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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Accepted Date:11/6/08
Version: 1.0
Page 1 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
Signature Page
Author’s Signature:
_Jeff Barker______________________
Typed Name of Author
_Not required______________________
Signature of Author
__________
Date Signed
Acceptance by Subcontracting Institution:
_Karl Klose_______________________
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 11/28/08_____
Freyja Lynn, Interim Project Officer
Not required_______________________ 1/26/09 Heidi Holley CO accepted
Signature of NIAID Interim Project Officer Date Signed
Page 1 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Accepted Date:11/6/08
Version: 1.0
Page 2 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
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 .................................................................................3
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location annotations) .................3
4.2
Interpretation ..............................................................................................................................................4
4.3
Quality Control ...........................................................................................................................................5
5 Deliverables Completed .............................................................................................................................................5
6 Appendices .................................................................................................................................................................7
6.1
Appendix 1: Original Data Tables and Figures .........................................................................................7
6.2
Appendix 2: Quality Assessment of Milestone Completion and Report ...................................................8
6.3
Appendix 3: Additional Data/Figures not included in the Text of the Milestone Completion Report
(Section 4) .............................................................................................................................................. 11
1
Milestone Summary
Milestone 48 examined the ability of the UV repair mutants to replicate in vitro in macrophages as
an indication and mechanism of attenuation and decreased virulence. UTSA successfully
constructed the uvrA, and uvrB mutants of F novicida in Milestone 39. In Milestone 48, UTSA
constructed the uvrA, uvrB double mutants of F novicida. UTSA also infected a macrophage cell
line with each of the 3 uvr mutants of F novicida and determined bacterial survival by counting
colony forming units. The results indicate that insertions in uvrA, uvrB and uvrAB do not affect
ability of U112 Francisella novicida to survive and grow in macrophages. Thus, loss of uvrAB can
be correlated to loss of DNA repair function only and not a loss of virulence, which may be critical
for an effective immune response.
2
Milestone Objectives
UTSA will characterize the uvrA, uvrB mutants in Ft subsp. novicida for intramacrophage growth
and virulence.
3
Methods, Critical Reagents and SOPs
To make a double mutant of UvrA and UvrB in U112, pKEK952 was cut with Bgl2 and BamHI.
FpKan (Kanamycin resistance gene under control of Francisella promoter) was isolated by cutting
pKEK898 with Bgl2 and BamHI. The two fragments, pKEK952 (Bgl2/BamHI) and FpKan
(Bgl2/BamHI), were ligated together. This construct was confirmed by sequence and designated as
pKEK1007.
Page 2 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Accepted Date:11/6/08
Version: 1.0
Page 3 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
pKEK1007 was cryotransformed into KKF71 (uvrB::ermC) and transformants were selected by
Kanamycin resistance. Colony PCR was performed to identify the correct mutants. The PCR
product was also sent for sequencing and the sequence was correct, KKF71 now has a Kanamycin
insertion in uvrA. This double mutant (uvrA, uvrB of F novicida) was designated as KKF100.
U112 Francisella novicida, KKF71 (uvrB), KKF72 (uvrA) and KKF100 (uvrAB) were grown
overnight in TSB + 0.1% cysteine. Overnight cultures were diluted in glycerol and frozen at –80
degrees Celsius. These frozen stocks were used to infect J774 Macrophages, in triplicate (SOP 50
– intramac#1B2CC1.doc). Bacterial survival was determined by comparing the plated CFU (colony
forming units) numbers at 24 hours post infection. The results are shown in Fig. 1 (saved as
uvrAB.jpg).
Critical Reagents include: Kanamycin (Sigma), cysteine (Sigma), J774 macrophage cell line
(ATTC)
SOP Number1
UTSA-50
SOP Title
Intramacrophage Bacterial Analyses
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)
Each uvr mutant as well as the double mutant need to be tested for their ability to grow and
survive in macrophages. Intramacrophage growth is a hallmark of Francisella virulence.
Showing that the uvr genes have no role in macrophage growth and survival is critical for
their potential use as a KMBA vaccine.
