Cerus - UNM Tech Call Minutes: 5/08/07
Prepared by: Barbara Griffith 5/8/07
Provided Draft to Justin: 5/8/2007
Reviewed and edited by: Justin Skoble 5/10/07
Provided to NIAID: 5/10/07
Present: Justin Skoble, Barbara Griffith, Rick Lyons, Marlene Hammer, Sam Kim, Vicki Pierson,
Caryl Trotter
Absent: Joe Breen, Kristin De Bord, Freyja Lynn,Tom Dubensky,
Action Items from May tech call:
Rick: will do literature search and will call Bernard regarding organ clearance post sc infections.
Rick/ Justin start an email thread and then schedule an off line call with Terry on KBMA LVS
vaccination and then SCHU S4 challenge experiment protocol.
Barbara: email Karl to send uvrB mutant of LVS to Justin. (completed email to Karl 5/8)
A. KBMA Tularemia Vaccine Progress
Justin presented a 24-slide Power Point presentation on Cerus’ progress: green are on going; red
are stopped or not open
1. SLIDE 2: Open Milestones:
a. Milestone 40: Phenotyping of F.t. novicida NER mutants
i. Milestone is completed; plan to provide MS completion report by end
of June
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. SLIDE 3: MS 40: Flow Diagram: Phenotyping of F.t. novicida NER mutants
3. SLIDE 4: MS 40: Summary of Previous Progress on Phenotyping of Ftn NER
mutants
4. SLIDE 5: MS 40: Biodistribution Studies
5. SLIDE 6: MS 40: Biodistribution Studies
a. 10x LD50 for mutant
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b. Grow rapidly for two days and then decrease by day 3 which was surprising
for a lethal dose. Didn’t expect the lethal dose to clear.
c. Virulence defect may be due to decrease ability to disseminate to lungs with
sc infection
d. Justin: asked Rick if virulence or time to death seems to correlate with lung
burden, rather than liver and spleen burden?
e. Rick: gives vaccinations via the lungs and Lungs always have 2 logs CFU
more than in liver or spleen, when you start with few organisms. The liver
has more organisms, but when you normalize per gram of tissue, the lungs
have more.
f. Justin: Didn’t keep a cohort that they didn’t sacrifice and plate out the CFU,
so we don’t know for sure whether the animals actually received a lethal
dose. Justin said the animals were a little bit under dosed but it wasn’t 2
logs under.
g. Dose was close to 1 or 2 e5 rather than 5e5 dose.
h. Rick: any literature on clearance after subcutaneous vaccination? Justin
didn’t know of any.
i. Rick: maybe the animals are dying of shock like a cytokine storm?
j. J: Would like to see the study repeated. When delivered IV, bacteria
stopped replicating but didn’t get cleared out like with the current SC.
k. Action Rick: will do literature search and will call Bernard too. Bernard
generally does lung infections rather than sc, but may have done s.c.
l. J: the uvrB mutants are not severely attenuated, which was the goal of the
study. So this mutation would not attenuate SchuS4 enough to be used as a
vaccine.
6. SLIDE 7: MS 40: Conclusions
a. Want to evaluate other mutations than NER
7. SLIDE 8: MS 40: Next Steps
8. SLIDE 9: MS 41: Flow Chart on Optimization of Photochemical Treatment
Regimen
a. Have made KBMA lot of UvrB F novicida
9. SLIDE 10: Progress on Optimization of Photochemical Treatment Regimen
10. SLIDE 11: MS 41: 400mL lots of KBMA Francisella tularensis ssp. novicida uv
11. SLIDE 12: MS 41: Metabolic Activity KBMA Ftn uvrB is stable for two months
a. 3 months stability time point
b. 1 month is lower and is probably due to experimental variation
c. May do next stability in 6 months rather than perform in 1 more month
12. SLIDE 13: MS 41: Conclusions
a. Plan to follow up that the NER genes are not induced; will look at uvrB gene
expression using pcr primers on mutant under photochemical treatment- was
discussed at the site visit
b. Plan to confirm that the lack of big difference in wt and mutant is due to the
lack of uvrB gene induction
13. SLIDE 14: MS 41: Next Steps
a. At 6 months and not at 3 months as listed on the slide
b. Have ordered some DNA damaging agents too.
