Transcript 8UTSA_Klose vacc cand Tues session 2008

University of Texas San Antonio
Choice of attenuating mutations for live vaccine candidates
10/6/08 Annual TVDC Meeting
Intracellular trafficking of Ft
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)
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.);
route of administration important
Ftt-specific Ag not being expressed by Ftn?
OAg Ab contribute to protection against Ftn,
role of Ab against Ftt is still debated, some evidence
suggests Ab is important for protection against Ftt
Question: Would Ftn FPI mutant with Ftt OAg
provide better protection against Ftt?
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.
Mutations that attenuate Ftn:
Trapped in phagosome:
mglA (txn regulator): poor protection against Ftn
iglC (other FPI mutations): good protection against Ftn
4 X Acid Phosphatases: good protection against Ftn
Others:
LipidA modifications (fimF/K): good protection against Ftn
Siderophore receptor (FTT0025): attenuated (protection?)
Signature Tag Mutagenesis screen (Monack):
164 genes required for i.p. infection (no protection data):
FPI, LPS, siderophore, capsule
Biosynthetic genes (purine/pyrimidine, biotin, transport)
Hypotheticals:
FTT0584, FTT0748: required for inhibition of ASC/casp1
(replicate normally in macs, induce high apoptosis)
Mutations that attenuate LVS:
sodB i.n. enhanced protection against i.n. Schuh4
i.n. protection (40%) against 14 CFU, prime/boost
42% protection against 103 CFU
FTL0552 (txn regulator) defective for macrophage growth,
“40% protection against Schuh4 challenge” (dose?)
LPS (OAg) mutant, attenuated, limited protection against
LVS
Question:
How relevant are attenuating/protective mutations in LVS?
Mutations that attenuate Schuh4:
FTT0918 (58 kD protein): slower growth in macs (1 log
increase over 24 h), attenuated by i.d. (LD50~105)
i.d. vaccine protects against i.d. challenge (500 CFU),
partial protection (33%) against low dose aerosol (10 CFU)
purMCD defective for macrophage growth, attenuated i.d.
and i.n., i.n. vaccination protects well against i.d. Schuh4,
less effectively against i.n. Schuh4 (14% no boost, 71%
w/boost against 100 CFU, no protection against 2000 CFU)
dsbB defective for intracellular growth, attenuated i.n. in
mice, no protection against low dose (13 CFU) i.n. Schuh4
Our experience so far:
iglC, iglD Schuh4 mutants highly attenuated by i.n. route,
not protective against low dose i.n. Schuh4 (~100 CFU)
iglC Ftn mutant i.g. provides some protection against
low dose i.n. Schuh4
Possible better attenuated vaccine candidates:
1. iglC Ftn with LVS OAg
2. pdpD Schuh4 (this shows some replication in macs)
3. metabolic Schuh4 mutants:
(possibilities glnA, nadM)
4. Mutants in interaction with Asc/casp pathway
(FTT0584, FTT0748)
nadM virulence (Ftn i.n. inoculation)
6
∆nadM 8.9 x 103
5
4
3
WT 1.1 x
2
∆nadM 8.9 x 105
103
1
DnadM/pnadM 9.7 x 103
0
1
2
3
4
5
6
7
8
9
Mouse virulence (Ftn i.n. inoculation)
murI
glutamate racemase (cell wall biosynthesis)
LRA
outermembrane protein (Bcell antigen)
glnA
glutamine synthetase (Gln auxotroph)
6
5
4
glnA 9.2 x 102
3
2
murI 1.34 x 103
1
LRA 1.28 x 103
WT 1.1 x 103
0
1
2
3
4
5
6
7
8
9
10
11
1. iglC Ftn with LVS OAg
The theory is that Ftn OAg contributes to Ftn-specific
response, changing to LVS OAg might stimulate Fttspecific response
2. pdpD Schuh4 (this shows some replication in macs)
we originally were going to make pdpD Schuh4, but
eliminated once we saw pdpD Ftn was not very
attenuated and grew in macs, this may be beneficial
3. metabolic Schuh4 mutants:
(predicted to escape phagosome)
glnA: contributes to intramac growth of Salmonella,
nadM: required for NAD synthesis, C-term mutation
attenuates Ftn
4. Mutants in interaction with Asc/casp pathway
(FTT0584, FTT0748): these Ftn mutants could grow
in macs, but were highly attenuated in mice.