Prepared by: Mindy 7/16/06
Edited by: Barbara 7/17/06, Justin Skoble 7/24/06
Sent to NIAID: 8/23/2006
Present: Justin Skoble, Jack Joseph, Tom Dubensky, Barbara Griffith, Joe Breen, Mindy Tyson
Absent: Vicki Pierson, Marlene Hammer, Rick Lyons
Action Items:

Action : Barbara – find out what OD is preferred by TVDC teams (both at UNM and DVC) so we can standardize (in progress 7/17/06)

Action : Justin / Jack : Barbara requests that Cerus present their Quality Assurance at the
August meeting. The brief QA report/slide would include the instrument maintenance, pipette calibrations, comparison of old and new lots of reagents etc. that have been performed in the prior 6 months.
A. Discussion of the progress of milestones
Justin presented an 11 slide PowerPoint presentation on Cerus’ progress
1. Active Milestones: Selection of agar and liquid media formulation has facilitated progress on milestones 40, 41, and 46. a. Milestone 40: Phenotyping of F.t. novicida uvr mutants i. Measure attenuation of uvr mutants in macrophages and in mice ii. Since F.t.
enters the phagosome and Ft can incur DNA damage in the phagosome, that perhaps the uvr mutants would actually be attenuated in the intracellular compartment. b. Milestone 41: Optimization of psoralen treatment and characterization of
KBMA F.t. novicida c. Establish photochemical inactivation regimen d. Measure metabolic activity of uvr mutants after photochemical treatment e. Determine the level of virulence/attenuation of KBMA F. novicida
2. Milestone 46: Scale-up of KBMA LVS vaccine production a. Optimize large –scale LVS culture conditions b. Establish 3L culture scale purification conditions c. Optimize 3L scale photochemical inactivation process d. Verify protective immunogenicity of vaccine candidates produced by optimized large-scale process
3. Progress on MS 40: Ft novcida

uvrB Mutant Has No Growth Defect in J774
Macrophages a. Assay optimization ongoing i. Infected with 2 different MOI: a low and a high multiplicity of infection ii. Plating determined that the MOI were 7 and 33 iii. Cerus measured the U112 (a wild type strain) and the uvrB mutant growing inside the J774macrophages – the usage of gentamicin in the culture media 2.5 hours after infection – assures that the extracellular bacteria are killed so Cerus is only seeing replication of intracellular bacteria iv. Both mutants grow at these multiplicities with identical relative characteristics, suggesting there is no defect in the growth of U112 and uvrB mutant of F. novicida in J774 macrophages b. More bactericidal macrophages to be tested i. J774 cells are not tremendously bactericidal
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ii. A more activated macrophage might reveal some differences, if there are any iii. In the next month Cerus will try a more bactericidal cell line and eventually will look at bone marrow derived macrophages c. Data suggest that attenuating mutation required for KBMA SCHU S4 i. In the long run we may need an attenuating mutation in conjunction with the DNA repair genes ii. In a later milestone, Cerus will look at double mutants (Igl or others, plus the uvr mutants in LVS) to see whether they maintain immunogenicity
1. we’ll be screening through 5 attenuating mutations produced by Karl
2. selections will depend on data with live attenuated vaccines
4. Progress on MS 41:

uvrB is More Sensitive to S-59 + UVA Photochemical
Inactivation than U112 a. Cultures were incubated with S-59 Continuously (switched from 1 hour incubation method) i. Cerus compared uvrB mutant in Fn to U112 (wild type)
1. uvrB is more sensitive and completely inactivated at a concentration of 5

M S-59
2. 20

M S-59 was required to inactivate the U112 strain
3. So there is a 4-fold increase in concentration between the uvrB mutant and the U112 srains, which is a little less difference than we’ve seen with some of our other bacterial strains
4. The difference in the S-59 concentration demonstrates the uvrB phenotype is as expected and increases sensitivity to photochemical treatment b. Exposed to 6.5 J/cm2 and plated on CHAH plates c. Inactivation profile still not as different as uvrAB mutants of Lm or Ba
5. MS 41: Photochemically Inactivated

