Transcript Delivery of Epitopes by the Salmonella Type III Secretion System for

Delivery of Epitopes by the
Salmonella Type III Secretion
System for Vaccine Development
Holger Rüssmann, Homayoun Shams,
Fernando Poblete, Yixin Fu, Jorge E. Galán,
Ruben O. Donis
By: Kita Scott and Anna Strongin
Question
Which Salmonella strain was unable
to translocate SptP chimeric proteins
into the host cell?
Salmonella as an Antigen
Delivery System
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Can be rendered avirulent
Expresses foreign proteins from pathogenic
organisms
Able to invade nonphagocytic cells
Uses type III secretion system for protein
translocation
Delivers antigens that can stimulate both
humoral and cellular immune responses
Type III Secretion System (TTSS)
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Encoded in a pathogenicity island at
centisome 63 of the Salmonella
chromosome
Pancetti and Galán, FEMS Micriobiology Letters, 197(2001), 203-208
Type III Secretion System (cont)
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Type III Secretion
System delivers
proteins to the host
cell cytosol
Importance of a type III secretion
system
Internalized bacteria is confined to a
membrane bound compartment within
the host cell
 This prevents protein delivery into host
cell cytosol
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Antigens must be present in the cytosol
to elicit a class I immune response
Alberts, Molecular Biology of the Cell, 4th edition
Some Proteins Secreted by the
TTSS
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SptP
– effector protein
– GTPase Activating Protein activity at the Nterminus
– tyrosine phosphatase activity at the C-terminus
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SipD
– essential for type III translocation but not secretion
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InvJ
– secreted but not translocated
– required for needle complex assembly
Fusion of TTSS Protein to
Class—I restricted Epitopes
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Chimera created by inserting influenza
virus epitope (IVNP366-374) and
lymphocytic choriomeningitis virus
nucleoprotein (LCMVNP118-126) into SptP
In Vitro Epitope Translocation to
the Host Cell
Chimeric proteins introduced into wildtype as well as sptP-/aroA- and SipDSalmonella mutant strains
 Cell fractions examined for presence of
SptP fusion proteins
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Results
 SipD- strain secretes SptP-IVNP366-374 but does not
translocate it to the cytosol
 Wild-type and aroA- sptP- strains deliver SptP-IVNP366-374
to host-cell cytosol
Conclusions
SipD is necessary for translocation into
host-cell by the type III system, but not
for secretion
 SptP can be used to deliver foreign
proteins to the host-cell cytoplasm
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Epitope Delivery to the Class-I
Antigen Presenting Pathway
Murine RMA thymoma cells infected
with different Salmonella strains
 Epitope delivery measured by
quantifying interleukin-2 secretory
response of infected cells using ELISA
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Strains Failing to Present Antigen
as Determined by low IL-2
Secretion
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SipD- mutant strain
SptP-IVNP366-374
InvJ-IVNP335-498
Peptide Transporter System
(TAP)
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Transports peptides from cytosol to the ER
 Aids with loading by class I molecules
Janeway, Immunobiology, 5th edition
Effect of TAP2-defective Mutants
on Antigen Presentation
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Impaired TAP system prevents class-I
molecule loading
 RMA-S mutants show impaired ability to
stimulate T-cell hybridomas
Conclusions
Antigen presentation depends on the
type III system delivering epitopes to the
cytosol
 Foreign peptides must be present in the
host cell cytosol for entry to the class I
antigen presenting pathway
 Functional TAP system is essential for
antigen presentation once epitope is in
the cytosol
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In Vivo Induction of Cytotoxic T
Lymphocytes (CTLs)
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Mice inoculated with different strains of S.
typhimurium
 Constant number of MHC class I-expressing
target cells stimulated by different numbers of
splenocytes
 Level of expression of CTLs determined in
terms of % cell lysis
 Most effective CTL response was to the strain
expressing SptP-IVNP366-374
Data Supports In Vitro Results
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Open squares: SptPIVNP366-374
Open circles: SptPLCMVNP118-126
Closed triangles: InvJIVNP335-498
Closed squares: Vaccinia
IVNP
Open triangles: Influenza
virus strain A/PR/8/34
Vaccine Effectiveness Challenged
Inoculated mice were infected with
lymphocytic choriomeningitis virus
(LCMV)
 Only mice immunized with the
Salmonella strain carrying the SptPLCMVNP118-126 protein exhibited 100%
survival
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Data Supporting Vaccine
Effectiveness
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Open squares: aroA
sptP mutant expressing
SptP-LCMVNP118-126
 Closed squares: aroA
mutant expressing SptPIVNP366-374
 Closed triangles: sipD
mutant expressing SptPLCMVNP118-126
 Closed Circles: mock
control
CTL Levels in Mice Infected
with LCMV
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Open squares: aroA
mutant expressing
SptP-LCMVNP118-126
 Closed Triangles:
aroA mutant
expressing SptPIVNP366-374
 Closed Squares: live
LCMV
Conclusions
Class I-restricted immune response
depends on antigen translocation to the
host cell cytoplasm
 Immune response induces antigenspecific CTLs
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Implications for the Future
The availability of the TTSS of
Salmonella can aid with further
development of effective and efficient
polyvalent vaccines
 Targeting tumor-specific antigens to the
host may combat cancers by inducing
cancer cell specific CTLs
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Suggestions
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Address the difference in CTL induction by
the SptP-LCMVNP118-126 expressing strain
versus live LCMV and the SptP-IVNP366-374
versus live influenza virus
 Address the difference in the survivability of
mice inoculated with a control and a SipDmutant strain