Cerus - UNM Tech Call Minutes: 8/8/06
Prepared by: Barbara 8/23/06
Reviewed and Edited by: Justin Skoble 9/19/06
Provided to NIAID: 9/20/06
Present: Justin Skoble, Vicki Pierson, Rick Lyons Barbara Griffith, Joe Breen, Mindy Tyson
Absent: Marlene Hammer, Jack Joseph, Tom Dubensky
Action Items:
 Action: Justin will do metabolic assay with heat killed LVS to show zero activity in the MTS
assay, since there is so little known about Francisella overall.
 Action: Justin will email Barbara an example excel spreadsheet showing the QA performed
 Action: Jack will draft the September report and provide it to Barbara on 9/7. Barbara won’t
submit the report until Justin reviews it by 9/11.
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.
Milestone 40: Phenotyping of F.t. novicida uvr mutants
Measure attenuation of uvr mutants in macrophages and in mice
Milestone 41: Optimization of psoralen treatment and characterization of KBMA
F.t. novicida
Establish photochemical inactivation regimen
Measure metabolic activity of uvr mutants after photochemical treatment
Determine the level of virulence of KBMA F. novicida
Milestone 46: Scale-up of KBMA LVS vaccine production
Optimize large–scale LVS culture conditions
Establish 3L culture scale purification conditions,
Optimize 3L scale photochemical inactivation process,
Verify protective immunogenicity of vaccine candidates produced by optimized largescale process
2. Milestone 40: Phenotyping of F.t. novicida uvr mutants- uvrB growth in
macrophages
Ft novcida uvrB Mutant Has No Growth Defect in J774 Macrophages
Repeated growth curve +/- 50 ug/ml gentamicin, results similar to last time, out to 37
hours. UTSA found same result with uvrB. UvrAB mutants aren’t attenuated in
Listeria either. Results suggest that nucleotide excision repair mechanism is not
required for intracellular growth of F novicida.
Cerus will determine growth rate of uvrA mutant vs U112.
Justin received Ft novicida uvrA mutant from Karl last week and will be determining
the growth curve, potentially in parallel with UTSA.
The other reason for these experiments is to determine whether a second attenuating
mutation will be needed in SCHU S4, where 100% must be attenuated.
Cerus will be testing virulence in mice too and checking for phenotype to compare to
prior tissue culture results with macrophages in vitro.
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Rick recommends BALB/c with intranasal delivery, but Cerus could do subcutaneous
infection route for wild type vs uvrB mutant comparison
uvrA is unlikely to attenuate alone, just as uvrB alone didn’t attenuate.
3. Progress on MS 41: Titration of UVA dose required for inactivation of U112 and
uvrB
Last month, determined the difference in S-59 concentration sensitivity (5uM for uvrB
and 20uM for wild type F novicida). For the S-59 concentration sensitivity testing,
Cerus kept the UVA dose constant and varied the S-59 concentration. Now
determining minimum UVA dose range to use, keeping S-59 constant at 5uM for uvrB
mutant and at 20uM for F novicida U112 wild type. Cerus wants optimal metabolic
activity with lowest necessary UVA dose and S-59 concentration.
U112 and uvrB were grown in CDM (Chamberlain’s defined media) +/- 20uM S-59
(U112) or 5uM S-59 (uvrB). Cultures were exposed to various doses of UVA,
ranging from 0-6.5 J/cm2
6.5 joules/cm2 was standard for Anthrax so started there first, using minimum S-59
concentrations for complete inactivation of uvrB and for U112.
Illumination was performed with doses ranging from 0 to 6.5 J/cm 2, with 0 as the
negative control and with 6.5 J/cm 2 expected to kill completely. The UVA dose
range experiment was performed twice and both times complete inactivation of the
wild type strain was achieved at 2, 4, and 6.5 J/cm 2. With the uvrB mutant some
residual colonies at 2 J/cm 2 but no residual colonies at 4 j/cm 2. So min UVA dose for
inactivation of uvrB mutant is 4 J/cm2. Then began testing metabolic activity, hoping
uvrB mutant would have good metabolic activity in 5uM S-59 with 4 J/cm 2 UVA dose.
