KBMA Tularemia Vaccine Progress
Cerus Update August 14th 2007
Tech Call, 14 Aug., 2007, Page 1
Cerus Milestones
•
Milestone 40: Phenotyping of F.t. novicida NER mutants
» Measure attenuation of live uvr mutants in vitro, in macrophages, and in mice
•
Milestone 41: Optimization of photochemical treatment regimen 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 42: Determine whether KBMA F.t. novicida vaccine protects against wild-type
F.t. novicida challenge in mice
» Vaccination route and regimen optimization, measure durability of protection
•
Milestone 43: Evaluation of genetically attenuated NER F.t. novicida strains as platform
strains for KBMA vaccine
» Screen 6 attenuated uvr double mutants for virulence attenuation and protective efficacy
•
Milestone 44: Formulation and evaluation of KBMA LVS
» Establish photochemical inactivation regimen of selected uvr mutant of LVS
» Measure metabolic activity and virulence of KBMA LVS
•
Milestone 45: Test vaccine efficacy of KBMA LVS in murine model
» Measure level and durability of protection against LVS challenge, send to UNM
•
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 large-scale process
Milestone 47: Develop KBMA protocols to transfer to UNM for SchuS4-based vaccine
Tech Call, 14 Aug., 2007, Page 2
MS 41: Optimization of Photochemical
Treatment and Characterization of KBMA Ftn
Milestone 41
Optimization of photochemical treatment regimen
and Characterization of KBMA Ft novicida
Determine minimal S-59 concentration
required for complete inactivation
uvrA, uvrB, uvrAB, vs U112
Determine the minimal UVA dose
required for complete inactivation
uvrA, uvrB, uvrAB, vs U112
Measure metabolic activity
after photochemical treatment
uvrA, uvrB, uvrAB, vs U112
Select optimal uvr candidate
for further study
uvrB
400mL scale inactivation process
optimized
Lot of KBMA uvrB produced
QC of KBMA Ftn vaccine lots
for sterility and metabolic activity
Stability testing is ongoing
Tech Call, 14 Aug., 2007, Page 3
KBMA Ftn vaccine is attenuated
in mice by IP, IV, and SC routes
Milestone 42: Determine whether
KBMA Ftn uvrB protects
against lethal Ftn challenge
MS 41: Progress on Optimization of
Photochemical Treatment Regimen
• Optimized S-59 and UVA doses at 3.5 mL scale
» Minimum S-59 concentration required to inactivate ~1 x 1010 cfu
– U112 = 40M
– uvrA, uvrB, + uvrAuvrB = 20 M
» 4 J/cm2 was the minimum dose of UVA required to achieve consistent
inactivation (at 3.5 mL scale)
» Metabolic activity profiles of all strains were similar
• Optimized 400 mL scale inactivation conditions for uvrB
» 40M S-59 + 7 J/cm2 UVA > 5x1010 inactivation
» Sterile lots produced that have metabolic activity
» MTS activity is stable at –80oC for 3 months (next time point is 6M)
• KBMA uvrB are highly attenuated
» >8 logs IP, ~8 logs IV, ~4 logs SC
Tech Call, 14 Aug., 2007, Page 4
MS 41: Analysis of the Sensitivity of U112 and
uvrB to Various DNA Damaging Agents
• Because there was only a minor difference between the uvrA, uvrB,
and uvrA/uvrB mutants compared to WT Ft novicida with regard to
sensitivity to photochemical inactivation with S-59 and UVA, we
have been investigating the the reason for this difference between
our experience with Listeria monocytogenes and Bacillus anthracis
and Francisella tularensis.
• Francisella may have a redundant DNA repair mechanism
• Francisella may have lost the ability to induce the nucleotide repair
genes
• To test these hypotheses we have looked at the sensitivity of Ft
novicida to various DNA damaging agents, and will measure the
transcriptional activity of the NER genes by rtPCR
Tech Call, 14 Aug., 2007, Page 5
MS 41: Analysis of the Sensitivity of U112 and
uvrB to Various DNA Damaging Agents
• We previously performed MIC assays using
» S-303
» mitomycin C
» cisplatin
» doxorubicin hydrochloride
» benzo[a]pyrene
» 4 nitroquinoline-N-oxide
• Of these,S-303, mitomycin C, cisplatin, 4 nitroquinoline-Noxide inhibited growth of Ft novicida,
• But only S-303 and 4 nitroquinoline-N-oxide inhibited the
growth of the uvrB mutant slightly more than U112
Tech Call, 14 Aug., 2007, Page 6
MS 41: Repeat testing of U112 and uvrB to
Various DNA Damaging Agents
Ftn sensitivity to DNA damage by S-303
0.7
U112 T16h
OD600 nm
0.6
0.5
uvrB T16h
0.4
U112 T0
0.3
0.2
uvrB T0
0.
9
1.
7
3.
4
6.
9
44
0.
0
22
0.
0
11
0.
