WP4 Serology method based on novel antigens to discriminate - PowerPoint Presentation

Slide1

WP4Serology method based on novel antigens to discriminate T. gondii infections acquired from oocysts

Frank SeeberDeputy leader WP4

seeberf@rki.de3.2.2020

Slide2

Previously identified antigens with source-attributing potential

2011

2015

2019

TgERP

(=TgLEA850)

TgCCp5A

TgCCp5A

TgOWP8

Slide3

WP4 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)

Slide4

Bioinformatic 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

Slide5

WP1-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)?

Slide6

Selection criteria for candidate proteins

8284 proteins

oocyst

-specific antigen(s)?

sources:

ToxoDB

;

UniProt

;

online predictors (e.g.

SignaP

5.0); literature

Slide7

present 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

Slide8

species-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)

Slide9

Plan B: antibody-profiling to identify patterns for cyst-derived infections

Slide10

Evaluation scheme for acute toxoplasmosisWellinghausen, N., Abele-Horn, M. &

Mantke, O. D. (2018) Immunological Methods for the Detection of Infectious Diseases.

instand-ev.de

Slide11

 

sporo

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 !

Slide12

Recombinant 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

Slide13

PCR-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

Slide14

Cloning of genes-of interest (GOI)

genomic DNA or

cDNA

Slide15

POI production, purification and ELISA

E

. coli MBP-tev

POI

AviTag-His

6

anti-His

6

Ab

MBP =

maltose-binding

protein

Slide16

Oriented 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

Slide17

100-500 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

Slide18

WP4-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

+

Slide19

WP4-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

Slide20

Pig

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

Slide21

Experimental

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

Slide22

Experimental

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

Slide23

Human 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

Slide24

Sera

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

Slide25

WP4-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

Slide26

Using 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

Slide27

To 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

Slide28

The 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

Slide29

Well-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

Slide30

seeberf@rki.defurio.spano@iss.it

gemaga@vet.ucm.es

luisucm@vet.ucm.esRebecca.Davidson@vetinst.noradu.blaga@vet-alfort.frHVN@ssi.dk