T gondii infections acquired from oocysts Frank Seeber Deputy leader WP4 seeberfrkide 322020 Previously identified antigens with sourceattributing potential 2011 2015 2019 TgERP ID: 932364
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Slide1
WP4Serology method based on novel antigens to discriminate T. gondii infections acquired from oocysts
Frank SeeberDeputy leader WP4
seeberf@rki.de3.2.2020
Slide2Previously identified antigens with source-attributing potential
2011
2015
2019
TgERP
(=TgLEA850)
TgCCp5A
TgCCp5A
TgOWP8
Slide3WP4 structure
Task 1
Identification and expression of a panel of oocyst/sporozoite-
specific proteins
Task 2
Task 3
Development of a novel
s
ource-attributing ELISA
Exploring the prevalence of
oocyst-driven infections
in animals and humans
sT1
sT2
sT1
Furio Spano/Frank Seeber
Gema Alvarez-Garcia/Luis Ortega Mora
Rebecca Davidson/Radu Blaga
sT3
sT2
(Rebecca Davidson)
(Radu Blaga)
(Henrik Vedel Nielsen)
(Gema Alvarez-Garcia)
(Luis Ortega Mora)
Slide4Bioinformatic selection
of oocyst/
sporozoite-specific antigens completed in WP4-T1 >>M28
Production
of
the
first
set
of purified soluble recombinant proteins (up to
100) by WP4-T1 for serological
assays
>>M36Evaluation of the source
attributing ability of the first sets
of stage-specific
antigens produced (
WP4
-
T2)
>>M36
SOP
for
a
novel
source-attributing
serological
method
(WP4-T2)
>> M48Report on the prevalence
of oocyst-derived infections
in humans
and animals (including
testing of
robustness, the ring trial, and
the pilot
studies) WP4-T2 >> M54
Milestones & Deliverables WP4
Slide5WP1-T1 Identification and production of T. gondii stage-specific antigens for source attribution
Task leader: Furio Spano (ISS); Deputy task leader: Frank Seeber (RKI)
or - finding the needle in the haystack
8284 proteins
oocyst
-specific antigen(s)?
Slide6Selection criteria for candidate proteins
8284 proteins
oocyst
-specific antigen(s)?
sources:
ToxoDB
;
UniProt
;
online predictors (e.g.
SignaP
5.0); literature
Slide7present in oocyst proteome (4 studies)
absent from tachyzoite / bradyzoite proteomes (many)
extracellular localization (signal peptide; exported proteins)abundance (peptide counts from proteome studies)stability (N-end rule)
solubility / expression level in E. coli
(# exons;
#
transmembrane
regions; length
; codon usage)luck
Selection criteria for candidate
oocyst proteins: > 50 criteria/data sets available
Slide8species-specificity (within/outside Coccidia)
diversity within T. gondii strains (polymorphism)
disorder / antigenicity (several algorithms)removal of apicoplast proteinspresence of protein modification (e.g. phosphorylation, acetylation)
Selection criteria for candidate proteins:
not included (yet)
Slide9Plan B: antibody-profiling to identify patterns for cyst-derived infections
Slide10Evaluation scheme for acute toxoplasmosisWellinghausen, N., Abele-Horn, M. &
Mantke, O. D. (2018) Immunological Methods for the Detection of Infectious Diseases.
instand-ev.de
Slide11sporo
Ag
early
brady Ag
late
brady Ag
early
tachy Aglate tachy Ag
Infection
via /status
IgM
IgG
/
avidity
IgM
IgG/
avidity
IgM
IgG
/
avidity
IgM
IgG
/
avidity
IgM
IgG
/
avidity
Oocysts
/
acute
+/ ++
+++
low
-high
-
-
-
-
-
-
-
-
Cysts
/
acute
-
-
+++
+
low
+
-
+
+
low
-
- / +
low
Cysts
/
early
chronic
?
?
-
(
+)
- (+)
low
- (+)
+
low
+
+
low
-
(+)
++low-mediumCysts/late chronic??+ (-)+medium-high+++medium-high+++medium-high-+++high
Preliminary scheme to show that infection was most likely caused by bradyzoites
>> requires input !
