Production and Evaluation of KBMA
Francisella tularensis Vaccines
Cerus TVDC Annual Update, October 8th 2007
TVDC Annual Conference, 8 Oct., 2007, Page 1
Killed But Metabolically Active (KBMA) Concept
NH2 . HCl
O
X
O
O
+
S-59 Psoralen
UVA Light
DNA crosslinked
uvrAB
Parental Strain
X
X
X
X
X
X
X
X
X
X
O
X
X
X
X
Expression Profile
Diminished
X
X
X
X
NER-
X
X
X
X
X
NER+
Expression Profile
Preserved (KBMA)
Theoretical “one crosslink per genome” inactivation
TVDC Annual Conference, 8 Oct., 2007, Page 2
Cerus Highlight of Key Achievements
•
Milestone 40: Phenotyping of F.t. novicida NER mutants – Milestone completed
» Measured 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
» Established photochemical inactivation regimen for Ftn
» Measured metabolic activity of uvr mutants after photochemical treatment
» Determined that KBMA Ftn are avirulent
•
Milestone 42: Determine whether KBMA F.t. novicida vaccine protects against wild-type
F.t. novicida challenge in mice
» Demonstrated that KBMA Ftn protects mice against a lethal WT Ftn challenge with a single high
dose or 2 low doses
» Demonstrated that the mechanism of protection is humoral
•
Milestone 43: Evaluation of genetically attenuated NER F.t. novicida strains as platform
strains for KBMA vaccine
» Received attenuated uvrB double mutants for virulence attenuation and protective efficacy
•
Milestone 44: Formulation and evaluation of KBMA LVS
» Determined the minimal concentration of S-59 to inactivate LVS uvrB
•
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
» Optimized large–scale LVS culture conditions
» Optimized medium scale photochemical inactivation process for WT LVS
» Demonstrated that KBMA LVS protect against LVS challenge, but not SchuS4 challenge
•
Milestone 47: Develop KBMA protocols to transfer to UNM for SchuS4-based vaccine
TVDC Annual Conference, 8 Oct., 2007, Page 3
MS 40: Summary
• Ft novicida NER mutants (uvrA, uvrB, and uvrAuvrB) have no growth
defects in vitro
» Identical growth rates in CDM
» Identical intracellular growth rates in J774 cells
• Ft novicida NER mutant virulence attenuation is very subtle
» No decrease in virulence when administered IP
» Slight decrease in virulence when administered or SC
» 1 log decrease in virulence when administered IV
• Ft novicida NER mutants have subtle growth defects in mouse organs.
» No growth defects in any organ when administered IV
» 1 log decrease in peak lung titer after SC administration
• 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
TVDC Annual Conference, 8 Oct., 2007, Page 4
MS 41: Progress on Optimization of
Photochemical Treatment Regimen for Ftn
• 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 for >12 h
» MTS activity is stable at –80oC for 3 months
• KBMA uvrB are highly attenuated
» >8 logs IP, ~8 logs IV, ~4 logs SC
TVDC Annual Conference, 8 Oct., 2007, Page 5
MS 41: Optimization of Photochemical
Treatment Regimen for Ftn
Photochemical Inactivation ofFt novicida Strains
1.E+10
cfu/ml
1.E+08
U112
uvrA
1.E+06
uvrB
1.E+04
uvrA uvrB
1.E+02
1.E+00
0
10
20
[S-59] uM
30
40
• All NER mutant strains were slightly more sensitive than U112
• Inactivation achieved at 20uM for NER-deficient strains, 40uM for U112
• With Lm and Ba the increase in sensitivity was 10-20 fold
TVDC Annual Conference, 8 Oct., 2007, Page 6
MS 41: Metabolic Activity of KBMA Ftn Strains
Metabolic Activity of KBMA Ft novicida Strains
1.6
1.4
Ftn uvrB KBMA (40uM S59, 4J)
1.2
Ftn uvrAB KBMA (40uM S59, 4J)
Ftn uvrA KBMA (40uM S59, 4J)
Ftn WT KBMA (80uM S59, 4J)
0.8
0
OD49
1.0
0.6
0.4
0.2
0.0
0
2
4
6
8
10
12
NB 963-054
Time
• All strains have similar levels of metabolic activity after inactivation
• Supports selection of uvrB mutant to move forward with
TVDC Annual Conference, 8 Oct., 2007, Page 7
MS 41: Conclusions/Statement of Problems
• Phenotype of all NER mutants were identical: supports
selection of single mutant (uvrB) for further development
• KBMA uvrB are highly attenuated for virulence
Potential Problems:
• 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 numerous other DNA damaging
agents
• Suggest that there is a redundant repair mechanism and that
the KBMA vaccine will not be very potent because inactivation
