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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 1 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
Author’s Signature:
Kathryn Sykes____________________
Typed Name of Author
Acceptance by Subcontracting Institution:
Kathryn Sykes, Ph.D__________________
Typed Name of Subcontracting PI
Acceptance by the University of New Mexico:
________________________________
C. Rick Lyons, MD. PhD
Acceptance by NIAID:
_Freyja Lynn__Reviewed on 3/26/09____
Typed Name of NIAID Project Officer
Heidi Holley__________________
Typed Name of NIAID Contract Officer
_3/26/2009__
Date Accepted
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 2 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
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 ................................................................................. 5
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location annotations) ................. 5
4.2
Interpretation .............................................................................................................................................. 7
4.3
Quality Control ........................................................................................................................................... 7
5 Deliverables Completed ............................................................................................................................................. 9
6 Appendices............................................................................................................................................................... 12
6.1
Appendix 1: Original Data Tables and Figures inserted in Section 4 ...................................................... 12
6.2
Appendix 2: Quality Assessment of Milestone Completion and Report ................................................. 13
6.3
Appendix 3: Additional Data/Figures not inserted into the Text of the Milestone Completion
Report (Section 4) .................................................................................................................................. 18
1
Milestone Summary
Arizona State University (ASU) is responsible for the generation of polypeptides that
collectively constitute the entire proteome of SCHU S4 F. tularensis. The goal of MS25
was to perform the necessary bioinformatic analyses on the SCHU S4 annotated sequence
database to enable the design of open-reading-frames (ORFs) for optimal performance as in
vitro transcription/translation (IVT) reaction templates, and for their encoded products to
optimally stimulate T cells. The ORF designs would then be used to predict
oligonucleotides (oligos) for use in our ORF production protocols. In the original SOW,
the entire library was planned to be generated by PCR-amplification of genomic DNA.
However, ASU held concern for possible technical problems in amplifying some sequence
stretches and for possible differences in ORF expression levels. Therefore, ASU moved to
a plan in which most ORFs would be PCR-amplified; however, ORFs would be evaluated
informatically to identify those likely to have amplification problems. A synthetic-based
ORF production would have been available if necessary for this set. This process would
ensure maximum production success and gene activity, while minimizing production costs.
For the synthetic ORF production, ASU held in reserve a new, patent pending gene building
method (SynBuild), which was being conceptualized and put into practice in our lab under
funding sources independent of TVDC. However, we were also improving our PCR primer
design methods for standard gene production by PCR-amplification from genomic
templates. In addition, the Francisella tularensis genome is relatively GC balanced and is
not highly repetitive as a number of pathogen genomes. These features together with good
primer designs led ASU not to need the SynBuild method
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 3 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
The design of the library of ORFs spanning all SCHU S4 coding sequences is completed.
The design of a set of PCR primers for PCR production and another set of overlapping gene
building oligos for synthetic production were both predicted. Pilot studies to validate the
gene assembly protocols are completed. With these informatics and protocols in place,
ASU has completed the design of an optimal SCHU S4 polypeptide library for our T cell
screening program.
2
Milestone Objectives
Design protein-fragment library corresponding to the SCHU S4 proteome.
1. Design a complete SCHU S4 library of polypeptides. ASU modified this
milestone to include the evaluation of all ORFs with respect to predicted
production and expression efficiencies. If these are compromised, the ORF will
be synthetically generated.
2. ASU suggested adding into subcontract after SOW: Design short synthetic
peptides (20-mers) for testing alongside the larger polypeptides during
development of the T cell activation assays.
3
Methods, Critical Reagents and SOPs
Methods for Objective #1:
Both PCR-amplification from genomic DNA and de novo synthesis of genetically
optimized ORFs were planned. As a component of the new gene building (Synbuild)
approach, a new computer program was written for analyzing the SCHU4 genome and
redesigning it as genetically-improved ORF modules for mass production and linking into in
vitro translation constructs. The design of a set of overlapping 500-600bp open-reading
frames (ORFs) spanning all SCHU S4 coding sequences is completed, and a list of oligos is
in hand. The ORFs comprising this library are designed for construction into an IVT system
and for production of a library of ~200 amino acid (aa) subproteins (polypeptides)
constituting the F. tularensis proteome. ORFs were evaluated as sufficiently naturally coded
to enable robust amplification from genomic DNA with efficient subsequent translation.
If any ORFs could not be amplified, they would be built by our new gene building protocol
called Synbuild.
