University of Texas San Antonio
Update on F. tularensis attenuated vaccine
strain construction and evaluation
TVD Team
4/19/10 site visit
Progress on milestones to date:
Milestone #16: Create luciferase expressing F. tularensis L
COMPLETE
Milestone #39: Creation of uvrA and uvrB mutant F.
tularensis subsp. novicida strains
COMPLETE
Milestone #43: Creation of uvrA and uvrB mutant F.
tularensis subsp. holarctica (LVS) strains
COMPLETE
Milestone #48: Characterize uvrA and uvrB mutant F.
tularensis subsp. novicida strains
COMPLETE
Milestone #51: Construction of F. tularensis subsp. novicida
uvrB + pdpD, iglA, iglB, iglC, iglD strains.
COMPLETE
2
Milestone #49: Construction of mutant F. tularensis subsp.
tularensis strains
COMPLETE
Milestone #50: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
COMPLETE
Milestone #52: Construction of mutant F. tularensis subsp.
tularensis strains containing recA mutations
ONGOING
Milestone #53: Immunologic characterization of F.
tularensis subsp. tularensis strains
ONGOING
Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
3
ONGOING
Our laboratory is focusing on the generation and characterization
of live attenuated F. tularensis subsp. novicida, tularensis, and
LVS strains for their vaccine potential
We have performed targeted disruption of various genes, including:
subsp. novicida:
uvrA, uvrB
These genes were inactivated for KBMA
component by Cerus
iglA, iglB, iglC, iglD, pdpD + uvrA/ uvrB same as above
LVS (subsp. holarctica):
uvrA, uvrB
(same as above)
4
subsp. tularensis:
vgrG,
iglC, iglD
These are virulence genes located in pathogenicity
island
nadM
involved in NAD synthesis, mutant isolation
unsuccessful
involved in LPS modification, mutant isolation
unsuccessful
lpxF
FTT0748
atpC
trpB
FTT1103
FTT1579
FTT0523
recA
transcriptional regulator important for virulence
in Fn
ATP synthase required for full virulence in Fn
required for Trp biosynthesis (in progress)
lipoprotein that contributes to LVS virulence (in progress)
restriction enzymes that inhibit genetic recomb.
in Ftt; inactivation will facilitate genetic manipulation
(also FTT1579 + FTT0523)
This gene facilitates genetic recombination; its
inactivation will stabilize potential vaccine strain
We have spent a considerable amount of time developing
and optimizing techniques for genetic manipulation of
Francisella tularensis with SUCCESS
Major accomplishments have included:
1. New optimized targeted mutagenesis of Ft novicida
2. New plasmid-based mutagenesis of Ft holarctica (LVS)
3. New technique to mutagenize Ft tularensis and
Ft holarctica “Tulatron”
6
Targetron mutagenesis in F. tularensis
•RNA loops EBS1 and EBS2
basepair with specific sequences
in target
•LtrA recognizes flanking sequences
•EBS1 and EBS2 can be “retargeted”
to recognize sites in target gene
•LtrA site preferences within target gene
are identified by Targetron computer
algorithm, which designs appropriate oligos
to retarget intron to your gene
Tulatron contains:
1. Ft promoters to drive
Antibiotic resistance
Intron RNP
2. Ft ori
3. ts mutation
4. Ec ori
5. KanR
6. lacZa “stuffer”
7
We continue to optimize and enhance mutant construction
in F. tularensis
 We are working with Jiri Perutka (Lambowitz lab) to
enhance targetron efficiency in F. tularensis
 He has refined algorithm to identify Ft-specific insertion
Sites
 He has designed new Ft-specific targetrons that
1. Introduce marker (Kan) into inactivated gene
2. Introduce transcriptional fusions (GFP, RFP) into
inactivated gene
 We are enhancing recombination in FTT by
1. Eliminating restriction barriers
2. Expressing lRed proteins to enhance recombination
8
Problems encountered (last year):
Inability to knockout nadM, lpxF genes in Schuh S4
We tried many different ways, times,
even (briefly) identified nadM mutant, only to ultimately fail to
isolate mutants
Corrective action: we abandoned attempts, but have
substituted with other mutations predicted to attenuate
We also are continually enhancing mutant strain construction
in Schuh4, including enhancing targetron efficacy
Conclusion: the nadM and lpxF mutations are lethal in
Schuh S4
We have been successful with all other Schuh4 mutations,
We anticipate our ever-improving mutagenesis strategies
and additional mutations will allow for identification of
best attenuated vaccine strains.
