KBMA Tularemia Vaccine Progress
Update May 13th 2008
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Cerus Milestones
Milestone 40: Phenotyping of F.t. novicida NER mutants – Milestone completed
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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
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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
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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
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Received attenuated uvrB double mutants for virulence attenuation and protective efficacy
Milestone 44: Formulation and evaluation of KBMA LVS
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Determined the minimal concentration of S-59 to inactivate LVS uvrB
Milestone 45: Test vaccine efficacy of KBMA LVS in murine model
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Measure level and durability of protection against LVS challenge, send to UNM
Milestone 46: Scale-up of KBMA LVS vaccine production
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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
May 13, 2008, Page ‹#›
vaccine
Overall Conclusions MS40-47:
• 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
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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
• MS 40-47 termination will allow for pursuit of alternative
strategies toward development of a more potent KBMA vaccine
based on a Listeria monocytogenes platform
May 13, 2008, Page ‹#›
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Cerus-Anza Milestones
Milestone 55: Compare Cellular Immune Responses Induced by Lm and Ft-Based
Tularemia Vaccines
• Measure cellular immunogenicity of live-attenuated vaccine platforms using model epitope
• Compare immunogenicity of KBMA tularemia vaccine platforms using model epitope
Milestone 56: Demonstrate that Lm Vaccines Induce Protective Cellular Immune
Responses to Ft Antigens
• Measure the T-cell response to IglC induced by live and KBMA Lm expressing IglC compared
with those elicited by Ftn or LVS vaccination
• Demonstrate that Live and KBMA Lm-IglC and Lm-KatG protect against an LVS challenge
• Demonstrate that Live and KBMA Lm-IglC and Lm-KatG protect against a SchuS4 challenge
Milestone 57: Optimization of KBMA Lm Vaccination Route and Regimen
• Compare various routes of administration including IV, IM, IN, ID and oral
• Optimize dosing regimen of most potent and tolerable route
• Confirm optimized route and regimen provides protection against SchuS4 at UNM
Milestone 58: Large Scale GMP-Like Production of KBMA Lm Tularemia Vaccine
• Optimize scalable KBMA vaccine production at 4L scale
• Produce up to a 30L lot of most potent vaccine under GMP-like conditions
• Develop quality assays to support release and stability testing of vaccine lots
• Perform toxicology studies using KBMA Lm platform
Milestone 59: Use Lm Platform For Delivery of Novel Ft Antigens Discovered by TVDC
• Cerus could potentially make available the Lm platform
• Clone up to 10 Ft antigens identified by TVDC group into Lm expression cassettes
• Characterize the intracellular expression levels of various Ft antigens (and SIINFEKL
immunogenicity)
