Transcript Anza/Cerus

Construction and Evaluation of LiveAttenuated and KBMA Listeria monocytogenes
Expressing Ft Antigens as Tularemia Vaccine
Candidates
TVDC Tech Call
June 8th, 2010
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Cerus/Aduro Milestones
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Milestone 55: Compare Cellular Immune Responses Induced by Lm-Based Tularemia
Vaccines
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Milestone 56: Demonstrate that Lm Vaccines Induce Protective Cellular Immune
Responses to Ft Antigens
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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
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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
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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
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MS 55: Lm Ft Vaccine Construct List
Strain
CRS-100/LM11
LM583
LM677
BH137
BH1222
BH2282
BH1228
BH1398
BH2094
BH2172
BH2098
BH2100
BH2180
BH2182
Genetic Background
actAinlB
actAinlBuvrAB
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
Antigen Cassette
none
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)
ActAN100-KatG-C250 (@tRNAarg)
ActAN100-SL8-KatG-C250 (@tRNAarg)
ActAN100-SL8-KatG-C250 (@tRNAarg)
ActAN100-IglC-B8R (@ comK)
ActAN100-SL8-KatG-C250 (@tRNAarg)
ActAN100-B8R-IglC-C175 (@tRNAarg)
ActAN100-B8R-IglC-C175 (@comK)
ActAN100-IglC-SL8 (@tRNAarg)
ActAN100-B8R-IglC-C175 (@comK)
ActAN100-KatG-SL8 (@tRNAarg)
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
Sequence verified
Remade and verified (BH2184
had point mutation in KatG)
BH2316
actAinlB
BH2292
actAinlBuvrABprfAG155S
BH2568
BH2594
BH2596
actAinlBuvrABprfAG155S
actAinlB
actAinlBuvrABprfAG155S
BH2608
actAinlBuvrABprfAG155S
BH2683
actAinlBuvrABprfAG155S
BH2697
actAinlBuvrABprfAG155S
BH2699
actAinlBuvrABprfAG155S
BH2932
actAinlBuvrABprfAG155S
ActAN100-KatG hyd2-SL8
Sequence verified
BH2934
actAinlBuvrABprfAG155S
ActAN100-KatGhyd1-SL8
Sequence verified
BH2936
actAinlBuvrABprfAG155S
ActAN100-KatGhyd1hyd2-SL8
Sequence verified
BH2938
actAinlBuvrABprfAG155S
Sequence verified
BH3014
actAinlBuvrABprfAG155S
ActAN100-IglC(no tag)
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-hyd2-SL8 (@tRNAarg)
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Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Sequence verified
Original Listeria Vaccine Candidates
actAp
ActAN100
KatG
SL8
BH2172
construct at tRNAArg
Kat G
SL8
actAp
ActAN100
IglC
B8R
BH2182
construct at comK
IglC
B8R
actAp
ActAN100
KatG
SL8
tRNAArg
construct at
B8R
BH2292
actAp
ActAN100
IglC
Kat G
IglC
B8R
construct at comK
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SL8
Evaluation of New IglC and KatG Constructs
1) Remove epitope tag from IglC construct
actAp
ActAN100
actAp
IglC
SL8
ActAN100
IglC
2) Remove hydrophobic regions from KatG
1
2
ActAN100
KatG (∆1-23)
• Intracellular expression of untagged IglC was equivalent to tagged
• KatG2 expression was 38 fold higher than full-length KatG
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SL8
Immunogenicity of New KatG Constructs
AS10-016
BH2172: KatG-SL8
BH2932: KatG2-SL8
BH2934: KatG1-SL8
BH2926: KatG12-SL8
NB#2000, pp 155-157
• Based on increased expression (last month) and equivalent SL8
immunogenicity (above), KatG2 allele was selected for further evaluation
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New Listeria Vaccine Candidates