Clarification by Rick:
For U112 in 20uM S59, since wild type retains DNA excision repair mechanisms, it
requires a higher concentration of S-59 to overwhelm their ability to repair the
crosslink. At 20uM S59 it takes very little UVA to result in inactivation. The uvrB is
more sensitive at a lower S-59 concentration because it is lacking one DNA repair
mechanism.
Cerus may repeat a UVA titration around 4 j/cm 2 and then vary S-59 around 5 uM for
uvrB mutant, to be sure they are using the minimum amount of both psoralen and
UVA. Assays were performed at the limit of detection of 10 CFU/ml. They could
plate more of the sample in future experiments to increase the sensitivity of detection
of the live bacteria. When they see 0 CFU from a 1010 culture, Cerus is confident that
the bacteria are all dead.
Bacteria plated for CFU on CHAH plates
4 J/cm2 required for inactivation of uvrB (LOD ~10 cfu/ml)
4. Progress on MS 41: Metabolic Activity of U112 After Decreasing UVA Dose
U112 cultures were grown in CDM +/- 20 uM S-59
Cultures were exposed to various doses of UVA
Bacteria serially diluted for MTS assay (data shown was for 1.64x 108 particles/ml)
Cell titer 96 assay is based on the reduction of MTS a tetrazolium compound to a
formazan that can be detected using a 96well plate reader. MTS is an acronym for
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt. MTS is reduced by enzymes in the electron transport chain
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and thus is correlate of metabolic activity. This reaction can be monitored for over 12
hrs. Higher MTS activity correlates with potency of other KBMA vaccines.
Positive control: live bacteria with no UVA and no S-59 (however these bacteria
replicate). The reagents alone are used as a blank and are subtracted from each
well. Live bacteria are capable of growing during the assay so 1.6 x 10 8 particles/ml
is only the starting number. Over time, the live bacteria increase in numbers while
killed cannot increase in CFU. So the difference in metabolic activity between the
live and KBMA become exaggerated over time. The values reach a plateau due to
the upper limit of the assay and thus it is performed with multiple concentrations of
input bacteria.
The 3 curves with killed bacteria (2, 4, 6.5 j/cm2) all have reduced activity compared
to live and overlap with each other, thus there is no correlation between UVA
exposure and the amount of metabolic activity remaining with wild-type Ft novicida.
If heat killed bacteria in the assay, there would be a flat line and would be zero.
Action: Justin will do metabolic assay with heat killed LVS to show zero activity in the
MTS assay, since there is so little known about Francisella overall.
5.
MS 41: Metabolic Activity of Ft novcida uvrB After Decreasing UVA Dose
uvrB cultures were grown in CDM +/- 5 uM S-59
Cultures were exposed to various doses of UVA and Bacteria serially diluted for MTS
assay (data shown were for 2.6 x 108 particles/ml)
Live controls had higher level of metabolic activity than the inactivated bacteria.
No dramatic increase in metabolic activity with decreasing UVA dose (even at 2 j/cm 2
where there were 2000 cfu/ml) thus the MTS assay is not sensitive to very low level
contamination of live bugs.
Cerus was hoping to see some dose response, but there isn’t in the uvrB mutant
either.
Most importantly, over the whole 12 hr MTS assay, the uvrB mutants maintain some
degree of metabolic activity. This may allow the KBMA bacteria to escapes from the
phagosome, persists long enough to signal to the host, and potentially induce an
appropriate immune response.
6. MS 41: Comparison of WT and uvrB Metabolic Activity After Photochemical
Inactivation
No dramatic difference in metabolic activity between photochemically inactivated WT
and uvrB mutant: surprising result.
Metabolic activity continues for at least 12h
With Listeria and anthrax double uvrAB mutant, Cerus see greater difference
between wild type and mutant. With novicida, still see similar levels metabolic activity
in WT and uvrB mutant. Suggests that wt francisella are not capable of repairing
DNA crosslinks efficiently.
