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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 1 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
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
____9/9/2009
Date Accepted
Page 1 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 2 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
Table of Contents
1
2
3
4
Milestone Summary ...................................................................................................................................................2
Milestone Objectives..................................................................................................................................................3
Methods, Critical Reagents and SOPs........................................................................................................................3
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 ..............................................................................................................................................9
4.3
Quality Control ...........................................................................................................................................9
5 Deliverables Completed .............................................................................................................................................9
6 Appendices ............................................................................................................................................................... 11
6.1
Appendix 1: Original Data Tables and Figures ....................................................................................... 11
6.2
Appendix 2: Quality Assessment of Milestone Completion and Report ................................................. 12
6.3
Appendix 3: Additional Data/Figures not included in the Text of the Milestone Completion Report
(Section 4) .............................................................................................................................................. 14
1
Milestone Summary
The goal of Milestone 44 was to determine the optimal photochemical inactivation conditions to
produce nucleotide excision repair (NER)-deficient killed but metabolically active (KBMA)
Francisella tularensis subspecies holarctica live vaccine strain (LVS) vaccine lots. These KBMA
vaccines were then to be tested in mice to determine whether they were less virulent than live LVS
and sent to UNM for evaluation of protective efficacy against a SCHU S4 challenge.
The sensitivity of uvrB LVS to photochemical inactivation with S-59 psoralen and ultraviolet
light (UVA) was evaluated. The S-59 psoralen concentration required to inactivate uvrB LVS
was the same concentration at which wild-type LVS was inactivated. This result led Cerus to
initiate experiments to determine whether this lack of increased sensitivity was general, or specific
to photochemical inactivation with S-59 and UVA. The uvrB LVS was not more sensitive to
other DNA damaging agents compared to wild-type LVS. These results suggested that there are
redundant and unidentified DNA repair mechanisms in Francisella species that function to repair
nucleotide crosslinks. This resistance to DNA crosslinking suggested that the inactivated uvrB
LVS vaccine would provide no advantage over photochemically inactivated wild-type LVS
vaccine, which had previously failed to protect mice against lethal a SCHU S4 challenge (see MS
46). These findings were consistent with results from NER mutants of Ft novicida (see MS 41 and
MS 42), and led Cerus to reevaluate the KBMA tularemia vaccine strategy. Cerus proposed to
terminate this milestone when only 5% complete, and instead focus efforts on developing liveattenuated and KBMA Listeria monocytogenes vaccines that express Ft antigens. UNM and
NIAID supported the termination of Milestone 44. Cerus focused new efforts on developing liveattenuated and KBMA L. monocytogenes vaccines that expressed Ft antigens in order to induce
and study the cellular immune response in new Milestones 55-59.
Page 2 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 3 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
2
Accepted Date:5/27/09
Milestone Objectives
Milestone 44 will determine the final conditions for psoralen treatment of the F. tularensis LVS
uvrB mutant. An SOP for photochemical inactivation will be delivered to UNM for review,
comments and approval.
The lack of increased sensitivity of ∆uvrB mutants of LVS and F. novicida to photochemical
inactivation suggests that Ft subspecies may have a redundant or alternate DNA repair mechanism
that prevents them from being amenable to formulation as KBMA vaccines. For this reason,
Milestone 44 was terminated when only 5% complete. Though originally planned, MS 44 1) will
not test the attenuation of the psoralen treated KBMA F. tularensis LVS uvrB compared to wild
type LVS in mice 2) will not produce a larger-scale protocol for photochemical inactivation and
3) will not determine the virulence of the KBMA ∆uvrB LVS. NIAID and UNM approved a plan
to change the focus from a KBMA ∆uvrB LVS vaccine to a Listeria monocytogenes-based vaccine
under other milestones.
3
Methods, Critical Reagents and SOPs
Handling of bacteria. Cultivation of Ft holarctica LVS 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
LVS strains, individual colonies or cell bank material 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.
Photochemical inactivation. Inactivation was carried out by following Cerus_Ft_protocol_005.