9
What will be the challenges in the next year?:
1. Challenges constructing mutant Schuh4 strains.
We do not anticipate any problems with any of the new
mutant strain constructions, but if we encounter any we
will substitute another potential attenuating mutation
for the “problem” mutation.
2. Problems with the proposed cloning of the LVS OAg.
This is a challenging goal for ourselves, we have never
successfully cloned such a large fragment. However,
new techniques have recently been developed, so we
think it is important to attempt this.
3. Problems with expressing fim genes in Ftn.
Again, expressing heterologous antigens can be challenging,
but if successful we may dramatically improve the efficacy
of oral Ftn vaccination, so we feel it is important to attempt
this.
10
We have three currently active milestones:
Milestone #50: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
Milestone #52: Create recA mutants in F. tularensis subsp.
tularensis
Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 52
Creation of recA mutant F.
tularensis subsp. tularensis mutant strains
Construct recA
mutagenesis plasmid
Transform into Schuh4,
isolate mutant
Verify mutants,
Pass on to Milestone 50
Generate, optimize
mutant strain construction
in Schuh4
Transform into iglC,
vgrG, iglD (other)
Schuh4 strains,
isolate mutants
12
Key Achievements in past year:
 Constructed recA and iglC1 iglC2 recA Schuh4 mutants
 Evaluated for virulence
 Identified, inactivated restriction enzymes in Schuh4
 Demonstrated enhanced transformation frequency in restriction minus Schuh4
 Cloned, expressed lRed proteins in F.t.
 Demonstrated enhanced recombination in Ftn/lRed
 Demonstrated attenuated phenotype of trpB Ft mutants, identification of trpB
as potential attenuating mutation
13
Increasing recombination frequency in Schuh4:
•Bacteriophage l encodes proteins (l Red recombinase) that
enhance recombination into bacterial chromosome
•We are adapting this system for use in Ftt Schuh4:
•Last month reported construction of Ft plasmid pKEK1327
that expresses lRed proteins from Ft promoter.
•We transformed into Ftn wildtype strain U112:
One transformed colony (lane
clearly has lRed genes by
PCR screen (compare with
control plasmid (lane 7)
•This strain, along with the wildtype strain (without plasmid), was
transformed with PCR product to knockout FTT1352 (ErmR)
•lRed stimulated 40X more transformant/knockouts
(ErmR colonies)!
•This works. We will next transform Schuh4 and test in Ftt.14
We are also testing requirement of tryptophan biosynthesis
for F. tularensis virulence:
•Last month we showed that trpB, but not trpA, mutant Ftn
was attenuated for virulence:
•trpA encodes second-to-last step in
Trp biosynthesis
•trpB encodes last step in Trp biosynthesis
are Trp auxotrophs
•i.n. vaccination with ~100 CFU
•We challenged surviving mice with 264 CFU WT Ftn, they
all succumbed to challenge (not protected), but vaccine dose
was low.
•IFNg shown to limit Trp inside treated cells (incl. macrophages)
•We tested growth of trpB mutant Ftn in IFNg-treated macs:
15
•Growth of wt, trpA, and trpB mutant Ftn in J774 +/- IFNg
•Note: no samples for 48 h trpA mutant (i.e. not tested).
•IFNg inhibits growth of wt and trpB mutant similarly
•No evidence of enhanced sensitivity of trpB mutant in
IFNg-treated J774 cells
•We will test primary (BMD) macrophages in case more sensitive
to IFNg
16
•We will introduce trpB mutation into Schuh4
Red: completed
Green: in progress
Blue: Steps in the milestone
Milestone 54
Creation of mutant F. tularensis
subsp. tularensis strains
Construct lpxF, atpC, 3 other
mutagenesis plasmids
Mate into Schuh4,
select for transconjugate,
Counterselect for mutant
Verify mutants,
Pass on to Milestone 50
17
Key Achievements in past six months:
 Created targetron plasmids to inactivate lpxF and atpC in Schuh4
 Successfully inactivated atpC in Schuh4
 Evaluated atpC Schuh4 mutant for virulence, no attenuation seen in
i.n. doses as low as 600 CFU
 Unable to inactivate lpxF in Schuh4 (essential gene?)
 Created targetron plasmids to inactivate FTT1103 in Schuh4
18
Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
Inactivation of lpxF, atpC in SchuhS4:
•We decided to discontinue construction of lpxF mutant, due
to unsuccessful isolation of pure mutant.
