Cerus - UNM Tech Call Minutes: 3/13/07
Prepared by: Barbara Griffith 3/15/07
Provided Draft to Justin: 3/15/07
Reviewed and edited by: Justin Skoble 3/15/07
Provided to NIAID: 3/16/2007
Present: Justin Skoble, Barbara Griffith, Rick Lyons, Marlene Hammer, Sam Kim,
Absent: Joe Breen, Kristin De Bord, Freyja Lynn,Tom Dubensky, Vicki Pierson,
Action Items:
Barbara- will improve site visit agenda appearance and then share with NIAID
Justin-The KBMA gene expression experiment could be discussed at the Cerus site visit
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 42: Determine whether KBMA F.t. novicida vaccine protects
against wild-type F.t. novicida challenge in mice
i. Vaccination route and regimen optimization, measure durability of
protection
ii. Newly started milestone this month
d. 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
e. Milestones 43, 44, 45, 47
i. not yet open; some dependent on reagents from UTSA
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
a. Pending: LD50 of uvrA and uvrAB mutants by SC
route
iv. Growth in mice
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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
with LD50 of around 1
d. WT, uvrA, uvrB, and uvrAuvrB strains were all highly virulent when
administered IV, but mutants may be ~1 log attenuated. First SC: LD50
uvrB mutant 2 logs attenuated compared to WT
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. 1st observation: uvrB was 2 logs attenuated by SC route (103 vs. 105)
h. 2nd SC study: all NER mutants have similar virulence by SC route
i.
But, WT was less virulent on second attempt (need to repeat study); Justin
thinks the U112 data is spurious in the second SC study; lost 1/3 animals at
many doses which impacts the data; serial dilutions went well; some mice
died and some didn’t although all lost weight; surprised to see same number
of animal deaths over a 4 log range. So will repeat the experiment. UvrB
data is consistent over 2 studies but control U112 appears variable.
j. Rick: no historical data on U112 with this route sc?
k. Justin: none available in his lab or literature
l. Justin: use frozen vial each time with same dilution scheme
5. MS 40: Biodistribution Studies
a. Goals
i. Determine whether NER mutants have growth defects in mouse
organs: lungs, livers, or spleens
ii. Ultimately this type of assay could be used to measure the degree
of protection vaccination provides by measuring the reduction in cfu
after lethal wild type infection instead of survival
iii. Want to know how quickly they grow and whether can use organ
dissemination as an end point rather than death.
b. AS07-034
i. Some differences in final cfu counts
ii. All NER mutants grew as well or better than WT (U112) in all organs
in this study
iii. Suggests strongly that NER mutants are not attenuated for growth in
any of these 3 organs after IV administration. LD50 by IV are also
about the same.
iv. See long lag in growth and see growth at day 2-3
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6. MS 40: Next Steps
a. Repeat SC LD50 to: 1) establish the level of virulence of U112 and 2)
determine whether NER mutants are attenuated
b. Maybe a stochastic , sc route
c. Primarily use sc for vaccination rather than for challenge.
d. If NER mutants are attenuated, we will measure the biodistribution after SC
administration to better understand the reason for virulence attenuation; but
wait to see difference in virulence before doing the biodistribution
7. MS 41: Optimization of Photochemical Treatment and Characterization of
KBMA Ftn
a. Made a lot of progress
8. MS 41: Previous Progress on Optimization of Photochemical Treatment
Regimen
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
c. KBMA uvrB are highly attenuated >8 logs IP, ~8 logs IV, ~4 logs SC; live
are progressively less virulent by these routes
d. Characterizing for stability and sterility
9. MS 41: 400mL lots of KBMA Francisella tularensis ssp. novicida uvrB
a. Lots stored in HBSS +10% sucrose at –80oC
b. Lot 948-164 failed QC (1 cfu/ml of final product)
i. MTS assays performed
ii. Produced with 6 j/cm2
iii. Mice challenged to demonstrate reduced virulence
iv. Stability ongoing
c. Lot 948-202 arm-2 (produced with 7J/cm2) was sterile
i. MTS assays performed
ii. Stability ongoing and is a sterile lot
iii. Will be doing animal studies in the future
iv. Need to repeat some LD50 studies
10. MS 41: Metabolic Activity KBMA Ftn uvrB is stable for two months
a. Lot 948-164 metabolic activity is equivalent after 1 month at –80oC
i. 2 month values are a little lower than T zero but within variation of
the MTS assay.
