Vaccine Candidates: Ft Antigens Presented
by Listeria monocytogenes Platform
Justin Skoble
Meredith Leong
TVDC Annual Meeting
Oct 6th, 2009
Oct 6, 2009, 1
Killed But Metabolically Active (KBMA)
Vaccines
NH2 . HCl
O
X
O
+
O
S-59 Psoralen UVA Light
Parental Strain
X
DNA crosslinked
uvrAB
X
X
X
X
X
X
X
X
X
O
X
X
X
X
X
X
X
X
NER-
X
X
X
X
X
NER+
Expression Profile
Diminished
Expression Profile
Preserved (KBMA)
Theoretical “one crosslink per genome” inactivation
Oct 6, 2009, 2
Rationale for Switching from Ft-Based
Vaccine to Lm-Based Vaccine
• Photochemically inactivated uvr mutants of Ftn and LVS were not more
metabolically active than parental strains
• Redundant repair mechanism in Ft strains preclude production of potent KBMA Ft vaccines
• KBMA Ft vaccines were not superior to heat killed vaccines
• KBMA Ftn protected against Ftn challenge, KBMA LVS protected against LVS challenge
• KBMA LVS did not provide significant protection against lethal SchuS4 challenge
• Immunity was humoral
• LVS did not induce proinflammatory cytokines or T cell responses
• IL6, IFN- IL-12, MCP-1, Il-10 not induced
• No Ft-specific cellular immune responses measured
• Co-administration of LVS and Lm inhibited IL-6 and MCP-1 elicited by Lm, and reduced
functionality of Lm-specific T cells
• Lm induces potent cellular immune responses to encoded antigens
• Horwitz Lab expressed Ft antigens (AcpA, Bfr, DnaK, GroEL, IglC, KatG, Pld) from Lm and
screened for for protection against LVS challenge
• 2 most protective antigens were IglC and KatG
Oct 6, 2009, 3
Exploiting the Intracellular Lifecycle of Listeria
monocytogenes to Make a Vaccine Platform
Attenuation w/o
loss of potency
Brockstedt et. al, 2004
Tilney and Portnoy, 1989
Oct 6, 2009, 4
MS 55: Key Achievements
• Compare Cellular Immune Responses Induced by Lm and
Ft-Based Tularemia Vaccines
• Lm-expressing epitope-tagged IglC or KatG were cloned
• 3 vaccine platforms
• Intracellular expression of IglC was higher than KatG
• CD8 T cell responses (SL8) were evaluated by B3Z assay, ICS, and ELISpot
• CD8 T cell responses were stronger when fused to IglC than KatG
• prfA* enhanced immunogenicity of IglC-SL8 vaccine
• Bivalent strains expressing both IglC and KatG were evaluated
• Intracellular expression and immunogenicity were similar to monovalent strains
• KBMA Lm-IglC primary responses were less than live
• LVS-pepO-SL8 did not induce SL8 response or boost Lm SL8 response
Oct 6, 2009, 5
MS 56: Key Achievements
• Demonstrate that Lm Vaccines Induce Protective Cellular
Immune Responses to Ft Antigens
• 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
• IglC-specific epitopes were mapped in C57BL/6 and Balb/c mice
• Lm-IglC induced stronger IglC responses than LVS
• Lm-IglC protected 100% of mice against 10 LD50 LVS challenge
• Lm-IglC did not protect against 100 LD50 LVS challenge
Oct 6, 2009, 6
MS 57: Key Achievements
• Optimization of Lm Vaccination Route and Regimen
• Single dose of Lm-IglC administered IM,SC,ID, and Orally induced
measurable cellular immune responses, but were lower than IV
• IV vs Oral route compared
• T cell responses in spleens were higher after IV administration
• Mucosal T cell responses were low, but similar after IV and oral