Methods for Objective #2:
In addition to designing the complete library of IVT polypeptides, a set of 500 peptides
from the SCHU S4 sequence database was informatically selected. Before ordering from LC
Sciences, Inc. we presented to UNM and NIH our proposal to design and synthesize 500 20mer peptides corresponding to F. tularensis protein-epitopes predicted to bind BALB/c MHC
I or MHC II molecules. We have performed MHC I and MHC II analysis using the website
(http://www.immuneepitope.org/home.do). ASU used two MHC prediction algorithms to
search the genomic coding sequence for consensus binding motifs. These outputs were
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 4 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
integrated to identify those epitopes predicted by both algorithms, and thereby their selection
assumes a higher level of confidence. MHC Class I binding analysis identified 2380 peptides
with half maximal (50%) inhibitory concentration (IC50 )< 50 . These high affinity binding
sites represent 397 distinct proteins. MHC Class II binding analyses identified 274 consensus
peptide sites with IC50 < 50.
From this predicted-epitope list, those MHCI or MHCII motifs mapping to the same
protein were identified and only the best scored of these were selected for the final list. In
this way any protein will only be represented once, thereby enabling the greatest number of
proteins to be sampled. The exception will be TUL4. All 3 experimentally-identified human
T cell stimulating peptides within this protein have been included in the list. We have also
included a fourth peptide from TUL4 that corresponds to the best epitope prediction for the
BALB/c mouse MHC haplotype. This final list was sent out for production. The plan will be
to test these peptides individually and in pools. These peptide pools simulate the multiplexing
plan for the full library will be used by the UNM team in pilot studies for their upcoming
high-throughput T cell assays.
Critical Reagents:
1. Peptide Manufacturer: LC Sciences/Alta Inc.
Alta Bioscience, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
UK Tel: 0121 414 5450. Fax: 0121 414 3376.
Overseas Tel: +44 121 414 5450. Fax: +44 121 414 3376.
websitewww.bham.ac.uk
Email: altabios@bham.ac.uk
2. Reagents for the PCR-amplification:
Polymerase- iProof High-fidelity DNA polymerase (Bio-Rad 1725302)
Life Science Research
2000 Alfred Nobel Drive
Hercules, CA 94547
Telex: 335-358
Toll Free: 1-800-4-BIORAD (1-800-424-6723)
Fax: 510-741-5800
Free Fax: 1-800-879-2289
www.bio-rad.com
PCR machine-Eppendorf Mastercycler epGradient S
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 5 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
One Cartiague Road
P.O. Box 1019
Westbury, NY 11590
1-800-645-3050 ext. 2101
www.eppendorfna.com
3. Primer Design Plus Program
The Primer Design Plus software is described in Section 4.3 under Quality Control.
The manual for the program is referenced in the QC section and is ASU SOP #P1. The
Primer Design Plus program was used for the TVDC MS#25, but the development of the
Primer Design Plus program was funded independently of TVDC.
SOP Number1
ASU- #P1 ver. 0.1
SOP Title
Primer Design Plus program manual
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)
Chip design files can all be found on the R server at ASU under the file path of
“R:\GeneVac\FTU\Contract\Proteome\Milestones\25\Block Design”
Initial design was for 55 +/- 1 C melting temperature with 500 oligos per chip (8
copies on a chip) and 6 oligos making up a complete block. Second design reduced
the number of copies on a chip (1000 oligos or 4 copies), but kept the other
parameters intact. The final design kept the chip at 1000 oligos (4 copies) and 55 +/1 C melting temp, but changed the design back up to 8 oligos to a complete block.
ASU has used this design approach to successfully assemble multiple subgene
“building blocks” of the intended 250bp size into full length, non-FTU genes with
high sequence fidelity. The gene quality is greater than that from standard oligos.
Also, ASU designed and successfully produced larger blocks of 500bp each that are
assembled into genes as large as 3kb. This is well beyond the size of any ORFs
designed for the library.
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 6 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
Files are stored at:
R:\CIM\GeneVac\FTU\Proteome Design
Notebook and File location:
a. All notebooks are kept in the file proof file cabinet located in the north end of the
laboratory in the Biodesign Institute (room number DBD220).
b. The files are located on the server R, in the following folders:
R:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT data
The list of 500 synthetic peptides covering MHC Class I and Class II epitopes, were
reviewed by Drs. Sykes, Johnston, Lyons, and Breen. The 500 synthetic
polypeptides were ordered from Alta Bioscience, received by ASU, and forwarded
to UNM. MHC binding prediction was done using the Immune Epitope website for
predicting binders to mouse MHC I (Dd) and MHC II alleles (Ad)
Files are located in “R:\GeneVac\FTU\Contract\Proteome\Milestones\25\MHC”
Returned peptides from the prediction software were sorted by their IC50
score. The 500 peptides with the lowest score from each of the predictions
were selected to create a top 500 list for that MHC molecule.