• Rank potency of each vaccine candidate by sharing with UNM for protection studies
• Determined the minimal concentration
of S-59 to inactivate LVS uvrB
May 13, 2008, Page ‹#›
Milestone 55: Compare Cellular Immune Responses
Induced by Lm and Ft-Based Tularemia Vaccines
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Measure cellular immunogenicity of live attenuated vaccine platforms
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Use model ovalbumin epitope to compare Lm-expressing IglC-SIINFEKL (SL8) and Lm
KatG-SL8 fusion proteins with Ftn-pepO-SL8 and LVS-pepO-SL8
• Measure the ability of each vaccine to stimulate a CD8 T cell response in vitro using a
B3Z assay
• Measure the cytokine responses elicited by vaccination with each platform in mice
• Compare the CD8 T cell response to SL8 after prime and boost vaccinations in mice
Compare immunogenicity of KBMA tularemia vaccine platforms
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Compare KBMA Lm-IglC-SL8 and Lm-KatG-SL8 fusion proteins with KBMA Ftn-pepO-SL8
and LVS-pepO-SL8
• Produce 400mL scale lots of each KBMA vaccine
• Measure metabolic activity of each lot of vaccine
• Measure the ability of each vaccine to stimulate a CD8 T cell response in vitro using a
B3Z assay
• Measure the cytokine responses elicited by vaccination with each platform in mice
• Compare the CD8 T cell response to SL8 after prime and boost vaccinations in mice
May 13, 2008, Page ‹#›
MS 55 Flow chart
Construct Lm Ft vaccine candidates
Receive Ft-SL8 vaccine
candidates from UTSA
Measure antigen expression in cells
Characterize immunogenicity of
Live attenuated vaccine candidates
Characterize immunogenicity of
Live attenuated vaccine candidates
Prepare stocks of KBMA vaccine
Prepare stocks of KBMA vaccine
Measure metabolic activity and
antigen expression in cells
Measure metabolic activity and
antigen expression in cells
Characterize immunogenicity of
KBMA attenuated vaccine candidates
Characterize immunogenicity of
KBMA attenuated vaccine candidates
May 13, 2008, Page ‹#›
Listeria monocytogenes
Attenuation
Without
loss of potency
Brockstedt et. al
2004
Tilney and
Portnoy,
1989
Live -Attenuated Platform actA inlB
KBMA Platform actA inlB uvrAB
May 13, 2008, Page ‹#›
Lm-Ft constructs B3Z and
expression in J774s
Molecular constructs at
tRNAArg:
actAp
IglC
ActAN100
actAp
SL8
or
ActAN100
KatG
SL8
Strain
bkgnd
construct
kDa
CRS-100
BH137
BH1222
BH1224
BH1226
BH1228
BH1230
BH1232
BH1398
BH1399
CRS100
CRS100
CRS100
CRS100
Lm583
Lm583
Lm583
Lm583
Lm583
ΔactA ΔinlB
ActAN100-OVA
ActAN100_iglC
ActAN100_KatG
ActAN100_katG(dl
ActAN100_iglC
ActAN100_KatG
ActAN100_katG(dl
ActAN100_katG(dl
ActAN100_katG(dl
37
33.5
93.8
91.5
33.5
93.8
91.5
91.5
91.5
SS) has mut'n*
SS) has mut'n*
SS) seq ok
SS) seq ok
* BH1226 & BH1232 contain a D->W mutation at position 524
of the fusion protein
B3Z assay 080907
Francisella constructs
Western blot in J774 cells
Anti-ActA polyclonal
0.6
OD 595
0.4
980.2
6249-
C
38-
B
R
S-
10
0
H
13
7
B
H
12
22
B
H
12
24
B
H
12
26
B
H
12
28
B
H
12
30
B
H
12
32
B
H
13
98
B
H
13
99
0.0
Listeria Strain
May 13, 2008, Page ‹#› 28-
NB919, pp67-71
PrfA regulon
Contains actAp and hlyp, two promoters used in
strain construction
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prfA
plcA
hly
mpl
actA
What is PrfA?
