Cerus - UNM Tech Call Minutes: 11/14/06
Prepared by: Barbara 11/20/06
Provided Draft to Justin: 11/20/06
Reviewed and Edited by: Justin Skoble11/20/06
Provided to NIAID: 11/21/2006
Present: Justin Skoble, Jack Joseph, Tom Dubensky, Vicki Pierson, Barbara Griffith, Sam Kim,
Tae Kim, Marlene Hammer
Absent: Rick Lyons
Action Items:
 Justin: Determine metabolic activity of uvrA uvrB double mutant in F. novicida
 Justin: Measure LD50 of all 4 strains by IV route and determine organ burdens
 Justin: Establish protocol using 2x minimum S-59 concentration for generation of KBMA
Vaccine.
 Justin: Initiate inactivation process scale-up from 3.5 ml to 400ml
 Justin: Perform 3L cultivation of LVS using Sigma Antifoam A in fermentor
 Justin: Test stability of different freezing medias with LVS
 Vicki: Explore possible funding for vitrification studies with UST
A. KBMA Tularemia Vaccine Progress
Justin presented a 12 slide PowerPoint presentation on Cerus’ progress
1. Active Milestones:
a. Milestone 40: Phenotyping of F.t. novicida uvr
i. Measure attenuation of uvr mutants in macrophages and in mice
b. Milestone 41: Optimization of psoralen treatment and characterization
of KBMA F.t. novicida
i. Measure metabolic activity of uvr mutants after photochemical
treatment
ii. Determine the level of virulence of KBMA F. novicida
iii. Establish photochemical inactivation regimen
c. Milestone 46: Scale-up of KBMA LVS vaccine production
i. Optimize large–scale LVS culture conditions
ii. Establish 3L culture scale purification conditions
iii. Optimize 3L scale photochemical inactivation process
iv. Verify protective immunogenicity of vaccine candidates produced by
optimized large-scale process
2. Milestone 40: Phenotyping of F.t. novicida uvr mutants: U112 and uvrAB
mutants
a. Growth in vitro is identical to wild-type
b. Growth in cells is identical to wild-type
c. Virulence of Ftn strains
i. Mice were infected IP with five serial 5 fold dilutions of Ftn strains
ii. Number of cfu injected was determined by growth on CHAH
iii. Weight and appearance of animals were monitored for 2 weeks
iv. All animal deaths occurred 3-4 days post-infection
v. All Ftn strains still highly virulent when administered IP
d. Summary
i. Ft novicida uvrA, uvrB, and uvrAuvrB mutants do not have
growth defects in broth
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ii. Ft novicida uvrA, uvrB, and uvrAuvrB mutants do not have
growth defects in J774 macrophages
iii. The LD50 of WT U112, uvrA, uvrB, and uvrAuvrB strains by
intraperitoneal infection were between 3 and less than one cfu. This
suggests that all strains are fully virulent. An LD50 in the one range
is insignificantly different, even at 3 cfu, and it suggests that a single
bacterium can kill a mouse; all strains had the same time to death,
no delay in UvrAB mutant despite having the highest LD50. IP is
most virulent route of administration; used BALB/c mice.
iv. Next Steps
1. LD50 of all 4 strains by IV route- expect 3-4 logs less
virulent, and may see differences if there are subtle
differences between ip and iv routes, intermediate (later
subc which is least virulent)
2. Growth rates in lungs, livers, spleens will follow the LD50
3. Justin: close to finished with phenotyping the nucleotide
excision repair (NER) F. novicida mutant strains; knocking
out NER doesn’t appear to impact the virulence. Justin:
uvrA and uvrB gene product form the complex; expect to
knock out NER if either is knocked out; since neither single
mutant was attenuated it is not surprising that the double
mutant isn’t either.
4. Tom: Is it important to look at the activation of the NER
pathway in wildtype in response to treatment? The mutant
observations are different than has been seen with Listeria.
5. Justin: This discussion will continue when we get to the
photosensitivity.