Action: will repeat psoralen concentration titration at 4 j/cm2, though don’t expect big
difference
With Listeria and anthrax, Cerus sees a bigger difference in metabolic activity
between the double mutants and the wild type. Cerus currently doesn’t see a huge
benefit to a uvrB mutation, but looks forward to testing the uvrA mutant and
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potentially, a double uvrB,uvrA double mutation in F novicida, which may lead to a
better difference in the metabolic activity.
Question: will a lower concentration of psoralen work at the lower j/cm2 exposure?
Since Cerus has done the lowest amount of psoralen at the higher j/cm 2, they don’t
expect a lower dose will work for inactivation at lower j/cm 2 of UV light.
Cerus is a little surprised that there isn’t a bigger difference between wild type and
mutant metabolic activity, and is different from other microorganisms.
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MS 41: KBMA WT and uvrB Have High Degree of Metabolic Activity
Comparison of metabolic activity of wild type and uvrB Ft novicida starting with 1x10 9
particles/ml
If start with a higher number of particles per ml, the uvrB mutant with psoralen and
UV at 4 j/cm2 looks much more like the live (0 j/cm 2). If Cerus starts with a higher
dose, the assay saturates at ~2.5 hours and there’s no difference between wild type
and the mutant. At the higher particle concentration, they are removing the
contribution of cell growth during the assay. It suggests that a psoralen inactivated
bacteria is a better way of making a killed vaccine, even if it isn’t a uvr mutant. This
is a little unusual and wonder if Cerus will see something different with the uvrA
mutant or uvrB,uvrA double mutant.
8. Next Steps for MS 41: Optimization of Photochemical Inactivation of Ft
novicida uvrA
Cerus has received Ft novicida uvrA mutant from UTSA
Have made frozen working cell banks have been prepared of uvrA, preparing to do
inactivation titrations, for both S-59 and UV dose.
We will optimize photochemical inactivation conditions for uvrA and in theory will be
same as uvrB mutant
S-59 and UVA doses will be optimized; expect it to be similar to uvrB.
Likely explanation is that Francisella is very inefficient at repairing DNA crosslinks
naturally and so may be highly sensitive to psoralen crosslinks to begin with. This
may explain the lack of big divergence between the strains. A redundant repair
mechanism is less likely than originally anticipated, since the metabolic activity of the
inactivated bacteria remains high for 12 hours. Justin thinks the KBMA approach will
still work but that a uvr mutation may not be necessary for making the killed vaccine.
We will still need an attenuating mutation but may not need a double mutant. Cerus
will check LVS mutants too.
UNM could look at SCHU S4 inactivated this way with sensitivity to UVA, as Cerus
gave UNM an identical light box for UVA exposure of other micro organism. UNM
has the protocol already for treating anthrax with psoralen and UV, for testing in
rabbits.
If Ft is very sensitive to psoralen and crosslinking, w/o uvrA and uvrB mutations,
UNM could begin to dissect this for SCHU S4. Could expose SCHU S4 to UVA w/o
introducing uvr mutants.
Last month, Cerus showed that LVS wild type were inactivated under same
concentrations of psoralen as the wild type U112 strain conditions. Again, suggests
that Ft novicida is a reasonable model for LVS. Also, if 20uM concentration is
sufficient for SCHU S4, then it also suggests that all the wild types are equally
sensitive to psoralen and UV.
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Rick: refers to paper, group used Listeria that is gamma irradiated, claimed no
metabolic activity… RAZ paper. Justin said that the gamma irradiated Listeria has no
metabolic activity, and they claimed a 1.5 log protection of growth of the wild type
bacteria. When Cerus uses KBMA in the same circumstances, Cerus sees 6 log
protection, so it is a much less potent vaccine but it also does not require genetic
engineering. Depending on the research article read, heat killed listeria don’t provide
protective immunity. If they titrate the lowest number of bacteria to kill in the wild type
challenge, they could see a little protective immunity with gamma irradiated bacteria
in RAZ paper. Cerus gets 100% protection in a wide of doses with KBMA and Cerus
gets a much more potent T cell response. The gamma irradiated listeria induce a
very low number of T cells, compared to the KBMA experiments. When Cerus
compares their raw data to Raz paper, KBMA vaccine works better than gamma
irradiated, though KBMA takes more manipulations in terms of engineering the
bacteria. RAZ;’s Listeria bacteria are eliciting small degree of T cell activity and
immunity and we don’t know what the immunity is against. With Anthrax, where we
know that you need secretion of PA and would not get this with gamma irradiation.
