Cerus - UNM Tech Call Minutes: 2/13/07
Prepared by: Barbara Griffith 2/15/07
Provided Draft to Justin: 2/15/07
Reviewed and Edited by: Justin Skoble 3/12/07
Provided to NIAID: 3/12/2007
Present: Justin Skoble, Vicki Pierson, Barbara Griffith, Rick Lyons
Absent: Joe Breen, Kristin De Bord, Frejya Lynn, Marlene Hammer, Tom Dubensky, Sam Kim,
Action Items

Justin will include the mean time to death in next presentation on the LD50 with the uvr
mutants of F novicida.
 Prior to administering an IN challenge Justin will talk to Bernard who may have used a
transposon mutant given SC to protect against an IN challenge. In animal models for F.
tularensis, there can be a problem trying to protect against lung challenge when
vaccinated by other routes like SC or IP.
A. KBMA Tularemia Vaccine Progress
Justin presented a 21-slide Power Point presentation on Cerus’ progress: green are on going; red
are stopped or not open
1. Open Milestones:
a. Milestone 40: Phenotyping of F.t. novicida NER mutants
i. Measure attenuation of live uvr mutants in vitro, in macrophages,
and in mice
b. Milestone 41: Optimization of photochemical treatment and characterization
of KBMA F.t. novicida
i. Establish photochemical inactivation regimen
ii. Measure metabolic activity of uvr mutants after photochemical
treatment
iii. Determine the level of virulence of KBMA F. novicida
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
d. Milestones 42, 43, 44, 45, 47
i. not yet open; some dependent on reagents from UTSA
ii. MS#42 will be opening next month to determine whether the KBMA
uvrB F novicida vaccination protects against wild-type Ft novicida
challenge
2. MS 40: Flow Diagram: Phenotyping of F.t. novicida NER mutants
a. Initially selected media for liquid and plate culture
b. Measured:
i. Growth in vitro: All strains have identical growth rate in CDM
ii. Growth in cells: All strains have identical growth rate in J774 cells
iii. Virulence:
1. All strains are still virulent by IP route
2. NER mutants slightly attenuated by IV
3. uvrB mutant 2 logs attenuated by SC
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a. Pending: LD50 of uvrA and uvrAB mutants by SC
route
iv. Growth in mice
1. Growth rate of U112 in lungs, livers, and spleens after IV
administration completed
a. Pending: Growth rates of NER mutants in lungs,
livers, and spleens
3. MS 40: Summary of Previous Progress on Phenotyping of Ftn NER mutants
a. Ft novicida uvrA, uvrB, and uvrAuvrB mutants do not have growth
defects in CDM
b. Ft novicida uvrA, uvrB, and uvrAuvrB mutants do not have growth
defects in J774 macrophages
c. WT, uvrA, uvrB, and uvrAuvrB strains are fully virulent by IP infection
d. WT, uvrA, uvrB, and uvrAuvrB strains were all highly virulent when
administered IV, but mutants may be ~1 log attenuated with the difference of
between 1 and 10 CFU. Cerus is also detecting a 2 log difference in SC
route between the mutant and wildtype.
4. MS 40: SC LD50 of Ftn strains
a. SC is the route by which Francisella is least virulent SC Dose range
b. 1x108 – 1x100 (9 groups at 3 animals/group)
c. Focused on WT U112 and uvrB at first to establish dosing range; never
seen LD50 publication for the SC route
d. All Ftn strains still highly virulent when administered IV or IP
e. Virulence of Ftn may not be as route dependent as LVS (in literature)
f. NER mutants may be ~1 log attenuated by IV route (caveat: very difficult to
determine accurate LD50 at this low dose level)
g. By SC route, U112 had LD50 of 1x103, and uvrB mutant of F novicida had
and LD50 of 1.28x105. So the mutant appears to be 2 logs attenuated.
h.
i. Will determine SC LD50 for uvrA and uvrA/B mutants in F novicida as well.
