Progress on Listeria monocytogenes-Based
Tularemia Vaccines
Justin Skoble
Meredith Leong
TVDC Annual Meeting
Oct 6th, 2008
Oct 6, 2008
Highlights of Key Achievements
• Milestone 55: Compare Cellular Immune Responses Induced by
Lm and Ft-Based Tularemia Vaccines
• Construction of Live-attenuated and KBMA Lm vaccine strains
expressing epitope-tagged Ft antigens
• Expression and immunogenicity of tagged antigens measured
• Extremely strong responses to IglC-SL8
• Weaker responses to KatG-SL8
• Milestone 56: Demonstrate that Lm Vaccines Induce Protective
Cellular Immune Responses to Ft Antigens
• T-cell response against IglC measured using overlapping peptide
library after vaccination with live Lm expressing IglC
• IglC epitopes mapped in multiple mouse strains
Oct 6, 2008
Highlights of Key Achievements
• Milestone 57: Optimization of KBMA Lm Vaccination Route and
Regimen
• Lm vaccine strains expressing an internalin A (inlA) allele with a
gain-of-function mutation that increases affinity for mouse Ecadherin have been constructed (inlAm). Initial studies do not show
enhanced immunogenicity.
Oct 6, 2008
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
Oct 6, 2008
Listeria monocytogenes intracellular
lifecycle
Attenuation w/o
loss of potency
Brockstedt et. al, 2004
Binds human but
not mouse Ecadherin
Lecuit et. al 1999
Tilney and Portnoy, 1989
Oct 6, 2008
PrfA Regulation of Virulence Genes
++
++
inlA
inlB
actA
plcB
++
prfA
plcA
hly
mpl
What is PrfA?
• Transcriptional activator of Lm virulence gene expression
• Normally “off” in vitro
• Highly upregulated in vivo
• prfA* are constitutively active alleles
Oct 6, 2008
Epitope-Tagged Lm-Ft
Tularemia Vaccine Candidates
actAp
actAp
Molecular constructs at tRNAArg:
ActAN100
IglC
actAp
SL8
ActAN100
actAp
ActAN100
IglC
SL8
A42R
C4L K3L
KatG
Molecular construct at comK:
ActAN100
IglC
B8R
B8R
Strain
CRS-100
Genetic Background
actAinlB
Antigen Cassette
none
Status
Sequence verified
BH137
actAinlB
ActAN100-Ova
Sequence verified
BH1222
actAinlB
ActAN100-IglC-SL8
Sequence verified
BH2282
actAinlB
ActAN100-KatG-SL8
Remade
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
BH2172
actAinlBuvrABprfAG155S
ActAN100-KatG-SL8
Complete
BH2098
actAinlB
ActAN100-IglC-VacQuad-SL8
Complete
BH2100
actAinlBuvrABprfAG155S
ActAN100-IglC-VacQuad-SL8
Complete
BH2180
actAinlB
ActAN100-IglC-B8R (@ comK)
Complete
BH2182
actAinlBuvrABprfAG155S
ActAN100-IglC-B8R (@ comK)
Complete
BH2316
actAinlB
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8 (@tRNAarg)
Remade
BH2292
actAinlBuvrABprfAG155S
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8 (@tRNAarg)
Complete
Oct 6, 2008
SL8
Lm-Ft Construct Expression in J774 cells
and B3Z Responses
Antigen expression cassettes at tRNAArg locus of Lm actA inlB
actAp
actAp
