Tularemia Vaccine Progress
Update Dec 9th 2008
12/09/2008
•
•
•
•
•
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
12/9/2008
2
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
12/9/2008
3
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
12/9/2008
4
Killed But Metabolically Active (KBMA)
Concept
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
Expression Profile
Diminished
X
X
X
X
NER-
X
X
X
X
X
NER+
Expression Profile
Preserved (KBMA)
Theoretical “one crosslink per genome” inactivation
Milestone 55: Compare Cellular Immune
Responses to Model Epitopes 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 (to SL8) 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-pepOSL8 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
12/9/2008
6
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
12/9/2008
7
Milestone 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
12/9/2008
8
Lm-Ft constructs
actAp
actAp
Molecular constructs at tRNAArg:
ActAN100
IglC
actAp
SL8
ActAN100
actAp
ActAN100
IglC
SL8
A42R
C4L K3L
Strain
CRS-100/LM11
LM677
BH137
BH1222
BH2282
BH1228
BH1398
BH2094
BH2172
BH2098
BH2100
BH2180
BH2182
BH2316
Genetic Background
actAinlB
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
Molecular construct at comK:
ActAN100
IglC
B8R
B8R
Antigen Cassette
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)
12/9/2008
9
Status
Sequence verified
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
Immunogenicity of Bivalent vs Monovalent
Live-attenuated Lm vaccines (ICS)
IglC-tag (B8R)
KatG-tag (SL8)
*
*
All strains vaccinated with 1e6
actAinlBuvrABprfAG155S
2172 = KatG-SL8
2182 = IglC-B8R
2292= KatG-SL8/IglC-B8R
½ dose 2172 + 2292
-Decrease in immunogenicity when dose decreased by ½ * p<.05,
12/9/2008
10
IglC and SL8 Responses Not Induced or
Boosted by LVS-PepO-SL8
IM08-090
• 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 does not induce measurable IglC or SL8 responses by itself
• Lm-induced responses not primed or boosted by LVS
12/9/2008
11
Live-attenuated vs. KBMA Primary
Response (ICS)
1222 ActA inlB
1228 ActAinlBuvrAB
• KBMA-Lm primary responses reduced by ~75%
• Likely to improve with prfAG155S and also after a boost,
12/9/2008
12
New Data: Dose Response to LiveAttenuated Lm
BH2098 (QuadVac-IglC in Lm11)
BH2098 (QuadVac-IglC in Lm11)
200
100
• SL8 Immunogenicity increased up to 1e6 dose
• 33-19 Immunogenicity peaked at 1e5 dose
13
1e3
BH2098 (QuadVac-IglC in Lm11)
IM08-098: Vaccinated C57BL/6 mice IV with BH2098 (actAinlB-IglC-quadvac)
Harvested splenocytes on D7 for ICS and ELISpot
12/9/2008
1e4
0
1e5
0
300
1e6
IFN- SFC/2e5 splenocytes
50
1e3
1e3
1e4
1e5
0
100
1e4
200
150
1e5
400
200
1e6
IFN- SFC/2e5 splenocytes
600
1e6
IFN- SFC/2e5 splenocytes
LLO190-201 responses
33-19 responses
SL8 responses
Dose Response to Lm Vaccines Varies
with Strength of Epitope
C4L responses
BH2098 (QuadVac-IglC in Lm11)
Conclusion:
More is not always
better
150
100
50
0
1e3
1e3
1e4
1e5
0
200
1e6
IFN- SFC/2e5 splenocytes
100
1e6
IFN- SFC/2e5 splenocytes
A42R responses
200
1e3
BH2098
(QuadVac-IglC
K3L
responsesin Lm11)
BH2098 (QuadVac-IglC in Lm11)
300
1e4
0
1e3
1e4
1e5
0
100
1e4
200
200
1e5
400
300
1e5
600
400
1e6
IFN- SFC/2e5 splenocytes
800
1e6
IFN- SFC/2e5 splenocytes
B8R responses
BH2098 (QuadVac-IglC in Lm11)
12/9/2008
14
100mL-scale Lm Vaccine Lots Produced
BH2172 Lm677:KatG-SL8
Type Titer
(CFU/mL)
Live 2.41 x 1010
BH2182 LM677:IglC-B8R
Live
1.96 x 1010
837-15-B CH-FR80-015
BH2292 Lm677:KatG-SL8/IglC-B8R Live
2.20 x 1010
837-15-C CH-FR80-015
BH2316 LM11: KatG-SL8/IglC-B8R Live
1.74 x 1010
837-15-D CH-FR80-015
Strain
Genotype
Lot#
Location
837-15-A CH-FR80-015
• Up to 50 aliquots available, 0.3mL each, ready to ship
12/9/2008
15
Milestone 55: Upcoming Experiments
• Optimize semi-quantitative Western blot:
• Confirm that p60 expression correlates with cfu by performing an
MOI dose response and compare western blot signal and cfu
• Initiated last week
• Evaluate the immunogenicity of KBMA strains after a prime
and boost vaccination
• KBMA lot production initiated, had some failures (due to cfu)
• Repeat Lm and LVS pepO-SL8 comparison using LVS at
higher doses
• Repeat dose-response study using prfAG155S platform
12/9/2008
16
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)
12/9/2008
17
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
12/9/2008
18
MS56: 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 wereCD4+, CD8+, or both depending on the haplotype of the mice
• IglC-specific CD8+ epitopes were identified in C57BL/6 and Balb/c mice
• IglC responses were also seen in C57BL/6 mice vaccinated with Lm-KatG
• Preliminary results suggest that Lm-IglC vaccine induces stronger IglC
and SL8 responses than LVS-pepO-SL8
• low-dose LVS was used
• 2 IV vaccinations with Lm-iglC protected 100% of mice against lethal LVS
challenge
• Lm KatG protected 40%, LVS and combination of LM-iglC and Lm-KatG protected
100%
12/9/2008
19
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
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
Lm-KatG Induced Responses Against IglC
Peptide in C57BL/6 Mice
IglC 33-10
12/9/2008
BH2172 = KatG-SL8
BH2182 = IglC-B8R
BH2292= KatG-SL8/IglC-B8R
½ dose 2172 + 2292
21
Lm Alone Does Not Induce IglC Response
• C57BL/6 and Balb/c Mice were vaccinated with Lm-expressing irrelevant
antigen: ELISpot performed with IglC peptide library and 33-10
100
IglC library in Balb/c mice:
P002-08-003, Notebook #2005, pp 54-56
90
IFNg SFC/2e5 cells
80
70
60
50
40
30
20
10
41
42
43
44
45
46
47
48
49
50
51
33-19
42
43
44
45
46
47
48
49
50
51
33-10
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
IglC library in C57BL/6 mice
P002-08-001 Notebook #2005, pp 57-59
90
80
70
60
50
40
30
20
10
12/9/2008
22
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
unstim
IFNg SFC/2e5 cells
41
100
unstim
0
Lm-IglC Vaccine Protects Balb/c mice
Against Lethal LVS Challenge
11/11/2008
23
MS56 Next Steps
• Increase stringency of LVS challenge (look at 10 vs 100 LD50)
• Anza will vaccinate mice with various live Lm vaccines to
determine whether IglC, KatG, or both protect against lethal
LVS infection
• Vaccinations initiated
• Balb/c mice will be vaccinated (2x) with Lm-IglC, then prior to
challenge antibodies will be injected IP to deplete T cell
populations: CD4, CD8, both, or irrelevant Ig
• Once MTA is approved, live and KBMA Lm lots will be sent to
UNM for evaluation in SchuS4 challenge model
12/9/2008
24
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
12/9/2008
25
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
12/9/2008
26
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
12/9/2008
Antigen Cassette
none
none
ActAN100-IglC-SL8
none
ActAN100-IglC-SL8
none
ActAN100-iglC-SL8
none
ActAN100-iglC-SL8
27
Status
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
MS56: 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)
12/9/2008
28
Preliminary Intranasal LD50
• Intranasal LD50 initiated (for use in route optimization)
• Doses of expanded DVC lot 16 LVS ranging from 5e2 to 1e5 IN
Grp # Mice
Strain
Dose
Route
Vx date
Survial
1
4
Ft-LVS
1e5
IN
11/5
3
2
4
Ft-LVS
5e4
IN
11/5
4
3
4
Ft-LVS
1e4
IN
11/5
4
4
4
Ft-LVS
5e3
IN
11/5
4
5
4
Ft-LVS
1e3
IN
11/5
4
6
4
Ft-LVS
5e2
IN
11/5
4
P001-08-001: LVS intranasal LD50. Notebook #2000, p45
• IN LD50 was > 1e5
• This is higher than reported by Horwitz lab
• LD50 may be influenced by use of inhaled isofluorane anesthetic
• Will repeat using Ketamine/xylosine
12/9/2008
29
MS57 Next Steps
• 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)
• Repeat LVS IN LD50 study using injected anesthesia
12/9/2008
30