Will also repeat the study with uvrB mutant in F novicida. Maybe with SC
injection, there are macrophages or dendritic cells that are activated or other
cells that are preventing Fn from disseminating to the primary organs. The
attenuation of the uvrB Fn mutant suggests that the blocking of
dissemination potentially involves DNA damage unable to be repaired by the
NER mutant.
j. RICK: intranasal infection with uvrB Fn mutant showed no difference in
virulence from wildtype Fn in Bernard’s lab. With iv, ip, and in, the cellular
recruitment is very different that with sc vaccination, which gives a major
inflammatory response and may impact the phenotype.
k. Justin: will repeat with uvrB Fn mutant and use uvrA mutant, in parallel.
l. Rick: Was the mean time to death different? Justin: fairly similar mean time
to death Action: Justin will include the mean time to death in next
presentation on the LD50 with the uvr mutants of F novicida.
m. NER mutations may attenuate Francisella in certain cells or organs
5. MS 40: Biodistribution Studies
a. Goals
i. Determine the growth rates of NER mutants in various organs and
compare to wild-type strains. There maybe organ specific differences
between the mutants and the wild-type strain.
ii. Measure the reduction in growth after vaccination as an alternative to
survival
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b. First pilot study to determine the growth rate of U112 and LVS after IV
administration of bacteria. AS07-002
i. Establish baseline parameters for future comparisons
ii. 100 cfu chosen for both strains
1. Equal cfu dose tofacilitate growth rate comparison
2. 100x LD50 of U112 (lethal dose)
3. ~.01xLD50 of LVS (sublethal dose)
4. In future, will also use a lethal dose of LVS too.
iii. LVS plateaus earlier than Fn U112 in growth in spleen, liver, and
lungs
1. Harvested spleens, livers and lungs from 3 mice per time
point and first harvest was 4 hrs after infection, to give some
time to replicate.
2. Harvested lungs at four hrs after infection. Didn’t detect in
lungs till day 2 and 3
3. Lethal dose of F novicida grew logarithmically and then the
animals died after day 3.
4. Sublethal dose of LVS grew logarithmically till day 2 in the
liver, day 3 in spleen and then stopped replicating. Maybe
the LVS is being killed at the same rate that they are
replicating or maybe they are in a persistent state.
5. In lungs, Cerus saw no colony forming units until day 3. So
as the LVS are slowing down in spleens and liver, the LVS
is just coming up in lungs.
6. As of day 4, no natural deaths in any group with LVS
infection.
6. MS 40: Next Steps
a. Compare growth of NER mutants (uvrA, uvrB, and uvrAuvrB) to U112
after IV administration.
i. Saw 1 log difference with IV; maytell us in which organs the
nucleotide excision repair machinery is needed. .
ii. Determine whether NER abrogation affects growth in specific organs
b. Measure the biodistribution after SC administration
i. Measure the rate of dissemination to organs
ii. Route with biggest log difference; attenuation
iii. Will measure the LD50 SC for uvrA and uvrA/Bmutantsas well; is the
2 log attenuation the same for all the NER mutants?
7. MS 41: Optimization of Photochemical Treatment and Characterization of
KBMA Ftn
a. Determine minimal S-59 concentration required for complete inactivation:
uvrA, uvrB, uvrAB, vs. U112 All the NER mutants were about 2 fold more
sensitive to psoralen inactivation.
b. Determine the minimal UVA dose required for complete inactivation: uvrA,
uvrB, uvrAB, vs. U112;
c. Measure metabolic activity after photochemical treatment: uvrA, uvrB, uvrAB,
vs. U112
d. Select optimal uvr candidate for further study: uvrB
e. 400mL scale inactivation process optimized, Lot of KBMA uvrB produced
i. QC of KBMA Ftn vaccine lot: Sterility, metabolic activity, stability
ii. Measure attenuation in mice: IP, IV, SC
iii. Move on to Milestone 42: Determine whether KBMA Ftn uvrB
protects against lethal Ftn challenge
8. MS 41: Previous Progress on Optimization of Photochemical Treatment
Regimen
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a. Optimized S-59 and UVA doses at 3.5 mL scale
i. Minimum S-59 concentration required to inactivate ~1 x 1010 cfu
1. U112 = 40M
2. uvrA, uvrB, + uvrAuvrB = 20 M
ii. 4 J/cm2 was the minimum dose of UVA required to achieve
consistent inactivation (at 3.5 mL scale)
iii. Metabolic activity profiles of all strains were similar
b. Optimized 400mL scale inactivation conditions
i. 40M S-59 (used twice the minimum) + 6 J/cm2 UVA > 1x109
inactivation
ii. Had moved from 4 j/cm2 and later up to 6 j/cm2 to get full inactivation.