Slide12Recombinant proteins to start with
protein
stageat handrec protein describedresponse expectedSAG1/SAG2A
tachy
yes
overall
BAG1
(early)
brady
soon
bradySporoSAGsporozoiteyes
sporo ??Crawford 2010SporoAMA1
sporozoite
yessporoERP (LEA850)oocystyes
oocyst LEA860oocystyesoocyst
CCp5Aoocyst
yessporo
OWP8
oocyst
no
yes
oocyst
generate antibodies against some of the published recombinant proteins
Slide13PCR-amplify GOI from gDNA or oocyst cDNA
(or synthesis)clone into pAvi(MBP)-
ccdB plasmid via SLiCE / Gibson cloningtransform, identify clones, confirm sequencere-transform into expression E. coli strain(s)expression in 24-deep-well plates at
<18-20°C
lysis
and direct purification by magnetic Ni-NTA beads
anti
-His
6
dot blot and/or SDS-PAGEwhen
successful >> initial testing for reactivity with animal sera by partnerswhen successful >> large-scale expressionwhen no (soluble) expression >> brain stormingExpression pipeline of POI
Slide14Cloning of genes-of interest (GOI)
genomic DNA or
cDNA
Slide15POI production, purification and ELISA
E
. coli MBP-tev
POI
AviTag-His
6
anti-His
6
Ab
MBP =
maltose-binding
protein
Slide16Oriented vs random coupling in ELISA – influence on antibody binding?
LEA850/860
AviTag-His
6
anti-His
6
Ab
LEA850/860
AviTag-His
6
>> test in small-scale experiment whether there is an influence
Slide17100-500 µg of purified protein initiallytested by Western Blot and/or ELISA with anti-His6 antibodies
What WP4-T1 will provide
constant supply of
new
proteins over 2 years
tons of protein
the magic bullet antigen(s)
What WP4-T1 can't guarantee
Slide18WP4-T2 Development of a novel stage-specific antigen-based ELISA to diagnose oocyst- and bradyzoite
-driven T. gondii
infectionsTask start month M29 - task end month M48
Task
leader
:
Gema
Álvarez
García,
UCM Deputy task leader: Luis Ortega Mora, UCMTask participants: UCM, ANSES, VRI, PIWET, RIVM
Summary of WP4- T2
Proteins
of interest (POIs) based ELISA from
WP4-T1Sera from
experimentally infected animals
with tachyzoites
,
bradyzoites
or
oocysts
Sera
from
humans
infected
congenitally
or
through
water/food/environment
Developing
process
POIs- based ELISA
proof of concept
Validation
process
+
Slide19WP4-T2- sT1 Standardization of a POI-based ELISA to diagnose oocyst-
and/or bradyzoite-driven
T. gondii infections using reference pig sera
Reference
for
the
e
xperimental
designOIE -
Manual of Diagnostic Tests for Aquatic Animals - 14/11/2019, chapter 1.1.2 “Principles
and methods
of validation of diagnostic assays
for infectious diseases
WP4-T2
WP4-T3
Slide20Pig
sera available
Laboratory
origin
Pig
(age
/
stage
, sex, breed/ lineage
)
Strains
Stage
of
the
parasite
Inoculation
dosis and
route
Sampling
days
post
inoculation
(pi
)
Serological
analysis
Any relevant clinical sign?
(yes/no)
Age
/
stage
Sex
Breed
/
lineage
Name
Genotype
Oocysts (O); Cysts
(
C
);
Tachyzoites (TZ)
Dosis
Route
Serological
technique
Time of seroconversion
post inoculation
(pi)
PIWET
12 weeks
Female
PLW
RH
Type I
TZ
10
7
SC*
Weekly until 91 days pi
Unknown
7 days pi
Unknown
VRI/VFU
Prepubertal
Female
Danhybrid-LY
CZ -Tiger
Type
II
O
250
Oral
Weekly, 7 weeks
PrioCHECK ELISA
Week 2 pi
Yes
VRI/VFU
Prepubertal
Female
Danhybrid-LY
CZ -Tiger
Type II
C
10
Oral
Weekly, 7 weeks
PrioCHECK ELISA
Week 2 pi
Yes
VRI/VFU
Prepubertal
Female
Danhybrid
-LY
Šimková
isolate
Type III
O
250
Oral
Weekly, 7 weeks
PrioCHECK ELISA
Week 2 pi
Yes
VRI/VFU
Prepubertal
Female
Danhybrid-LY
Šimková
isolate
Type
III
C
10
Oral
Weekly, 7 weeks
PrioCHECK ELISA
Week 2 pi
Yes
ANSES
9 months
Female
Unknown
ME49
Type II
O
1000
Oral
Weekly from 3 to 9 months
Unknown
Unknown
Unknown
ANSES
9 months
Female
Unknown
ME49
Type II
C
1000
Oral
Weekly from 3 to 9 months
Unknown
Unknown
Unknown
* SC:
Subcutaneus
; MAT:
microagglutination
test;
sera
already
submitted
to UCM
group
WP4-T2- sT1
Standardization of a POI-based ELISA to diagnose
oocyst
-
and/or
bradyzoite
-
driven
T.
gondii
infections using reference pig
sera
Slide21Experimental
design
ELISAs
of
lyophilized
tachyzoites
and
sporozoites
Comercial ELISA
Western Blot and immunofluorescence
Pig
sera
characteri
z
ation
Specificity
of
proteins
of
interest
(
POIs
)
Western
Blot
for
Neospora
caninum
and
Besnoitia
spp
.