requires a higher number of cross links
Corrective measures
• Investigate alternative vaccine platforms
TVDC Annual Conference, 8 Oct., 2007, Page 8
MS 42: Determine Whether Mice Vaccinated with KBMA
Ftn Vaccine are Protected Against Lethal Challenge
AS07-009
HBSS
HK Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
Vaccination
dose (particles)
1.00E+09
1.00E+09
1.00E+08
1.00E+09
1.00E+08
1.00E+09
1.00E+08
Route
IV
IP
IP
IP
IV
IV
SC
SC
Vaccination
survivors
5
5
2
5
0
2
5
5
100 x IP LD50
challenge survivors
0
5
2
3
0
2
3
0
Protection
0%
100%
100%
60%
100%
60%
0%
Mean time
to death
3d
NA
NA
4d
NA
NA
4.5d
4d
• 100% protection with KBMA observed with IP and IV vaccination
• 100% protection with KBMA observed only at doses that were near LD50
• HK control was less virulent and protected 100% (at 1 x 109 dose)
• Suggests humoral response
• Difficult to evaluate various platform strains and formulations (MS 43)
TVDC Annual Conference, 8 Oct., 2007, Page 9
MS 42: Mechanism of KBMA Ftn Protection
Against Ftn Challenge is Humoral
Survival of mice vaccinated twice with 1e7 KBMA IV and challenged with 100 x IP LD50 U112
Vaccination
No Depletion
CD4
CD8
CD4 + CD8
HBSS
0% (MTD 3d)
100%
80% (MTD 6.5d)
100%
90% (MTD 7d)
KBMA uvrB
Anti-FT ELISA
7000
AS07-045 Survival After Passive Transfer of Serum
Naive serum
KBMA uvrB immunized
KBMA imunized -CD4
KBMA imunized -CD8
KBMA imunized -CD4/-CD8
80
60
40
6000
5000
Titer
Percent survival
100
4000
3000
2000
20
0
0.0
1000
0
2.5
5.0
7.5
10.0
12.5
15.0
Day
TVDC Annual Conference, 8 Oct., 2007, Page 10
Naive
Mock
depleted
CD4
depleted
CD8
CD4+CD8
depleted depleted
MS 42: Summary of 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 1-2 day increase in MTD with
low titer serum or 20% survival with higher anti-FT titer serum
TVDC Annual Conference, 8 Oct., 2007, Page 11
MS 42: Conclusions/Statement of Problems
• KBMA Ftn provides protection against U112 challenge with a single
high (1x LD50) dose or two 0.1x LD50 doses
• Immunity appears to be largely humoral
Problems and Corrective Measures
• KBMA Ftn are not be superior to heat killed Ftn
• Cannot compare efficacy of KBMA attenuated strains by protection
assays
• In order to measure cellular immunity, we asked Karl Klose to
construct an epitope-tagged strain of Ftn expressing an
immunodominant CD8 epitope from ovalbumin (SIINFEKL)
• We recently received U112-pepO-SIINFEKL and LVS -pepOSIINFEKL strains
TVDC Annual Conference, 8 Oct., 2007, Page 12
MS 44: Formulation and Evaluation of KBMA
LVS
Summary
• LVS uvrB mutant inactivated with same dose of S-59 as WT
• LVS uvrB is not more sensitive to DNA damage by cisplatin or S303 with than WT
Conclusions
• The data we get with KBMA uvrB LVS may be similar to data
obtained with KBMA WT LVS
Problems
• This suggests that there is a redundant repair mechanism that
precludes formulation of highly potent KBMA LVS vaccine
Corrective measures
• Investigate alternative vaccine platforms
TVDC Annual Conference, 8 Oct., 2007, Page 13
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 Live LVS cryopreservation in 8% DMSO or 10% sucrose
» Up to 4 month stability
• Live 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
TVDC Annual Conference, 8 Oct., 2007, Page 14
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
» Used 10nM S-59 and 6J/cm2 UVA
•
KBMA LVS maintained metabolic activity for >12 hours after PCT
» Metabolic activity stable for > 3 months
•
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
» KBMA LVS sent to Terry Wu did not provide protection against SchuS4 challenge
•
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
TVDC Annual Conference, 8 Oct., 2007, Page 15
MS 46: Stability of Metabolic Activity of KBMA
LVS
OD (490nm)
Nominal 1e8 particle/mL (KBMA) F. tularensis holarctica LVS
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
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
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
TVDC Annual Conference, 8 Oct., 2007, Page 16
MS 46: KBMA-LVS Did Not Protect BALB/c Mice
Against i.n. SCHU S4 Challenge
TVDC Annual Conference, 8 Oct., 2007, Page 17
MS 46: Suppression Experimental Design
HBSS
1x105 cfu Listeria-OVA (Lm)
Day 0
