UNM TVDC
ASU - UNM Tech Call Minutes 6/27/06
Prepared 6/27/06: Barbara Griffith
Revised 6/28/06: Kathy Sykes
Present: Kathy Sykes, Stephen Johnston, Mitch Magee, Rick Lyons, Vicki Pierson, Mindy Tyson,
Absent: Joe Breen, Marlene Hammer, Kristin DeBord
Action Items
 Kathy/Stephen-Write up strategy, with list and send to Joe Breen and cc Rick and Vicki.
Give Joe opportunity to review for approximately one week before the order is placed.
 Kathy/Stephen Should have the 500 20mers within a month (~7/28/06).
 Stephen will send Rick information on Europium (noble element). Companies are using
for their T cell based assays.
 Kathy will send Rick preferably 20-25 ug for the first couple of subproteins being
produced for Rick; approximately 3 subproteins will be produced.
 Rick will send Stephen information to plan the time points for the infection experiments.
Discussion of the Progress of Milestones
Milestone 25 description: Design protein-fragment library based on SCHU S4 sequence.
Milestone 25 progress (Dr. Kathy Sykes):
 Designing ORFs this month, in particular to get the DNA/ORFs to input in the IVT to get
the proteins. ASU is designing 600 BP ORF’s for subproteins of 200 amino acids.

They are also having synthesized on a chip, 500peptides’ 20 mers. They have the list of
20mers. They have used tools available at http://www.immuneepitope.org/home.do and
other free software for MHC 1 and 11 predictions and then cross compared to
publications to determine how derivative antigens/proteins compare.

Luckily, the 3 peptides from 204, 17Kd proteins, are fact and are known T cell stimulating
epitopes/peptides within Tularensis proteins. They are all in 204, and they do come out
of Kathy’s lab predictions.From published material, there is a list of what is a virulence
factor, a membrane protein, and lipoprotein and should be immunogenic.

ASU incorporated these thoughts into a few 200 amino acid IVTs, then designed and
produced subproteins to optimize the assays. So Kathy is providing Rick with two
different kinds of material to optimize the assays 1) antigenic material, a small number of
200 amino acid peptides and 2) pooling of material

Stephen said they are ready to order peptides this week. So the strategy was to take the
predictions and make 500 20 mers. Take the 3 proteins in the literature and take
multiples from there and then take multiples from other high ones and then one of each of
everything else, down to 500. So we will have some proteins with multiple
representations in the pooled material and other proteins with just one representation in
the pooled material, knowing they score above the minimal for MHC binding. This is their
strategy.
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
Action: Kathy/Stephen-Write up strategy, with list and send to Joe Breen and cc Rick and
Vicki. Give Joe opportunity to review for approximately one week before the order is
placed.

Regarding the timeline, Stephen will check with Rich at Alta, and can do >100 per week;
possibly 300-400/week. Rich sends mass spec data on the peptides and ASU still
checks solubility etc. Action: Kathy/Stephen should have the 500 20mers within a month
(~7/28/06)
Milestone 26 description: Confirmation of gene expression (Design HTP SOPs, Test HTP
SOPs, ORF library production, confirm gene expression).
Milestone 26 progress (Dr. Kathy Sykes):
 Kathy spent time with a better ORF design; much less complex and hits important
aspects of generating lots of high quality ORFs very fast. They have a much less goal in
recoding, but much more important goal in eliminating problem areas of the genome.
Tularemia genome is extremely AT rich with long strings of A that are challenging and
has the transposable element which results in many repeated sequences.

They now have the ORF design and have code written, with these challenges in mind.

Technically, makes this genome easier to build. Not worrying so much about codon
usage per se, but with fixing the genome so it is easier. It is easy to punch out all the
Oligos that they want. Will use the designs to make subproteins for Rick. Kathy thinks
that Rick needs a larger mass of a smaller number of subproteins, to use for optimizing
the assays.

Rick’s Strategy in mice: Vaccinate with LVS, challenge with SCHU S4, harvest lymph
nodes from draining sites, use those cells as reporter cells, then get antigens from ASU
to define what is the lowest amount of antigen concentration usable in the assay and
what are the cell conditions (# antigen presenting, # of T cells etc). Want optimized in
mouse before moving to primates. Want at least 1 antigen that has been shown in the
past to respond to an LVS vaccination, like TUL4 response. It may not be the best, but
no data differentiates whether it is the best. Would like a couple more as well, because
randomly others may work too. Use TUL4 because they have taken in the peptides as
well as taken in the full length proteins at 10ug /ml and LVS and use them in T cell
proliferative assays with cytokine secretions. Both materials have worked for stimulation.

