KBMA Tularemia Vaccine Progress
Update Aug 20th 2008
Aug 20, 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 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
July 8, 2008, Page 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
B8R
Aug 20, 2008, Page 3
KatG
SL8
Molecular construct at comK:
ActAN100
IglC
B8R
Milestone 55: Upcoming Experiments
• We will construct the bivalent IglC/KatG expressing Lm
strain on the KBMA background
• We will evaluate the immunogenicity of the bivalent Live
attenuated Lm strain expressing IglC-B8R and KatG-SL8
fusion proteins
• We will compare the immunogenicity of each monovalent
strain with the bivalent strains
• We will produce a lot of live Ftn-PepO-SL8
• The SL8 responses induced by Ft-PepO-SL8 and LM-iglCSL8 will be compared
Aug 20, 2008, Page 4
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)
Aug 20, 2008, Page 5
Search for IglC Immune Responses after
Vaccination with Lm-IglC
Mice
Balb/c
C57BL/6
C3H/HeJ
FVB/NJ
SJL/J
Vaccination
H2-d
H2-b
H2-k
H2-q
H2-s
ELISpot with peptide library
51 peptides (2 pools)
LmactA inlBuvrAB prfA* IglC-SL8
7 days
(BH2094)
Harvest spleens
1x 106 cfu IV
IM08-059 NB#2000 pp11-14
Aug 20, 2008, Page 6
Pool 1 : Peptide #s 1-26
Pool 2: Peptide #s 27-51
400
200
200
0
SJL
C3H
FVBN
C57BL/6
0
400
SJL
iglC pool2
C3H
600
LLO pool
FVBN
iglC pool1
unstim
600
C57BL/6
unstim
LLO pool-specific response
Balb/c
800
IFN-g SFC per 2e5 splenocytes
IglC responses
Balb/c
IFN-g SFC per 2e5 splenocytes
Responses to IglC Peptide Pools in All
Strains of Mice by IFN-g ELIspot
•IglC responses were detectable in all 5 strains of mice (weak with SJL)
•Responses were mostly to pool2 peptides
•Responses were strongest in FVBN Mice
IM08-059
Aug 20, 2008, Page 7
IglC Immune Responses Were Also
Detected by ICS
iglC pool2
CD4
0.5
SJL
C3H/HeJ
FVB/N
C57BL/6
-0.5
Balb/c
SJL
C3H/HeJ
FVB/N
C57BL/6
Balb/c
0.0
2
0
-2
C3H/HeJ
1.0
FVB/N
0.00
1.5
CD8
4
C57BL/6
0.05
CD4
CD8
Balb/c
0.10
2.0
% IFN- g T cells
% IFN- g T cells
% IFN- g T cells
6
CD4
CD8
0.15
-0.05
LLO pool
2.5
0.20
•Responses to IglC pool1 peptides were weak in all 5 strains of mice
•Responses to IglC pool2 peptides were CD4 (Balb/c, FVBN), CD8
(C57BL/6) or both (C3H/HeJ), SJL had very weak responses
•Responses were strongest in FVBN Mice
IM08-059
Aug 20, 2008, Page 8
SJL
iglC pool1
Mapping IglC Responses in Balb/c and C57BL/6 mice
BH2094 (actAinlBuvrAB prfA*-iglC) in Balb/c mice
180
Balb/c
160
Peptide #33, 34
QEYKTDEAWGIMIDL
TDEAWGIMIDLSNLE
CD4+
140
IFNg SFC/2e5 cells
120
100
80
60
Peptide #9
NCRLFIDSLTIAGEK
?
40
20
C57BL/6
51
50
49
48
47
46
45
44
43
42
41
40
39
38
37
Peptide #34, 35
TDEAWGIMIDLSNLE
WGIMIDLSNLELYPI
CD8+
40
IFNg SFC/2e5 cells
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
BH2094 (actAinlBuvrAB prfA*-iglC) in C57BL/6 mice
60
50
17
16
15
14
13
12
11
9
10
8
7
6
5
4
3
2
1
0
30
20
10
51
50
49
48
47
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
IM08-059
Mapping IglC Responses in FVBN and C3H/HeJ mice
BH2094 (actAinlBuvrAB prfA*-iglC) in FVB/N mice
800
FVB/NJ
700
Peptide #37, 38
NLELYPISAKAFSIS
YPISAKAFSISIEPT
CD4+
600
IFNg SFC/2e5 cells
500
400
300
Peptide #24
VLIKSNVRTKIEEKV
200
100
47
48
49
50
51
48
49
50
51
46
47
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
9
10
8
7
6
5
4
3
2
1
0
BH2094 (actAinlBuvrAB prfA*-iglC) in C3H mice
90
C3H/HeJ
80
70
IFNg SFC/2e5 cells
60
50
40
Peptide #33, 34
QEYKTDEAWGIMIDL
Peptide #35, 36
TDEAWGIMIDLSNLE
WGIMIDLSNLELYPI
CD8+
IDLSNLELYPISAKA
Peptide #23,24
CD4+
ITLGVLIKSNVRTKI
VLIKSNVRTKIEEKV
CD4+
30
20
10
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
9
8
7
6
5
4
3
10
IM08-059
2
1
0
Mapping IglC Responses in SJL mice
BH2094 (actAinlBuvrAB prfA*-iglC) in SJL mice
120
SJL
100
Peptide #37, 38
NLELYPISAKAFSIS
YPISAKAFSISIEPT
IFNg SFC/2e5 cells
80
60
Peptide #46
DGLTTSQGSLPVCCA
Peptide #50
STDKGVAKIGYIAAA
40
20
IM08-059
