of
photochemically inactivated Ft
novicida strains
NB963 p.54
File name 963-054 data JS.
C:\Documents and
Settings\jskoble.DELL-D630-009\My
Documents\tularemia docs\francisella
papers\Data
C drive is located on Justin Skoble laptop
PC. All data are backed up on to a
second hard drive.
T-1
Optimization of photochemical
inactivation conditions at the
400mL scale
NB 948 p.136,
p155, p164,
p202
File name: Notebook 948. C:\Documents
and Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\francisella papers\Contract reporting
info\notebooks
Whole scanned Notebook is located on
Justin Skoble’ laptop PC. All data backed
up on to a second hard drive.
F-3
Comparison of KBMA vaccine lots
of Ft novicida uvrB produced with
6 or 7 J/cm2 UVA.
NB968 p.50
File name: Notebook 968. C:\Documents
and Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\francisella papers\Contract reporting
info\notebooks
Whole scanned Notebook is located on
Justin Skoble’ laptop PC. All data backed
up on to a second hard drive.
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 16 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Table/
Figure1
Title
Notebook
Location2
Accepted Date:7/7/09
Electronic Location2 (Full Path & File
Name)
(Notebook # and
page numbers)
F-4
Stability of metabolic activity of
KBMA lot 948-202 Arm-2 over
time.
NB968 p.72
File name: Notebook 968. C:\Documents
and Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\francisella papers\Contract reporting
info\notebooks
Whole scanned Notebook is located on
Justin Skoble’ laptop PC. All data backed
up on to a second hard drive.
T-2
Reduced virulence of KBMA Ft
novicida ∆uvrB
AS07-009
File name: AS07-009 KBMA Ft LD50
and Protection SummaryJS1.
C:\Documents and
Settings\jskoble.DELL-D630-009\My
Documents\tularemia docs\animal data
F-5
Sensitivity of Ft novicida wild type
and ∆uvrB strains to DNA damage
induced by S-303 and cisplatin
NB920 p.189
File name: 920-198 DNA damaging
agents killing curve
C:\Documents and
Settings\jskoble.DELL-D630-009\My
Documents\tularemia docs\francisella
papers\Data
C drive is located on Justin Skoble laptop
PC. All data are backed up on to a
second hard drive.
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 17 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
6.2
Accepted Date:7/7/09
Appendix 2: Quality Assessment of Milestone Completion and Report
Assessment Criteria for Milestone Completion
Evaluation of Milestone Completion Report
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?
Are salient negative and positive original data included in the
Milestone Completion Report?
Has the Deliverables Table been completed?
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?
Evaluation of Data included
Yes No N/A Comment
X
X
X
X
X
X
X
Yes No N/A Comment
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 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?
X
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?
X
X
X
X
X
C drive is located on
Justin Skoble laptop PC.
All data are backed up on
to a second hard drive.
Whole scanned Notebook
is located on Justin
Skoble’ laptop PC. All
data backed up on to a
second hard drive.
Notebook, scanned
notebooks, and electronic
files are maintained by
Justin Skoble
The files are no longer
stored in a secure location
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 18 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
Assessment Criteria for Milestone Completion
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
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?
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?
Do UNM and the subcontractor agree that the data supports the
scientific interpretation of the milestone?
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?
due to the closure of
Anza, but electronic
copies will be provided to
UNM for storage.
No statistical analysis was
performed.
X
X
X
Yes No N/A Comment
X
X
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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
Page 19 of 20
Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
Assessment Criteria for Milestone Completion
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.
Evaluation of SOPs
X
Yes No N/A Comment
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?
X
X
SOP 005 was accepted by
UNM in association with
MS 42 MSCR. SOP 010
and SOP 006 were
accepted on 7/7/09.
Appendix 3: Additional Data/Figures NOT included in the Text of the
Milestone Completion Report (Section 3 or 4)
6.3.1
Stability metabolic activity of KBMA lot948-164 over a 2 month period.
Lot 948-164 (Nominal 1.5e8 particles/mL)
1.40
Pre-Freeze
t=0
t=1 Month
t= 2 Month
1.20
OD (490nm)
6.3
Minimum number of vials
to UNM is 4; Minimum #
of vials at Cerus is 2.
X
1.00
0.80
0.60
0.40
0.20
0.00
0
1
2
3
4
5
6
7
Time (hours)
8
9
10
11
12
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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 # 41
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:3/2/06
MS End Date:6/15/08
Report Date: 4/17/09
Version: 2.0
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Reviewed by : Barbara Griffith 4/20/09, 6/29/09,
7/2/09, 7/7/09, 9/24/09, 9/25/09, 10/1/09
Accepted Date:7/7/09
6.3.1 Stability of metabolic activity of KBMA lot 948-164 over time. The metabolic activity of
lot 948-164 was determined using the MTS assay before freezing, after storage for 1 day (pink), 1
month (yellow), or 2 months (blue) in 10% sucrose cryo-preservation media at -80oC. The assays
were performed on serially diluted samples of bacteria and the dilution corresponding to 1.5 x108
cfu/mL is presented. Results from samples run at the different time points were then plotted on the
same graph. Notebook 968-050
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