1x103 cfu LVS (LVS)
1x105 cfu Listeria-OVA + 1x103 cfu LVS (Lm+LVS)
Day 7
Harvest spleens, single
cells suspension. ICS +
ELISpot
B6
1000
LV
S
VS
0
ac
tA
+L
LV
S
VS
ac
tA
+L
ac
tA
1e
5
H
B
SS
0
2000
ac
tA
50
24H MCP-1
1e
5
100
3000
B
SS
24H IL-6
H
IL-6 @ 24 HPI (pg/mL)
MCP-1 @ 24 HPI (pg/mL)
24H Serum Cytokines
150
• LVS does not induce Th1 inflammatory cytokine response
•1x103 cfu LVS suppress Lm-elicited inflammatory cytokines
TVDC Annual Conference, 8 Oct., 2007, Page 18
IL-2 production within LLO190 specific CD4s
+
Lm
20
S
LV
S
0
30
LV
2
40
+
4
50
Lm
6
60
Lm
%IL-2+ of LLO190-201 -specific
IFN-+CD4+ T cells
8
Lm
%LLO190-201 specific CD4+ T cells
MS 46: LVS Coinfection Suppresses The
Function of Listeria-Induced CD4+ T cells
• Coinfection with 1x103 cfu LVS does not suppress the magnitude
of the Lm-elicited T cell response
• But does impair the capacity of 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
TVDC Annual Conference, 8 Oct., 2007, Page 19
Overall Conclusions:
• Francisella tularensis strains behave differently than other bacterial
pathogens studied by Cerus to date
» NER mutants of Ftn and LVS are not significantly more sensitive to
photochemical inactivation than wild type
• We have produced lots of KBMA Ftn and LVS vaccine that can
protect mice against lethal Ftn and LVS challenge
»
»
»
»
These lots are killed, metabolically active, and stable for months
The mechanism of protection appears to be humoral
Protection is not significantly different than heat killed vaccines
KBMA LVS did not protect against SchuS4 lethal challenge in mice
• We have preliminary data demonstrating that LVS impairs
inflammatory immune responses which may ultimately impair a
strong memory T cell response
• We will present alternative strategies toward development of a more
potent KBMA vaccine based on a Listeria monocytogenes platform
tomorrow morning
TVDC Annual Conference, 8 Oct., 2007, Page 20
Questions after the presentation
•
•
•
•
•
•
•
•
•
Bernard: thinks humoral protection plays a role in responding to a SCHU S4
challenge.
Karl: quotes paper that shows protection can be transferred with serum
challenge
Stephen: ASU has looked at Ricksettia and detected a high serum antibody
which can protect against Ricksettia. Ricksettia is an intracellular bacteria
too.
Justin: low dose model can result protection via humoral response, but
Cerus isn’t detecting humoral protection in a high challenge dose model
Kathy: maybe the antibodies produced to a live inactivated Ft are the wrong
antibodies for protection against a high challenge dose?
Stephen: A biothreat vaccine has different requirements than a traditional
vaccine. For a biothreat, we want the vaccine to be very effective for a
transient exposure of the microorganism.
Justin: probably federal agencies would prefer a long term effective vaccine
Bernard: For a biothreat, we need quick neutralization of the microorganism
and thinks there is a role for antibody in this process.
Justin: For long term vaccination, T cell immunity is more potent than
antibody immunity.
TVDC Annual Conference, 8 Oct., 2007, Page 21
Questions after the presentation
•
•
•
•
•
•
•
Rick: A high titer of preformed antibody could be protective , but TB
protective antibody doesn’t work well and organism has an extracellular
phase as well. Mouse, rat, and humans may be more different. May not be
able to make a true sterilizing vaccine, but we need a vaccine to FT to
control symptoms of infection rather than a fully sterilizing vaccine.
Kathy: could the host have a truly separated T and B cell immune
response? Does seem unimaginable to shut down one or the other aspects
of the immune system.
Rick: antibody is made, but probably need T cell responses to Ft too. The
research community needs one vaccine against a true intracellular bacteria
for proof of principal.
Justin: Lm is good example of an intracellular bacteria which we can protect
against, but it isn’t a lung pathogen
Karl: Our team shouldn’t discard the antibody data for protection.
Chuck: why is LVS not a good vaccine? It has been very effective in
humans
Rick: the attenuation of LVS is not understood and may be unstable.
TVDC Annual Conference, 8 Oct., 2007, Page 22