KBMA Tularemia Vaccine Progress
Cerus Update July 10th 2007
Tech Call, 10 July 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, 10 July 2007, Page 2
MS 40: Flow Diagram
Milestone 40
Phenotyping of Ft novicida NER mutants
Selection of Media for Growth of Ftn
CDM for liquid
CHAH for Agar
Measure growth in vitro
uvrA, uvrB, uvrAB vs U112
Measure growth in cells
uvrA, uvrB, uvrAB vs U112
Measure virulence in mice
uvrA, uvrB, uvrAB vs U112
Measure growth in mice
uvrA, uvrB, uvrAB vs U112
All strains have identical
growth rate in CDM
All strains have identical
growth rate in J774 cells
All strains are still highly virulent
by IP route
Growth rate of U112
in lungs, livers, and spleens
after IV administration
completed
NER mutants highly virulent by IV route
but are slightly attenuated
NER mutants not attenuated for growth
in lungs, livers, and spleens
after IV infection
NER mutants are all ~1 log attenuated
for virulence by SC route
uvrB mutant appears to be attenuated
for growth in lungs after SC administarion
Tech Call, 10 July 2007, Page 3
MS 40: Conclusions
• Ft novicida NER mutants have no growth defects in vitro
• Virulence attenuation is very subtle (at most 1 log)
• All three NER-deficient strains of Ft novicida are indistinguishable
• These data support selection of a single (uvrB) NER mutant for
further development as a vaccine candidate
• Limited attenuation supports the evaluation of secondary
attenuating mutations (in MS 43) that would add safety feature to
KBMA SchuS4-based Vaccine
Tech Call, 10 July 2007, Page 4
MS 40: Next Steps
• Prepare milestone completion report by end of next week
Tech Call, 10 July 2007, Page 5
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, 10 July 2007, Page 6
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, 10 July 2007, Page 7
MS 41: Analysis of the Sensitivity of U112 and
uvrB to Various DNA Damaging Agents
Highest
Solubility
DNA damaging agent concentration
Solvent
in media
S-303
13300 uM
media
yes
Benzo pyrene
33.3 ug/ml dichloromethane
no
Doxorubicin
hydrocholoride
0.83 ug/ml media/ethanol 1/1
no
Cisplatin
50000 ug/ml
DMSO
yes
Mitomycin
3333 ug/ml
H2O
yes
4 nitroquinoline-N-oxide 33333 ug/ml
hot acetone
no
Tech Call, 10 July 2007, Page 8
Growth Difference between
inhibition
U112 and uvrB
minor
yes
no
no
no
no
no
yes
no
yes
minor
yes
MS 41: Analysis of the Sensitivity of U112 and
uvrB to Various DNA Damaging Agents
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
U112 T 0
U112 T 16h
U112 uvrB T 0
U112 uvrB T 16h
33
33
.0
83 00
3.
2
20 50
8.
31
52 3
.0
7
13 8
.0
20
3.
25
5
0.
81
4
0.
20
3
0.
05
1
0.
01
3
OD600
M itomycin
ug/ml
DNA damage induced by mitomycin C inhibits growth of U112 and uvrB
at similar concentrations
Tech Call, 10 July 2007, Page 9
MS 41: Analysis of the Sensitivity of U112 and
uvrB to Various DNA Damaging Agents
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
U112 T 0
U112 T 16h
U112 uvrB T 0
U112 uvrB T 16h
13
30
0.
33 00
25
.0
0
83
1.
2
20 5
7.
81
51
.9
5
12
.9
9
3.
25
0.
81
0.
20
0.
05
0.
01
OD600
S303
um
uvrB mutant appears to be slightly more sensitive to crosslinks induced by
he synthetic psoralen S-303
Tech Call, 10 July 2007, Page 10
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, 10 July 2007, Page 11
MS 41: Next Steps
• Repeat some of the MIC assays using a 2-fold dilution series
• Measure the induction of the uvrB gene by rtPCR after DNA
damage
Tech Call, 10 July 2007, Page 12
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, 10 July 2007, Page 13
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, 10 July 2007, Page 14
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
Tech Call, 10 July 2007, Page 15
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, 10 July 2007, Page 16
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: Formulation and evaluation of KBMA
LVS
• UvrB mutant of LVS arrived
• Frozen stocks prepared
Next month
• Will evaluate sensitivity of uvrB mutant to S-59 and UVA
inactivation
• Will compare uvrB and LVS MICs to panel of DNA damaging
agents
Tech Call, 10 July 2007, Page 17
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, 10 July 2007, Page 18
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, 10 July 2007, Page 19
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, 10 July 2007, Page 20
MS 46: Suppression of T Cell Potency
• Protective immunity to intracellular pathogens requires the lytic activity
of antigen-specific CD8+ T cells
• The priming of a protective CD8+ T cell population following vaccination
requires the combination of antigen presentation, cytokines, and CD4+ T
cell help
• Recent data from our lab using another intracellular pathogen, Listeria
monocytogenes, has shown that attenuated strains of this bacterium
which do not elicit protective CD8+ T cell responses are not simply nonimmunogenic, but actively suppress the CD8+ T cell response.
» The attenuation of these Listeria strains limits their escape into the
host cell cytosol
• Because LVS demonstrates a delayed egress from the phagocytic
vacuole relative to other intracellular pathogens (40 minutes versus <1
minute for Listeria), we questioned if LVS would have a similar negative
impact on inflammation and CD8+ T cell priming
Tech Call, 10 July 2007, Page 21
MS 46: Supression Experimental Design
Day 0
HBSS
1x105 cfu Listeria-OVA
1x103 cfu LVS
1x105 cfu Listeria-OVA + 1x103 cfu LVS
Day 7
B6
4H
24H
Serum cytokines
Tech Call, 10 July 2007, Page 22
Harvest spleens, single cells
suspension. ICS + ELISpot
MS 46: 1x103 cfu LVS suppress Lm-elicited
inflammatory cytokines
24H MCP-1
24H IL-6
Tech Call, 10 July 2007, Page 23
LV
S
ac
tA
+L
VS
0
ac
tA
LV
S
VS
ac
tA
+L
ac
tA
1e
5
H
B
SS
0
1000
1e
5
50
2000
B
SS
100
3000
H
MCP-1 @ 24 HPI (pg/mL)
IL-6 @ 24 HPI (pg/mL)
150
Tech Call, 10 July 2007, Page 24
ac
tA
+L
4
2
0
S
VS
0
6
ac
tA
+L
V
5
8
ac
tA
10
%LLO190-201 specific CD4+ T cells
15
ac
tA
%OVA257-264 specific CD8+ T cells
MS 46: 1x103 cfu LVS do not suppress the magnitude
of the Lm-elicited CD4+ and CD8+ T cell response
MS 46: 1x103 cfu LVS impair the capacity of
CD4+ T cells to produce IL-2
60
50
40
30
Tech Call, 10 July 2007, Page 25
ac
tA
+L
VS
20
ac
tA
%IL-2+ of LLO190-201 -specific
IFN-+CD4+ T cells
IL-2 production within LLO190 specific CD4s
MS 46: Next Steps
• Awaiting results from Terry’s SchuS4 challenge study to know
whether KBMA wt LVS protects against lethal challenge
• 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
vaccine-elicited CD8+ T cells
Tech Call, 10 July 2007, Page 26
Action Items:
• No new action items from Cerus 7/10/07 Tech call
Tech Call, 10 July 2007, Page 27