KBMA Tularemia Vaccine Progress
Update July 8th 2008
July 8, 2008, Page 1
Business Progress
• Service agreement between Cerus and Anza has been
fully executed
• Anza is no longer working “at risk”
• Cerus signed the subcontract modification 4r2 with UNM
• MTA draft was sent to UNM and UCLA to facilitate transfer
of vaccine candidates expressing UCLA peptides
• Some revisions from UCLA were incorporated into a second draft
that is currently under review at UCLA and UNM
July 8, 2008, Page 2
•
•
•
•
•
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
July 8, 2008, Page 3
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
July 8, 2008, Page 4
MS 55 Flow chart
Construct 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
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
July 8, 2008, Page 5
Milestone 55: Summary
• Cellular immunogenicity of live-attenuated Lm vaccine platforms were
measured
• Lm-expressing IglC-SL8 and Lm KatG-SL8 fusion proteins were cloned
• The ability of each to stimulate a CD8 T cell response to SL8 were 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
July 8, 2008, Page 6
New Lm-Ft constructs
actAp
actAp
Molecular constructs at tRNAArg:
ActAN100
IglC
actAp
SL8
ActAN100
actAp
ActAN100
IglC
SL8
A42R
C4L K3L
KatG
SL8
Molecular construct at comK:
ActAN100
IglC
B8R
B8R
Strain
Genetic Background
Antigen Cassette
Status
CRS-100
actAinlB
none
Sequence verified
BH137
actAinlB
ActAN100-Ova
Sequence verified
BH1222
actAinlB
ActAN100-IglC-SL8
Sequence verified
BH2106
actAinlB
ActAN100-KatG-SL8
Sequence verified
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
BH2184
actAinlB
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8 (@tRNAarg)
Complete
July 8, 2008, Page 7
New vaccine lots produced
• 400mL scale lot of Live LVS was produced according to
the TVDC SOP
• NB# 2001-012
• 400mL scale lot of live LVS PepO-SL8 was produced using
same method
• NB# 963-092
• 400mL scale KBMA 2094 was produced:
LMactAinlBuvrABprfAG155S ActAN100-IglC-SL8
• NB#2001-026B
Titer of live lots and log kill for KBMA lot need to be
completed
July 8, 2008, Page 8
Milestone 55: Upcoming Experiments
• We will construct the bivalent iglC/katG strain on the KBMA
background
• We will evaluate the immunogenicity of the bivalent Lm
strain expressing iglC-B8R and katG-SL8 fusion proteins
• We will compare the immunogenicity of each monovalent
strain with the bivalent strains
• We will produce a lot of live Ftn-PepO-SL8
• The SL8 responses induced by Ft-PepO-SL8 and LM-iglCSL8 will be compared
July 8, 2008, Page 9
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)
July 8, 2008, Page 10
MS 56 Progress
• Overlapping peptide library has been synthesized
• 51 peptides > 80% purity
• Arrived lyophilized as individual peptides and as a pool of all 51
• Next month
• Master plate will be arrayed with concentrated stock of each
• Mice of various haplotypes (BALB/c, C57BL/6, FVBN, C3H/HEJ,
SJL) will be vaccinated with live BH2094:
(actAinlBuvrABprfAG155S ActAN100-IglC-SL8)
• Splenocytes will be incubated with diluted peptides to determine
whether there are t-cells responsive to any iglC epitopes by IFN-g
ELISpot
• Anza will vaccinate mice with various Lm vaccines to determine
whether IglC, KatG, or both protect against lethal LVS infection
• Once MTA is approved, live and KBMA Lm lots can be sent to
UNM for evaluation in SchuS4 challenge model
July 8, 2008, Page 11
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
July 8, 2008, Page 12
Milestone 57: Progress
• 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
Genetic Background
Antigen Cassette
Status
CRS-100
actAinlB
none
Sequence verified
BH2130
actAinlBinlAWT
none
Sequence verified
BH2164
actAinlBinlAWT
ActAN100-IglC-SL8
Sequence verified
BH2170
actAinlBinlAM
none
Sequence verified
BH2194
actAinlBinlAM
ActAN100-IglC-SL8
Sequence verified
BH2132
actAinlBuvrABprfAG155SinlAWT
none
Sequence verified
BH2166
actAinlBuvrABprfAG155SinlAWT
ActAN100-iglC-SL8
Sequence verified
BH2134
actAinlBuvrABprfAG155SinlAM
none
Sequence verified
BH2168
actAinlBuvrABprfAG155SinlAM
ActAN100-KatG-SL8
Sequence verified
• Virulence and immunogenicity of inlAWT and inlAM expressing strains
will be evaluated in the coming month/months
July 8, 2008, Page 13
Action Items:
• Barbara: follow-up with Nancy Carr on the UNM review of
the 5 way MTA between UCLA, Anza, UNM, UST and
LBERI. (NOTE after 7/8/08 call: Nancy Carr has UNM legal
reviewing the MTA and is scheduling a teleconference with
Anza/UNM/Cerus for Monday 7/14 at 1:30pm MT)
July 8, 2008, Page 14