Briefly, 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. When the
growth rate slowed to ½ the maximal rate (early stationary phase) in Plate A, plate B was
illuminated with 6.5 J/cm2 of UVA light. To determine the degree of inactivation, bacteria from
plate B were distributed on CHAH plates to determine if there were any remaining viable cfu 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 by following QCP-01-003
protocol.
Minimum inhibitory concentration (MIC) assay. To determine the sensitivity of the NER
mutant LVS to alternative sources of DNA damage, 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 µl/well
were transferred to duplicate 96 well plates to which 100 µl/well of diluted overnight cultures of
Page 3 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 4 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
LVS or uvrB LVS were added. The OD600 nm was read at Time 0 and again after incubation at
37oC for 16h without shaking.
List of Critical Reagents
Cystine Heart Agar (Difco)
Bovine Hemoglobin (Becton, Dickinson)
Chamberlains Defined Medium (Teknova cat# C0715)
LVS (DVC lot 16)
∆uvrB LVS (UTSA KKF 303 strain; MS#43)
Amotosalen-HCL (S-59 psoralen) (Cerus)
SOP Number1
PRP01 ver.002
Cerus_Ft_protocol_005
QCP-01-003
SOP Title
Preparation and Quality Control of Bacterial Culture Media
Photochemical Inactivation of Francisella tularensis in a 6-well
Microplate
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. NOTE: Two of above procedures (PRP01 v .002 and QCP01.003) were accepted by UNM and NIAID in association
with Cerus MS 40 MSCRs and are not provided in conjunction with the MS 44 MSCR. Ft 005 is submitted in association
with Cerus MS44 MSCR.
4
Salient Original Data, Results, Interpretation, Quality Control
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location
annotations)
Rationale. Francisella tularensis (Ft) subspecies holarctica live vaccine strain (LVS) was
produced by serial passage and is attenuated for unknown reasons. While it is used as a live
attenuated vaccine to protect laboratory personnel, it’s pathway to FDA licensure is
obstructed due to the uncharacterized nature of the attenuation. The aim of Milestone 44 was
to produce and evaluate a killed but metabolically active (KBMA) vaccine using a
photochemically inactivated nucleotide excision repair (NER)-deficient strain of LVS.
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 S59 to crosslink nucleic acids and prevents the replication of the bacterial chromosome.
Photochemical inactivation is a process used commercially by Cerus for pathogen
Page 4 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 5 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
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 generally are exquisitely
sensitive to the process, and replication can theoretically be prevented with a single crosslink.
Photochemical inactivation with randomly distributed infrequent crosslinks allows NERdeficient 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.
Because LVS is more difficult to genetically manipulate than Ft novicida and required
development of novel tools by UTSA, initial proof of concept studies were done using Ft
novicida NER mutants, with the intent of identifying a single mutant that could then be
developed in LVS. In M S41the ∆uvrB mutant of Ft novicida was found to be equally
sensitive to photochemical inactivation as a ∆uvrA strain or a double mutant strain
∆uvrA/∆uvrB. Thus the ∆uvrB LVS strain was selected for evaluation as a KBMA LVS
vaccine candidate. Because engineering of this strain was expected to take a significant
amount of time, initial conditions for cultivation and inactivation of wild-type LVS were
established in MS 46 and these conditions were replicated for the ∆uvrB mutant in Milestone
44.
Defining photochemical inactivation conditions for uvrB LVS. In MS 46 Cerus
determined that chamberlains defined medium (CDM) was an excellent medium for
cultivation of LVS, and was used as the medium in which photochemical inactivation would
be performed. In MS 41 Cerus 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 strains to photochemical inactivation (Cerus_Ft_protocol_005). To determine
the sensitivity of ∆uvrB LVS to photochemical crosslinking, increasing concentrations of S59 were added to each well of 6 well dishes containing one bacterial strain. The dishes were
illuminated with 6.5 J/cm2 of UVA light and ~1x109 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 and are presented below (F-1). Complete inactivation was achieved at 5µM S-59. This
is the exact same concentration of S-59 at which wild-type LVS was inactivated (MS 46).