ActAN100
BH1224
Ova
KatG
SL8
actAp
actAp
ActAN100
IglC
SL8
BH1226
ActAN100
KatG
SL8
B3Z Assay
Antigen Expression in J774 Cells
0.6
98-
0.4
KatG SS
62-
0.2
4938-
IglC
Listeria Strain
NB919, pp67-71
26
H
12
B
H
12
B
B
H
12
22
7
H
13
R
S-
10
0
28-
24
0.0
C
Ova
OD 595
BH1222
ActAN100 SS
B
BH137
Live Lm-IglC-SL8 Vaccine Candidates Elicit
Strong CD8+ Response Against SL8
SL8 responses
SL8 responses
30
% IFN-g CD8+ T cells
% IFN-g CD8+ T cells
30
20
20
10
10
0
0
BH1228 = actAinlBuvrAB -IglC-SL8
BH1398 = actAinlBuvrAB-KatG-SL8
BH1222 = actAinlB-IglC-SL8
BH1228 = actAinlBuvrAB-IglC-SL8
BH2094 = actAinlBuvrABprfAG155S-IglC-SL8
BH2098= actAinlB-IglC-VacQuad-SL8
BH2100= actAinlBuvrABprfAG155S-IglC-VacQuad-SL8
• IglC-SL8 fusion elicits more T-cells than KatG-SL8
• prfAG155S increases immunogenicity of Lm-IglC-SL8 Vaccines
• VacQuad reduces SL8 immunogenicity, and is not helped by prfA*
Oct 6, 2008
Milestone 55: Summary of Achievements
• Cellular immunogenicity of live-attenuated Lm vaccine platforms were
measured
• Lm-expressing IglC-SL8 and 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
Oct 6, 2008
Milestone 55: Future Directions
• Evaluate the immunogenicity of the bivalent live-attenuated
prfA* Lm strain expressing IglC-B8R and KatG-SL8 fusion
proteins
• Compare the immunogenicity of each monovalent strain
with the bivalent strains, vs co-administration
• Compare SL8 responses induced by Ft-PepO-SL8 and
Lm-IglC-SL8
• Evaluate the immunogenicity of KBMA Lm-IglC vaccines
and compare to live-attenuated vaccine
• Measure cytokine responses after vaccination with Lmand Ft-based vaccines using multiplex CBA assay
Oct 6, 2008
Milestone 56: Demonstrate that Lm Vaccines Induce
Protective Cellular Immune Responses to Ft Antigens
• Compare T-cell responses to IglC induced by live and KBMA Lm
expressing IglC to 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/or LmKatG-SL8 protect against a SchuS4 challenge
• Produce lots of KBMA vaccine and send to UNM for testing in animal models
(mice and rats)
Oct 6, 2008
MS56: Evaluation of 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 and ICS
with IglC peptide library
Lm actA inlB uvrAB prfA* IglC-SL8
7 days
(BH2094)
1x 106 cfu IV
Harvest
spleens
IM08-059 NB#2000 pp11-14
Oct 6, 2008
51 peptides in 2 pools
(15mers overlapping by 11 aa)
Pool 1: Peptide #s 1-25
Pool 2: Peptide #s 26-51
400
200
SJL
C3H
FVBN
C57BL/6
0
400
200
0
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
Different Mouse Strains
• 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
Oct 6, 2008
IM08-059
IglC Immune Responses Detected by ICS
IglC pool2
2.5
0
-2
C3H/HeJ
SJL
C3H/HeJ
0.0
2
FVB/N
0.5
4
C57BL/6
1.0
-0.5
CD8
% IFN-g T cells
1.5
FVB/N
SJL
C3H/HeJ
FVB/N
-0.05
C57BL/6
0.00
CD4
CD8
C57BL/6
0.05
2.0
Balb/c
