Construction and Evaluation of LiveAttenuated and KBMA Listeria monocytogenes
Expressing Ft Antigens as Tularemia Vaccine
Candidates
TVDC Tech Call
Jan12th, 2010
1
Cerus/Aduro Milestones
•
Milestone 55: Compare Cellular Immune Responses Induced by Lm-Based Tularemia
Vaccines
•
•
•
•
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 LVS vaccination
Demonstrate that Live and KBMA Lm-IglC and/or Lm-KatG protect against an LVS challenge
Demonstrate that Live and KBMA Lm-IglC and/or Lm-KatG protect against a SchuS4 challenge
Milestone 57: Optimization of Vaccination Route and Regimen
•
•
•
•
Construct vaccine candidates
Measure cellular immunogenicity of live-attenuated vaccines using model epitopes
Compare immunogenicity of KBMA tularemia vaccines using model epitopes
Compare various routes of administration including IV, IM, ID and oral
Optimize dosing regimen of most potent and tolerable route
Confirm optimized route and regimen provides protection against SchuS4 at UNM
Milestone 59: Use Lm Platform For Delivery of Novel Ft Antigens Discovered by TVDC
•
•
•
•
Clone up to 5 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
Construct multivalent vaccine candidate
2
MS 55: Construct and Measure Cellular
Immunogenicity of Lm-Ft Vaccines
Construct epitope-tagged Lm vaccine
candidates expressing IglC or KatG
Measure antigen expression from
live-attenuated strains in cells
Prepare stocks of KBMA vaccines
Characterize immunogenicity of
Live-attenuated vaccine candidates
Measure metabolic activity and
antigen expression in cells
Construct bivalent vaccine candidates
Characterize immunogenicity of
KBMA vaccine candidates
Characterize immunogenicity of bivalent
Live-attenuated vaccine candidates
Work in progress
Work to be initiated
3
MS 55: Key Achievements
• Compare Cellular Immune Responses Induced by Lm and FtBased Tularemia Vaccines
• Lm expressing epitope-tagged IglC or KatG were cloned
• 3 platforms: actAinlB, actAinlBuvrAB, actAinlBuvrABprfAG155S
• Intracellular expression of IglC was higher than KatG
• CD8 T cell responses were evaluated by B3Z assay, ICS, and ELISpot
• CD8 T cell responses to SL8 were stronger when fused to IglC than KatG
• prfAG155S enhanced immunogenicity of IglC-SL8 vaccine
• LVS-PepO-SL8 did not induce SL8 response or boost Lm SL8 response
• Bivalent strains expressing both IglC and KatG were evaluated
• Intracellular expression and immunogenicity were similar to monovalent strains
• KBMA Lm-IglC-SL8 primary responses were lower than live after prime
• Boost pending
• Membrane-targeted KatG and IglC expression cassettes were constructed
• Surface-targeted KatG immunogenicity was not improved
4
Lm Bivalent Vaccine Candidates
actAp
construct at
tRNAArg
ActAN100
KatG
SL8
actAp
construct at comK
ActAN100
IglC
IglC
B8R
B8R
BH2292
Kat G
SL8
ActAN100-KatG-SL8
at tRNAArg:
ActAN100
KatG
SL8
IglC
B8R
Kat G
SL8
CTD
ActAN100-B8R-IglC-C175
at comK:
ActAN100
BH2699
B8R
IglC
ActAC175
• Potential advantages of surface-associated proteins:
1) Pre-loading of antigen that is expressed by the bacteria in culture
• KatG appeared to be stuck in the membrane without tm domain
• Currently evaluating KBMA with IglC targeted to the membrane
5
Vaccination with Lm-IglC/KatG Induces
Cytotoxic T Cells that Respond to Both Tags
GB2
400
SL8
HBSS
• In vivo cytotoxicity performed in
C57Bl/6 mice after a single IV
vaccination with HBSS or
BH2292 Lm677::IglCB8R-KatGSL8
• In vivo killing of Naive splenocytes
pulsed with indicated peptides
B8R
# Cells
300
200
100
0
0
250
10 3
CFSE
10 4
10 5
BH2292
B8R: 97.8 ± 0.2%
SL8: 98.2 ± 0.3%
# Cells
200
10 2
150
100
50
AS09-018
0
0
10 2
10 3
CFSE
10 4
10 5
6
IglC-Specific Cytotoxicity is Lower than TagSpecific Cytotoxicity
SL8
100
15
% IglC-specific killing
B8R
100
IglC-33-19
50
0
% Killing
% Killing
10
50
0
5
0
-5
HBSS
AS09-018
Lm-IglC
AS09-013
7
MS55 Next Steps
• Compare immunogenicity of live and KBMA bivalent Lm
vaccine after a prime and a boost vaccination
• AS09-015 ELISpot failed due to contamination
• Need to repeat experiment will take ~1.5 months
• Evaluate expression of membrane-targeted IglC constructs by
B3Z assay, and intracellular western
• Determine whether shorter ActA C-terminal membrane-targeting
sequences are less prone to proteolysis
• Stocks of KBMA strains expressing membrane-targeted IglC
have been made
• will evaluate metabolic activity and immunogenicity compared with
secreted IglC
8
MS 56: Demonstrate that Lm Vaccines Induce
Protective Cellular Immune Responses to Ft Antigens
Construct IglC 15/11 overlapping peptide library
Vaccinate mice with Lm-IglC and
screen for IglC responses by ICS and ELISpot