Page 3 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Accepted Date:11/6/08
Version: 1.0
Page 4 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
FIG. 1
Figure 1. Growth in J774 macrophages at 24 hours post infection (log10 cfu/ml). Samples
were performed in triplicate, data shown as an average of the three with error bars.
Comparisons were done with the wildtype strain U112. (saved as uvrAB.jpg located in
Jeff’s folder/Graphs on Mac Powerbook G4)
The results indicate that insertions in uvrA, uvrB and uvrAB do not affect ability of U112
Francisella novicida to survive and grow in macrophages.
4.2
Interpretation
uvrAB are not required for intramacrophage growth. This indicates that all the virulence
determinants within Francisella will still be expressed despite the uvr mutation and will be
available for immune processing, an important characteristic of any live vaccine.
Page 4 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Version: 1.0
Page 5 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
4.3
Accepted Date:11/6/08
Quality Control
F novicida mutant constructs made have been verified by DNA sequencing and PCR
verification.
The PCR product of the uvrA,uvrB double mutant was sent for sequencing and the sequence
was correct; KKF71 now has a Kanamycin insertion in uvrA. This double mutant was
designated as KKF100.
The bacterial frozen stocks were used to infect J774 Macrophages, in triplicate, for the CFU
assay
5
Deliverables Completed
Report that includes the data, interpretation and summation of results regarding intramacrophage
growth of F. novicida uvrA or uvrB
The pKEK898 (Francisella promoter controlled kanamycin plasmid); pKEK1007 (UvrA deletion
plasmid with Kanamycin marker); KKF100 (Ft subsp. novicida UvrAB double mutant) have been
stored at UTSA.
Bacterial strain KKF100 (uvrB::ermC; uvrA::Kan in F novicida) is stored at UTSA and at UNM.
Bacterial strains have been banked at UTSA and backup stocks and aliquots have been received by
UNM for long term storage. UNM requested that all bacterial stocks be received as long term
frozen stocks rather than stabs that need to be grown and then frozen.
Deliverable Reagents
Bacterial
Strain
# of
vials
Vial
Concentra
-tion
Storage
media
Date Stored
or Date
Transferred*
Storage location*
UTSA-BSE 3.242, -80
freezer, shelf 5, sleeve 1,
box KKF82UNM- BRF G72
UNM- BRF G72, -80,
E1,E2
KKF100
2
109 cfu/ml
glycerol
12/6/2006
KKF100
KKF100
2
2
109 cfu/ml
109 cfu/ml
Stab
Glycerol
12/6/2006
11/6/2008
(Institution, room, shelf, etc)
Page 5 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Version: 1.0
Page 6 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
Tissue
identifier
# of
blocks
RNA/DNA/Pl
asmids
# of
vials
Tissue
Type
Vial
Concentra
-tion
~ Mass
per
block
Storage
media
Date Stored
or Date
Transferred*
Storage location*
Date Stored
or Date
Transferred*
Storage location*
UTSA- BSE 3.242 -80
freezer, shelf 2, sleeve 1,
box KEK947UTSA- BSE 3.242 -80
freezer, shelf 2, sleeve 1,
box KEK866UNM- BRF G72, 80freezer, bottom shelf,
rack J4, slot 2
UNM- BRF G72, 80freezer, bottom shelf,
rack J4, slot 2
UNM- BRF G72, 80freezer, bottom shelf,
rack J4, slot 2
UNM- BRF G72, 80freezer, bottom shelf,
rack J4, slot 2
pKEK1007
2
109 cfu/ml
in Ecol
12/6/2006
pKEK898
2
109 cfu/ml
in Ecol
12/6/2006
pKEK1007
4
109 cfu/ml
In Ecoli
8/14/08
pKEK898
3
109 cfu/ml
In Ecoli
8/14/08
pKEK1007
1
DNA
midi prep
8/14/08
pKEK898
1
100600ug/ml;
400ul
100600ug/ml;
400ul
DNA
midi prep
8/14/08
Polypeptide
#
Concentra
-tion
Storage
media
Accepted Date:11/6/08
Date Stored
or Date
Transferred*
(Institution, room, shelf, etc)
(Institution, room, shelf, etc)
Storage location*
(Institution, room, shelf, etc)
*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 6 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Version: 1.0
Page 7 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
6
Accepted Date:11/6/08
Appendices
6.1
Appendix 1: Original Data Tables and Figures
Table/
Figure1
F-1
Title
Notebook Location2
(Notebook # and page
numbers)
(saved as uvrAB.jpg).
