KBMA Tularemia Vaccine Progress
Cerus Update June 18th 2007
Tech Call, 18 June 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, 18 June 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, 18 June 2007, Page 3
MS 40: Summary of Progress on Phenotyping
of Ftn NER mutants
• Ft novicida uvrA, uvrB, and uvrAuvrB mutants do not have
growth defects in CDM
• Ft novicida uvrA, uvrB, and uvrAuvrB mutants do not have
growth defects in J774 macrophages
• WT, uvrA, uvrB, and uvrAuvrB strains are fully virulent by IP
infection
• WT, uvrA, uvrB, and uvrAuvrB strains were all highly virulent
when administered IV, but mutants were ~1 log attenuated
• Ft novicida uvrA, uvrB, and uvrAuvrB mutants do not have
growth defects in mouse organs when administered IV
• By SC administration uvrA, uvrB, and uvrAuvrB are
attenuated by approximately 1 log compared to WT
• Decreased virulence of uvrB may be due to decreased ability to
disseminate to lungs, but replication rate is similar to WT
Tech Call, 18 June 2007, Page 4
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, 18 June 2007, Page 5
MS 40: Next Steps
• Prepare milestone completion report by end of June
Tech Call, 18 June 2007, Page 6
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, 18 June 2007, Page 7
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, 18 June 2007, Page 8
MS 41: Conclusions
• NER mutants of Ft novicida are only slightly more sensitive to
PCT than WT
• Phenotype of all NER mutants were identical: supports
selection of single mutant (uvrB) for further development
• KBMA uvrB are highly attenuated for virulence
Tech Call, 18 June 2007, Page 9
MS 41: Next Steps
• Continue QC on 400mL lot lot 948-202 of Ftn uvrB
» Measure stability of lot at –80o C by MTS assay at 6 months
• Because all strains have high degree of metabolic activity
after PCT, suggests that NER genes are not induced
» Will follow up by
• Measure sensitivity to other DNA damaging agents (mitomycin
C, doxorubicin, benzo[a]pyrene, and 4 nitroquinoline-N-oxide).
• measuring uvrB gene induction after treatment with various
• Initiated Milestone 42: Determine whether KBMA Ftn protect
against wilt-type Ftn challenge
Tech Call, 18 June 2007, Page 10
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, 18 June 2007, Page 11
Measure the durability of protection
using optimized route and regimen
MS 42: Progress on POC Studies to Determine
Whether Vaccination with KBMA Ftn is Protective
Single Dose Ftn uvrB
• LD50 of KBMA Ftn uvrB
» SC >1x109, IP ~1x109, IV~1x108
• 100% protection against 100 x IP LD50 Ftn challenge (MTD 3d)
» SC >1x109, IP 1x109, IV 1x108
Multiple dose Ftn uvrB
• 100% protection against 100 x IP LD50 Ftn challenge after vaccination
with 1x107 KBMA if administered 2 x separated by 3 weeks
» 100% protection when CD8+ cells depleted prior to challenge
» 80% protection when CD4+ cells depleted prior to challenge (MTD 6.5d)
» 90% protection when CD4+ and CD8+ cells were depleted (MTD 7d)
• These data suggest that CD4+ T cells contribute to protection
» May provide help for humoral immunity
• Passive transfer of 300ul serum resulted in 20% protection of group that
received CD8-depleted serum and 1-2 day increase in MTD
Tech Call, 18 June 2007, Page 12
Whole-Bug ELISA to measure humoral
immunity to KBMA Ftn
Anti-FT ELISA
7000
6000
Titer
5000
4000
3000
2000
1000
0
Naive
Mock
depleted
CD4
depleted
CD8
CD4+CD8
depleted depleted
NB: 980-031
Tech Call, 18 June 2007, Page 13
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, 18 June 2007, Page 14
MS 42: Next Steps
• In order to rank cellular immunity we have asked Karl Klose to
construct an epitope-tagged strain of Ftn expressing an
immunodominant CD8 epitope from ovalbumin (SIINFEKL)
• Using this strain we can measure the ability of Ftn to induce
cellular immunity
• When we receive this strain we will compare Ftn
immunogenicity to historical Lm immunogenicity
• We will also use this tag to rank double mutant strains
Tech Call, 18 June 2007, Page 15
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, 18 June 2007, Page 16
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, 18 June 2007, Page 17
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
Tech Call, 18 June 2007, Page 18
MS 46: Stability of Metabolic Activity of KBMA
LVS
Nominal 1e8 particle/mL (KBMA) F. tularensis holarctica LVS
1.0
0.9
OD (490nm)
0.8
0.7
0.6
0.5
0.4
T=0 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
T=1 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
T=2 698-040 Arm-1 (10uM S-59, 6J/cm2 UVA) "
T=3 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
0.3
0.2
0.1
0.0
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (hours)
NB968-105
• No decrease in metabolic activity after 3 months of storage
Tech Call, 18 June 2007, Page 19
MS 46: LVS Cellular Immunogenicity
• Goal: measure T-cell response to vaccination with KBMA LVS
• Dr. Jeff Frelinger's group isolated a C57Bl/6 mouse CD4+ T cell
clone that reacted to the Ft Tul4 protein (RLQWQAPEGSKCHDTS).