9. MS 41: Production of 400mL lot of KBMA Francisella tularensis ssp. novicida
uvrB
a. 1st attempt to produce lot failed QC (1 cfu/ml of final product) out of 4x10 10
i. cfu determination repeated (with same result)
ii. MTS assays performed
iii. animals were challenged
b. 2nd attempt under same conditions also failed (1 cfu/ml of final product out
of 4x1010/ml). This is almost complete inactivation, but not 100% inactivation.
c. 3rd lot produced with 7J/cm2 was sterile; there was complete killing; 0 CFU
out of 5x1010 colonies is complete inactivation.
d. 6-7 j/cm2 has been used for anthrax and Listeria, so 7 j/cm 2 for Ft is
reasonable.
e. Justin thinks is a reasonable dose of UVA light
10. MS 41: Metabolic Activity KBMA Ftn uvrB
a. Lot 948-164 has metabolic activity (1cfu lot)
b. 1 day after freeze (in 10% sucrose) metabolic activity is comparable to
prefrozen metabolic activity; T=0 is 16 hrs after the lot was frozen at -80C.
c. Will use 10% sucrose since it it behaves similarly to “freezing media” in LVS
stability study
d. This lot has 1 cfu/3.4 x1010 particles (not likely contributing to MTS)
11. MS 41: Virulence of KBMA Ftn uvrB
a. This lot has 1 cfu/3.4 x1010 particles (not likely contributing to virulence)
b. Two doses (1x108 or 1x109 ) and iv, ip, and sc routes
c. Used a heat killed uvrB Ftn as a control and just ip, which is the route that
live Ftn is most virulent.
d. SC: lost no animals in 10x108 or 10x109 group
e. IV group: lost 3 of 5 animals in 10x108 group and lost all animals in 10x109
group. So the LD50 is probably close to 10x108. It is surprising that the IV
group has a lower LD50 than the IP group with KBMA but not for live.
f. The LD50 for KBMA Listeria iv is in 10x108 range also.
g. KBMA deaths all within 24 hrs, like a toxic shock response while the live Ft
infection results in death in 3-4 days post administration. The KBMA deaths
may be related to the input dose.
h. KBMA uvrB is dramatically attenuated: 4 logs SC, ~8 logs IV, and > 8 logs
IP; this is what want to see; KBMA makes the vaccine much safer
i. SC KBMA LD50 is >109 and lost no animals
ii. SC live LD50 is 105
iii. IP heat killed Ftn uvrB, saw no animal deaths at 108 group but did
see some animal deaths in the 109 group with KBMA Ftn uvrB.
i. So KBMA Ftn uvrB mutant is a little more virulent than heat killed Ftn uvrB
mutant
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j.
Survivors will be challenged with 100cfu U112 IP 1M after vaccination
12. MS 41: Next Steps
a. Complete QC on 400mL lot lot 948-202 of Ftn uvrB (sterile lot)
i. Measure degree of metabolic activity using MTS assay
ii. Measure stability of lot at –80o C over time
iii. Establish degree of attenuation of KBMA vaccine
1. In BALB/c mice via IV, and IP routes; won’t do SC since
were avirulent and can’t give a dose much higher than 109
dose.
b. Initiate Milestone 42: Determine whether KBMA Ftn protect against wild-type
Ftn challenge
i. Have many vials of KBMA Ftn uvrB mutant at 1010 and higher
particles, which allows start MS#42.
ii. Challenge mice from LD50 study (1 cfu lot) which have been
vaccinated 1 time, to determine which routes are most protective; will
challenge at 1 month after the prior vaccination; Will get protection
data with challenge in ~1 week; Will accrue data on whether the sc iv
or ip vaccination routes work for protection.
iii. Also will vaccinate mice with 0.1 LD50 KBMA Ftn uvrB IV and then
challenge with lethal dose of wildtype Ftn U112 after a month. Will
run multiple arms thus
1. Will do T cell depletion studies (CD4, CD 8 or both)
2. Will compare to non-depleted animals, to determine whether
KBMA vaccine is recruiting T cells
iv. Rick: take step back, prior to immunology studies, to review the
multiple vaccination routes and determine which vaccination route
gives the best protection.