b. Slight decrease in metabolic activity after 2 months, but within error
c. This lot has 1 cfu/3.4 x1010 particles (not likely contributing to MTS)
11. MS 41: Metabolic Activity of Sterile KBMA Ftn 7J/cm2 lot is comparable to
6J/cm2 lot
a. Little “ lot to lot “variability
b. Lot 948-164 was produced with 6 J/cm2 has 1 cfu/3.4 x1010 particles
c. Lot 948-202 produced with 7 J/cm2 and is sterile and has high metabolic
activity; no negative impact for increasing uv dosage.
d. Metabolic activity between lots is comparable
12. MS 42: Determine whether KBMA Ftn protect against wild-type Ftn challenge
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a. New flow chart for this newly opened milestone
b. Optimize routes of KBMA and wild type delivery, optimize dose, determine
number and timing of vaccination, may need multiple doses to get optimal
protection, will determine durability
13. MS 42: Determine whether mice vaccinated with KBMA Ftn are protected
against lethal Ftn challenge
a. Challenged mice from LD50 study (ASO7-009) to determine which routes are
protective
i. Doses of 1 x 109 and 1 x 108 KBMA Ftn uvrB (lot 948-164) were
injected into BALB/c mice SC, IV, IP
ii. Used 1x109 heat killed as control too
iii. This lot has 1 cfu/3.4 x 1010 particles
iv. Mice were challenged with 100 x IP LD50 dose of U112 after 1
Month after vaccination (~100 CFU dose)
v. 100% protection with KBMA observed only with IP and IV
vaccination
vi. 100% protection with KBMA observed only at doses that were near
LD50
vii. HK control was less virulent and protected 100% (at 1 x 109 dose);
maybe heat killed looks better as vaccine than a KBMA vaccine.
viii. Only 60 % protection at 0.1 LD50 dose IP, which is not optimal
ix. At 1 x LD50: get protection but really want protection at a lower dose!
x. Don’t know the LD50 dose by the sc route but 1x109 is about the
highest dose that Cerus can achieve.
xi. Need to improve regimen; can get protection 100% by iv and ip but
only with vaccine delivered at an LD50 dose, which is quite high.
Possibly 2 doses would be more protective than single dose (maybe
will mimic Listeria)
14. MS 42: Next Steps
a. AS07-017
i. Vaccinated mice IV with 1 x 107 KBMA uvrB (~ 0.1 x IV LD50) Lot
948-202,
ii. Vaccine administered 2 times separated by 3 weeks
iii. Mice will be challenged 1 month post-boost with 100 x IP LD50 dose
of live wild-type Ft novicida
iv. We will also determine whether CD4+, CD8+, or CD4+ and CD8+ T
cells contribute to this protection by doing antibody depletion studies
by depleting for 3 days prior to the challenge to look at T cell mediate
immunity vs. other mediate immunity.
v. Justin: T cell depletion is a blunt tool, but works to establish whether
we are eliciting T-cell mediated protection. The converse experiment
would be to performadoptive transfer of serum to show it is antibody
mediated
vi. Rick: can also show t-cell dependence by adoptive transfer of cells
vii. Justin: one caveat to this experiment is that Karen Elkins saw a
double negative population
viii. Rick: LVS may look more like F novicida
ix. Justin: starting to look at immunity associated with vaccination
15. 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
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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
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
16. 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
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 was very similar whether IV or IP route and this is
similar to the observations with wild type F novicida, in Cerus’
experiments.