actAp
ActAN100
IglC
BH2938
construct at comK
IglC
actAp
KatG ∆2
ActAN100
SL8
BH2932
construct at tRNAArg
KatG
SL8
actAp
ActAN100
KatG∆2
SL8
tRNAArg
construct at
B8R
BH3014
actAp
ActAN100
IglC
Kat G
IglC
B8R
construct at comK
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SL8
Immunogenicity of New Listeria Vaccine
Candidates
AS10-018
BH2938: IglC untagged
BH2932: KatG2-SL8
BH2172: KatG-SL8
BH3014: IglC-B8R + KatG2-SL8
BH2292: IglC-B8R + KatG-SL8
AS10-018, Notebook 2000 pp. 171-173
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New Vaccine Lots Prepared and Shipped to
UNM
• New lots of Lm vaccine candidates were prepared
• Shipped to UNM 6/7/10
Strain #
Expression
cassette
Background
Lot #
Titer
BH2182
IglC-B8R
actAinlBuvrABprfAG155S
837-067-A
1.7 x1010
cfu/mL
BH2932
KatG2-SL8
actAinlBuvrABprfAG155S
837-067-B
2.1 x1010
cfu/mL
BH2938
IglC-untagged
actAinlBuvrABprfAG155S
837-067-C
1.6 x1010
cfu/mL
BH3014
IglC-B8R
KatG2-SL8
actAinlBuvrABprfAG155S
837-067-D
1.8 x1010
cfu/mL
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MS 55 Next Steps
• Continue to characterize new IglC and KatG constructs
• Repeat expression and immunogenicity studies
• LVS challenge after vaccination with new KatG2 strain (MS56)
• Plan SchuS4 rat protection studies with UNM
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MS56: 2nd Rat SchuS4 Protection Study
Design (Ftc91.2)
Regimens
Groups (1-2) Prime → → → → → 10→ → → → SchuS4 IT Challenge
Groups (3-5,7) Prime → → 6 wk → Boost → 4 wk → SchuS4 IT Challenge
Groups (6)
Prime → 2 wk → Boost → 4 wk → SchuS4 IT Challenge
Groups
1) PBS – SC
2) 5x107 LVS (No boost) – SC
3) 1x108 BH2182 (Lm677:IglC) – IM
4) 1x108 BH2182 (Lm677:IglC) – ID
5) 1x106 BH2182 (Lm677:IglC) – IM
6) 1x108 BH2182 (Lm677:IglC) – IM 2 week p/b
7) 1x108 BH1222 (Lm11:IglC) – IM
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Two Vaccinations with Lm-IglC Are
Insufficient to Confer High-Level Protection
One month after second vaccination, Fisher 344 rats were challenged
with ~200 cfu SchuS4 IT
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UNM Data provided by Terry Wu and Gloria Statom
(FTC91.2 UNM Notebook 128 pp 170-173, 181, 186
and notebook 147 pp 3,13-15)
Modifications to 3rd Rat Protection Study
(Ftc91.3)
Regimens
Group 1
Prime → → → → → 14wk→ → → → SchuS4 IT Challenge
Groups(2-4) Prime→6wk→Boost 6wk→Boost→ 4wk→SchuS4 IT Challenge
Groups
1) PBS - IM
2) 1x108 Live BH2182 (Lm677:IglC) -IM, -IM - IV
3) 1x108 Live BH2182 (Lm677:IglC) -IM, -IM - IM
4) 1x108 Live BH2182 (Lm677:IglC) -IM, -IM, 5x107 LVS SC
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Design for KBMA Lm Rat SchuS4 Protection
Study (Ftc91.4)
Regimens
(Groups1-2) Prime → → → → → → →
→ 8wk→ SchuS4 IT Challenge
(Groups 3-6) Prime→2wk→Boost→2wk→Boost→4wk→SchuS4 IT Challenge
Groups
1) PBS - SC
2) 5x107 LVS (No boost) - SC
3) 1x108 Live BH2182 (Lm677:IglC) - IM
4) 1x108 KBMA BH2182 (Lm677:IglC) - IM
5) 2x109 KBMA BH2182 (Lm677:IglC) - IV
6) 2x109 KBMA BH2182 (Lm677:IglC) - IM
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LVS Protection Study in C57Bl/6 mice
AS10-013: NB#2000, pp 104, 126, 175, 186
Mice vaccinated IV with 5e6 Lm or 1e3 LVS IV on D0 and D28
All mice challenged 5 weeks after boost with 4.3e5 LVS IV
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MS 56 Next Steps
• Evaluate ability of new KatG2 allele to provide protection
against lethal LVS challenge using monovalent and bivalent
vaccine strains
• Repeat LVS protection study in C57BL/6 mice
