UNM TVDC UTSA - UNM Tech Call Minutes: 1/23/2007
Prepared 1/26/2007: Barbara Griffith
Sent to UTSA for review: 1/26/07
Reviewed: Karl Klose 1/26/07 and Bernard Arulanadam 1/29/07
Distributed to NIAID on: 1/ 29/2007
Present: Karl Klose, Barbara Griffith, Vicki Pierson, Marlene Hammer, Bernard Arulanandam,
Rick Lyons
Absent: Joe Breen, Kristin DeBord
Action Items:
 Barbara has milestone completion reports for MS 16 &39 from UTSA and will be reviewing
 Karl will ask Jeff Barker to provide UNM the MS 48 completion report (gave to Jeff 1/23/07)
 Barbara- Justin/Karl/Rick- setup a call to discuss double mutants in Fn. (completed 1/25/07)
 Karl/Bernard- add Quality Assurance slides to the 2/20/07 UTSA Tech call presentation
 Karl/Bernard- add Flow diagram slides for each active milestone to the 2/20/07 UTSA Tech
call presentation
The meeting was recorded for the purposes of the minutes.
A. Active milestones during last reporting period:
a. Milestone #43: Creation of uvrA and uvrB mutant F. tularensis subsp. holarctica
(LVS) strains
b. Milestone #49A: Construction of iglC F. tularensis subsp. tularensis strain
c. Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida,
subsp. tularensis, and LVS strains
d. Milestone #51: Construction of F. tularensis subsp. Novicida uvrA + pdpD, iglA,
iglB, iglC, iglD and uvrB + pdpD, iglA, iglB, iglC, iglD strains.
B. Milestone #43: Creation of uvrA and uvrB mutant F. tularensis subsp. holarctica
(LVS) strains
a. We have created new low copy vector for mutant construction in LVS/Schu S4,
pKEK1090, this is conjugative, conditionally replicative requiring special strains to
get replication, with FT groELp to drive sacB (counterselective marker) and CmR
b. This vector should work in Schu S4 and LVS; mob assures will be inserted into
the chromosome
c. pds132 is the parent vector to pKEK1090
d. uvrB::Kan was inserted into this plasmid to create pKEK1114.
e. pKEK1114 was transformed into dapA E. coli mating strain, and mated with LVS
(1:10 ratio). dapA is a great mating strain.
f. Selection done for CmR which is on the plasmid, then counterselection with
KanR and SucR. When grown on sucrose, the plasmid has to leave the strain
otherwise the cells containing the sacB marker will die. Sucrose is toxic in the
presence of the sacB marker.
g. Single colony resulted was Kan and sucrose resistant, evaluated by PCR
h. This strain has uvrB::Kan in chromosome! Conjugation worked and is in correct
site. This is UTSA’s first mutant made in LVS.
i. However, the plasmid backbone remains, and UTSA will eliminate NaCl from
media (assists in sucrose counterselection) and repeat again. It is now a matter
of eliminating plasmid from this strain. (documented in UTSA TVD notebook #2)
j. Question: Rick- could sac B have acquired a mutation? Maybe per Karl. First
time grew up LVS on sucrose (LVS is worst grower of all the strains of Ft strains).
Karl- used modified TS media to grow the mutant strains but in the future will
order individual components and eliminate NaCl from TS media composition.
C. Milestone #49A: Construction of iglC F. tularensis subsp. tularensis strain
a. Several concurrent strategies:
b. Strategy 1: create plasmid similar to published (colE1 ori)
1. Recall we successfully utilized this previously, but were unable to
remove plasmid due to lack of counterselection)
2. We have introduced sacB into our Schuh4 mutagenesis vector
downstream of Ftp to provide a counterselectable marker:
3. Previously used a high copy number plasmid but created Kan
under Ft promoter in colE1 plasmid and needed to add the sacB
as counterselectable marker. Ft promoter driving Kan resistance
and sacB.
4. Currently, Crystal is adding iglC mutant to this plasmid.
c. Strategy 2: use new mating plasmid (oriR6K)
1. We have performed ligation of iglC into mating plasmid
pKEK1090, currently evaluating resultant colonies.
d. Strategy 3: (Value added) Targetron system
1. We have adapted Targetron (intron targeting system of Sigma)
to work in Ft
2. We have created plasmid with RNA +LtrA, required for Targetron
in Ft, into this (in RNA) we introduce fragment that “retargets”
intron to specific gene.
3. We have designed and ordered the oligos to knockout iglC,
PCR fragment has been amplified, we are in process of inserting
into Targetron plasmid (documented in UTSATVD Notebook 3).
D. Milestone #51: Creation of uvrA + pdpD, iglA, iglB, iglC, iglD and uvrB + pdpD, iglA,
iglB, iglC, iglD mutant F. tularensis subsp. novicida strains
a. We are constructing all ten of these strains, by moving uvrA::Kan and uvrB::Kan
mutations into strains with single ermC insertions in pdpD, iglA, iglB, iglC, and
iglD
1. We have all the single mutant strains except iglB, so we have
constructed a iglB::ermC mutant
2. iglB::ermC has been constructed by overlapping PCR, (as we
have made all other mutants in novicida), ligated into pGEMT
(pKEK1118)
3. pKEK1118 transformed into U112, selection for ErmR, cycling to
eliminate plasmid backbone (as for all other Ftn mutants),
resulting in creation of KKF235 (iglB::ermC) (documented in
UTSATVD Notebook 2).