MHC I
http://www.immuneepitope.org/analyze/html/mhc_binding.html
“how it was run.txt”
First run of Protein Data
Species: Mouse
MHC allele: H-2 Dd
Peptide length: 9
show IC50 below 500
Second run of Protein Data
Species: Mouse
MHC allele: H-2 kd
Peptide length: 9
show IC50 below 500
MHC II
http://www.immuneepitope.org/tools/matrix/iedb_input
Matrix: H-2 I-Ad
Linear
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 7 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
500 cutoff
top 1000 displayed
Files are stored at
R:\GeneVac\FTU\Contract\Proteome\Milestones\26\MHC\ftu_mhc_output.xls
The ASU and UNM groups held a conference call to plan use, testing, and storage of
the peptides.
Based on the decisions made at the phone conference, the 500 designed 20-mers were
sent from ASU to UNM as lyophilized samples (~4 mg) in 96 well microtiter plates
for testing. These peptides correspond to tularemia protein sequences that have been
predicted to be MHC Class I or Class II epitopes.
4.2
Interpretation
Objectives # 1 and #2 have been fully met. In addition ASU has added a component
to each. Firstly, a complete SCHU S4 library of production and expression optimized
polypeptides was designed. Secondly, a set of 500 short synthetic peptides (20-mers)
are in hand. These will be tested alongside the larger polypeptides during
development of the T cell activation assays.
Problems/Issues:
No issues are apparent at this time.
Files are stored at
R:\GeneVac\FTU\Contract\Proteome\Milestones\26\MHC\ftu_mhc_output.xls
Next Steps:
The next steps will be to test the ORFs in possible expression modules and develop
high throughput protocols for in vitro transcription and translation in Milestone 26.
4.3
Quality Control
The primers for the FTU project were designed by using our Primer Design Plus
program which ASU developed internally and applied to the FTU genome file
downloaded from the NCBI website. .
The Primer Design Plus program incorporates standard primer design criteria (such
as melting temperature, GC content, hairpin and primer-dimer avoidance, repeat
region avoidance) along with newer scientific concepts developed at our center.
Newer concepts applied to primer design include things such as rare codon avoidance
and stop codon suppression.
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 8 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
More details about the program can be found in a manual written for it, the location
of which is described in the section below.
Location of the program, codes, user manuals
The software used for designing primers for FTU was written in-house by Preston
Hunter, who works as a bioinformatics computer programmer for the ASU Center for
Innovations in Medicine.
The source code is stored on a shared network drive which is mirrored in multiple
back-up locations both within the Biodesign Institute building as well as outside of
the building. The file server is preserved to tape weekly, with tapes being stored in a
secure external location.
The source code is located on the Biodesign Shared server at:
peptide\Shared\CIM\software\source code\Primer Design Plus sf
Because the subsection of this server for the Center for Innovations in Medicine
(CIM) is automatically mapped for CIM users, the location typically appears to us in
the following format:
S:\software\source code\Primer Design Plus sf
The actual source code file is:
Primer_Design_Plus.rbp
This is a Real Basic source code file. Older versions of the source code, representing
multiple snapshots of the development of the software at various intervals over time,
are stored in the sub-folder named "old source".
The Primer Design Plus program has been compiled to executable files that run either
on Windows or Mac OS operating systems. The compiled applications consist of a
single executable file and require no outside files, libraries, etc. The compiled
application can be found at:
S:\software\custom software\Primer Design Plus f
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 9 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
A manual for the Primer Design Plus software was written by Yusuke Uemura and is
stored as folder with a small number of related MS Word files in the following
folder:
S:\software\custom software\Primer Design Plus f\Primer Design Plus Manual
Quality control on the PCR primers and IVT production is planned as follows.
a. The PCR products and LEE construction are tested by running the products
on gel after amplification of the LEE. The presence or absence of the PCR
product is assessed in Milestone 26.
b. IVT and amount peptide production are analyzed by TCA precipitation and
S35 scintillation counts. The purity and proper size is determined by
acrylamide gel in Milestone 26.
