Selection of Vaccine Candidates From
Proteome / Transcriptome / Genetic Mutant
Approaches
Justin Skoble , Stephen Johnston, Kathy
Sykes, Mitch Magee, Karl Klose
TVDC Annual Meeting
Oct 6th, 2009
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Listeria monocytogenes Vaccine Candidates
• Lm-IglC
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•
•
•
High-level expression, measured cellular immunogenicity
Complete protection against 10 LD50 LVS challenge
Horwitz Lab demonstrated Lm-IglC protection against SchuS4 challenge
SchuS4 challenge studies underway at UNM
• Lm-KatG
•
•
•
•
Low-level expression
Incomplete protection against 10 LD50 LVS challenge
Lm-KatG did not protect against SchuS4 Challenge (Horwitz strain)
SchuS4 challenge studies underway at UNM
• Lm-IglC/KatG Bivalent Strain
• High-level expression of IglC, low-level expression of KatG
• Up to 5 New Antigens in MS59
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Expression of Antigens from Listeria
monocytogenes
actAp
Secreted
ActAN100
Antigen X
SL8
actAp
Surface Targeted
ActAN100 SL8
Antigen X
ActACTD(390-629)
Insert into chromosome of vaccine strain using integration vector
SL8 Antigen X
Lm ∆actA∆inlB∆uvrABprfAG155S
CTD Antigen X SL8
• Can be formulated as live or KBMA vaccine
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Practical Considerations for Antigen
Selection with Lm Platform - Hydrophobicity
ActAN100 SS
KatG (fl)
SL8
KatG (∆1-23)
ActAN100
SL8
Antigen Expression in J774 Cells
KatG fl
KatG∆SS
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Decisions to be Made for New Vaccine
Candidates in MS59
• Antigen Selection
• Protection studies with peptides (ASU/UNM)
• In silico expression analysis
• Surface targeted or secreted
• Base decision on KatG and IglC immunogenicity and protection
• Live-attenuated or KBMA
• Base decision on KatG and IglC protection studies
• Use of ∆actA∆inlB∆uvrABprfAG155S platform allows both
• Production of multivalent vaccine candidates
• Perform initial immunogenicity as single antigens
• Combination of new antigens
• Add new antigens to IglC-expressing strain or KatG/IglC
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Sample of data from ELISpots of pooled polypeptides
against immune NHP lymphocytes
Set , pool
lymph
lymph
spleen
spleen
1,A01
1
2
29
11
1,A02
2
2
0
3
1, A03
1
1
92
9
1, A07
3, B09
3
20
6
0
0
170
28
26
4, E04
5
6
7
7
3, E08
4
2
61
33
3, E10
6
5
35
117
3, E11
9
8
44
205
3, E12
12
11
53
293
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Individual analysis of polypeptides from 3 selected pools
Against frozen lymph node derived lymphocytes
Media
HK LVS
1:10 1:100
Pool E08
Pool E10
Pool E11
Controls
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Set 3 Pool Library
source
well
position
Positive
tissue, Rd #1
Gene name
Pool E10
A10
Spleen
FTT1079 conserved hypothetical protein
Pool E10
D10
Spleen
FTT0323 fusA
elongation factor G (EF-G)
peptide #171: SGQTIISGM
Pool E10
G10
Spleen
FTT1130 cphA
cyanophycin synthetase
Pool E11
B11
Spleen
FTT0955 gor
Pyruvate/2-oxoglutarate dehydrogenase
complex,dihydrolipoamide dehydrogenase
Pool E11
D11
Spleen
FTT0323 fusA
elongation factor G (EF-G)
peptide #171: SGQTIISGM
Pool E8
B8
Spleen
FTT1531 fadA
3-ketoacyl-CoA thiolase
Pool E8
C8
Spleen
FTT1377 fabH
3-oxoacyl-[acyl-carrier-protein] synthase II
peptide #131: SGIGGIETL
Pool E8
F8
Spleen
FTT1269 Chaperone protein dnaK (heat shock protein
family 70 protein)
Pool E10
E10
Spleen
FTT0087 acnA
aconitate hydratase
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Transcriptome Specific Aims
• Is there a relation between expression of genes in vivo
and antigenicity?