This finding is unusual for organisms that are NER-deficient (e.g. ∆uvrAB mutants of L.
monocytogenes and B. anthracis), but is consistent with the observation that Ft novicida NER
mutants were inactivated with similar concentrations of S-59 and were not more
metabolically active than the wild-type strain (MS 41). Photochemical inactivation of LVS
was investigated despite the lack of sensitivity of Ft novicida to confirm that the subspecies
Page 5 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 6 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
would behave in a similar manner. The lack of increased sensitivity of ∆uvrB mutants of
LVS and F. novicida to photochemical inactivation suggests that Ft subspecies may have a
redundant or alternate DNA repair mechanism that prevents them from being amenable to
formulation as KBMA vaccines. For this reason, Milestone 44 was terminated when only
5% complete.
Page 6 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 7 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
F-1
Accepted Date:5/27/09
Photochemical Inactivation of LVS uvrB
NB 963-081
1.E+10
1.E+08
CFU
LVS uvrB
1.E+06
1.E+04
1.E+02
1.E+00
0
1
2
3
4
5
6
7
8
9
10
[S-59] M
F-1. 5 µM S-59 required to kill uvrB mutant LVS. uvrB mutant LVS were grown in 6
well dishes containing 3.5mL CDM and increasing concentrations of S-59. Dishes were then
illuminated with 6.5 J/cm2 of UVA and plated on CHAH plates for cfu enumeration.
Notebook 963, page 081.
Additional investigations into sensitivity of uvrB LVS to DNA damaging agents: To
determine whether the uvrB mutant’s lack of increased sensitivity to DNA crosslinking was
specific to S-59 and UVA treatment or was more general, Cerus initiated experiments to
compare the sensitivity to alternative DNA damaging agents of uvrB LVS with wild-type
LVS. Minimum inhibitory concentration (MIC) assays were performed using the DNA
damaging agents S-303 and cisplatin which had been shown in MS 41 to inhibit the growth of
Ft novicida. Both uvrB LVS clones received from UTSA (clone 1-A and 1-B) were
compared with DVC lot 16 LVS. As with S-59, the uvrB LVS mutants were not more
sensitive to growth inhibition by S-303- or cisplatin-induced DNA damage (F-2, A and B,
respectively). Surprisingly, growth of the wild-type LVS strain appeared to be inhibited to a
greater extent at intermediate concentrations of both S-303 and cisplatin than either of the two
uvrB clones. One possible explanation for this apparent paradoxical increase in resistance
of the uvrB mutants to DNA damaging agents may be that the process of gene deletion
required serial passages that may have selected for clones that replicate more rapidly than the
minimally passaged DVC lot 16 LVS. If this were the case, then the bacteria might achieve
a higher optical density at concentrations that do not completely inhibit growth (as seen
below).
Page 7 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 8 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
F-2
LVS and uvrB sensitivity to S-303
A
OD600 nm
0.45
0.4
0.35
uvrB 1-A T16h
uvrB 1-B T16h
0.3
0.25
LVS 16 h
0.2
0.15
uvrB 1-A T0
LVS T0
uvrB 1-B T0
0.
9
1.
7
3.
4
6.
9
13
.8
27
.5
55
.0
44
0.
0
22
0.
0
11
0.
0
0.1
LVS and uvrB
sensitivity to Cisplatin
uM S-303
uvrB 1-A T16h
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
uvrB 1-B T16h
LVS 16h
LVS T0
uvrB 1-A T0
2.
3
4.
6
9.
3
uvrB 1-B T0
11
85
.0
59
2.
5
29
6.
3
14
8.
1
74
.1
37
.0
18
.5
OD600 nm
B
ug/ml cisplatin
F-2. Minimum inhibitory concentration (MIC) assays with S-303 and cisplatin show
that ∆uvrB mutants of LVS are not more sensitive to DNA damage than LVS. Serial
dilutions of S-303 and cisplatin were made in 96 well dishes and diluted liquid cultures of Ft
strains were then added. The OD600 of the plates was read at t0 and after incubation at 37oC
for 16 hours. Notebook 920, page 198.