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Transcriptional activator of hly and actA expression
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Normally “off” in vitro
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Highly upregulated in vivo
May 13, 2008, Page ‹#›
plcB
Vacccina Quadrorotpe Tag
May 13, 2008, Page ‹#›
G155S PrfA* Improves Cellular
Immunogenicity of KBMA Vaccines
prfA* G155S allele increases the number of CD8+ T cells
May 13, 2008, Page ‹#›
G155S PrfA* Improves Cellular
Immunogenicity of KBMA Vaccines
• prfA* G155S allele increases the cytolytic activity of T cells
• Based on these results we are constructing KBMA vaccine
candidates that contain the prfA* G155S allele
May 13, 2008, Page ‹#›
Lm vaccine candidates
Strain
Genetic Background
Antigen Cassette
Status
BH1222
actAinlB
ActAN100-IglC-SL8
Sequence verified
BH1226
actAinlB
ActAN100-KatG-SL8
Sequence error
BH1228
actAinlBuvrAB
ActAN100-IglC-SL8
Sequence verified
BH1398
actAinlBuvrAB
ActAN100-KatG-SL8
Sequence verified
actAinlBuvrABprfAG155S
ActAN100-IglC-SL8
In process
actAinlBuvrABprfAG155S
ActAN100-KatG-SL8
In process
actAinlB
ActAN100-IglC-VacQuad-SL8
In process
actAinlBuvrABprfAG155S
ActAN100-IglC-VacQuad-SL8
In process
May 13, 2008, Page ‹#›
Milestone 55: Upcoming experiments
• Complete construction of enhanced KBMA Lm vaccine candidates
• Expressing iglC-SL8 and katG-SL8 and iglC-VacQuad-SL8
• Measure cellular immunogenicity of live attenuated vaccine candidates
• Measure the ability of each vaccine candidate to deliver SL8-tagged antigens
to cultured DCs using using a B3Z assay
• Enumerate the magnitude of the CD8 T cell response to SL8 epitope after
single IV vaccination in mice
• Compare the effect of the Vac-quatrotope tag vs single SL8 epitope tag
May 13, 2008, Page ‹#›
Milestone 56: Demonstrate that Lm Vaccines Induce
Protective Cellular Immune Responses to Ft Antigens
• Demonstrate that Cerus strains of Live and KBMA Lm-IglCSIINFEKL and Lm-KatG-SIINFEKL protect against a SchuS4
challenge
• Produce lots of KBMA vaccine and send to UNM for testing in animal models
(mice and rats)
• Measure the T-cell response to IglC induced by live and KBMA
Lm expressing IglC compared with those elicited by Ftn or LVS
vaccination
• Produce IglC overlapping peptide library 15aa overlapping by 11aa (211
amino acid long protein)
• Use IglC peptide library for ELISpot assays to measure the IglC-specific T
cell responses induced after vaccination with live and KBMA Lm-IglC and
compare to live and KBMA Ftn and LVS vaccination
• Demonstrate mechanism of protection induced by Lm vaccines is cellular by
depletion of T cell populations and passive transfer studies
May 13, 2008, Page ‹#›
Milestone 57: Optimization of KBMA Lm
Vaccination Route and Regimen
• Compare various routes of administration including IV, IM, IN, ID and
oral
• For oral, IN, and ID administration we will first mutate the inlA gene of Lm to
allow for binding of murine E-cadherin in order to mimic the human interaction
• We will compare the potency of the inlA gain of function mutants to our
traditional platform strain
• Routes will be ranked by ability to induce a cellular immune response: Elispot,
in vivo cytotoxity, and ICS
• Optimize dosing regimen of most potent and tolerable route
• Lm expressing IglC and/or KatG will be used
• Initial evaluation will be performed by immunogenicity
• Optimized route and regimen will be confirmed by SchuS4 protection
studies at UNM
May 13, 2008, Page ‹#›
Milestone 58: Large Scale GMP-Like
Production of KBMA Lm Tularemia Vaccine
• Optimize scalable KBMA vaccine production at 4L scale
• Produce a 30L lot of most potent KBMA Lm vaccine under
GMP-like conditions at a contract manufacturer for
protection studies in non-human primates
• Develop quality assays to support release and stability
testing of KBMA Lm vaccine lots
• Perform toxicology studies using KBMA Lm platform
• Histopathology of administration sites
• Biodistribution studies (using PCR based methods)
May 13, 2008, Page ‹#›
Milestone 59: Use Lm Platform For Delivery
of Novel Ft Antigens Discovered by TVDC
• Cerus could potentially make available the Lm platform
• Clone up to 10 Ft antigens identified by TVDC group into
Lm expression cassettes
• Characterize the intracellular expression levels of various
Ft antigens (and SIINFEKL immunogenicity)
• Rank potency of each vaccine candidate by sharing with
UNM for protection studies
May 13, 2008, Page ‹#›