Transcript Anza-cerus TVDC tech call minutes 1-13-09 final

Tularemia Vaccine Progress
Update Jan 13th 2009
1/13/2009
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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 SL8
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
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MS 55: Flow Chart
Construct epitope-tagged 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
Characterize cytokine profile 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
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Milestone 55: Summary of Key
Accomplishments
• Lm-expressing epitope-tagged IglC or KatG were cloned
• 3 vaccine platforms (Lm:actAinlB, actAinlBuvrAB, actAinlBuvrABprfAG155S)
• Intracellular expression of IglC was 60-180x higher than KatG
• CD8 T cell responses (against SL8) were evaluated using a B3Z assay,
ICS, and ELISpot
• CD8 T cell responses were stronger when fused to IglC than KatG (~ 2 fold)
• prfA* enhanced immunogenicity of IglC-SL8 vaccine (~ 2 fold)
• Quadrotope tag decreased immunogenicity
• Bivalent strains expressing both IglC and KatG were evaluated
• Intracellular expression of each was similar to monovalent strains
• Immunogenicity (ICS and ELIspot) were similar to monovalent strains and better than
coinjection of ½ dose of monovalent strains
• KBMA Lm-IglC induced primary response that was 25% of live
• Only single-dose evaluated, without prfA*
• LVS-pepO-SL8 did not induce SL8 response or boost Lm SL8 response
• Only low-dose LVS used
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Lm-Ft constructs
Molecular constructs at tRNAArg:
actAp
ActAN100
IglC
actAp
ActAN100
SL8
actAp
Molecular construct at comK:
actAp
ActAN100
IglC
SL8
A42R
C4L K3L
Strain
CRS-100/LM11
LM583
LM677
BH137
BH1222
BH2282
BH1228
BH1398
BH2094
BH2172
BH2098
BH2100
BH2180
BH2182
BH2316
Genetic Background
actAinlB
actAinlBuvrAB
actAinlBuvrABprfAG155S
actAinlB
actAinlB
actAinlB
actAinlBuvrAB
actAinlBuvrAB
actAinlBuvrABprfAG155S
actAinlBuvrABprfAG155S
actAinlB
actAinlBuvrABprfAG155S
actAinlB
actAinlBuvrABprfAG155S
actAinlB
BH2292
actAinlBuvrABprfAG155S
KatG
ActAN100
IglC
B8R
B8R
Antigen Cassette
none
none
none
ActAN100-Ova
ActAN100-IglC-SL8
ActAN100-KatG-SL8
ActAN100-IglC-SL8
ActAN100-KatG-SL8
ActAN100-IglC-SL8
ActAN100-KatG-SL8
ActAN100-IglC-VacQuad-SL8
ActAN100-IglC-VacQuad-SL8
ActAN100-IglC-B8R (@ comK)
ActAN100-IglC-B8R (@ comK)
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8 (@tRNAarg)
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8 (@tRNAarg)
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Status
Sequence verified
Sequence ve
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Remade and verified (BH2184 had
point mutation in KatG)
Sequence verified
SL8
p60 Expression is a Surrogate Indicator of
Intracellular Lm in DC2.4
CFU
1.00E+08
1.00E+07
1.00E+06
1.00E+05
0.00
Lane
1
2
3
4
5
6
7
8
9
10
5.00
10.00
p60
11
10
HPI
0
2.5
5.0
9.5
CFU
4.7x105
1.47x106
1.14e7
1.68x107
p60
0
0.05
0.70
1.67
IglC
0
1.09
29.29
33.20
IglC/p60
0
21.80
41.79
19.88
1
0.1
0.01
0.00
2.00
4.00
6.00
8.00
10.00
• DC2.4 were infected with BH2180 at an MOI=1.5.
• Lysates of infected cells were harvested in triplicate, plated for intracellular CFU and examined
for p60 (shown in green) and iglC (red) expression by multiplex western.
