Tularemia Vaccine Progress
Update February 10, 2009
2/10/2009
Cerus-Anza Milestones
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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
2/10/2009
2
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
2/10/2009
3
MS 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
2/10/2009
4
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)
2/10/2009
5
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
New Lm-Ft Constructs: KatG linked to
bacterial surface
BamHI
ActAN100-KatG-ActA(390-629)
SpeI MfeI
KatG
ActAN100
ActACTD(390-629)
Strain
Genetic Background
Antigen Cassette
Status
Notebook page
BH2562
∆actA∆inlB
ActAN100-KatG-SL8-anchored
Not sequenced
NB208, p62.
BH2568
∆actA∆inlB∆uvrAB G155SprfA*
ActAN100-KatG-SL8-anchored
Not sequenced
NB208, p62.
BH2564
∆actA∆inlB
ActAN100-KatG-SL8-anchored
ActAN100-IglC-B8R @comK
Not sequenced
NB208, p62.
BH2566
∆actA∆inlB∆uvrAB G155SprfA*
ActAN100-KatG-SL8-anchored
ActAN100-IglC-B8R @comK
Not sequenced
NB208, p62.
•Potential advantages of surface linked proteins:
1) Pre-loading of antigen that is expressed by the bacteria in culture
2) Potentially increasing expression of poorly secreted hydrophobic
antigens due to proximity to the membrane
• Strains constructed, but not sequence verified yet
• Expression and immunogenicity experiments to be planned
2/10/2009
6
100mL-scale Live Attenuated and
400mL-scale KBMA Lm Vaccine Lots Produced
BH2172
Genetic
Background
Lm677
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
KBMA
9.9 x 109 P/mL
963-104a
Strain
Antigen Cassette
Type
KatG-SL8
Live
Titer (cfu/mL
or Particles/mL)
2.41 x 1010 cfu/mL
Lot#
837-15-A
• Live and KBMA vaccine lots available for vaccination and shipment to UNM
• All KBMA lots listed had 0 cfu/mL
2/10/2009
7
MS 55: Upcoming Experiments
• Evaluate immunogenicity of KBMA strains compared to live-attenuated
strains after one or two vaccinations
• Have primed, waiting for boost
• Strains with surface-linked expression:
• Determine expression and immune responses of Lm expressing surface-linked
KatG protein
• Determine if presence of KatG on bacterial surface impacts expression and
immunogenicity of secreted IglC.
• Repeat Lm and LVS pepO-SL8 comparison using LVS at higher doses
2/10/2009
8
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 LmKatG-SL8 protect against a SchuS4 challenge
• Produce lots of KBMA vaccine and send to UNM for testing in animal
models (mice and rats)
2/10/2009
9
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
2/10/2009
10
MS 56: 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 were 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
• low-dose LVS was used
• Two IV vaccinations with Lm-IglC protected 100% of mice against lethal
LVS challenge
• 40% protection from Lm-KatG, 100% protection from LVS, Lm-IglC, and combination
of Lm-IglC and Lm-KatG
2/10/2009
11
100x LD50 of LVS Is too Strong for
Challenge Dose
Percent survival
Survival after 100x LVS challenge
HBSS
LVS
Lm677
BH2172 Lm-KatG
BH2182 Lm-IglC
BH2292 Lm-KatG-IglC
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
Time (days post-challenge)
• None of the animals given 100x LD50 LVS iv survived
• Animals vaccinated twice with LVS died first
• Will use 10x LD50 LVS challenge for next challenge experiments
2/10/2009
12
Protection Experiments Initiated
• P006-08-003
• Balb/c mice have been prime and boost vaccinated iv with Lm-IglC
strain (BH2182) or LVS
• T cell populations will be depleted and the animals will receive a
10x LD50 iv LVS challenge (mid-February)
• P009-006
• Balb/c mice will be prime and boost vaccinated iv with live or
KBMA Lm-IglC (BH2182) or LVS
• 10x LD50 iv LVS challenge is scheduled for mid-March
2/10/2009
13
MS 56: Next Steps
• Once MTA is signed by UNM/Cerus/Anza/LBERI/UCLA, vaccine lots of
live and KBMA Lm will be sent to UNM for evaluation in SchuS4
challenge model
• Optimize protocol for IN administration of LVS for challenge studies
• Currently planning up-coming challenges as IV with 10x LD50 dose of LVS
2/10/2009
14
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 mutated inlA gene of Lm to allow for better
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 potent cellular immune response:
ELISpot, ICS, and in vivo cytotoxicity