NA
Electronic
Location2 (Full
Path & File Name)
Jeff’s folder/Graphs
on Mac Powerbook
G4 ( BSE building
room 3.242)
N/A
Sequencing data confirmed ; file name is
“KKF200PCR.seq”
Jirong/Sequencing folder
Power Macintosh
G3( BSE building
room 3.242)
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 7 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Version: 1.0
Page 8 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
6.2
Accepted Date:11/6/08
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?
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
been 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 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?
Is the data storage location secured either in a locked fireproof
X
X
X
X
2 plasmids in e coli were
sent to UNM on
approximately 8/14/08,
for back up storage and 2
were sent on 11/6/08
PKEK1007 and pKEK
898 plasmid DNA midi
preps were also sent
Yes No N/A Comment
X
X
X
X
X
X
X
Electronically
The data is stored only on
Page 8 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Accepted Date:11/6/08
Version: 1.0
Page 9 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
Assessment Criteria for Milestone Completion
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?
a Mac Powerbook , and an
external hard drive but
not behind a firewall.
Computer coding for
MacIntosh is not in the
public domain and thus
MacIntosh computers are
unlikely to be hacked.
Both MacIntoshes are
password protected and
program changes require
the password. 7/25/08 BG
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?
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
X
DNA sequencing and
restriction digests
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
Evaluation of Deliverables, as outlined in the
Statement of Work
Yes No N/A Comment
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
X
UNM received edited
SOP#50 for
intramacrophage growth
on 7/25/08
Page 9 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Accepted Date:11/6/08
Version: 1.0
Page 10 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
Assessment Criteria for Milestone Completion
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
X
X
X
2 plasmids in e coli were
sent to UNM on
approximately 8/14/08,
for back up storage.(
PKEK1007 and pKEK
898 plasmid DNA midi
preps were also sent
NIAID and Rick agreed
that at least 6 vials of each
bacterial stock will be
made. 2 will be kept at
UTSA and 4 will be sent
to UNM. Then at the end
of the contract, UNM will
retain 2 vials at UNM and
send 2 vials to NIAID.
UTSA sent total of 4 vials
of mutant bacterial stocks
as of 11/6/08; Rick
doesn’t want all plasmid
stocks to make the
mutants so 3 vials of
pKEK898 is okay
Evaluation of SOPs
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
UNM received edited
SOP#50 for
intramacrophage growth
on 7/25/08
X
UNM received edited
SOP#50 for
intramacrophage growth
on 7/25/08; NIAID
accepted SOP#50 on
Page 10 of 11
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 # 48
Institution: UTSA
Author: Jeff Barker
MS Start Date:9/1/06
MSEndDate:10/29/06
Report Date: 10/3/07
Accepted Date:11/6/08
Version: 1.0
Page 11 of 11
Reviewed by : Barbara Griffith 11/02/07,4/4/08,
7/3/08, 7/25/08, 8/15/08,9/19/08, 10/30/08, 11/6/08,
11/17/08
Assessment Criteria for Milestone Completion
9/16/08
6.3
Appendix 3: Additional Data/Figures not included in the Text of the Milestone
Completion Report (Section 4)
6.3.1 DNA sequencing original data is electronic only and is an extensive file. This file has not
been included in the text of the Milestone Completion Report and the file is too large to
provide as a separate attachment. The original DNA sequencing data in a file named
KKF100PCR and is located in the Jirong/Sequencing folder on the Power Macintosh
G3 and is backed up on an external hard drive.
6.3.2
Page 11 of 11