3. Milestone 41: Optimization of Photochemical Treatment regimen for Ftn
i. 1ml of culture plated (increasing LOD 10x to 1 cfu/ml)
ii. All NER mutant strains were slightly more sensitive than U112
iii. Inactivation achieved at 20uM for NER-deficient strains, 40uM for
U112
iv. Did simultaneous head to head comparison of wild type and 3
mutants and did a dose titration of S59 to get more accurate
sensitivity to photochemical inactivation. With plating 100ul, the limit
of sensitivity was previously 10 CFU/ml but with plating 1ml, Cerus
has a sensitivity down to 1 CFU/ml. Cerus was seeing only subtle
differences between the NER mutants and the wild type. Wanted the
head to head comparison to be performed with the same batch of
reagents and at the same time for all 3 mutants and the wild type.
v. With plating 1ml, the wild type is fully inactivated at 40uM and 3
mutants are at 20uM; This is an average of 2 experiments and
results in slightly higher concentrations needed, due to increasing
sensitivity to 1 CFU. Getting fully sterile inactivation down to 1 CFU
sensitivity.
vi. Hoped to see increase in sensitivity of uvrAB double mutant, but is
no more sensitive that uvrA or uvrBalone. This makes sense if you
think about them as part of the same complex so that if you knock
out any one of the NER genes, you knock out the complex and
knocking out two NER genes would be no more effective.
vii. Justin: Surprising that it is only 2 fold more sensitive than wild type;
Listeria and Anthrax are usually 20 fold different in sensitivity to S59.
viii. Justin: We have to keep in mind that they are not tremendously more
sensitive to S59 than the wild type strain is. One possibility is that
there is a redundant mechanism for repair so not made very
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sensitive or that the uvrA uvrB genes are not turned on under these
conditions. The later is the hypothesis we favor at this time because
the mutants maintain such high metabolic activity for 12 hrs after
inactivation suggesting that they have very few DNA crosslinks.
ix. Tom: In your preliminary data, how does the metabolic activity of the
single mutant compare to the double mutant and the wildtype?
x. Justin: The level of metabolic activity of the uvrA and uvrB single
mutants is very similar to the wildtype. We get more reproducible
killing at the lower concentration of S59. It makes sense to move
forward with a single mutant as our prime candidate because it is
more sensitive and more reproducibly inactivated at lower
S59(psoralen) concentrations and double mutant actually has two
antibiotic resistance (kan and erm) markers. Since the goal is to
generate a KBMA LVS or ShuS4 strain a single mutant maybe most
acceptable. Justin will use the uvrB mutant in F novicida for proof of
concept of the KBMA vaccine work.
xi. MS 41 summary
 Optimization of S-59 dose and UVA dose
 4 J/cm2 is the minimum dose of UVA required to
achieve consistent inactivation.
 Minimum S-59 concentration required to inactivate 1 x
1010 cfu
-U112 = 40uM
-uvrA, uvrB, + uvrAuvrB = 20uM

Next Steps
 Establish protocol using 2x minimum S-59
concentration for generation of KBMA
Vaccine.
 Perform MTS metabolic activity assays on
all strains inactivated under these
conditions.
 Determine the LD50 of KBMA vaccine IP.
 Initiate process scale-up from 3.5 ml to
400ml.
xii.
1. Use 2x min psoralen dose, which provides a buffer in case
there is slightly higher optical density one day or some
difference in culture conditions so that we get consistent
inactivation process.
2. Tae: We don’t see much change in metabolic activity with 2x
min S-59 dose so there is no downside to using 2X as the S59 minimum inactivation concentration.
3. Tom: It usually has almost no influence on metabolic activity.
10 fold has impact on metabolic activity but 2 fold has no
measurable impact.
4. Cerus will establish this protocol using 2X minimum
concentration of S-59 and then we will perform metabolic
activity assays looking at all strains under this condition.
5. Cerus will examine S-59 inactivated KBMA bacteria (urvB
mutant F. novicida) and determine if they are avirulent using
the IP route of infection in BALB/c mice. The IP LD50 for
these bacteria is about 1 CFU. Plating will be used to
determine whether the KBMA bacteria are dead.
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6. Ready to scale up to intermediate 400ml scale inactivation
process. inactivate in a UV transparent blood bag, and
freeze aliquots which can be used for vaccination and
challenges from the same lot of bacteria.
