Transcript Anza-cerus tech call minutes 06-10-08 final

KBMA Tularemia Vaccine Progress
Update June 10th 2008
June 10, 2008, Page 1
•
•
•
•
•
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
June 10, 2008, Page 2
Milestone 55: Compare Cellular Immune Responses
Induced by Lm and Ft-Based Tularemia Vaccines
•
Measure cellular immunogenicity of live attenuated vaccine platforms
•
•
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
•
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
June 10, 2008, Page 3
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
June 10, 2008, Page 4
Lm-Ft constructs
Molecular constructs at tRNAArg:
actAp
actAp
ActAN100
actAp
IglC
ActAN100
IglC
SL8
or
ActAN100
KatG
SL8
SL8
A42R
C4L K3L
B8R
Strain
Genetic Background
Antigen Cassette
Status
CRS-100
actAinlB
none
Sequence verified
BH137
actAinlB
ActAN100-Ova
Sequence verified
BH1222
actAinlB
ActAN100-IglC-SL8
Sequence verified
BH1226
actAinlB
ActAN100-KatG-SL8
Sequence error
BH2106
actAinlB
ActAN100-KatG-SL8
Complete
BH1228
actAinlBuvrAB
ActAN100-IglC-SL8
Sequence verified
BH1398
actAinlBuvrAB
ActAN100-KatG-SL8
Sequence verified
BH2094
actAinlBuvrABprfAG155S
ActAN100-IglC-SL8
Complete
BH2096
actAinlBuvrABprfAG155S
ActAN100-KatG-SL8
Does not express Ag
BH2098
actAinlB
ActAN100-IglC-VacQuad-SL8
Complete
BH2100
actAinlBuvrABprfAG155S
ActAN100-IglC-VacQuad-SL8
Complete
June 10, 2008, Page 5
Lm-Ft Constructs Stimulate B37 Cells
B3Z 052308
0.8
0.4
0.2
IglC
KatG
Except BH2096, which will be recloned
June 10, 2008, Page 6
06
B
H
21
96
B
H
20
98
B
H
13
00
B
H
21
98
B
H
20
94
H
20
B
B
H
12
22
7
H
13
B
R
S1
00
0.0
C
OD 595
0.6
IglC Fusion Elicits More T-cells than KatG
BH1228 = actAinlBuvrAB ActAN100-IglC-SL8
BH1398= actAinlBuvrAB ActAN100-KatG-SL8
3
2
1
98
H
13
B
H
12
B
98
B
H
13
28
H
12
28
0
0
unstim
SL8
1000
500
0
IFN- SFC/2e5 splenocytes
10
4
1500
LLO 190 responses
800
600
400
200
0
Likely due to better secretion of IglC
June 10, 2008, Page 7
unstim
LLO 190
BH1398
20
IFN- SFC/2e5 splenocytes
% IFN- CD4+ T cells
5
B
% IFN- CD8+ T cells
30
SL8 responses
BH1398
LLO 190 responses
BH1228
SL8 responses
IM08-042 ELISpot (NB2000 p1-5)
BH1228
IM08-042 ICS (NB2000 p1-5)
Constitutive prfA Allele (G155S) Increases
Immunogenicity of LM-IglC-SL8 Vaccines
BH1222 = actAinlBActAN100-IglC-SL8
BH1228 = actAinlBuvrAB ActAN100-IglC-SL8
BH2094 = actAinlBuvrABprfAG155S ActAN100-IglC-SL8
BH2098= actAinlBAct AN100-IglC-VacQuad-SL8
BH2100= actAinlBuvrABprfAG155S ActAN100-IglC-VacQuad-SL8
LLO190 responses
SL8 responses
6
% IFN-  CD4+ T cells
20
10
4
2
BH2100
BH2098
BH2094
BH2100
BH2098
BH2094
BH1228
BH1222
BH1228
0
0
BH1222
% IFN-  CD8+ T cells
30
VacQuad reduced SL8 immunogenicity, and was not helped by prfA*
BH1222: actAinlB-iglC
June 10, 2008, Page 8
Milestone 55: Upcoming Experiments
• Strain 2096 (LmactAinlBuvrABprfAG155SKatGSL8)
will be recloned.
• We will clone a bivalent vaccine strain that expresses both
IglC and KatG.
• We will clone the gain of function mutations into the inlA
gene in order to increase the affinity for murine E-cadherin
and this inlA allele will be introduced into vaccine strains.
These strains will be used to evaluate immunogenicity of
Lm vaccines by various routes of administration.
June 10, 2008, Page 9
Action Items
• Barbara- will edit MS 56 start date to 8/1/2008 in the TVDC MS
Project file.
• Anza will develop a 3 way MTA to allow UNM and Anza to
work with materials developed by Dr. Horowitz
June 10, 2008, Page 10