14. SLIDE 15: MS 42: Flow Chart to Determine Whether KBMA Ftn Protect Against
Wild-Type Ftn Challenge
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a. Had to deliver 1x LD50 dose as vaccination to get protection with single
dose, from Ftn challenge
15. SLIDE 16: MS 42: Progress on POC Studies to Determine Whether Vaccination
with KBMA Ftn is Protective
a. The report in semiannual report was premature- got 100% protection from
the mock depleted group, and 100% protection when CD8 cells were
depleted, but 2 of the mice with CD4 depletion died on day 6 and day 7
which is a little longer to death. So got 80% protection when CD4 cells were
depleted at the time of the challenge. When CD4 CD8 cells were depleted,
then 1 of 10 mice died at day 7. Were in the middle of the experiment when
the semiannual report was due. Data suggest that CD4 T cells contribute to
protection but not a major player at 80-90%. Humoral immunity and that the
CD4 help at the time of the challenge may contribute to increased antibody
responses. Clearly not a CD 8 response.
b. Took serum from mice in this experiment and adoptively transferred into
naïve mice and then gave them a lethal challenge with 100x LD50 IP
challenge of F novicida. Saw a 1-2 day increase in the mean time to death
but didn’t get much protection as 1 mouse out of 20 survived after adoptive
transfer with 300 ul of serum. This was the max sera volume that they
could use. Seeing humoral immunity but not enough to fully protect the
cohort.
c. Bernard has shown that protection against F novicida can be humoral and so
are protection studies really relevant in F novicida model?
d. Decided to study less on protection studies and more on ability to elicit CD8
positive T cell response using the OVA tagged strain.
e. Marlene Question: when transfer serum? Justin: transferred 200ul serum 1
day before challenge and then 100ul 4 hrs before the challenge. Delivered
serum iv and challenge ip.
16. SLIDE 17: MS 42: Conclusions
a. Challenge studies not meaningful when know that humoral immunity protects
b. Karl has the sequence of the tag and is making the ova tagged strain. Justin
will contact Karl for estimated time to completion. R: Karl said not major
thing to make the ova tagged strain.
c. May delay optimization/hold this milestone until have reagent from Karl
d. Rick: optimizing the ability of the KBMA to induce the best cellular immunity
that it can.
17. SLIDE 18: MS 46: 3L-Scale Propagation of LVS
18. SLIDE 19: MS 46: Summary of Progress on LVS Scale-Up
a. Are making KBMA lot of LVS too
19. SLIDE 20: MS 46: Cryopreservation of 3L lot of LVS
a. At 4 months, the viability is back up, so 3 month may be aberrant
b. Using sucrose for all F novicida work and with LVS too
20. SLIDE 21: MS 46: Preparation of KBMA Lots of WT LVS
a. Will focus on lot 968-040 Arm 1
21. SLIDE 22: MS 46: Stability of Metabolic Activity of KBMA LVS
a. 2 month MTS is higher than 1 month or 0 month
22. SLIDE 23: MS 46: Virulence of KBMA LVS
a. KBMA LVS is dramatically attenuated relative to live LVS
b. None of HK LVS vaccinate animals died
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KBMA and HK LVS vaccinations both protected the animals at 1e7 low dose
vaccination but may not be dependent on metabolic activity because the heat
killed protected. So, literature suggests that immunity to LVS infection may
also be humoral.
R: Rick Titbal confirmed this; Rick said that cell mediated immunity is more
important in LVS than F novicida. The humoral immunity enhances the cell
mediated immunity.
Can’t base decision on survival when HK and KBMA are both protective.
J: could UNM do a SCHU S4 challenge with animals protected with the
KBMA LVS lot? R: yes
R: heat killed protection seems to be very short lived, like a month and then
the protection is gone. Justin could wait 4 more weeks and then challenge
with live LVS. This study was done at 1 month post HK LVS vaccination. So
Cerus may have been on the edge of protecting the animals with the HK.
Action: Rick/ Justin have off line call with Terry on SCHU S4 challenge
experiment protocol.
J: Does live LVS only work with lung administration?
R: The route of challenge is also the best way to vaccinate. So if challenge
sc, vaccinate sc. If challenge by aerosol, should vaccinate by aerosol. SC is
more forgiving than aerosol challenge and could use higher doses with SC
than aerosol too because of the delivery systems.
R: can give 50ul i.n.
J: live LVS is difficult unless do it SC. For KBMA vs. Heat killed, can use any
route.
R: UNM has never looked at iv route
J: which route to use?
Do 2 routes of KBMA LVS vaccination and a couple of challenge routes; may
take two vaccination doses as is done for Listeria.
R: boost didn’t make any difference at UNM, for providing protection using
low number of LVS as the vaccination dose.
J: KBMA particles get cleared more quickly than the live LVS particles.
R: has whole bug ELISA working for humoral response to antigens; can see
if boost enhances the response via ELISA.
J: protection against LVS is not humoral
R: higher titer via ELISA doesn’t mean
Action: start an email discussion with Justin, Terry, and Rick- for
experimental design and then follow-up for a telecom when design is close.
J: has the KBMA LVS ready to use
23. SLIDE 24: MS 46: Next Steps
Additional Discussion:
Action Barbara: email Karl to send uvrB mutant of LVS to Justin. (completed email to Karl 5/8)
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