• Intracellular CFU and p60 expression increase at similar rates
• IglC expression, when normalized to p60, generally remains constant
Notebook #2006 pp. 91-97
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100mL-Scale Live Attenuated and
400mL-Scale KBMA Lm Vaccine Lots Produced
BH2172
Genetic
Antigen Cassette
Background
Lm677
KatG-SL8
Live
Titer (cfu/mL
Lot#
or Particles/mL)
2.41 x 1010 cfu/mL 837-15-A
BH2182
LM677
IglC-B8R
Live
1.96 x 1010 cfu/mL
837-15-B
BH2292
Lm677
KatG-SL8/IglC-B8R Live
2.20 x 1010 cfu/mL
837-15-C
BH2316
LM11
KatG-SL8/IglC-B8R Live
1.74 x 1010 cfu/mL
837-15-D
BH2172
Lm677
KatG-SL8
KBMA
8.9 x 109 P/mL
2002-070
BH2182
LM677
IglC-B8R
KBMA
9.7 x 109 P/mL
2002-060A
BH2292
Lm677
KatG-SL8/IglC-B8R KBMA
9.6 x 109 P/mL
2002-060B
BH2100
LM677
IglC-VacQuad
9.9 x 109 P/mL
963-104a
Strain
Type
KBMA
• Live and KBMA vaccine Lots available for vaccination
• All KBMA lots listed had 0 cfu/mL
• Need to perform MTS assay on KBMA lots
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Milestone 55: Upcoming Experiments
• Evaluate the immunogenicity of KBMA strains after a prime
and boost vaccination
• KBMA lot production initiated, had some failures (due to cfu)
• Repeat Lm and LVS pepO-SL8 comparison using LVS at
higher doses
• Repeat dose-response study using prfAG155S platform
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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
• 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
• Demonstrate that strains of Live and KBMA Lm-IglC-SL8 and Lm-KatGSL8 protect against a SchuS4 challenge
• Produce lots of KBMA vaccine and send to UNM for testing in animal models
(mice and rats)
1/13/2009
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MS 56: Flow Chart
Construct IglC 15/11 overlapping peptide library
Inject various strains of mice with Lm-iglC
and screen for IglC responses by ICS and ELISpot
Compare Lm and Ft -induced IglC specific
T cell responses
Prepare stocks of KBMA Lm vaccine
Compare Live and KBMA IglC responses in mice
Perform LVS challenge studies to determine
whether KBMA Lm vaccines protect
Perform LVS challenge studies to
determine whether live Lm vaccines protect
Perform T cell depletion studies
to determine mechanism of protection
Prepare stocks of live attenuated vaccine
Send KBMALm vaccines
To UNM for SchuS4 challenge studies
Send Live-attenuated Lm vaccines
To UNM for SchuS4 challenge studies
1/13/2009
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MS56: Summary of Key Accomplishments
• A single IV vaccination with Lm-IglC induced cellular immune responses
to IglC peptides in Balb/c, C57BL/6, FVBN, and C3H/HeJ mice
• Responses wereCD4+, CD8+, or both depending on the haplotype of the mice
• IglC-specific CD8+ epitopes were identified in C57BL/6 and Balb/c mice
• IglC responses were also seen in C57BL/6 mice vaccinated with Lm-KatG
• Preliminary results suggest that Lm-IglC vaccine induces stronger IglC
and SL8 responses than LVS-pepO-SL8
• low-dose LVS was used
• 2 IV vaccinations with Lm-iglC protected 100% of mice against lethal LVS
challenge
• Lm KatG protected 40%, LVS and combination of LM-iglC and Lm-KatG protected
100%
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Protection Experiments Initiated But not
Completed
• p006-08-001
• Balb/c mice were prime and boost vaccinated with Lm-IglC and
Lm-KatG monovalent and bivalent strains (BH2172, BH2182,
BH2292) or LVS
• 100 LD50 IV LVS challenge is scheduled for Mid-January.
• P006-08-003
• Balb/c mice were vaccinated with Lm-IglC strain BH2182 or LVS
by the IV route.
• We will boost vaccinate this month
• Next month T cell populations will be depleted and the animals will
receive a lethal LVS challenge dose.
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MS56 Next Steps
• We will initiate prime-boost vaccinations with KBMA Lm-IglC
vaccines and perform LVS lethal challenge to determine
whether KBMA vaccines can protect mice.
• Once MTA is approved by NIAID and signed by
UNM/Cerus/Anza/LBERI/UCLA, live and KBMA Lm lots will be
sent to UNM for evaluation in SchuS4 challenge model.