Though the RAZ author calls it a broadly applicable strategy, Justin thinks that the
gamma irradiation would not work at all with other organisms like anthrax while Cerus
has demonstrated that KBMA works well with anthrax. Gamma irradiation would
eliminate the metabolic activity and PA would not be produced. Rick said gamma
irradiation induces weak protection and for a short time frame with low memory cells
production. Cerus has gamma irradiator access and would take a higher power
gamma irradiator , otherwise using the UC Berkeley irradiator would take 4 days to
achieve the same number of Rads. Cerus would like to do a “head to head”
comparison, if they can locate a more powerful gamma irradiator.
9. MS 46: Large-Scale Propagation of LVS
Cerus now has DVC lot 16 LVS.
Last month, Cerus tried to grow LVS in Chamberlain’s medium and it didn’t grow in
fermentor. LVS didn’t grow in the fermentor again this month.
Cerus used a starter culture grown in same Chamberlains media as is in the
fermentor, so there’s no difference in media.
Cerus is concerned that starting with 2 consecutive overnight passages in CDM, to
get large enough starting volume of bacteria, may be problematic. One possibility
was that there may be a limit to the number of passages in CDM after going from
hemoglobin plates and maybe the bacteria stop growing as well in Chamberlains’
after 2. They have tested this in shaker flasks and there it is not a problem in shaker
flasks, but it could be a problem for the fermentor culture.
When they followed the protocol with the starter culture, the culture ends up being in
very late stationary phase which may not be good. If they follow the same culture
scheme using shaker flasks, they find a doubling time of 4 hours, instead of about 2
hours. Because they are only doing 1:10 dilutions, it is only basically 3 doublings in
the fermentor before reaching saturation again. If they are doubling every 4 hours in
the fermentor, then this may be part of the explanation for not getting decent growth
in the fermentor.
Q: often do 1/10 back dilutions for the starter culture but impact varies with growth
phase of starter culture. Cerus starts from a frozen stock and put it into
Chamberlains media for the seed lots and see doubling every 2 hrs. Then Cerus
inoculates that into another starter culture, and they grow fine with doubling every 2
hrs. But if let sit 16 hrs while they get the fermentor started and then use the starter
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culture for the inoculum then they double at 4 hrs. Justin thinks they should dilute
more so they are not in stationary phase as long, as they may switch their
metabolism over when they are in stationary phase too long. Cerus is developing the
optimal dilution scheme. The issue is to start with enough bacteria so they don’t
need to be fermented long but not so many that they take a long time to reach log
phase growth.
Another possibility is that there is some residue in the fermentor causing the growth
inhibition. Cerus uses high quality soap like Alconox but there may be a residue that
Francisella is specifically sensitive too. Cerus will test this by making media, running
it through the fermentor and then culture the bacteria in this “fermentor exposed”
media in a shaker flask. If they don’t grow in the shaker flasks, then some residue
may be coming from the fermentor and Cerus will change their cleaning methods.
Cerus has never had this trouble culturing other organism in the fermentor but
Francisella may be more sensitive.
The agitation speed and dissolved oxygen level in the fermentor could also be
optimized, but need some growth to enable optimization detection. Cerus has the
DVC protocol for the agitation speed and dissolved oxygen level and Cerus is trying
to use these conditions now, but DVC wasn’t growing in Chamberlains media. Maybe
the two different medias have different agitation speed and dissolved oxygen
requirements. Cerus has a blood gas analyzer to measure the dissolved oxygen in
the media in the shaker flask. Cerus will determine the dissolved oxygen level from
the shaker flask with Chamberlains and then use this level in the fermentor, to avoid
hypoxic shock or too much oxygen.