0
55
.0
27
.5
13
.8
0.1
S-303 uM
uvrB mutant appears to be slightly more sensitive to crosslinks induced by
the nitrogen mustard S-303
Again, no differences in sensitivity to mitomycin or cisplatin
Tech Call, 14 Aug., 2007, Page 7
MS 41: Conclusions
• Phenotype of all NER mutants were identical: supports
selection of single mutant (uvrB) for further development
• KBMA uvrB are highly attenuated for virulence
• NER mutants of Ft novicida are only slightly more sensitive to
inactivation with S-59 and UVA than WT
• This appears to be true with other DNA damaging agents
• May suggest that there is a redundant repair mechanism
Tech Call, 14 Aug., 2007, Page 8
MS 41: Next Steps
• Measure the induction of the uvrB gene by rtPCR after DNA
damage
Tech Call, 14 Aug., 2007, Page 9
MS 42: Determine Whether KBMA Ftn Protect
Against Wild-Type Ftn Challenge
Milestone 42
Determine whether KBMA Ftn protect against wilt-type Ftn challenge:
Vaccination route and regimen optimization
Durability of protection established
Compare protective efficacy of KBMA vaccine
delivered by various routes
Select optimal roue
Determine optimal dose of KBMA vaccine
required for complete protection
Select dose
Determine number and timing of vaccinations
that provide highest degree of protection
Select dosing regimen
Determine the highest challenge dose for which protection is 100%
Using optimal regimen
Tech Call, 14 Aug., 2007, Page 10
Measure the durability of protection
using optimized route and regimen
MS 42: Conclusions
• KBMA Ftn provides protection against U112 challenge with a
single high (1x LD50) dose or two 0.1x LD50 doses
• This may not be superior to Heat Killed
• Immunity appears to be largely humoral,
» Heat killed Ftn provide protection
» CD4 T-cell depletion has a modest effect on survival that is
CD8 independent
» Survival after passive transfer of serum correlates with
antibody titer
Tech Call, 14 Aug., 2007, Page 11
MS 42: Next Steps
• In order to measure cellular immunity we have asked Karl
Klose to construct an epitope-tagged strain of Ftn expressing
an immunodominant CD8 epitope from ovalbumin (SIINFEKL)
• When we receive this strain we will compare Ftn
immunogenicity to historical Lm immunogenicity
• MS is currently on hold until we can use a cellular immune
assay to optimize the dosing regimen/potency
Tech Call, 14 Aug., 2007, Page 12
MS 44: Formulation and evaluation of KBMA
LVS
Milestone 44
Optimization of photochemical treatment regimen
and Characterization of KBMA LVS
Determine minimal S-59 concentration
required for complete inactivation
uvrB vs LVS
Determine the minimal UVA dose
required for complete inactivation
uvrB vs LVS
Measure metabolic activity
after photochemical treatment
uvrB vs LVS
400mL scale inactivation process
optimized
Lot of KBMA uvrB LVS produced
QC of KBMA uvrB LVS vaccine lots
for sterility and metabolic activity
Tech Call, 14 Aug., 2007, Page 13
Measure the virulence of KBMA uvrB LVS vaccine
in mice by IP, IV routes
compare to WT LVS
Milestone 45: Determine whether
KBMA uvrB LVS protects
against lethal LVS challenge
MS 44: uvrB mutant inactivated with 5uM S59
Photochemical Inactivation of LVS uvrB
NB 963-081
1.E+10
1.E+08
CFU
LVS uvrB
1.E+06
1.E+04
1.E+02
1.E+00
0
1
2
3
4
5
6
7
8
9
10
[S-59] M
• 5 uM S-59 is the same concentration that was required to inactive WT LVS
• Will repeat dose titration with uvrB and WT at same time
Tech Call, 14 Aug., 2007, Page 14
MS 44: Formulation and evaluation of KBMA
LVS
uvrB 1-A T16h
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
uvrB 1-B T16h
LVS 16h
LVS T0
uvrB 1-A T0
2.
3
4.
6
9.
3
uvrB 1-B T0
11
85
.0
59
2.
5
29
6.
3
14
8.
1
74
.1
37
.0
18
.5
OD600 nm
LVS and uvrB sensitivity to Cisplatin
ug/ml cisplatin
• uvrB mutants appear to be more resistant to cisplatin than WT
Tech Call, 14 Aug., 2007, Page 15
MS 44: Formulation and evaluation of KBMA
LVS
LVS and uvrB sensitivity to S-303
OD600 nm
0.45
0.4
0.35
uvrB 1-A T16h
0.3
0.25
LVS 16 h
0.2
0.15
uvrB 1-A T0
uvrB 1-B T16h
LVS T0
uvrB 1-B T0
0.
9
1.
7
3.
4
6.
9
13
.8
27
.5
55
.0
44
0.
0
22
0.
0
11
0.