• Identify genes or gene islands regulated during the
time course of infection
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Primate Selected Top 5
FTT0323
FTT1079c
FTT1130c
FTT0955c
FTT0323
FTT1079c
FTT1130c
FTT0955c
fusA
hypothetical protein
cphA
gor
elongation factor G
cyanophycin synthetase
glutathione reductase
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Pathogenicity Island Expression Patterns
Rat
Mouse
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Intracellular trafficking of Ft
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FPI: Duplicated in Ftt and Fth, single copy in Ftn
FPI is required for phagosome escape, intramacrophage
growth, induction of apoptosis, and virulence
Mutants in FPI are highly attenuated for virulence
Ftn FPI mutants are effective as live vaccines against
homologous (Ftn) challenge
Ftn FPI mutants do not protect against Ftt challenge*
*Ftn i.g. can protect against Ftt challenge (Arul)
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Ftt FPI mutants do not protect against Ftt challenge (!)
Question #1:
Why does Ftn not protect against Ftt?
Answer: Ftn (i.g., not i.n.) can protect against Ftt (i.n.)
in mice; route of administration important
Ftn (i.t.) can protect against Ftt (i.t.) in rats
Ftt-specific Ag not being expressed by Ftn?
OAg Ab contribute to protection against Ftn,
evidence suggests Ab is important for protection
against Ftt
Question: Would Ftn FPI mutant with Ftt OAg
provide better protection against Ftt?
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Question #2:
Why does Ftt FPI mutant not protect against Ftt?
Possible answer: failure to escape phagosome
Phagosomal escape predicted to facilitate
Ag presentation by MHC-I
Indicates fundamental difference in protective immune
response to Ftn vs. Ftt
Question: Are there attenuating mutations that
would still allow phagosomal escape?
Answer: yes.
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•
We have opened new milestone (54) to create new
attenuated Schuh S4 mutants based on literature,
contract.
Our first two mutations: lpxF and atpC
LpxF is a Lipid A 4’-phosphatase (Wang et al PNAS
104:4136).
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•
•
Mice infected s.c. with 106 CFU Ftn, all lpxF infected
animals survived (highly attenuated), attracted more
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neutrophils, higher pro-inflammatory cytokines.
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•
•
AtpC is ATP synthase subunit
(Kraemer et al IAI 77:232).
atpC Ftn mutant is attenuated for
virulence via aerosol route
(FTN1645):
We have begun constructing
lpxF and atpC mutations in
Schuh S4 strain.
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(A. Charbit)
ggt (g-glutamyl transpeptidase) important for acquisition
of cysteine inside cells, can escape phagosome but
replicates poorly
Lipoproteins
(J. Stulik, Czech Republic)
FTT1103 (Lipoprotein) in holarctica: attenuated for mac
replication, virulence in mice, protective against homologous
challenge
Suggested additional mutations for inactivation in SchuhS4:
aroE
ggt
FTT1103
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Metabolic mutations:
Predicted to escape phagosome, slowly die within host,
facilitating good immune response:
(A. Charbit, France)
purF, D, L, M, B, H, A (purine metabolism) attenuated in LVS
(Zahrt et al, Wisconsin)
purMCD Schuh S4 highly attenuated, but did not protect
against Schuh S4 via i.n. in mice (not recommended?)
(A. Charbit)
aroG, E, (aromatic aa) attenuated in LVS
(Frank et al, Wisconsin)
aroE attenuated for intracellular growth (LVS)
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List of defined Schuh S4 mutants (milestone 54):
lpxF
atpC
aroE
ggt
FTT1103
Additional useful strain?
iglC Ftn with LVS OAg
The theory is that Ftn OAg contributes to Ftn-specific
response, changing to LVS OAg might stimulate Fttspecific response, in mice (esp. in rats?)
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10/6/09 Action Items
• UNM could do the 5 peptides as a test run against the
vaccinated NHP and vaccinated human PBMC.
• Kathy, Philip, Rick Terry Gloria: will develop a plan to
rescreen with fresh cells against the peptides.
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