5
Deliverables Completed
ASU is responsible for two major components of the contract , 1) Transcriptome analysis
of F. tularensis SCHU S4 RNA during in vivo infection and 2) Generation of peptides
that represent the entire proteome of SCHU S4 F. tularensis. ASU accomplished the
following:
Proteome approach:
Starting from the annotated sequence of F.tularensis SCHU S4, Milestone 25
performed the necessary bioinformatics on the SCHU S4 annotated sequence to determine
the oligonucleotide sequences that generated peptides of antigenic length. All parameters
used for database mining were recorded during the process including the date, database
identifier, etc and are provided in this MS#25 MSCR report and the associated appendix
files.
The default parameters for Primer Design Plus were used for FTU primer design.
These include:
- maximum ORF length: 1000 bp
- ORF fragmentation overlap: 99 bp
- maximum upstream and downstream primer search region length: 99 bp
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 10 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
- Allowable primer lengths: 18 to 30 bp
- Allowable GC% range: 30 to 65%
- Melting Temp. range: 40 to 65 F
- Max TM difference between forward and reverse primers: 5 degrees
- Target TM: 55 F
- Maximum allowable rare codons: 1
- Maximum number of duplicate consecutive nucleotides: 4
- GC Clamping: end should be G or C
- Primer-Dimer (complementarity to other primer) maximum length: 3
- Haripin maximum length (self-complementarity): 3
FTU primer design files are located on the Research ("R") network drive.
R:\GeneVac\FTU
QC controls were performed
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 11 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
Deliverable Reagents
Bacterial
Strain
# of
vials
Vial
Concentra
-tion
Storage location*
Date Stored
or Date
Transferred*
Storage location*
(Institution, room, shelf, etc)
# of
blocks
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*
lyopholiz
ed
lyopholiz
ed
11/28/06
UNM
6/11/2008
Returned to ASU
Storage
media
Date Stored
or Date
Transferred*
Storage location*
\\peptide\
Shared\C
IM\softw
are\custo
m
software\
Primer
Design
Plus f
12/1/2006
ASU
SCHU S4
pilot set
SCHU S4
pilot set
500
~4 mg/well
500
~4 mg/well
Software
Purpose
Primer
Design
Oligo prediction for
high fidelity synthetic
gene production
~ Mass
per
block
Date Stored
or Date
Transferred*
Tissue
identifier
Polypeptide
Tissue
Type
Storage
media
(Institution, room, shelf, etc)
(Institution, room, shelf, etc)
(Institution, room, shelf, etc)
(Institution, room)
*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.
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 12 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
6
Appendices
6.1
Appendix 1: Original Data Tables and Figures inserted in Section 4
Table/
Figure1
Title
Notebook Location2
(Notebook # and page
numbers)
Electronic
Location2 (Full
Path & File Name)
N/A
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
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 13 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
6.2
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?
Yes No N/A Comment
X
All questions were resolved by
conference call with Kathy and Alex on
9/4/08 and final edits were received from
Kathy on 9/19/08
X
ASU edited post 4/08 and add a
concluding statement
X
ASU added the “critical reagents” list to
section 3 of this MS completion report
so this is done as of 7/25/08
The critical software tool is Primer
Design Plus, which is a custom
executable application created using the
Real Basic IDE (Integrated Development
Environment) tool. Development and
testing of this program was done
principally by in-house senior
programmer Preston Hunter, assisted by
team members Kevin Brown and Yusuke
Uemura.
X
MS 25 includes no original
laboratory bench data
After initial programmer-performed tests,
Primer Design software was given
usability tests in an organized event in
which our center's students and many
staff members tested it and met after
testing to discuss their experience.
Input/output testing was performed by
development team as individual functions
and modules were designed and added to
system.
Primer design output has been performed
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 14 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
Assessment Criteria for Milestone Completion
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
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?
X
X
X
by extensive comparisons of software
package's primer designs to actual
genome, using BioPerl-based tests
conducted by Kevin Brown. Actual
primers ordered based on Primer Design
were used to generate DNA sequences
which were tested for proper length and
quality/volume. These wet bench tests
were done using gel-based analysis and
volume testing such as with PicoGreen.
Lab bench Results will be reported with
MS 26 MSCR.
ASU completed
UNM has the 3 attachment excel files
MS 25 includes no original laboratory
bench data
Yes No N/A Comment
X
X
X
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 crossreferenced 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?
Data is stored on network drive servers which
are continuously replicated onto multiple
mirrored locations: a separate server within the
building and one outside of this building.
Weekly tape backups are stored in a secure,
high-security location. Every week a new copy
is stored permanently. The Biodesign Institute
X
Tables are listed as attachments in
appendix 6.3 and electronic locations are
provided by ASU
X
MS 25 contains no original lab bench
data in appendix 1
Electronically. The Primer design
software is stored in two locations; one in
CIM and one external secure site.