• 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
2/10/2009
15
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
2/10/2009
16
MS 57: 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
2/10/2009
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
MS 57: 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)
2/10/2009
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Comparison of Immune Responses
Induced by Different Routes
800
unstim
LLO 190
600
400
200
• Vaccination by IV route induces highest immune responses
• IM is next best route
• For high avidity T cell responses, IV and IM are comparable
• Noticed slight scarring (4/5) and necrosis (1/5) at the site of the ID injection
• possibly due to constitutive expression of virulence determinants (prfA*)
2/10/2009
19
oral (1e9)
id (1e8)
sc (1e8)
im (1e7)
im (2e6)
0
iv (2e6)
oral (1e9)
id (1e8)
0
IFN- SFC/2e5 splenocytes
25
sc (1e8)
oral (1e9)
id (1e8)
sc (1e8)
im (1e7)
im (2e6)
0
33-19
200
100
50
im (1e7)
50
LLO 190 responses
unstim
im (2e6)
350
100
350
300
iv (2e6)
unstim
iglC pool2
33-19 responses
IFN- SFC/2e5 splenocytes
400
iv (2e6)
IFN- SFC/2e5 splenocytes
iglC pool2 responses
Comparison of Immune Responses
Induced by Non-IV Routes
LLO 190
600
400
200
oral (1e9)
id (1e8)
0
sc (1e8)
oral (1e9)
0
unstim
im (1e7)
10
800
im (2e6)
20
IFN- SFC/2e5 splenocytes
30
id (1e8)
oral (1e9)
id (1e8)
sc (1e8)
im (1e7)
0
33-19
40
sc (1e8)
10
unstim
im (1e7)
20
50
im (2e6)
unstim
iglC pool2
LLO 190 responses
33-19 responses
IFN- SFC/2e5 splenocytes
30
im (2e6)
IFN- SFC/2e5 splenocytes
iglC pool2 responses
• Vaccination by IV route induces highest immune responses
• IM is next best route
• For high avidity T cell responses, IV and IM are comparable
• Noticed slight scarring (4/5) and necrosis (1/5) at the site of the ID injection
• possibly due to constitutive expression of virulence determinants (prfA*)
2/10/2009
20
Comparison of Routes: 100x LD50 LVS
Challenge
Percent survival
Survival after 100x LVS challenge
HBSS
LVS
Lm677
BH2182 iv
BH2182 im
BH2182 sc
BH2182 id
BH2182 oral
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
Time (days post-challenge)
• None of the animals given 100x LD50 LVS iv survived
• Animals vaccinated with LVS 2x died first
• Will use 10x LD50 LVS challenge for other experiments
2/10/2009
21
MS 57: Next Steps
• Repeat challenge study comparing different routes of administration
with lower challenge dose of LVS
• Compare IV and IM routes after single and prime-boost vaccinations
• Optimize conditions for intranasal challenge in mice for alternate way
to compare potency of different routes. Repeat LVS IN LD50 study.
• 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)
2/10/2009
22
Other News
• Anza re-organization
• Anza has signed Multiparty MTA
• Will ship reagents when MTA is fully executed by all parties
• Move to Emeryville is planned for February 14&15
• After clean test results from sentinel mice, can bring mice in to
begin experiments.
2/10/2009
23
Action Items
• Meredith: 4 new LmFt constructs with the KatG linked to the bacterial surface will be sequenced
• Barbara: will follow-up with Nancy Carr on the status of the Anza/UNM/Cerus/UCLA/LBERI MTA (At
Cerus on 2/11/09; has been signed by Anza)
• Cerus/Anza will use i.v. challenge at 10x LD50 LVS for future challenges rather than 100X LD50 LVS
i.v. challenge dose.
• Meredith will determine an i.v. LVS LD50 on LVS vaccinated mice
• Meredith: When looking at immune response at LVS expressed IgLC vs. LM expressed IglC,
Meredith should consider trying to normalize against the amount of immunogenic protein made by
the two strains rather than normalizing to CFU. Cerus/Anza has never tried to quantitate protein from
LVS, so not sure how straightforward or difficult this might be.
• Cerus is letting Anza finish on-going animal experiments at Cerus site, then will start new animal
experiments at Anza after sentinel animals are observed.
• Meredith will start longer animal experiments at Anza so animals are only at one site during the
experiment.
• Cerus/Anza site visit is 4/16/09 Thursday: Rick and Barbara arrive in Oakland at 9:35am 4/16
Thursday and depart Oakland at 7:25pm at 4/16 Thursday, to take advantage of non-stop flights.
2/10/2009
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