Page 8 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 9 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
5
Accepted Date:5/27/09
4.2
Interpretation
The uvrB mutant of LVS was not more sensitive than expanded DVC lot 16 LVS to DNA
damage induced by photochemical inactivation with S-59 and UVA or with chemical DNA
damage inflicted by treatment with S-303 or cisplatin. This suggested that the uvrB LVS
mutant would not be any more effective as a KBMA vaccine candidate than photochemically
treated LVS. Since photochemically inactivated LVS did not protect mice against a lethal
challenge with the Ft tularensis SchuS4 strain in Milestone 46, it seemed unlikely that this
strategy was going to produce an effective vaccine using uvrB LVS. These findings were
also consistent with results from NER mutants of Ft novicida (see MS 41 and MS 42), and
led Cerus to reevaluate the KBMA tularemia vaccine strategy. Cerus proposed to terminate
this milestone when only 5% complete, and instead focus efforts on developing liveattenuated and KBMA Listeria monocytogenes vaccines that express Ft antigens. UNM and
NIAID supported the termination of Milestone 44. Cerus focused new efforts on developing
live-attenuated and KBMA L. monocytogenes vaccines that expressed Ft antigens in order to
induce a cellular immune response in new Milestones 55-59.
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.
Deliverables Completed
The deliverables for Milestone 44 were to be 1) a protocol for treating F. tularensis LVS ∆uvrA or
∆uvrB to produce optimally killed but metabolically active bacteria and 2) provide a report
documenting their virulence compared to F. tularensis LVS challenge.
A protocol for small scale inactivation of LVS ∆uvrB using 5µM S-59 and 6.5J/cm2 UVA is
provided in Cerus_Ft_protocol_005. The milestone was terminated prematurely at approximately
5% completion because of the lack of sensitivity of ∆uvrB LVS to photochemical inactivation, and
hence an inability to be formulated as an effective KBMA vaccine. Since there was an approved
plan to change the focus from a KBMA ∆uvrB LVS vaccine to a Listeria monocytogenes-based
vaccine, a larger-scale protocol for photochemical inactivation was not produced, nor was the
virulence of the KBMA ∆uvrB LVS determined.
Page 9 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 10 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
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 10 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 11 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
6
Accepted Date:5/27/09
Appendices
6.1
Appendix 1: Original Data Tables and Figures (those included in the above
sections 3 and 4)
Notebook
Location2
Electronic Location2 (Full
Table/
Figure1
Title
F-1
5 µM S-59 required to kill uvrB mutant
LVS.
Notebook
963, page 081
963-081 lvs uvrb kill curve:
C:\Documents and
Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\francisella papers\Data
F-2
Minimum inhibitory concentration
(MIC) assays with S-303 and cisplatin
show that ∆uvrB mutants of LVS are not
more sensitive to DNA damage.
Notebook
920, page 198
920-198 DNA damaging agents
killing curve: C:\Documents and
Settings\jskoble.DELL-D630009\My Documents\tularemia
docs\francisella papers\Data
Path & File Name)
(Notebook #
and page
numbers)
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 11 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 12 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
6.2
Accepted Date:5/27/09
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
X
X
X
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?
.BG reviewed protocol
.005 and UNM accepted
it.
X
Both electronic and in
notebooks
Backup copies will be
provided to UNM for
storage by 9/1/09.
X
Page 12 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 13 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
Assessment Criteria for Milestone Completion
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?
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.
X
Yes No N/A Comment
X
X
X
X
X
Page 13 of 14
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 # 44
Institution: Cerus Corporation
Author: Justin Skoble
MS Start Date:6/18/07
MS End Date:6/15/08
Report Date: 5/11/09
Version: 2.0
Page 14 of 14
Reviewed by : Barbara Griffith 5/19/09, 5/27/09,
6/25/09, 8/7/09
Accepted Date:5/27/09
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
X
Appendix 3: Additional Data/Figures NOT included in the Text of the
Milestone Completion Report (Section 3 or 4)
N/A
Page 14 of 14