•(last month) We tested virulence of atpC Schuh4 mutant
in Balb/C mice via i.n. route
•All mice died at lowest i.n. dose (~600 CFU)
•We will test at even lower dose
•We will also combine this mutation with others (trpB, etc)
to determine if combination of mutations attenuates Schuh4
Inactivation of FTT1103 and ggt in Schuh4:
•FTT1103 encodes lipoprotein shown to contribute to virulence
of Fth (J. Stulik)
•ggt encodes g-glutamyltransferase, facilitates acquisition of
cysteine inside macrophages (A. Charbit)
•Arul lab tested Ftn mutants in FTT1103 and ggt for intramac
replication, the FTT1103 mutant was defective.
•We are creating targetron to inactivate FTT1103 in Schuh4:
We have 2 targets in gene,
270 and 466
270 targetron plasmid (lanes4,5)
466 targetron plasmid (lanes6,7)
show correct digestion pattern vs.
parent plasmid (lanes 3,8):
Upper band decrease in size in correct
clones
•These plasmids will be sequenced to confirm they are
correct
•Plasmids will be transformed into Schuh4 to generate
FTT1103 mutant
20
Plan for following six months:
Milestone #16: completed.
Milestone #39: completed.
Milestone #48: completed.
Milestone #43: completed.
Milestone #49: completed.
Milestone #52:
1. Characterize recombination frequency in lRedexpressing Schuh4
2. Characterize recombination frequency in restriction
mutants (FTT1579 and FTT0523)
3. Enhance oral delivery of Ftn vaccine strains by
expression of fim genes
4. Enhance vaccine efficacy of Ftn strains by expression
of LVS OAg genes
Milestone #54:
1. Create FTT1103 Schuh4 mutant, characterize
virulence in mice, macrophages
2. Create ggt Schuh4 mutant, characterize
virulence in mice, macrophages
3. Create trpB Schuh4 mutant, characterize
virulence in mice, macrophages
Milestone 53-A
Immunologic characterization of F.
tularensis SCHU S4 mutant strains
recA
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Green: in progress
Blue: Steps in the milestone
Red: completed
recA double mutants
In vitro Growth
In vivo Bacterial Burden
LD50 determination
nadM, FTT0748,
atpC, FTT1103, FTT1181
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Further immunological characterization
based on initial screen
Key Achievements
 Evaluate the potential use of an SCHU S4 atpC mutant as a candidate vaccine
 Evaluate the infectivity of SCHU S4 restriction enzyme mutants
 Evaluate the potential use of additional F. novicida mutants as candidate
vaccines
 Evaluate antigen-specific cell mediated immune responses by oral and intransal
U112 DiglB (KKF235) immunization using ELISPOT technology
24
Intramacrophage growth of SCHU S4 atpC mutant (KKT-32)
 AtpC is the epsilon subunit of FoF1-ATP synthase catalyzing synthesis of ATP from ADP
and inorganic phosphate
 KKT-32 has a similar replicative profile as the wild-type strain
Intramacrophage growth of SCHU S4 restriction enzyme
mutants: KKT-28 (FTT0523 mutant) and KKT-29
(FTT0523/FTT1579 double mutant)
A.
B.
 FTT0523 and FTT1579 function as restriction enzymes. Mutation of these genes allows
stabilization of the foreign plasmid DNA thus enhancing transformation efficiency when
generating various SCHU S4 mutants.
 KKT-28 and KKT-29 replicate to similar levels as SCHU S4, and this may be advantageous
when UTSA studies the virulence of the subsequent mutations generated in the KKT-29
background.
Intramacrophage growth of F. novicida lipoprotein (FTN0771)
and gamma-glutamyltranspeptidase (FTN1159) mutants
 Both mutants are moderately attenuated.
 The SCHU S4 orthologies of FTN0771 (FTT1103) and FTN1159 (FTT1181) will be
generated and evaluated as potential vaccine candidates.
Cellular cytokine recall responses to i.n. and oral
KKF235 (F. novicida DiglB) vaccination
A.
B.
 Cell mediated immune responses are induced by KKF235 vaccination via both routes.
