KBMA Tularemia Vaccine Progress
Update Nov 11th 2008
Justin Skoble
11/11/2008
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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
11/11/2008
2
Milestone 55: Summary
• Cellular immunogenicity of live-attenuated Lm vaccine platforms were
measured
• Lm-expressing IglC-SL8 and Lm KatG-SL8 fusion proteins were cloned
• The ability of each to stimulate a CD8+ T cell response to SL8 was
evaluated using a B3Z assay, ICS, and ELISpot
• CD8 T cell responses against SL8 were stronger when fused to IglC than
KatG
• prfA* enhanced immunogenicity of IglC-SL8 vaccine ~ 2 fold
• Quadrotope tag decreased immunogenicity and will not be used
actAp
actAp
Molecular constructs at tRNAArg:
ActAN100
IglC
actAp
ActAN100
SL8
actAp
ActAN100
IglC
SL8
A42R
C4L K3L
KatG
Molecular construct at comK:
ActAN100
B8R
11/11/2008
SL8
3
IglC
B8R
Lm-Ft constructs
actAp
actAp
Molecular constructs at tRNAArg:
ActAN100
IglC
actAp
SL8
ActAN100
actAp
ActAN100
IglC
SL8
A42R
C4L K3L
Strain
CRS-100/LM11
LM677
BH137
BH1222
BH2282
BH1228
BH1398
BH2094
BH2172
BH2098
BH2100
BH2180
BH2182
BH2316
Genetic Background
actAinlB
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
Molecular construct at comK:
ActAN100
IglC
B8R
B8R
Antigen Cassette
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)
11/11/2008
4
Status
Sequence verified
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
Strains Evaluated for Intracellular Expression
by Multiplex IR-fluorescence Western Blot
Insert
Lane
Sample
Parent
tRNA-Arg
1
SeeBlue Plus2
2
Mock
3
BH1029
CRS-100
NS5b
4
CRS-100
CRS-100
5
CRS207
CRS-100
6
BH2292
Lm 677
7
BH2182
Lm 677
8
BH2172
Lm 677
katG-SL8
9
BH2316
CRS-100
katG-SL8
10
BH2180
CRS-100
11
BH2282
CRS-100
comK
inlB
mesothelin
katG-SL8
iglC-B8R
iglC-B8R
iglC-B8R
iglC-B8R
katG-SL8
NB2006_053
11/11/2008
5
Overlay of 700nm and 800nm Images
188
< katG
62
< p60
49
< iglC
38
28
1
2
3
4
5
6
7
iglC
+
+
katG
+
8
+
9
10
+
+
+
11
+
Overlay image with boxes showing
integrated intensities of selected proteins
NB2006_053:
1
2
3
4
5
6
7
iglC
+
+
katG
+
11/11/2008
8
+
7
9
10
+
+
+
11
+
Relative IglC and KatG Expression Levels
Insert
Strain
iglc
katG
BH2292
+
+
BH2182
+
BH2172
BH2316
+
BH2180
+
BH2282
katG*
iglC*
p60*
iglC/p60 katG/p60
0.26
33.68
5.98
5.63
0.04
0
30.06
4.01
7.50
0.00
+
0.19
0
4.76
0.00
0.04
+
0.31
11.02
3.66
3.01
0.08
0.05
30.87
3.61
8.55
0.01
0.41
-0.03
3.01
-0.01
0.14
+
BH1029
NS5b
52.97
3.12
16.98
ANZ207
Meso
0.36
3.13
0.12
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8
Immunogenicity of Bivalent vs Monovalent
Live-attenuated Lm vaccines (ICS)
IglC-tag
KatG-tag
Lm-control
*
*
***
**
*
• C57BL/6 Mice immunized IV with 1e6 prfA* or 5e6 actA/inlB
• Compared bivalent strain to dosing with ½ dose of both monovalent strains
-Slight decrease in T cell frequency with bivalent strain
-Decrease in immunogenicity was larger when dose decreased by ½
* p<.05, **P<.005, ***p<.005
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9
IglC and SL8 Responses not Induced or
Boosted by LVS-PepO-SL8
IM08-090
• 1e6 Lm-IglC administered IV, 1e4 LVSPepO-SL8 delivered ID
• 3 weeks between prime and boost, spleens harvested d6 post-boost
• LVS-pepO-SL8 does not induce measurable IglC or SL8 responses by itself
• Lm-induced responses not primed or boosted by LVS
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10
Live-attenuated vs. KBMA primary
response (ICS)
1222 ActA inlB
1228 ActAinlBuvrAB
• KBMA-Lm primary responses reduced by ~75%
• May improve after a boost
11/11/2008
11
Live-attenuated vs. KBMA primary
response (ELISpot)
1222 ActA inlB
1228 ActAinlBuvrAB
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12
Milestone 55: Upcoming Experiments
• We will confirm that p60 expression correlates with cfu by
performing an MOI dose response and perform western
blot and cfu analysis in parallel
• We will evaluate the immunogenicity of KBMA strains after
a prime and boost vaccination
11/11/2008
13
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)
11/11/2008
14
Previous work
• A single IV vaccination with Lm-IglC induces cellular
immune responses to IglC in Balb/c, C57BL/6, FVBN, and
C3H/HeJ mice
• Responses are CD4+, CD8+, or both depending on the
haplotype of the mice
• IglC-specific CD8+ epitopes were identified in C57BL/6
and Balb/c mice
• Preliminary results suggest that Lm-IglC vaccine induces
stronger IglC and SL8 responses than LVS-pepO-SL8
11/11/2008
15
Lm Induces Responses Against IglC Peptide
by ICS and ELISpot in C57BL/6 Mice
ICS
ELISpot
•Vaccination with Lm-KatG induced response against IglC peptide (33-10).