1/13/2009
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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
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MS 57: Flow Chart
Construct inlA gain of function vaccine candidates
that have enhanced mouse E-cadherin Binding
Measure cellular infectivity
Compare immunogenicity of live-attenuated Lm
after vaccination by various routes
using ICS and ELISpot
Prepare stocks of KBMA Lm vaccine
Select non-IV route
Compare Live and KBMA responses in mice
Perform LVS challenge studies to determine if
alternative routes of administration are protective
Perform LVS challenge studies to determine
whether KBMA Lm vaccines protect
Optimize vaccination regimen by
Varying time between prime and boost
UNM to performSchuS4 challenge studies after
vaccination by alternate route
UNM to performSchuS4 challenge studies after
vaccination by alternate route and regimen
1/13/2009
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MS57: Strain Construction for Route
Optimization
• To facilitate route optimization, the inlA gene of our platform Lm strains
has been altered to allow for binding to murine E-cadherin
• The sequence of the wild-type EGDe inlA gene was synthesized and the
inlA gene in our platform strain was replaced (inlAWT) in our wild-type and
KBMA platform strains
• 2 point mutations S192N and Y369S were incorporated into the EGDe inlA
sequence (inlAM) and inserted into the chromosome of our wild-type and
KBMA platform strains
• As published in Wollert et al., Cell 2007
Strain
CRS-100
BH2130
BH2164
BH2170
BH2194
BH2132
BH2166
BH2134
BH2168
Genetic Background
actAinlB
actAinlBinlAWT
actAinlBinlAWT
actAinlBinlAM
actAinlBinlAM
actAinlBuvrABprfAG155SinlAWT
actAinlBuvrABprfAG155SinlAWT
actAinlBuvrABprfAG155SinlAM
actAinlBuvrABprfAG155SinlAM
1/13/2009
Antigen Cassette
none
none
ActAN100-IglC-SL8
none
ActAN100-IglC-SL8
none
ActAN100-iglC-SL8
none
ActAN100-iglC-SL8
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Status
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
MS57: Summary of Key Accomplishments
• inlAm gain of function did not enhance invasion of CaCo2 cells
as reported by Wollert et al.
• We have identified mouse epithelial cell lines for further testing
• IV vs Oral route comparison initiated
• T cell responses in spleens were higher after IV administration
• Mucosal T cell responses (IEL) were low, but similar after IV and oral
administration
• InlA Gain-of-Function mutation did not significantly enhance splenic
immunogenicity either by oral or IV route
• InlA Gain of function may slightly increase immune responses after Oral
administration (less than 2-fold increase)
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Routes of Administration
• In order to evaluate which route of administration is the
most effective at conferring protection, we have initiated a
prime-boost vaccination regimen with the live attenuated
Lm-IglC strain BH2182. Mice were vaccinated with 2x106
cfu IV or IM, 1x108 cfu SC or ID, and 1x109 orally. These
animals were boosted in December and will be challenged
with a 100 IV LD50 dose of LVS in Mid January.
1/13/2009
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MS57 Next Steps
• Mucosal immunity will be evaluated again after oral
immunization to determine whether the >2fold increase in
mucosal immunity seen with the inlAM strain is reproducible
• Invasion assays will be performed in murine epithelial cell
line (CT-26)
• Repeat LVS IN LD50 study using alternate method of
anesthesia
1/13/2009
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Other News
• Update on Status of Multiparty MTA
• Anza move to Emeryville is scheduled for Feb15
1/13/2009
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Action Items
• Justin requested that Barbara share Dr. Horwitz’s recent electronic journal article with
NIAID and TVDC. Action: Barbara will do this (done 1/13/09)
• Multiparty MTA will be signed by UNM, Cerus, Anza, UCLA, and LBERI. Nancy Carr of
UNM will send the original MTAs for signature.
• Justin will send IglC live and KBMA Lm constructs to Terry at UNM for protection studies
against a SCHU S4 challenge at UNM. (after the MTA is signed)
• Barbara will re-email site visit dates to Justin (done 1/13/09)
• Anza/Cerus has a 5 kgm batch of Teknova Chamberlains and could share some with
UNM and LBERI if desired.
1/13/2009
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