4. Know that iglB is highly attenuated.
5. Will have 10 specific strains with uvr mutations and attenuating
mutations.
E. Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida,
subsp. tularensis, and LVS strains
a. Results Update #1:
1. Isotyping of anti-Δiglc antibodies in sera of mice immunized with
Δiglc + LVS LPS Groups of BALB/c mice (6 mice per group)
were vaccinated with Δiglc (106 CFU per mouse via intranasal
(i.n.) route) and purified LVS LPS (50μg per mouse via
intraperitoneal injection). These mice received an additional
boost with Δiglc (106 CFU, i.n.) + LVS LPS 3 weeks after the first
immunization. Mice were bled after vaccination and sera were
analyzed by isotype-specific ELISAs using UV-inactivated Δiglccoated microtiter plates. The combination of the Δiglc (106 CFU,
i.n.) + LVS LPS was not protective.
2. UTSA had previously shown that the Δiglc Fn mutant alone was
not protective against a SCHU S4 challenge.
3. Fig. 1 Serum antibody profiles after vaccination i.n. with Ft
novicida Δiglc and LPS (LPS boosts immunity)
4. Question; Rick- What is the Antigen on the Elisa plate? Bernard:
uv inactivated iglc coating on the plate.
5. Mice primed with Δiglc+LPS exhibit the induction of specific total,
IgG1 and IgG2a antibodies
6. They were not protected after this vaccination and boost with
LPS.
7. Already published that iglC mutation Fn as a vaccine is highly
protective with challenge by wildtype F novicida.
b. Results Update #2:
1. Determine the kinetic growth and clearance of the Ft novicida
Δiglc mutant in target organs after i.n. vaccination
2. BALB/c mice were vaccinated with Δiglc Fn mutant (106 CFU)
intranasally. Lungs, liver, spleen, and pooled cervical draining
lymph nodes were collected from the vaccinated mice at a three
day interval (3 mice per time point). Numbers of bacteria in each
organ were determined by dilution plating.
3. Fig. 2 Kinetic growth and clearance of the Ft novicida Δiglc
mutant in target organs after i.n. vaccination. (day 3 to 15)
Bacterial burdens were determined from lungs, liver and spleen
of individual mouse and from pooled lymph nodes at each time
point (3 mice per time point). Numbers of mice without
detectable bacterial burden are indicated in the figure.
4. By day 30, with immuno fluorescence with anti LPS, they have
detected a minimal number of organisms.
b. Results Update #3: Summary
1. As shown in Fig. 2, there was heightened replication of the
organism in the lungs within the first 12 days, with reduction
noted at day 15.
2. There were lower levels of replication within the liver and spleen,
with minimal detection of replicating organism by day 15. There
were organisms recovered from the draining lymph nodes, but at
much lower levels than that seen with the other target organs.
3. Although Ft novicida Δiglc is highly attenuated in mice, there is
detectable replication of the organism up to 15 days. This
replication pattern may account for the robust priming of the
immune system, with this attenuated vaccine candidate.
4. Karl: interesting that Δiglc Fn protects against challenge with
wildtype F novicida and Δiglc Fn persists for 15 days in the lung,
which makes it a good vaccine candidate. Good for inducing a
good cell response. Generally challenge with wildtype Fn 30
days after vaccination with Δiglc Fn. Karl predicts that longer
persistence of the vaccination strain will correlate with better
protection.
5. Rick: in general, vaccine candidate persistence correlates with
better protection but protection may wane in 50 days in mouse
and guinea pig, almost back to a non-immune state, which is not
well understood. Persistence helps with initial protection but not
with long term immunity.
F. Plan for following month:
a. Milestone #16: completed.
b. Milestone #39: completed.
c. Milestone #48: completed.
d. Milestone #43:
1. Modify counterselection technique to remove uvrB::Kan
plasmid from LVS to create uvrB::Kan LVS mutant
2. removing NaCl from media used during sucrose selection
3. Clone uvrA::Kan into pKEK1090, mate into LVS
e. Milestone #49A: 3 different strategies
1. Clone iglC into colE1 mutagenesis vector
2. Clone iglC into R6K mutagenesis vector
3. Clone iglC targeting fragment into Targetron vector
f. Milestone #51:
1. Construct uvrB::Kan iglA::ermC and uvrB::Kan iglC::ermC
double mutants
g. Milestone #50:
1. Determine the LD50 of Ft subsp. novicida iglB mutant –
predicted to be highly attenuated.
2. Monitor Ft subsp. novicida iglB mutant replication and
dissemination in mice – will determine the attenuation
h. Rick: which Fn double mutant should be targeted or prioritized?
Action: Barbara- Justin/Karl/Rick- setup a call to discuss double mutants in Fn.
(completed 1/25/07)
Discussion:
Karl gave seminar to Cerus in December 19&20, met scientists in their group, Justin
and group interacted with Karl. In person, is more productive than long distance
discussions. Karl appreciated the excellent caliber of science at Cerus; small
company is positively influenced by lead scientists. Many scientists are from
Portnoy lab at UC Berkeley. This was Karl’s first visit to Cerus.
G. Next UTSA Tech Calls:
a. February 20, 2007, noon-1pm MT, 1-2pm CT, 2-3pm ET