0
0.1
uM S-303
• uvrB mutants appear to be more resistant to S-303 than WT
Tech Call, 14 Aug., 2007, Page 16
MS 44: Formulation and evaluation of KBMA
LVS
Summary:
• uvrB mutant of LVS does not appear to be more sensitive to
DNA damage than WT
• This suggests that the data we get with KBMA WT LVS may
be similar to data
Tech Call, 14 Aug., 2007, Page 17
MS 44: Formulation and evaluation of KBMA
LVS
Next Steps:
• Directly compare S-59 + UVA inactivation profile of WT and
uvrBLVS
• Compare metabolic activity of WT and uvrBLVS after
photochemical inactivation
Tech Call, 14 Aug., 2007, Page 18
MS 46: 3L-Scale Propagation of LVS
Milestone 46
Scale-up of KBMA LVS vaccine production
Select agar and liquid media that support
robust growth and viability of LVS
CHAH and CDM
Develop 3L scale fermentation conditions for LVS
CDM Sigma antifoam A
Develop cryopreservation conditions for LVS
8% DMSO + 1% sucrose vs 10% sucrose
Confirm preservation of LVS virulence
Develop 3L scale
photochemical inactivation conditions
Monitor stability of frozen LVS
Develop 3L scale purification conditions
optimize TFF for LVS
Demonstrate KBMA LVS is avirulent
Demonstrate KBMA LVS protect against
Lethal LVS challenge
Confirm protective efficacy of KBMA LVS
Produced by 3L-scale methods
Tech Call, 14 Aug., 2007, Page 19
Demonstrate KBMA LVS protect against
Lethal Schu S4 challenge
at UNM
MS 46: Summary of Progress on LVS Scale-Up
• High efficiency of LVS cfu recovery on CHAH agar plates
• Robust growth of DVC lot 16 LVS in CDM in shaker flasks
» LVS expanded and frozen
• 3L LVS grown in fermentor using CDM and Sigma antifoam A
• Efficient LVS cryopreservation in 8% DMSO or 10% sucrose
» Up to 4 month stability
• LVS virulence established by 3 routes
» Cerus IP LD50 range 1x103-3x104 v.s. Green et. al 2005: 4x100
» Cerus expanded LVS is ~10x more virulent than DVC lot 16
» Cerus IV LD50 range 3x103-7x104 v.s. Green et. al 2005 2.2x104
» Cerus SC LD50 > 1.26 x108 v.s. Green et. al 2005 1.3x109
Tech Call, 14 Aug., 2007, Page 20
MS 46: Summary of KBMA WT LVS Data
• Produced 400mL lot of KBMA LVS (968-040) for proof of concept studies
prior to receiving NER mutant from UTSA
• KBMA LVS maintained metabolic activity for >12 hours after PCT
» Stability of metabolic activity ongoing
• KBMA LVS IV LD50 is 6.8x108
» attenuated for virulence by 4-5 logs compared with live
• Single dose of KBMA LVS provided 100% protection against 100xLD50
IP LVS challenge with doses as low as 1x107
» Heat killed was equivalent, suggesting that LVS protection was humoral
» Sent vials of KBMA LVS to Terry Wu for SchuS4 challenge studies
• There was no significant tul-4 peptide-specific increase in IFN-producing
cells by ICS or ELIspot
» Neither Live nor KBMA LVS induced a response
» May be a very weak peptide
» May be due to LVS inducing a poor T-cell response
Tech Call, 14 Aug., 2007, Page 21
MS 46: KBMA-LVS did not protect BALB/c mice
against i.n. SCHU S4 challenge
Tech Call, 14 Aug., 2007, Page 22
MS 46: Suppression of T Cell Potency
• LVS suppressed pro-inflammatory response induced by Listeria
monocytogenes (e.g. MCP-1 and IL-6)
• LVS coinfection with Lm did not reduce the magnitude of the Lm-incuced
T cell response, but…
• LVS impaired the capacity of Lm-induced CD4+ T cells to produce IL-2
which is required for priming of CD8 memory
• Conclusion: LVS may actively suppress a productive T cell memory
response and may be a sub-optimal vaccine platform
Tech Call, 14 Aug., 2007, Page 23
MS 46: Next Steps
• Produce 400mL-scale lot of KBMA uvrB LVS and compare to WT
• Use increasing doses of live and KBMA-LVS in combination with
Listeria to evaluate suppression of inflammation and adaptive
immunity
• Discuss alternative strategies for improving the potency of vaccineelicited CD8+ T cells
» We have constructed Lm expressing Ft antigens and would like
permission to pursue this line of investigation
» We would like compare KBMA LVS and KBMA Lm based tularemia
vaccines
Tech Call, 14 Aug., 2007, Page 24
Action Items
• Justin will provide Barbara and Rick an outline of a plan to
redirect efforts toward the KBMA Lm based tularemia
vaccines
• Rick: will talk to Karl at the 8/21 UTSA Tech call about the
status of the OVA construct
• Justin may discuss the usage of overlapping peptide libraries
for immune response detection at the annual meeting.
• Justin: Will prepare RNA from the chemically exposed
samples to determine whether the NER genes just are not
induced, using quantitative RT PCR
Tech Call, 14 Aug., 2007, Page 25