Work plans for the coming six months milestone
53A
 Determination of LD50 of the FTN_0771 and FTN_1159 mutants using
pulmonary challenge mouse model
 Evaluation of additional SCHU S4 mutants generated in MS 52 and MS54 as
vaccine candidates
29
Milestone 53-B
Characterization of protective immunity against
pulmonary tularemia via oral vaccination in the F344 rat model
Characteristics of oral
vs. i.d. vaccination of
LVS/survival
Correlates of humoral
and cellular immunity
of LVS vaccination
Protective efficacy of
2 attenuated SCHU S4
strains
Intramacrophage survival
Vaccination/challenge
Bacterial dissemination
Histological analyses
CD4+ T cell
responses
Serum antibody responses
Secreted, BAL antibody
responses
Intramacrophage survival
vaccination/challenge
antibody responses
Bacterial dissemination and
histology
Red: completed
Green: in progress
Blue: Steps in the milestone
Key Achievements
We have evaluated the Francisella tularensis subsp. novicida wild-type
strain (U112) and a defined mutant strain (DiglB) for protective efficacy in
the Fischer 344 rat model.
Intratracheal challenge of F344 rats with F. novicida U112 leads to significant
antigen-specific humoral and cellular responses and is protective against subsequent
F. tularensis SCHU S4 challenge
Oral vaccination with either strain leads to significant antigen-specific IFN-g
production
Oral vaccination with either U112 or the DiglB defined mutant strain induces
significant antigen-specific systemic and mucosal humoral responses
 Oral vaccination with either strain is highly protective against subsequent F.
tularensis SCHU S4 challenge
31
Cellular Responses to Oral Vaccination with F. novicida
U112 and DiglB
A
F. novicida U112
B
F. novicida U112 DiglB
Humoral Responses to Oral Vaccination with F. novicida
U112 and DiglB and Protective Efficacy Against SCHU S4 Challenge
A
Systemic
C
B
Mucosal
Work plans for the coming six months milestone
53B
 Isolate primary alveolar macrophages from Fischer 344 rats
 Determine intracellular replication profiles of Francisella strains in primary
alveolar macrophages
 Continue evaluation of both defined F. novicida and F. tularensis mutant strains
as vaccine candidates using the Fischer 344 rat model
34
University of Texas San Antonio
Choice of attenuating mutations for live vaccine candidates
4/19/10 Site Visit
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 in mice
Ftn FPI mutants do not protect against Ftt challenge in mice*
*Ftn i.g. can protect against Ftt challenge (Arul)
Ftt FPI mutants do not protect against Ftt challenge in mice
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
Our evidence demonstrates Ftn as a potentially
excellent live vaccine candidate against Ftt. iglD Ftn
protected rats as well as iglD Ftt (Arul).
Question: Can we “tweak” Ftn to make it more efficacious
as a live vaccine?
Answer: Yes.
1. Ftt-specific OAg not being expressed by Ftn—
evidence suggests OAg Ab is important for protection
against Ftt
We are attempting to clone entire OAg gene cluster from
LVS and get it expressed in Ftn.
2. Targeting Ftn to Mcells should enhance protective
efficacy of i.g. administration
We are attempting to clone and express fimH operon from
S. typhimurium in Ftn to determine if this enhances
oral efficacy:
FimH fimbriae in St interact with GP-2 on Mcells, enhance
oral vaccination. Hase et al. Nature 462:226-230
39
Question #2:
Why does Ftt FPI mutant not protect against Ftt in mice?
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.
Non-FPI mutations predicted to allow phagosome escape:
lpxF: Lipid A modification enzyme, makes Ftn more
resistant to AMPs, knockout attenuates Ftn (Raitz)
atpC: ATP synthase component, knockout attenuates Ftn
(Manoil)
FTT0748: transcriptional activator suppresses immune
response to Ftn, knockout attenuates Ftn (Monack)
FTT1103: lipoprotein with undefined function, knockout
attenuates Fth (Stulik)
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)
(A. Charbit)
ggt (g-glutamyl transpeptidase) important for acquisition
of cysteine inside cells, can escape phagosome but
replicates poorly
(Klose)
trpB required for Trp biosynthesis, knockout attenuated
for virulence in Ftn in mice (slightly)
glnA required for Gln biosynthesis, knockout attenuated
For virulence in Ftn in mice (slightly)
•
•
•
•
•
•
•
•
•
We have already attempted 4 different (non-FPI)
potential attenuating mutations in Schuh4:
lpxF: LipidA modification enzyme required for
virulence in Ftn
nadM: required for NAD biosynthesis, C-terminal
mutation is attenuating in Ftn
Unsuccessful at knocking either gene out in Schuh4
These may be essential genes in Ftt
FTT0748: identified by Monack et al. as transcriptional
activator involved in downregulating immune
response in Ftn, attenuated for virulence
We knocked out FTT0748 in Schuh4, Ftt mutant is not
significantly attenuated for virulence in mice.
atpC: encodes ATPase that contributes to Ftn
virulence identified by Manoil et al.