7. Mutants are, reproducibly, slightly more sensitive than wild
type but still need to measure metabolic activity on uvrAB
double mutant
xiii. Vicki: requested description of transfusion blood bag: Tae- 1 liter
PL24 bag from Baxter, a UV-transparent plastic; it also allows limited
oxygen exchange across the plastic. Shaker flask immersed in ice bath
to stop replication and decrease metabolic activity; culture is pumped
into the bag for the UV exposure on the illuminator for 7 min; then the
culture is processed at 7C in centrifuge. Historically, not seen significant
loss of CFU for the brief period in the bag.
xiv. Vicki- have you ever grown a bacteria, susceptible to phase shift, in
these bags? Justin: really don’t grow the bacteria in the bag, as the bag
is only used for 7 min for the UV light inactivation step. Tae: Bag has a
short light path; illuminated from top and bottom both; Illuminator shakes
contents too so every bacteria passes near the surface of the bag to get
UV dose. Have used this process for Anthrax and Listeria so should be
easy transition for Francisella. Justin- at research level inactivation
occurs in 6 well plates, they use a different illuminator, w/o shaking, but
with larger volumes need the shaking and bags for even exposure.
4. MS 46: Large-Scale Propagation of LVS
a. Cerus examined multiple antifoams to replace the prior antifoam which
prevented growth in the Cerus fermentor.
b. Sigma antifoam 204 (used for Listeria fermentation) prevents LVS growth in
the fermentor
i. Shakers w/o antifoam grew Ft well but in fermentor with antifoam
204 inhibited growth
c. Evaluate Sigma Antifoam A (LBERI) and BASF Industrol DF 204 (DVC)
i. Found that Sigma Antifoam A didn’t inhibit growth at 0.01%
ii. Found BASF Industrol at 0.01% inhibited badly; BASF antifoam used
by DVC is used at 30 fold lower in DVC’s fermentor than tested at
Cerus. DVC uses 1 drop per 13 liter fermentor and is not inhibitory
in DVC’s hands at this lower concentration.
d. Sigma A antifoam does not prevent LVS growth: Cerus will use this one at
0.01% or less.
i. We have established that antifoam was the inhibitory material
preventing LVS growth in our fermentor
ii. We have identified Sigma Antifoam A as a non-inhibitory antifoam for
LVS in shaker flasks
iii. We will perform a 3L fermentor run using CDM + no more than
0.01% Sigma antifoam A
iv. Growth of LVS will be measured by OD600 and by CFU
v. pH and dissolved O2 will also be measured
vi. Samples will be formulated in Cerus “freeze buffer” or 10% sucrose
and viability at –80 degrees will be monitored
1. won’t use glycerol as LVS is sensitive to it
2. Cerus’ freeze buffer: salt solution containing DMSO and
sucrose
3. 10% sucrose is used by DSTL and NRC
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4. Vicki- In clinic LVS lyophilization trials with various sugars,
the 10% sucrose worked very well. Other sugars didn’t work
well.
e. Vitrification
i. At the Woods Hole TVDC pre-meeting the question was raised
whether vitrification may be superior to lyophilization
ii. At Cerus we have found that vitrified live Listeria monocytogenes are
viable at room temperature for greater than 1 year. Tae offered to
share the data.
iii. We are currently working with Dr. Victor Bronshtein at Universal
Stabilization Technologies (UST) to prepare a vitrified KBMA Anthrax
vaccine
iv. Dr. Bronshtein has expressed interest in working with us or with
NIAID to develop a formulation that preserves viability of LVS at
elevated temperatures; would work with Cerus, UNM or NIAID
directly. Vicki: This is not product specific, so will explore other
funding that could benefit multiple products on multiple contracts.
Will get “yes/no” answer from NIAID before asking Cerus/UNM for
approximate costs after Thanksgiving break.
v. Vicki: Does UST have viral data on vitrification? Justin- possibly on
small pox, which may be in progress for NIAID
B. Other Topics:
1) Next Cerus Tech Call will be Tuesday, December 12th. 11am-12:00pm PST, 12:00pm1:00pm MST, 2:00pm-3:00pm EST.
2) January Cerus call : will be on January 16th ( we are shifting to the 2nd to 5th Tuesdays in
January since Jan 2 is a UNM holiday)
C. Comments:
1) Barbara and Vicki thanked Justin for the excellent quality and quantity of data, making
progress on the Cerus milestones.
2) Tae Kim and Jack Joseph: both on their last TVDC call; Tae is moving to Bayer and Sam
Kim is replacing Jack Joseph, as a full time project manager for Cerus’ vaccine efforts.
3) Tom: Will have to replace Tae, in medium term. Tae has a team of competent professionals
who will be reporting to Justin.
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