administration
• InlA Gain-of-Function mutation did not significantly enhance
immunogenicity either by oral or IV route
Oct 6, 2009, 7
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
Oct 6, 2009, 8
26
B
H
12
24
B
H
12
22
H
12
B
B
H
13
0
28-
7
0.0
10
Ova
C
R
S-
BH1222
ActAN100 SS
OD 595
BH137
NB919, pp67-71
Live Lm-IglC-SL8 Vaccine Candidates Elicit
Strong CD8+ Response Against SL8
SL8 responses
SL8 responses
20
10
30
% IFN- CD8+ T cells
% IFN- CD8+ T cells
30
20
10
0
BH1228 = actAinlBuvrAB -IglC-SL8
BH1398 = actAinlBuvrAB-KatG-SL8
0
BH1222 = actAinlB-IglC-SL8
BH1228 = actAinlBuvrAB-IglC-SL8
BH2094 = actAinlBuvrABprfAG155S-IglC-SL8
• IglC-SL8 fusion elicits more T-cells than KatG-SL8
• prfAG155S increases immunogenicity of Lm-IglC-SL8 Vaccines
Oct 6, 2009, 9
Construction of Bivalent Vaccine Strain and
Analysis of Intracellular Expression
Molecular construct at tRNAArg:
actAp
ActAN100
actAp
<katG
KatG
SL8
Molecular construct at comK:
ActAN100
IglC
B8R
<p60
Insert
Strain
IglC
BH2172
<iglC
KatG
+
BH2182
+
BH2292
+
+
KatG
IglC
p60
IglC/
p60
KatG/
p60
0.19
0
4.76
0.00
0.04
0
30.06
4.01
7.50
0.00
0.26
33.68
5.98
5.63
0.04
• Expression of IglC and KatG is Similar in Bivalent Strain
Oct 6, 2009, 10
Immunogenicity of Bivalent vs Monovalent
Live-Attenuated Lm Vaccines
*
*
All mice vaccinated with 1e6
actAinlBuvrABprfAG155S
2172 = KatG-SL8
2182 = IglC-B8R
2292= KatG-SL8/IglC-B8R
½ dose 2172 + 2292
• Immunogenicity of IglC and KatG is Similar in Bivalent Strain
• Decrease in immunogenicity when dose decreased by ½ * = p<.05
Oct 6, 2009, 11
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
IM08-059
Oct 6, 2009, 12
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
9
31NCRLFIDSLTIAGEK45
DSLTIAGEK 9.7
IDSLTIAGE
FIDSLTIAG
LFIDSLTIA
RLFIDSLTI
CRLFIDSLT
NCRLFIDSL
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-  SFC/2e5 cells
• Low CD8+ responses to IglC peptide 9 (IglC31-45)
• Stronger response to 9mers 9.3 (IglC33-41) and 9.4 (IglC34-42)
Oct 6, 2009, 13
IglC responses
200
unstim
IglC pool1
150
SL8 Responses
30
IglC pool2
IglC116-124
100
50
0
% IFN-  CD8+ T cells
IFN-  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, 2009, 14
BH1222 LVS-SL8
Days 7 and 9
Measure immune
responses
HBSS
IglC and SL8 Responses Not Induced or
Boosted by LVS-PepO-SL8
IM08-090
SL8 responses
400
unstim
IglC pool2
300
200
100
50
% IFN CD8+ T cells
IFN SFC per 2e5 splenocytes
IglC pool2 responses
40
30
20
10
0
0
• 1e6 Lm-IglC administered IV, 1e4 LVS-PepO-SL8 delivered ID
• 3 weeks between prime and boost, spleens harvested d6 post-boost
• LVS-pepO-SL8 did not induce measurable IglC or SL8 responses by itself
• Lm-induced responses not primed or boosted by LVS
Oct 6, 2009, 15
Lm-IglC Vaccine Protects Balb/c mice
Against Lethal LVS Challenge
Oct 6, 2009, 16
100x IV LD50 of LVS Is Too High a 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 and 50x LD50 LVS challenge for next challenge experiments
• Have initiated SchuS4 challenge studies in mice and rats at UNM
Oct 6, 2009
Listeria-Based Tularemia Vaccine Proof of
Concept by Jia et.al
Vaccine. 2009 Feb 18;27(8):1216-29
Mice immunized i.d. at weeks 0 and 4 with 1 × 107 CFU rLmΔactA, rLm/iglC or rLm/katG
or with 1 × 104 CFU LVS. Challenge was 6 weeks post-boost.