Question: what is the cysteine content of the Chamberlains media? Justin was
unsure but Chamberlains contains cysteine but he didn’t know the concentration.
Vicki commented that during vaccine fermentation, periodically they would encounter
a slow growth curve and it would trace back to the cysteine being inadequate or old.
At Cerus, bacteria grows fine in shaker flasks and then doesn’t grow in the same
media in fermentors so Justin doesn’t think it is the media itself.
Question: DVC definitely grows LVS in fermentors for vaccine stocks, so fermentors
alone can’t be the problem. Maybe the Chamberlains in the fermentors is a bad
combination but Cerus is having trouble getting the DVC media to dissolve. Some
particulate matter in the DVC media that wasn’t dissolving so Cerus is trying again
and also consulting with Andrew Hoover at DVC. Using DVC media was their
planned control but they need advice on the DVC media preparation. When Cerus
tried growing LVS in the not fully dissolved DVC media, the LVS didn’t grow in the
shaker. Cerus needs help from DVC. This is a technical problem to overcome.
Question: Does the LVS passage 3 times in Chamberlains when grown in the
shaker? It grows but poorly if they follow the procedure that Cerus used to set up the
fermentor. Cerus plans to change to get the starter culture in early stationary phase
rather than in late, late stationary phase for the back dilution. They have isolated this
as one of their problems; however, with suboptimal passaging performed, Cerus gets
a 4 hr doubling time in shaker flasks and a greater than 9 hour doubling time in the
fermentor. So this is not the only problem.
Semi-Annual QA report
 June 1, 2006 Cerus Laboratory notebooks were sent out for microfilming
o All completely filled notebooks and those issued for more than 1 yr were signed
and microfilmed and then returned to the scientific staff.
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

All Cerus equipment is calibrated regularly. Calibration is overseen by our QA
department. Equipment calibrated since March 2006 include:
o Hematology Analyzer, Analytical Balances, Top Loading Balances, Biological
Safety Cabinets, Bottletop Dispensers, Digital Calipers, Centrifuges, Chart
recorders, Electrophoresis Devices, Freezers, Incubated and Refrigerated
Shakers, Incubators, Incubator Shakers, CO2 Incubators, Magnehelic Gauges,
Electronic Pipetters, Multi-Channel Pipetters, Repeat Pipetters, Single Channel
Pipetters, Refrigerators, Thermometers, Weight Sets, WinSpectral Liquid
Scintillation Counter.
o Justin has a spreadsheet for each instrument
o Undergoing ISO certification in prep for selling instruments/products in Europe so
all manufacturing and research equipment have to be ISO certified.
o Certificates available upon request and all is done by professional calibration
company.
Lot # c071516F601 of Chamberlain’s Defined Medium (CDM) from Teknova was
compared to lot #c071110D601.
o Growth of LVS was identical in both lots. Samples of lot # c071516F601 were
distributed to LBERI, DSTL, and NRC for testing.
o Had been concerned that visual appearance of two lots was slightly different but
both lots are darkening over time, ( lot 1 and lot 2). The change in color does not
have an adverse effect on the growth of LVS.
Barbara said the QA summary is fine
Action: Justin will email Barbara an example excel spreadsheet showing the QA performed
B. Other Topics: Next Cerus Tech Call will be Tuesday, October 10th. 11am-12:00pm PST,
12:00pm-1:00pm MST, 2:00pm-3:00pm EST.
C. September Report: Justin requested that Sept report be sent late: Justin is away on 7 th, back
in country on 9th; Justin wished to submit on the 11th. Barbara requested that Jack could draft
and submit draft on 7th; Barbara would not send to NIAID until Justin reviewed and approved
by 9/11/06. Justin agreed to this arrangement. Action: Jack will draft the September report
and provide it to Barbara on 9/7. Barbara won’t submit the report until Justin reviews it by
9/11.
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