Prepare stocks of KBMA
Lm vaccine
Compare Lm and Ft -induced IglC specific
T cell responses
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 KBMA Lm vaccines
To UNM for SchuS4 challenge studies
Send Live-attenuated Lm vaccines
To UNM for SchuS4 challenge studies
9
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
• Live and KBMA Lm vaccine lots were produced and shipped to UNM
and are being used for SchuS4 challenge studies
• Lm IglC did not protect against an ~80 LD50 SchuS4 challenge
10
AS09-001: Live and KBMA Lm Vaccination LVS Challenge, Challenge Dose Too Low
• 10 Balb/c mice per group vaccinated IV twice separated by 1month with
HBSS, 1x103 LVS PepO-SL8
2x106 live Lm ANZ-100, BH2172, BH2182, BH2292
1x108 KBMA Lm BH2172, BH2182, BH2292
• 1 month after boost, all mice challenged with ~5e5 Lot837-032 Lot-16 LVS
• KBMA IglC strains may
provide some benefit
Weight (%)
• LVS challenge was underdosed
Only 1.7 x 105 cfu administered
No animal death observed, but
many lost significant weight
Maximum weight lost
30
20
10
0
AS09-001, NB #2005: pp. 130-131, 139, 184-185
11
New Lot of Anti-CD8 Antibody Not Depleting
CD8+ T cells
Anti-CD4 antibody (GK1.5)
10 5
No Depletion
10 4
10 5
13.3%
10 4
10 5
10 4
0.17%
10 3
Regimen #1:
14.8%
200 mg ab d-6, -3
Bleed: d0
22.2%
10 3
8.44%
25.3%
10 3
10 2
10 2
0
0
0
10 2
CD8
Anti-CD8 antibody (2.43)
10 2
10 3
10 4
10 5
10 5
0
10 2
10 3
10 4
10 5
10 5
0
15.5%
10 4
0
10 2
10 3
10 4
CD4
10 4
200 mg ab d-3, -2,-1
Bleed: d3
10 5
0.07%
10 3
AS09-021#1: NB#2005, pp 173-175
Regimen #2:
10.5%
10 2
10 2
0
0
0
10 2
10 3
10 4
20.1%
10 3
10 5
0
10 2
10 3
10 4
10 5
• Old lot of anti-CD8 2.43 Mab will be tested for depletion this month
• Once demonstrated, vaccinated mice will be depleted and LVS challenged
12
Lm Monovalent Vaccines Failed to Protect
Against SchuS4 Challenge in Mice
Balb/c mice immunized twice separated by 6 weeks with:
2x106 cfu BH2172 (Lm-KatG) or 2182 (Lm-iglC) IV, 1x104 cfu LVS IN or PBS IV
1 month after boost, animals were challenged with ~10 cfu SchuS4 IV or IN
UNM Data provided by Terry Wu and Gloria Statom
(UNM Notebook 128, pp. 119-126, 130-132, 138-143, 145-146, 148-14)
Rats vaccinated with same strains were challenged this week
MS56 Next Steps
• Repeat LD50 with Lot 837-032 DVC Lot-16 LVS
• Stock was retitered with calibrated pipetmen
• Repeat comparison of live and KBMA prime-boost LVS
protection study with 10 LD50 LVS challenge
• Deplete T-cell populations in mice vaccinated with bivalent
Lm and perform 10 LD50 LVS challenge (AS09-002)
• If old lot of anti-CD8 antibody depletes
• Initiate heterologus prime-boost experiments
• DNA, DC + IglC Peptides, IglC protein + adjuvant as prime followed
by LM boost.
14
MS 57: Optimization of Lm Vaccination
Route and Regimen
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
15
MS 57: Key Achievements
• Optimization of Lm Vaccination Route and Regimen
• 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
• Single dose of Lm-IglC administered IM,SC,ID, and Orally induced
measurable cellular immune responses, but were lower than IV
• After boost vaccination, IM appears to be comparable to IV by ELIspot
16
Immunogenicity of Lm-IglC-KatG Administered
IM is Equivalent to IV After a Boost Vaccination
AS09-010
ELIspot Assay
NB 2005 pp 145-149
17
By ICS Analysis, Immunogenicity by Route
Appears Epitope Dependent
AS09-010
ICS Assay
NB 2005 pp 145-149
18
Multifunctionality of CD8+ T Cells by Route
AS09-010
ICS Assay
NB 2005 pp 145-149
19
Multifunctionality of LLO Specific CD4+
T Cells by Route
SC may induce more CD4 cells
20
MS57 Next Steps
• After LVS LD50 is repeated, will evaluate whether mice
vaccinated via different routes are protected against 10x
LVS challenge
• Will us1x106 IM vaccination for regimen optimization
• Vaccinate 2x Q1M, Q2M, Q3M and perform ICS and ELIspot
analysis
21
Additional Points
Deliverables completed for each active milestone:
MS55: Live and KBMA Lm vaccine lots delivered to UNM for testing
MS57: IM route identified as potential non-IV ROA
List of relevant publications from the past month:
None
MSCR status
MS 40, 41, 44: Completed and accepted by NIAID
MS 42: final edits under NIAID review (1/5/2010)
MS 46: UNM reviewing MSCR (Cerus/Aduro sent edits to BG on 12/18/09)
MS 55,56,57: milestones are active
MS 59: milestone not started yet
MS 43, 45, 47, 58: Terminated (not initiated), no MSCR to write
22
Action Items
• Aduro/Cerus will check whether the anti CD 8 antibody is
staining CD 8 T cells.
• Review rat model data within the week. Rat model data
impacts future mouse and rat experiments with Aduro/Cerus
vaccine constructs