Oct 6, 2009, 18
Comparison of Immune Responses Induced
by Lm Administered Via Various Routes
800
unstim
LLO190-201
600
400
200
• Vaccination by IV route induces highest immune responses
• IM is next best route
• For high avidity T cell responses (LLO), IV and IM are comparable
• Noticed slight scarring (4/5) and necrosis (1/5) at the site of the ID injection
Oct 6, 2009, 19
oral (1e9)
id (1e8)
sc (1e8)
im (1e7)
im (2e6)
0
iv (2e6)
oral (1e9)
id (1e8)
0
LLO190-201 responses
IFN- SFC/2e5 splenocytes
25
sc (1e8)
oral (1e9)
id (1e8)
sc (1e8)
im (1e7)
im (2e6)
0
200
100
50
im (1e7)
50
unstim
IglC117-124
im (2e6)
350
100
350
300
iv (2e6)
unstim
IglC pool2
IFN- SFC/2e5 splenocytes
IglC117-124 responses
400
iv (2e6)
IFN- SFC/2e5 splenocytes
IglC pool2 responses
Live-attenuated vs. KBMA Primary Response
BH1222 actA inlB
BH1228 actAinlBuvrAB
• KBMA-Lm primary responses reduced
• Likely to improve with prfAG155S and also after a boost,
Oct 6, 2009, 20
New Lm-Ft Constructs: KatG Anchored to
Bacterial Surface
BamHI
ActAN100-KatG-ActA(390-629)
ActAN100
SpeI MfeI
KatG
SL8
ActACTD(390-629)
IglC
CTD
B8R
Kat G
SL8
• Potential advantages of surface-associated 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
• Expression and immunogenicity experiments planned
Oct 6, 2009, 21
Example of Surface Expression
Western blot from infected DC2.4 cells
1
2
3
4
5
< surface anchored
< secreted
NB2006_103 Gel2: p60-normalized expression levels
1
2
3
4
5
MW
mock
BH2224
BH2454
BH2412
PfCSP-secreted
0
5.49
6.92
-
PfCSP-surface
0
-
12.86
22.03
Oct 6, 2009, 22
Problems Encountered, Corrective Actions
• Economic downturn forced closure of Anza in Feb and all
technical progress stopped
• Corrective Actions
•
•
•
•
TVDC team members hired by Aduro Biotech in July
Laboratory and Animal facility renovations completed
Service agreement between Aduro and Cerus in place
Contract modification to reduce scope of work approved in September
• MS57 Lm vaccine strains expressing an internalinA allele with
increased affinity for mouse E-cadherin (inlAm) did not show
enhanced immunogenicity, and did not enhance invasion of
CaCo2 cells as reported by Wollert et al
• Corrective Actions
• Milestone modified, will now focus on strains with wild-type inlA allele
Oct 6, 2009, 23
Plan for Next 6 Months
• MS56 Determine whether monovalent and bivalent vaccine
strains protect against 50x IV LD50 LVS challenge and work
with Terry to establish protection against SchuS4 challenge in
mice and rats
• MS57 Evaluate IV, ID, SC, IM and IN routes for immune
responses after boost vaccination
• MS55/56 Evaluate KBMA Lm vaccine immunogenicity after a
boost vaccination and determine whether KBMA vaccines can
protect against LVS challenge
• MS55/56 We will evaluate whether surface-anchored
antigens increase immunogenicity of live and KBMA vaccines
Oct 6, 2009, 24