17. 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
18. MS 46: Cryopreservation of 3L lot of LVS
a. Cells suspended in “freeze buffer” or 10% sucrose
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b.
Cells Samples thawed, diluted, and plated for cfu at times indicated Stable
for > 2 months
c. Each sample diluted twice and plated for cfu on 5 plates (pre-freeze)
d. cfu counts were stable for 2 months
e. Drop in cfu at 3 months in both solutions (could be experimental error)
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
assay and the viability may go down within 3 months. Will determine
empirically.
19. MS 46: Stability of Cryopreserved LVS
a. MTS assays performed on frozen Lot 948-119 Arm-1 (DMSO stabilized) and
Arm-2 (10% sucrose stabilized)
b. Metabolic activity of both arms similar over 3 months Suggests drop in cfu
may be experimental. Will be monitoring the trend and hope is stable for
more than 2 months.
c. Rick: LBERI has stored LVS many months in glycerol- lost spray factorperhaps due to cryopreservation stocks diminishing in stability over time.
d. Justin: 3 fold drop is well within error of plating and this is much less harsh
than sprays. So Cerus will continue to monitor the frozen stocks of LVS.
e. With Listeria, MTS assays dropped before the titers were lost
f. Will keep monitoring CFU in the frozen LVS stocks
20. MS 46: Preparation of KBMA lots of LVS
a. Metabolic activity of NER mutants of Ftn were similar to wild-type
b. Suggests that KBMA vaccine could be produced without NER mutation using
S-59 and UVA; have made 2 lots of KBMA LVS w/o mutations
c. LVS maintained metabolic activity for >12 hours after PCT
d. Decided to produce 400mL lot of KBMA LVS for proof of concept studies
prior to receiving NER mutant from UTSA
e. Used 2x and 4x minimum S-59 concentration in 3.5 mL scale
f. Both lots are sterile
g. Increasing the S-59 dose didn’t negatively impact the metabolic activity
21. MS 46: Metabolic activity of KBMA lots of LVS
a. Similar metabolic activity with both arms
b. Suggests drop in cfu may be experimental
22. MS 46: Next Steps
a. We will continue to monitor stability of fermentor-grown live LVS in “freeze
buffer” vs. 10% sucrose
i. Monthly plating for CFU
ii. Monthly MTS assay (can’t plate for CFU because they are sterile and
won’t grow)
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 monitor the stability of photochemically inactivated lots of LVS in 10%
sucrose
i. Monthly MTS assays
c. We will determine virulence of photochemically inactivated LVS
i. Administered by SC, IV, IP and IN routes; probably won’t get usefully
data sc but Iv and ip routes may be more useful. Techs are doing i.n
vaccinations and gaining confidence based on protocols from UNM.
ii. Survivors will be challenged with lethal dose of LVS
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iii. Rick: i.n. LVS vaccination is more optimal for protecting against
SCHU S4 challenge
iv. Justin: if T cell mediated, then may get a better systemic response
than if the response if combination of T cell and B cell mediated.
v. Rick: i.n.- very useful tool before get some other mutants from Karl.
vi. Justin: little disappointed that protection may require a boost, but not
too unexpected since it would be like Listeria KBMA
vii. Rick: usually inject KBMA Listeria IV?
viii. Justin: yes
Additional Discussion:
1. Site visit: draft agenda looks okay to Justin
Action: Barbara- will improve site visit agenda appearance and then share with NIAID
2. Justin commented on the Biodefense meeting: Mark Bolanowski and Justin Skoble both
attended and discussed the KBMA vaccine approach. Justin asked if we could look at
transcriptional profile of KBMA vs. live organism using ASU gene expression microarray slides?
Rick: look at broth culture gene expression first? Justin: may differ from in vivo but agrees that
may be harder to detect KBMA LVS gene expression in presence of animal organ RNA… can
use GDP primers though. Rick: If KBMA protects, then gene expression is more intellectually
challenging and interesting. Action: JustinThe KBMA gene expression experiment could be
discussed at the site visit.
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