Rick will use new read out proliferation assays which do not include radioactivity; use
luciferase proliferation assay kits which are highly sensitive. Lucerifase is more sensitive
than tritium. Rick is also talking to Larry Sklar who has a sensitive flow based assay for
detecting intracellular IL2 as a readout.

Stephen has seen a Europium assay which is cell based detection for proliferation;
Europium has the advantage of radioactivity but not the disposal problem of radioactivity.
It is as convenient as the enzyme based assays like luciferase. Action: Stephen will send
Rick information on Europium (noble element). Companies are using for their T cell
based assays.

Kathy asked Rick how many wells and what volumes will you use in the assays. Rick
guessed that they will use similar conditions to detecting Tetanus toxoid, using 100ul
media and 500ng/ml of antigen, which equals 50ng of protein. 50ng is an optimal
amount, though UNM may even see responses at lower concentration, using a “yes or
no” type of response detection. So in triplicate, need 150ng total. Kathy will send 10ug
at minimum and will try to send 20ug for the initial assay developmental experiments
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
Action: Kathy will send Rick preferably 20-25 ug for the first couple of subproteins being
produced for Rick. Kathy will make subproteins (~3) for Rick to test in mice first.
Milestone 32 Description: Oligos selected for microarray production; Oligos list refined, 70mer
Oligos procured, GDP Oligos defined, Based on SCHU S4 sequence.
Milestone 32 Progress (Dr. Stephen Johnston):

Stephen redid analysis using latest 2005 annotations rather than using Southwestern’s
dated sequences on their arrays; new Oligos have been predicted using 2005
annotations; ready to have synthesized by Invitrogen, which now owns Illumina; $1112,000 cost for the Oligos and these are optimized now. Will be delivered next week.

Rick asked who is responsible for updating genome annotations for SCHU S4? Stephen
said there is no centralized annotation for Tularemia sequence data, rather it depends
on the lab generating data.

Stephen described the challenges of the genome directed primers (want to prime across
region that matches the oligo). When you optimize the 70mer to detect the transcript that
puts a constraint on the GDP primer location as you want to prime across where the oligo
is made . Also, the genome is AT rich. These constraints leave not too many options
for location of GDP to amplify 99% of genome. Currently, it may take 367 GDP primers,
which is a record. Mitch says they continue to work on optimization algorithms to try to
reduce the number of GDP needed.

Stephen said that they are not going to order the 367 yet, but the number seems high
due to AT richness and positioning. MTB only took 37 GDP and Tularemia is taking 10x
the number for a genome that is half the size. These GDP primers aren’t critical until
they get samples and want to start amplifying. They can test out the microarrays with
the RNA already received from UNM, and are using direct labeling and priming in the
mean time.

Rick asked if ASU is planning on enriching the bacterial RNA using standard columns
Stephen responded that they are not. They tested the columns with plague and saw
loss of sequence representation using the columns . It was better to rely on GDP and
the arrays, than use the subtraction columns. They were theoretically good but not
technically advantageous

Rick will start planning the infection experiments and will send information to Stephen off
line to plan time points. Action: Rick will send Stephen information to plan the time points
for the infection experiments.

Mitch pulled the power points together for today’s discussion.

Rick’s lab will be testing antigen memory: comparing UV, heat and formalin fixedantigens which can be different depending on inactivation method. This testing will
parallel developing immunoassays to determine immunization, protective correlates, etc.
Jason will be doing the T cell assays at UNM.

Kathy asked if the psoralen safe enough for inactivation. Rick said that the problem is
reproducibility more than safety for the immunoassays. Psoralen good for vaccine but
another method of inactivation is better for immunoassays.
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Problems solved


New Microarrayer is working well
New quantitation of protein is working well. Stephen has vetted a new technology from
Invitrogen for quantifying peptide mass and it works! This has been a problem in the past,
for normalizing responses relative to the peptide mass.
Goals for the next month

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

ASU is designing 600 BP ORF’s for subproteins of 200 amino acids.
Get Joe’s review and comment within the next week for the 500 20mers.
Kathy will be making ~ 3 subproteins at >20-25ug for optimizing assays at UNM
Oligos are ordered and will try printing with 2 different arrayers, using corning ultragaps
slides, and will test with RNA received from UNM. Ultragaps are giving good arrays and
PLL has worked well in the best. UNM also has seen that PLL and corning Ultragaps
work equally well.
Mitch/Preston will continue improving the GDP primer selection in hopes of decreasing
the numbers of GDP needed.
Rick and Stephen will be discussing the time points of infection which will be used for the
RNA isolations.
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