uvrB Mutant Maintains Metabolic Activity a. Cerus used the same number of bacteria (1x10 8 ) of uvrB mutant and treated either with i. 5uM S-59 plus UVA ii. UVA alone iii. No UVA b. The uvrB mutant maintained a high degree of metabolic activity after S-
59/UVA exposure despite this organism being incapable of replication, suggesting that the KBMA concept is working ( killed but metabolically active vaccine strain) c. Slight decrease in metabolic activity observed upon exposure to UVA or S-59 and UVA, suggesting that bacteria are responding and changing their metabolic profile in response to just the ultraviolet light illumination by itself d. Cerus is optimistic that a KBMA vaccine can be developed in F novicida, as a model
6. Next Steps for MS 41: Next Steps for MS 41: Optimization of Photochemical
Inactivation of Ft novicida a. Optimize UVA illumination dose (ranges between 2-7 J/cm2) b. Compare metabolic activity of inactivated U112 and
 uvrB using MTS assay i. Goal is to show that uvrB mutant has a higher degree of metabolic activity c. Should receive
 uvrA mutant from UTSA this month
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i. Optimize photochemical inactivation conditions for
 uvrA ii. Compare with
 uvrB and U112 – will do a kill-curve with optimal concentrations of UV light as determined with uvrB .
1. assumption is that phenotypes will be the similar between uvrA and uvrB
7. Progress on MS 46: Update on Media for F. tularensis a. New 5 Kg lot (# c071516F601) of Chamberlain’s Defined Medium (CDM) was ordered from Teknova. Had slightly different appearance than 1st lot – was much lighter in color. b. Growth of LVS was identical to original lot (#c071110D601) i. found that the old lot had actually gotten darker in the bottle ii. darker appearance may be the result of some oxidation of some ferrous material in the powder, but it doesn’t seem to affect growth parameters. c. Samples of lot # c071516F601 were distributed to LBERI and DSTL – they will test on type A and LVS strains d. Question: What wavelength is optimal for Ft (540, 600, 620)? i. At Cerus, typically do OD600 for bacteria ii. Literature is divided on what wavelength to use for measuring optical density (OD540 – OD620) iii. Might be a good idea to get a consensus on this and use the same
OD for measuring growth parameters iv. At OD600, Cerus finds that the number of colony forming units is not entirely linear to optical density over time at early vs. later log – we see a slight difference in CFU to OD v. Action : Barbara – find out what OD is preferred by TVDC teams
(both at UNM and DVC) so we can standardize (in progress 7/17/06)
8. MS 46: Selection of DMSO as Cryopreservation Agent for LVS a. Typically use frozen KBMA bacteria as our vaccine stocks b. When growing up large scale cultures Cerus usually uses frozen seed stocks to initiate the culture because it’s a more standard approach c. DMSO preserves higher degree of viability than glycerol (at 2.5 weeks and -
80) d. Additional supplements (sucrose, Mg++) will be evaluated e. Typically Cerus freezes bacteria down at early stationary phase because you get higher CFUs and better viability - in general it’s a more standard practice
9. MS 46: Preliminary Photochemical Inactivation of LVS a. Complete inactivation of LVS observed at S-59 concentration of 20

M b. This concentration is identical to Ftn U112 c. Suggests Ftn capsule is not a confounding factor d. Cerus will continue to better define the optimal S-59 dose range for LVS
(between 10 and 20

M, and will include ~5uM too)
B. Problems that have arisen and approaches that need to be discussed

Still waiting on DOD federal rate agreement
C. Goals for the next month
1. Repeat Cultivation of LVS in the fermentor a. Monitor the pH, O2, OD600, CFU in Chamberlain’s defined medium (CDM) b. Compare growth characteristics and viability of LVS in CDM vs. DVC medium
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c. The lack of growth of the initial fermentor culture may have been due to technical error.
D. Summary
1. uvrB gene is not required for growth in vitro or in J774 cells
2. Demonstrated increased sensitivity of Ftn
 uvrB mutant to photochemical treatment
3. Photochemically treated Ftn
 uvrB maintain metabolic activity after inactivation
4. LVS appears to have an inactivation profile similar to U112
5. This suggests that work with novicida should continue and is likely to be predictive of
LVS results.
E. Other Topics: Next Cerus Tech Call will be Tuesday, August 8 th . 11am-12:00pm PST,
12:00pm-1:00pm MST, 2:00pm-3:00pm EST . NOTE ADDED AFTER MEETING : Barbara requests that Cerus present their Quality Assurance at the August meeting. The brief QA report/slide would include the instrument maintenance, pipette calibrations, comparison of old and new lots of reagents etc. that have been performed in the prior 6 months.
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