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MS57: Comparison of Routes Followed by
Stringent LVS Challenge
AS10-006: NB#2000, pp 91, 127, 153, 187
Balb/c mice vaccinated on D0 and D28 with
1e3 LVS IV, 2e6 BH2182 IV or IM, or 2e7 BH2182 or UCLA-IglC ID
One month later mice challenged with 1.22e6 LVS IV
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MS57: Heterologous Prime/Boost Regimens
to Try to Increase IglC Immunogenicity
Regimens
(Groups1-5) Prime→ → 5wk → → Boost→ 6d→ harvest spleens for ELISpot
(Group6)
Prime wk→ Boost→ 6d→ harvest spleens for ELISpot
(Group7)
Prime 1wk → harvest spleens for ELISpot
BH2182 = Lm677:IglC-B8R
Groups
1) 2x106 BH2182 -IM,-IM
2) 1x109 BH2182-Oral, 2x106 BH2182-IM
3) 25ug IglC peptide pool + 50uG polyi:c-IV, 2x106 BH2182-IM
4) 25ug IglC peptide pool + 25uG CpG-IV, 2x106 BH2182-IM
5) 100ug pCDNA3-IglC plasmid DNA-IM, 2x106 BH2182-IM
6) 100ug IglC peptide 33-17 + 100ug PLGA microspheres-IV, 2x106 BH2182-IM
7) 2x106 BH2182 (Lm:IglC) –IM (single dose)
AS10-007 NB#2000, pp 92-95, 131, 142-146, 150-151
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MS57: Heterologous Prime/Boost Regimens
Can Increase IglC Immunogenicity
AS10-007 NB#2000, pp 92-95, 131, 142-146, 150-151
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-10
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IFNg SFC/2e5 cells
130
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IFNg SFC/2e5 cells
Immune Responses to IglC Peptides After
Heterologous Prime/Boost Vaccination
150
BH2182 IM-IM
110
90
70
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150
CpG + BH2182 IM
110
90
70
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MS 57 Next Steps
• Repeat comparison of live Lm-IglC via multiple routes
followed by 15x LD50 LVS challenge (AS10-006)
• IV, IM, ID
• Compare KBMA Lm-IglC via multiple routes followed by 15x
LD50 LVS challenge
• IV, IM, ID
• IN LD50
• Determine whether low-dose prime/high-dose boost regimen
with live provides protection against LVS challenge
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MS 59: New Antigens
• Goal is to construct 5 new vaccine candidates and compare
to IglC and KatG expression, immunogenicity, and protection
• Progress on new antigen list?
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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
Abstract submitted for Chemical and Biological Defense Science and
Technology Conference hosted by DTRA
MSCR status
MS 40, 41, 42, 44: Completed and accepted by NIAID
MS 46: Under NIAID review (UNM submitted to NIAID on 3/2/10)
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
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Action Items
1. Justin will add the regimen associations to the groups for the minutes for
slide 11.
2. Justin will insert the correct study protocol into slide 13.
3. Terry/Rick- on 6/10/10, will discuss options for UNM training Aduro on
i.t. challenges in mice. I.v. challenge dosing doesn’t work very
consistently at Aduro.
4. Terry will send the reference to Justin on the head to head comparison
of mouse strains for i.t. challenge.
5. Justin will provide more details in the minutes for the arms using the
adjuvants on slide 18.
6. Justin will present both CFU and LD50 values for challenges each time
when describing challenges so the reader can understand and compare,
from study to study. Sometimes Aduro uses HBSS or naïve as the
control nomenclature. Justin will use same /consistent nomenclature.