Bovine and
ovine sera
Selection of
the most specific
POIs
From pigs
experimentally infected with
TZ, oocysts or
cysts
Pool pig reference sera
Sporozoites vs.
bradyzoites POIs- based
ELISA
To be continued
Standardization and
optimization of the new
POIs
-
based
ELISA
Conjugate
dilution
Sera
dilution
Concentration
of
POIs
Mixture of
POIs
?
Determination
of
cross-reaction
POIs
from
sporozoites
and
bradyzoites
WP4-T2- sT1
Standardization of a
POIs-based
ELISA to diagnose
oocyst
- and/or bradyzoite-driven T. gondii infections using reference pig seraSub-task leader: Gema Álvarez García, UCM Sub-task participants: ANSES, VRI, PIWET
Slide22Experimental
design
A
nalytical
sensitivity
Diagnostic
performance
New
validaded
T. gondii oocyst/cyst
POIs- based ELISA for
pig sera
R
epeatability
Data
collection
and
TG ROC
analysis
Diagnostic
Se and
Sp
Contingency
plan:
Detection
of
IgM
IgG
avidity
Sporozoites
vs.
bradyzoites
POIs
-
based
ELISA
Risky Plan
B!
WP4-T2- sT1
Standardization of a POI-based ELISA to diagnose
oocyst
-
and/or
bradyzoite
-
driven T. gondii
infections using reference pig sera
Slide23Human sera
available
Laboratory
origen
Sampling
information
Serological tests employed
Relevant
clinical
signs
Country
Adquired
or
congenital
infection
ELISA (kit
employed
)
WB (
antigen
employed
)
RIVM
VS
Atlanta
outbreak
(
water
)
In-
house
IgG
In-house
WB IgM/ISAGA IgM
-
Laboratory
origen
Specie (age, sex/ stage, breed/ lineage)
Strains
Stage
of
the
parasite
Inoculation
dosis and
route
Sampling
days
postinoculation
(pi)
Serological
analysis
Any relevant clinical sing
presented? (yes/no)
Sheep
(S
)
Cat
(C)
Goat
(G)
Age
/
stage
Sex
Breed
/
lineage
Name
Genotype
Oocysts (O); Cysts (Q); Tachyzoites (TZ)
Dosis
Route
Serological technique
Time of seroconversion
postinoculation (pi)
UCM
S
Adult
Female
Churra (pregnant)
ShSp1
Type II
O
10
Oral
3,8, 12, 14, 22 and 27
In house ELISA with TZ extract*
Analysis in process
Yes
UCM
S
Adult
Female
Rasa Aragonesa (pregnant)
ME49 y ShSp1
Type II
O
500, 50
and
10
Oral
3,5, 7, 10 and weekly until abortion or birth
In house ELISA with TZ extract*
21 days pi
Yes
Anses
S
3 months
Female
Pré Alpes du Sud
ME49
Type II
O
1000
Oral
Weekly from 3 to 52 days pi
MAT*
12 days pi
Yes
* SC:
Subcutaneus
; MAT:
microagglutination
test;
Other
animals
sera
available
Cat sera could be available
WP4-T2- sT2:
Validation of a novel stage-specific antigen based ELISA to diagnose
oocyst
-
and/or
bradyzoite
-driven
T.
gondii
infections using reference sera from several relevant host species including
humans
Slide24Sera
from
humans
Sera
from
other
species
Characteri
zation of all
sera
with a reference technique
ELISAs of lyophilized tachyzoites and sporozoites
Commercial ELISA etc.