% IFN-g T cells
0.10
Balb/c
% IFN-g T cells
0.15
6
CD4
CD8
Balb/c
CD4
0.20
LLO pool
• 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
Oct 6, 2008
IM08-059
SJL
IglC pool1
Mapping IglC Responses in Balb/c and
C57BL/6 mice
180
Balb/c
160
Peptide #33, 34
QEYKTDEAWGIMIDL
TDEAWGIMIDLSNLE
CD4+
140
120
IFNg SFC/2e5 cells
100
80
60
Peptide #9
NCRLFIDSLTIAGEK
40
20
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
60
C57BL/6
50
Peptide #34, 35
TDEAWGIMIDLSNLE
WGIMIDLSNLELYPI
CD8+
IFNg SFC/2e5 cells
40
30
20
10
IglC peptide library
Oct 6, 2008
IM08-059
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
Mapping IglC Responses in Balb/c and
C57BL/6 mice
180
Balb/c
160
Peptide #33, 34
QEYKTDEAWGIMIDL
TDEAWGIMIDLSNLE
CD4+
140
120
IFNg SFC/2e5 cells
100
80
60
Peptide #9
NCRLFIDSLTIAGEK
40
20
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
60
C57BL/6
50
Peptide #34, 35
TDEAWGIMIDLSNLE
WGIMIDLSNLELYPI
CD8+
IFNg SFC/2e5 cells
40
30
20
10
IglC peptide library
Oct 6, 2008
IM08-059
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
Fine Mapping of IglC-Specific CD8+ Immune
Response in Balb/c Mice
NCRLFIDSLTIAGEK
DSLTIAGEK
IDSLTIAGE
FIDSLTIAG
LFIDSLTIA
RLFIDSLTI
CRLFIDSLT
NCRLFIDSL
9
9.7
9.6
9.5
9.4
9.3
9.2
1-1
9.1
1-2
unstim
0
10
20
IFN- g SFC/2e5 cells
Oct 6, 2008
30
Fine Mapping of IglC-Specific CD8+ Immune
Response in Balb/c Mice
NCRLFIDSLTIAGEK
DSLTIAGEK
IDSLTIAGE
FIDSLTIAG
LFIDSLTIA
RLFIDSLTI
CRLFIDSLT
NCRLFIDSL
9
9.7
9.6
9.5
9.4
Predicted H2-Kd
9.3
9.2
1-1
9.1
1-2
unstim
0
10
20
30
IFN- g SFC/2e5 cells
• Low CD8+ responses to IglC peptide 9
• Stronger response to 9mers 9.3 and 9.4
• Ordered 10mer peptides RLFIDSLTIA and LFIDSLTIAG
Oct 6, 2008
Mapping IglC Responses in Balb/c and
C57BL/6 mice
180
Balb/c
160
Peptide #33, 34
QEYKTDEAWGIMIDL
TDEAWGIMIDLSNLE
CD4+
140
120
IFNg SFC/2e5 cells
100
80
60
Peptide #9
NCRLFIDSLTIAGEK
40
20
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
60
C57BL/6
50
Peptide #34, 35
TDEAWGIMIDLSNLE
WGIMIDLSNLELYPI
CD8+
IFNg SFC/2e5 cells
40
30
20
10
IglC peptide library
Oct 6, 2008
IM08-059
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
Fine Mapping IglC-Specific CD8+ immune
Response in C57BL/6 Mice
WGIMIDLSNLELYPI
TDEAWGIMIDLSNLE
QEYKTDEAWGIMIDL
LSNLELYPI
DLSNLELYP
IDLSNLELY
MIDLSNLEL
IMIDLSNLE
GIMIDLSNL
WGIMIDLSN
AWGIMIDLS
EAWGIMIDL
DEAWGIMID
TDEAWGIMI
KTDEAWGIM
YKTDEAWGI
EYKTDEAWG
QEYKTDEAW
35
34
33
33.15
33.14
33.13
33.12
33.11
33.10
33.9
33.8
33.7
33.6
33.5
33.4
33.3
33.2
33.1
unstim
2-1
2-2
0
20
40
60
IFN-g SFC/2e5 cells
Oct 6, 2008
80
Fine Mapping IglC-Specific CD8+ immune
Response in C57BL/6 Mice
WGIMIDLSNLELYPI
TDEAWGIMIDLSNLE
QEYKTDEAWGIMIDL
LSNLELYPI
DLSNLELYP
IDLSNLELY
MIDLSNLEL
IMIDLSNLE
GIMIDLSNL
WGIMIDLSN
AWGIMIDLS
EAWGIMIDL
DEAWGIMID
TDEAWGIMI
KTDEAWGIM
YKTDEAWGI
EYKTDEAWG
QEYKTDEAW