Aug 20, 2008, Page 11
51
50
49
48
47
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
9
10
8
7
6
5
4
3
2
1
0
ICS with Individual Peptides
C57BL/6
CD4
CD8
2
1
•Balb/c response to peptide 34 is CD4
•C57BL/6 responses to peptides 34 and 35 are CD8
•Robust FVBN response to peptides 37 and 38 are CD4
IM08-059
Aug 20, 2008, Page 12
LLO pool
38
0
37
% IFN- g T cells
LLO pool
LLO 190
SL8
LLO pool
LLO 91
0
34
1
CD8
35
2
3
CD4
34
3
30
28
26
24
22
20
10
8
6
4
2
1.0
0.8
0.6
0.4
0.2
0.0
unstim
CD8
% IFN- g T cells
4
CD4
unstim
% IFN- g T cells
5
FVBN
unstim
Balb/c
ICS with Individual Peptides
C3H
1.0
0.2
0.2
LLO pool
36
35
34
33
24
23
0.0
unstim
0.0
0.4
LLO pool
0.4
0.6
38
0.6
0.8
37
CD8
0.8
CD4
CD8
unstim
CD4
% IFN- g T cells
% IFN- g T cells
1.0
SJL
•C3H/HeJ responses to peptides 23 and 24 are CD4
•C3H/HeJ responses to peptides 33 and 34 are CD8
•C3H/HeJ responses to peptides 35 and 36 are CD4
•SJL responses to peptides 37 and 38 are weak
IM08-059
Aug 20, 2008, Page 13
MS56 Summary
• A single IV vaccination with Lm-IglC induces a cellular
immune response 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
Aug 20, 2008, Page 14
MS56 Next Steps
• Finer mapping of optimal IglC peptides in regions of
potential CD8 responses (because 9mers may stimulate a
stronger response than 15mers)
• We will order seven 9mers (overlapping by 8) spanning peptide # 9
to determine whether the weak response in Balb/c mice in that
region is a secondary region of immunogenicity
• We will order fourteen 9mers (overlapping by 8) that span peptides
#33-35 to map the optimal CD8+ epitopes in that region in C57Bl/6
and C3H/HeJ mice.
• Comparison of Lm-IglC and Ft vaccines:
• Direct comparison of the celllular immune response to an
endogenous Ft antigen by ELIspot and ICS
Aug 20, 2008, Page 15
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
Aug 20, 2008, Page 16
Milestone 57: Progress
• 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
Genetic Background
Antigen Cassette
Status
CRS-100
actAinlB
none
Sequence verified
BH2130
actAinlBinlAWT
none
Sequence verified
BH2164
actAinlBinlAWT
ActAN100-IglC-SL8
Sequence verified
BH2170
actAinlBinlAM
none
Sequence verified
BH2164
actAinlBinlAM
ActAN100-IglC-SL8
Sequence verified
BH2132
actAinlBuvrABprfAG155SinlAWT
none
Sequence verified
BH2166
actAinlBuvrABprfAG155SinlAWT
ActAN100-iglC-SL8
Sequence verified
BH2134
actAinlBuvrABprfAG155SinlAM
none
Sequence verified
BH2168
actAinlBuvrABprfAG155SinlAM
ActAN100-iglC-SL8
Sequence verified
• Virulence, immunogenicity, and cellular infectivity of inlAWT and inlAMexpressing strains will be evaluated in the coming month/months
Aug 20, 2008, Page 17
Anza QA performed in past 6 months
• All pipetmen are calibrated twice per year (March and Sept)
• All incubators, refrigeration equipment, and freezers are
calibrated once per year (Sept)
• All BSCs, and fume hoods are calibrated once per year (Oct)
• All centrifuges are checked and calibrated once per year
(var)
• Spectrophotometer, thermometers, balances, are calibrated
once per year (var)
• All freezers are on backup power and are alarmed (via
protection 1), but we are investigating a new wireless system
that is connected through the web
Aug 20, 2008, Page 18
Action Items
• Barbara: Add Dr. Meredith Leong to TVDC email routing lists (done 8/21/08)
• Terry will contact Justin for the peptide library pools (done 8/21/08)
• Justin: use IV route with the construct strain first rather than optimizing many
different routes
• Justin: will test iglC strain or mix the two, and test now with iv vaccination first,
to determine whether it elicits protection. Get information sooner rather than
later. Anza/Cerus can send them to UNM as soon as MTA is completed.
• Barbara: joining Anza/UNM teleconference on 8/21 regarding multiparty MTA
with UCLA
• Justin: Anza will move sites in January 2009
Aug 20, 2008, Page 19