In-house POIs
-
based
ELISA (
from
sT1)
Experimental
design
Experimental
design
adapted
from
sT1
Standardization
and
optimization
of
the
new
intended
POIs
-
based
ELISA
Conjugate
dilution
Sera dilution
A
nalytical
sensitivity
and
repeatability
Diagnostic
performance
New validated
T
.
gondii
oocyst
/
cyst
POIs
-
based
ELISA
for
humans
and
other
species
Detection
of
IgG
and
IgM
IgG
avidity
if
necessaryWP4-T2- sT2: Validation of a novel stage-specific antigen based ELISA to diagnose oocysts and/or bradyzoite-driven T. gondii infections using reference sera from several relevant host species including humansSub-task leader: Luis Ortega Mora, UCM Sub-task participants: ANSES, RIVM, SSI
Slide25WP4-T3 Exploring the prevalence of oocyst-derived T.
gondii infections in animals and humans
Task start month M40 - task end month M54Task leader:
Rebecca Davidson, NVI Deputy task leader:
Radu Blaga,
ANSES
Task
participants
: NVI, ANSES, PIWET, SSI, BRCT, RIVM, INIAV, WBVR, VRI, BIOR, INSA
Summary of WP4-T3
Screening sera from domestic animals and wildlife used for human consumption with validated POI ELISA
POI-ELISA from WP4-T2-sT1: interlaboratory validation using panel of positive and negative reference sera
Subtask
1: lead Klevar, NVINVI, ANSES, PIWET, RIVM, INSA, BRCT
Subtask 2: lead Blaga, ANSESANSES, NVI, PIWET, INIAV, WBVR, VRI, BIOR, SSI
Subtask 3: lead Nielsen, SSI
Screening well defined positive sera from humans from different EU regions with validated POI ELISA
SSI, RIVM, INSA, BRCT/NRCT
Slide26Using the POI-based ELISA from WP4-T2-sT1
Inter-laboratory validation as a ring trial using standardised panel of positive and negative reference sera.Evaluate accuracy and precision of test
Estimate test sensitivity and specificityEstimate interlaboratory reproducibility and variationOnce validated the POI-ELISA will be offered to all consortium partners for use in screening existing collections of positive sera
All studies to be performed in year 2-3 of this project (year 4-5 of EJP)
Sub-Task Leader:
Siv Klevar, NVI
Sub-Task participants: NVI, ANSES, PIWET, RIVM, INSA, BRCT
WP4-Task 3 subtask 1
Interlaboratory
validation of the POI-based ELISA
Slide27To reveal differences in the relative prevalence of oocyst-driven infections between animal species, regions and management systems, available T. gondii
positive sera from natural infections from at least Northern (NVI), Eastern (PIWET) and Western (ANSES) Europe will be analysed
Using the POI-based ELISA from WP4-T2 The work will consist of 2 steps:All studies to be performed in year 2-3 of this project (year 4-5 of EJP)
Sub-Task Leader: Radu BLAGA, ANSES, France
Sub-Task participants: ANSES, NVI, PIWET, INIAV, WBVR, VRI, BIOR, SSI
WP4-Task 3 subtask 2
Exploring
the prevalence of
oocyst
-driven infections in domestic animals (pigs, small ruminants) and wildlife (wild-boar, wild ungulates) used for human consumption
Slide28The work will consist of 2 steps:
A panel of 50-100 sera per animal species from domestic pigs (indoor & outdoor production systems), wild boars, small ruminants (ovine, goats) and wild ungulates will be tested by three core labs (ANSES, PIWET, and NVI).
Subsequently, the validated test will be offered to other laboratories in the consortium (including INIAV, WBWR, VRI, BIOR, SSI) for implementation and testing of existing collections of positive sera.
WP4-Task 3 subtask 2
Exploring the prevalence of oocyst-driven infections in domestic animals (pigs, small ruminants) and wildlife (wild-boar, wild ungulates) used for human consumption
Slide29Well-defined positive sera from T. gondii-infected humans available from different European regions will be tested
Using the POI-based ELISA from WP4-T2
The work will consist of 3-4 pilot studiescongenital infections (tachyzoite-derived) (n=50–100 samples from children aged <13 months)Sera from older children
(n=50–100, aged >2 years
)
N
ewly/recently
infected adults (n=200–500
)
Sera from possible European outbreaks if identified
All studies to be performed in year 2-3 of this project (year 4-5 of EJP)Sub-Task Leader: Henrik Vedel Nielsen, SSISub-Task participants: SSI, RIVM, INSA, BRCT/NRCT
WP4-Task 3 subtask 3 Exploring the prevalence of oocyst-driven infections in humans
Slide30seeberf@rki.defurio.spano@iss.it
gemaga@vet.ucm.es
luisucm@vet.ucm.esRebecca.Davidson@vetinst.noradu.blaga@vet-alfort.frHVN@ssi.dk