• We had this peptide synthesized and attempted to measure the
number of T-Cells that respond to this peptide by intracellular
interferon-gamma (IFN-) cytokine staining (ICS) or ELISpot assay.
Tech Call, 18 June 2007, Page 20
MS 46: LVS Cellular Immunogenicity
IM07-058
Day 0
200 ul HBSS
1e3 Live LVS 934-070
1e8 KBMA LVS 968-040 arm1
Day 7
B6
Tech Call, 18 June 2007, Page 21
Harvest spleens, single cells
suspension. ICS + ELISpot
ICS Data
Ft peptide CD4
1.0
1.0
0.8
0.8
%IFNg+CD4+
%IFNg+CD4+
Unstim CD4
0.6
0.4
0.2
0.6
0.4
0.2
0.0
0.0
HBSS
LVS
KBMA
HBSS
Immunization
KBMA
Immunization
Unstim CD8
Ft peptide CD8
1.0
1.0
0.8
0.8
%IFNg+CD8+
%IFNg+CD8+
LVS
0.6
0.4
0.2
0.6
0.4
0.2
0.0
HBSS
LVS
Immunization
Tech Call, 18 June 2007, Page 22
KBMA
0.0
HBSS
LVS
Immunization
KBMA
ELISpot Data
Unstim
Ft peptide
100
SFU per 2e5 cells
SFU per 2e5 cells
100
80
60
40
20
80
60
40
20
0
0
HBSS
LVS
Immunization
KBMA
HBSS
LVS
KBMA
Immunization
• LVS appears to non-specifically increase the number of IFN-producing cells
Tech Call, 18 June 2007, Page 23
MS 46: Cellular Immunity Conclusions
• 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
• Will loaded DCs with peptide to see if peptide can be presented
• Also will perform heterologous prime-boost with peptide loaded DC
Tech Call, 18 June 2007, Page 24
MS 46: Next Steps
• We will continue to monitor stability of fermentor-grown live LVS in
“freeze buffer” vs 10% sucrose
» At 6 and 12 months
» We will compare the virulence of fermentor-grown LVS stored in
“freeze buffer”or 10% sucrose, to expanded and freshly
suspended DVC lot 16
• We will monitor the stability of photochemically inactivated lot of
LVS in 10% sucrose
» Monthly by MTS assays (at 6,12 months)
• Will vaccinate with lower doses to determine whether KBMA LVS
provides any benefit over HK against LVS challenge
Tech Call, 18 June 2007, Page 25
Action Items: Cerus 6/18/07 Tech call
• Action: Barbara contacted Karl Klose on 6/18/07 and Cerus
thereafter did receive the uvrB LVS mutant from UTSA.
(completed)
• Action: Justin will prepare the MS 40 Milestone completion
report by the end of June, or even middle of July
• Action: Barbara ask Karl if willing to let Justin screen the
SINFEKL in parallel with Karl’s Western. (Note: Karl
responded that he prefers to fully test the construct in his lab
before sharing with other labs) (completed)
• Action: Barbara ask Rick if we wish to ship aliquots of ASU’s
predicted immunodominant peptides to Cerus. (discussed
with Rick at 7/6 Internal Tech Meeting)
Tech Call, 18 June 2007, Page 26