v. Justin: The best immunization route for protection will occur before
the immunology studies. The vaccinated animals will be challenged
IP , using 100 CFU of wildtype Ftn U112. This challenge dose is
100x the LD50 and will occur 1 month after the vaccination.
vi. Rick: In animal models for F. tularensis, there is a problem trying to
protect against lung challenge when vaccinated by other routes like
SC or IP. Action: Justin will talk to Bernard who may have used a
transposon mutant given SC to protect against an IN challenge.
vii. Justin agrees will need route optimization and trying to get proof of
concept study initially.
viii. Rick: Review this information while performing pilot study. Don’t start
much immunology until review the route of immunization vs route of
challenge.
ix. Justin: In the literature, the immunological characterization of an
immune response to an Ft systemic infection is sparse.
13. MS 46: 3L-Scale Propagation of LVS
a. Scale up of KBMA LVS vaccine production
i. Select agar and liquid media that support robust growth and viability
of LVS CHAH and CDM
1. Develop 3L scale fermentation conditions for LVS CDM
Sigma antifoam A
a. Confirm preservation of LVS virulence
b. Develop 3L scale photochemical inactivation
conditions
i. Develop 3L scale purification conditions
optimize TFF for LVS
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1. Confirm protective efficacy of KBMA
LVS Produced by 3L-scale methods
ii. Demonstrate KBMA LVS is avirulent
iii. Demonstrate KBMA LVS protect against
lethal LVS challenge
2. Develop cryopreservation conditions for LVS 8% DMSO +
1% sucrose vs. 10% sucrose
a. Monitor stability of frozen LVS
14. MS 46: Summary of Previous Progress on LVS Scale-Up
a. High efficiency of LVS cfu recovery on CHAH agar plates
b. Robust growth of DVC lot 16 LVS in CDM in shaker flasks
i. LVS expanded and frozen; see prior reports in parallel LVS with Fn
ii. Optimized the media as CDM with sigma antifoam A
c. 3L LVS grown in fermentor using CDM and Sigma antifoam A
d. Efficient LVS cryopreservation in 8% DMSO or 10% sucrose
i. Stability ongoing- if works for live LVS should work for KBMA LVS in
the future
e. LVS virulence determination ongoing
i. Cerus LVS IP LD50 range 1x103-3x104 vs. Green et. al 2005: 4x100;
Cerus range is very different from the prior literature reports and
Cerus results are consistent with UNM results.
ii. Cerus expanded LVS is ~10x more virulent than DVC lot 16 by the
IP route; the expanded Cerus LVS is 5 logs attenuated by the IP
route relative to literature reports.
iii. Cerus IV LD50 range 3x103-7x104 vs. Green at al 2005 at 2.2x104;
Cerus IV LD50 is similar to the IV literature but is very different from
the literature on IP route.
iv. The LVS LD50 were very similar whether IV or IP route and this is
similar to the observations with wildtype F novicida, in Cerus’
experiments.
15. MS 46: SC Virulence of LVS
a. Attempted to determine the LD50 of Cerus expanded lot 16 LVS via the SC
route
b. Because DVC LVS virulence appeared to be less route sensitive
i. Broad 10-fold serial dilutions ranging from 1.26 x108 to 1.26 x 102
administered, which is an intentionally broad range
c. At highest dose of 1.26 x 108, got 33.3% lethality was achieved (1 of 3 died)
no other mortalities. So the Cerus LD50 will be probably be greater than
1x108 and be similar to Green et. al, 2005 published LVS SC LD50 of 1.3
x109.
d. Cerus needs a higher titer stock to determine actual SC LD50 (but virulence
appears similar to published data) to start at a higher dose.
e. LVS is dramatically attenuated when delivered by the SC and maybe due to
recruitment of the inflammatory cells to the SC site, and or prevention of
systemic dissemination
16. MS 46: Cryopreservation of 3L lot of LVS
a. Cells suspended in “freeze buffer” or 10% sucrose
b. Suspension diluted twice and plated for cfu on 5 plates (pre-freeze)
c. Cells frozen at –80oC
d. Samples thawed, diluted, and plated for cfu at times indicated
e. Stable for > 2 months
f. At least 100 % viability; maybe freeze/ thaw cycle results in small chains of
the bacteria, causing increases in CFU or maybe within range of error of the
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assay and the viability may go down within 3 months. Will determine
empirically.
g. Rick: Are you flash freezing the bacteria?
h. Justin: don’t flash freeze; just place in the -80C to freeze. Prior studies
showed flash freezing of other organisms was not advantageous.