• GIMIDLSNL sequence not present in KatG
• GIMIDLS is present in Lm genome- hypothetical protein lmo0368
11/11/2008
16
Lm-IglC Vaccine Protects Balb/c mice
Against Lethal LVS Challenge
11/11/2008
17
MS56 Summary
• Endogenous Lm antigen is cross-reactive with IglC in
C57BL/6 mice
• Should not use this strain for further IglC immunogenicity
• OK to use for SL8 or B8R studies
• Will focus on Balb/c mice
• IV vaccination with LM-IglC protects 100% Balb/c against
10x LD50 LVS challenge
• Lm-KatG only protects 40% of animals
• Vaccination with1/2 dose of both Lm strains also protected 100%
• as did LVS vaccination
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MS56 Next Steps
• Increase stringency of LVS challenge (look at 10 vs 100
LD50)
• Anza will vaccinate mice with various live and KBMA Lm
vaccines to determine whether IglC, KatG, or both protect
against lethal LVS infection
• Entire IglC peptide library will be tested with Lm expressing
an irrelevant antigen to determine if there is cross-reactivity
between Lm and IglC in Balb/c mice as well
• Once MTA is approved, live and KBMA Lm lots will be sent
to UNM for evaluation in SchuS4 challenge model.
11/11/2008
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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
11/11/2008
20
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
Antigen Cassette
none
none
ActAN100-IglC-SL8
none
ActAN100-IglC-SL8
none
ActAN100-iglC-SL8
none
ActAN100-iglC-SL8
11/11/2008
21
Status
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
inlAM Allele Does Not Increase Invasion of
CaCo2 Cells
CaCo2 Invasion NB2006-024
10 6
CFU/mL
10 5
10 4
10 3
10 2
10 1
11/11/2008
H
21
66
B
B
B
22
H
21
94
H
21
64
H
21
B
• CaCo2 cells are of human origin not mouse
• But was the cell line used in Wollert at al.
68
10 0
Name
Description
Antigen
BH 2164
BH 2194
BH 2166
BH 2168
actAinlBinlAwt
actAinlBinlAM
actAinlBuvrprfA*nlAwt
actAinlBuvrprfA*inlAM
ActAN100-iglC-SL8
ActAN100-iglC-SL8
ActAN100-iglC-SL8
ActAN100-iglC-SL8
MS57 Previously Reported
• Route comparison initiated
• C57BL/6 mice were vaccinated with BH2164 and BH2194 IV and orally
to determine whether the inlAm gain of function contributes to
immunogenicity
• InlA Gain-of-Function mutation did not significantly enhance splenic
immunogenicity either by oral or IV route
11/11/2008
23
Oral Administration Induces Lower Splenic
but Equal Mucosal Immunity as IV
BH1228: actAinlBuvrAB-iglC-SL8, 5e6 CFU-IV, 1e9cfu Oral
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24
Repeat of (1e9) Oral Administration with inlAm
vs inlAwt, Splenic responses by ELISpot
Name
Description
BH 2164
BH 2194
BH 1228
actAinlBinlAwt
actAinlBinlAM
actAinlBuvr
Antigen
ActAN100-iglC-SL8
ActAN100-iglC-SL8
ActAN100-iglC-SL8
• No significant
differences with inlAM
• 2194 trending higher
than 2164 after oral
11/11/2008
25
IEL Responses after Oral Administration
• IEL Responses to IglC and LLO epitopes were below LOD
• inlAm gain of function may increase SL8 mucosal immune
response after oral administration, but needs repeating
11/11/2008
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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.
• An intranasal LD50 will be performed with DVC lot16 LVS
as this route of infection may be more relevant for
investigation of tularemia vaccines.
• Murine epithelial cell line will be purchased for invasion
assays
Action Items
• Barbara will contact Nancy Carr at UNM HSC Contracts
and grants to urge completion of the MTA (done 11/12/08).
BG offered to organize a teleconference call also.
• Justin- do intranasal vaccination route at some time. IV
vs. IN may be different enough due to targeting differences
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