We knocked out atpC in Schuh4, Ftt mutant is not
significantly attenuated for virulence in mice.
44
List of defined Schuh S4 mutants (milestone 54):
lpxF (attempted, not successful)
nadM (attempted, not successful)
atpC (successful, not highly attenuated)
FTT0748 (successful, not highly attenuated)
FTT1103 (in progress)
trpB (in progress)
aroE (planned)
ggt (planned)
glnA (suggested)
Also worth considering: Combination strains
e.g. trpB + atpC, glnA + trpB, FTT1103 + trpB, etc
UTSA Milestones and Milestone Completion Reports:
Schedule for next 18 months
Completed Milestones: MSCR Status
16, 39, 43, 48: finalized and accepted by NIAID
50: under review at NIAID
49, 51: being written or revised at UTSA
None: under review at UNM
Active Milestones
52,53,54: scientifically in progress
46
Publications:
Biotechniques. 2007 Oct;43(4):487-90, 492.
Construction of targeted insertion mutations in Francisella
tularensis subsp. novicida.
Liu J, Zogaj X, Barker JR, Klose KE.
University of Texas San Antonio, San Antonio, TX 78249, USA.
PLoS One. 2010 Apr 1;5(4):e9952.
The Fischer 344 rat reflects human susceptibility to
francisella pulmonary challenge and provides a new
platform for virulence and protection studies.
Ray HJ, Chu P, Wu TH, Lyons CR, Murthy AK, Guentzel MN,
Klose KE, Arulanandam BP.
Vaccine. 2009 Sep 18;27(41):5554-61. Epub 2009 Aug 3.
Vaccination with a defined Francisella tularensis subsp. novicida
pathogenicity island mutant (DeltaiglB) induces protective immunity against
homotypic and heterotypic challenge.
Cong Y, Yu JJ, Guentzel MN, Berton MT, Seshu J, Klose KE, Arulanandam BP.
47
Clin Vaccine Immunol. 2009 Apr;16(4):444-52. Epub 2009 Feb 11.
Oral live vaccine strain-induced protective immunity against pulmonary
Francisella tularensis challenge is mediated by CD4+ T cells and antibodies,
including immunoglobulin A.
Ray HJ, Cong Y, Murthy AK, Selby DM, Klose KE, Barker JR, Guentzel MN,
Arulanandam BP.
We intend to publish several additional manuscripts, our ideas:
1. Requirement for Trp biosynthesis for F.t. virulence
2. Effect of UVR/Rec mutations on FT virulence
3. Modifications to enhance genetic engineering in FTT (if they
work)
4. Further examination of rat model
5. Identification of protective live Ftn vaccines against FTT
In rat model
48
Discussion later in site visit day
• F novicida is a BSL2 level
• LBERI plans to get rid of SIV positive NHP. Could vaccinate with F novicida
various doses subcutaneous and look at site reactogenecity
• Action: talk to Julie Hutt about bronching in lung, plating and histopathology, could
compare data directly to SCHU S4 and LVS. LVS doesn’t nothing when SCHU S4
is destroying the lung.
• Most lipoproteins are in the outer membrane.
• Jieh showed a FOPC (integral lipo protein) that is IFN gamma dependent and
broad effects are reversible by genetic complenation. The mutants releases many
proteins into the supernatant. FopC mutant makes the membranes very leaky.
Jieh is working to publish this data on lipoproteins. This work was performed
under the group’s PPG. Rick wonders if is enhancing normal secretion or if
causing proteins to be secrted that are not normally secreted?
• Karl analyzed the UTSA Felgner data. Found 8 genes up across all species, 4 up
in all except holartica, 10 that came up in all except , 14 genes only up in SCHU
S4 primary infected animals, has long list (>30) of only up in
vaccinated/challenged animals including some FPI genes; some were
49 specific to
oral vaccination and others to intradermal vaccination.
Felgner analysis: post call discussion
• Cross species: proteins up in all species with vaccination are
interesting.
• Vaccinated/survived challenge- proteins up with challenge
that were not up just with vaccination.
• LVS vaccination across species pre-vaccination and LVS
vaccination post-vaccination across species (ASK RICK)
• One UTSA rat survived SCHU S4: unique up pattern in this
rat
• NHP : If two NHP are vaccinated, one survives challenge and
one succumbs to challenge; what is the difference in the pre
challenge sera?
Oct 6, 2009