17. MS 46: Stability of Cryopreserved LVS
a. MTS assays performed on frozen Lot 948-119 Arm-1 (DMSO stabilized) and
Arm-2 (10% sucrose stablilized)
b. Compared with lyophilized DVC lot 16 LVS
c. Metabolic activity preserved for greater than 2 months
d. When normalized to OD600 of 0.5, the live vaccine has much more metabolic
activity than the DVC lot 16 LVS , which has much less metabolic activity; By
this measure, lyophilization decreases metabolic activity. This maybe
explained by dead bacteria in the lyophilized vial.
e. Suggests that DVC lyophilized lot 16 LVS has many dead bacteria.
f. The colony forming units are substantially below the OD600 expectation.
g. When normalized to CFU, then DVC lyophilized lot 16 LVS has greater
metabolic activity than the frozen Cerus cryopreserved LVS; By this
measure, lyophilization may result in bacteria that have higher metabolic
activity or that the “dead” bacteria are high in number and have residual
metabolic activity.
h. Vicki: like a KBMA essentially (dead bacteria in lyophilized vial have
metabolic activity)
i. Justin: Yes, suggests that DVC lot 16 has many dead bacteria, but perhaps
the dead bacteria also retain some degree of metabolic activity
j. Justin: About a 100 fold difference exists for the OD to CFU ratio, with the
lyophilized DVC lot 16 LVS.
k. Rick: QC on lyophilization usually done on CFU recovered while the
metabolic activity maybe more objective and may give an overview of the
population health of the preserved bacteria.
l. Justin: It is harder to make the metabolic activity truly quantitative; it is a
relatively qualitative assay. Can’t currently determine an absolute number of
bacteria. MTS is more quantitative than examining only CFU, since can
measure the metabolic activity of bacteria that can’t form a colony.
m. Vicki: Could the MTS assay determine what kind of metabolites are being
made by the dead or dying bacteria? Could Quorum sensisngor something
being occurring here?
n. Justin: No, not with the MTS assay. May need to lease very expensive
Biolog equipment to measure what the bacteria are metabolizing or growing
on (in minimal media with addition of different Nitrogen/carbon sources to
sustain growth) but equipment does not measure the metabolites that are
being produced. Cerus will lease the equipment when have a KBMA assay to
test. Cerus budgeted to lease the equipment for 1 year only.
18. MS 46: Next Steps
a. We will continue to monitor stability of fermentor-grown LVS in “freeze buffer”
vs 10% sucrose
i. Monthly plating for CFU
ii. Monthly MTS assay
iii. We will compare the virulence of fermentor-grown LVS stored in
“freeze buffer”or 10% sucrose, to expanded and freshly suspended
DVC lot 16
b. We will prepare a 400mL batch of photochemically inactivated LVS – without
an NER mutant.
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i. Trying for proof of principal of KBMA LVS, since don’t have uvr
mutations of LVS from UTSA yet
ii. Grown in shaker flasks (both glass and plastic)
iii. With Sigma antifoam A
iv. With 10, 20,and 40uM S-59 : range of doses
v. Will illuminate with 6J/cm2UVA or increase to 7 J/cm 2
vi. QC for Sterility and Metabolic activity
vii. If lots pass QC, we will determine virulence and vaccinate mice
B. Other Topics:
1. Next Cerus Tech Call will be Tuesday, March 13th. 11am-12:00pm PST, 12:00pm1:00pm MST, 2:00pm-3:00pm EST
2. Justin is attending the ASM Biodefense meeting in D.C. to present Anthrax data
and not presenting the Ft data. Justin is presenting on the Wednesday session.
3. Vicki may attend some sessions of the ASM meeting and Rick will not be attending
this year. Vicki will tell Kristin about Justin’s presentation.
C. Comment:
Vicki, Rick and Barbara: praised Cerus for the quality and quantity of work shared
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