35
34
33
33.15
33.14
33.13
33.12
33.11
33.10
33.9
33.8
33.7
33.6
33.5
33.4
33.3
33.2
33.1
unstim
2-1
2-2
0
20
40
60
IFN-g SFC/2e5 cells
Oct 6, 2008
80
Fine Mapping IglC-Specific CD8+ immune
Response in C57BL/6 Mice
IMIDLSNL
GIMIDLSNL
WGIMIDLSNLELYPI
33-19
Predicted H2-Kb
33-10
35
unstim
0
20
40
60
IFN- g SFC/2e5 cells
Oct 6, 2008
80
IglC responses
200
unstim
iglC pool1
150
SL8 Responses
30
iglC pool2
iglC 33-10
100
50
0
% IFN- g CD8+ T cells
IFN- g SFC per 2e5 splenocytes
Comparison of Lm-IglC and LVS Vaccines
20
10
0
BH1222
LVS-SL8
HBSS
Day 0
Vaccinate C57BL/6 with:
BH1222 (Lm actAinlB-IglC-SL8): 5e6 cfu iv
LVS-PepO-SL8: 1e4 cfu id
HBSS
Oct 6, 2008
BH1222 LVS-SL8
Days 7 and 9
Measure
immune
responses
HBSS
MS56 Summary
• 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
Oct 6, 2008
MS56 Next Steps
• Repeat comparison of Lm-IglC and Ft vaccines
• Assess immune responses induced by LVS when used
with Lm in heterologous prime-boost regimen
• Prime-Boost-LVS-Challenge
• Vaccination with Lm-IglC or Pep-O SL8 LVS
• Challenge with LVS 1M after boost
• Compare IglC responses from live and KBMA vaccines
• Distribute vaccines to Terry for SchuS4 challenge studies
• If protection is seen, then initiate depletion studies. If no
protection, then investigate heterologus prime-boost
strategy.
Oct 6, 2008
Milestone 57: Optimization of 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
Oct 6, 2008
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
Oct 6, 2008
Status
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
InlA Gain-of-Function Mutation May Not
Enhance Immunogenicity in Mice
iglC-pool#2
iglC 33-10
150
100
50
0
600
SFU per 1e5 cells
50
SFU per 2e5 cells
SFU per 2e5 cells
200
LLO190 response
40
30
20
10
0
IV
Oral
BH2164
IV
Oral
BH2194
400
200
0
IV
Oral
IV
BH2164
BH2194
Day 0: Vaccinate C57BL/6
BH2164 (actAinlBinlAwt-IglC): 5e6 cfu iv
BH2164 (actAinlBinlAwt-IglC): 1e9 cfu oral
BH2194 (actAinlBinlAM-IglC): 5e6 cfu iv
BH2194 (actAinlBinlAM-IglC): 1e9 cfu oral
Oct 6, 2008
Oral
IV
Oral
BH2164
Day 7
Measure immune
responses in spleen
IV
Oral
BH2194
MS57 Next Steps
• Route comparison initiated
• Will repeat experiment and also test prfA* vaccines
• Will evaluate ID, SC, IM and IN routes for systemic and
mucosal immune responses
• Routes that provide best responses will be tested in INchallenge model
Oct 6, 2008
Problems Encountered, Corrective Actions
• Vac-quad epitope tag reduced immunogenicity:
• This strategy was dropped in favor of single SL8 tag
• Since we were able to identify IglC responses the need for other
epitopes is not great
• Have been unable to share Lm-IglC and KatG with UNM
because original MTA with UCLA did not include UNM
• A multiparty MTA with UCLA, Cerus/ANZA, UNM, LBERI and
others is in process
Oct 6, 2008