Hardcopy notebooks are located in
fireproof cabinet in lab (see section 4.1 of
report). Server is protected.
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 15 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
Assessment Criteria for Milestone Completion
has a full time staff of IT professionals
dedicated to maintaining data integrity and
security.
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?
ASU has described QC on the software
design and usage in section 4.3 of this
report
X
After initial programmer-performed tests,
Primer Design software was given
usability tests in an organized event in
which our center's students and many
staff members tested it and met after
testing to discuss their experience.
Input/output testing was performed by
development team as individual functions
and modules were designed and added to
system.
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
X
Primer design output has been performed
by extensive comparisons of software
package's primer designs to actual
genome, using BioPerl-based tests
conducted by Kevin Brown. Actual
primers ordered based on Primer Design
were used to generate DNA sequences
which were tested for proper length and
quality/volume. These wet bench tests
were done using gel-based analysis and
volume testing such as with PicoGreen.
MS 25 is for peptide design and not for
peptide production; therefore, this review
criteria is not applicable
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 16 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
Assessment Criteria for Milestone Completion
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.
X
Yes No N/A Comment
X
Has the Milestone Completion Report been
written by subcontractor, reviewed by UNM,
revised by subcontractor as requested, and
accepted by UNM?
X
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?
X
ASU provided a Primer Design Plus
manual (P_1), A manual (created as a
Microsoft Word document and saved
separately as a PDF file) is available for
the Primer Design Plus software. This
manual can be found on the Center's main
Shared server:
\\peptide\SHARED\CIM\software\custom
software\Primer Design Plus f\Primer
Design Plus Manual.
This manual includes descriptions of how
to design primers with the software and
can be regarded as SOP for this process.
ASU drafted in 12/06, BG entered into
this template 10/07. BG sent edits in
ver0.2 to ASU in 11/07, got ASU edits
back on 12/21/07 on ver. 0.2 and BG
returned Ver. 0.3 to ASU for a few more
edits around 3/31/08. KS returned edits
on 4/18/08. BG reviewed on 7/25/08 and
completed a conference call with Kathy
and Alex on 9/4/08 to discuss the minor
edits in ver. 0.4 and create final ver. 0.5
on 9/5/08; Final edited version is 0.6
9/19/08. BG and KS discussed
NIAID/MH questions by phone on
10/2/08; KS also sent emails to BG on
10/2 and 10/3 regarding NIAID
questions. New version is 0.8 10/22/08
Final version is 2.0 on 3/26/09
In monthly tech reports
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 17 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
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?
X
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
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
Yes No N/A Comment
X
X
These will not be standard lab bench
protocols, but will be software design and
testing protocols. Primer design software
manual was received at UNM on 4/14/08
from Preston Hunter.
Yes; MH said Joe Breen accepted the
SOP 9/30/08
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 # 25
Institution: Arizona State University (ASU)
Author: Kathryn Sykes
MS Start Date:3/2/06
MS End Date:12/1/06
Report Date: 10/7/07
Version: 2.0
Page 18 of 18
Reviewed by : Barbara Griffith 10/23/07, 3/31/08,
Accepted
Date:9/19/2008
8/6/08, 9/19/08, 10/1/2008, 10/22/08, 1/28/09, 3/5/09,
3/25/09
6.3
Appendix 3: Additional Data/Figures not inserted into the Text of the
Milestone Completion Report (Section 4)
6.3.1 SCHU S4 protein list is provided as an attachment in excel. ORFs to encode
protein fragments (polypeptides) covering all F. tularensis coding sequences
(proteome)R:\GeneVac\FTU\Contract\Proteome\Milestones\25\ftu_proteins_se
gs.xls
6.3.2 500 informatically selected peptides predicted to have high probability of being
immunogenic in T cell assays.
R:\GeneVac\FTU\Contract\Proteome\Milestones\25\MHC\ftu_mhc_output.xls
R:\GeneVac\FTU\Contract\Proteome\Milestones\26\MHC\ftu_mhc_output.xls
*updated file name
6.3.3 Informatic partitioning of the F. tularensis (SCHU4) coding sequences into 500
bp fragments, optimized for ORF synthesis and polypeptide production.
R:\CIM\GeneVac\FTU\Proteome Design. *updated location:
R:\GeneVac\FTU\Primer design\FTU output PCR products with
masksf\FTU_PCR_product_details.xls