Developing two in vitro assays to measure antibody

mediated protection against intracellular bacteria

Senior Scientist Jes Dietrich,

Dept. of Infectious Disease Immunology,

Division of Vaccine,

Statens Serum Institut,Artillerivej 5,

DK- 2300 Copenhagen S

CHLAMYDIA TRACHOMATIS

Chlamydia, caused by infection with Chlamydia trachomatis,

More than 100 million chlamydial infections are estimated annually

Cause serious damage in the upper genital tract which can lead to infertility.

CHLAMYDIA LIFE CYCLE

Which antigen?

ANTIBODIES

Induction of phagocytosis Neutralization

Prevent pathology in upper genital tract

FINDING THE RIGHT ANTIBODY

MOMP protein

MOMP

SCREENING FOR PROTECTIVE ANTIGENS

Produce the antigens

Vaccinate animals with the

antigens and test for

Protection against infection

Select protective antigen

Antigen 1

Antigen 2

Antigen 3

Antigen 4

Antigen 5

Antigen 6

In vitro experiments using bacteria and cell lines

Exchange with in vitro assays

SCREENING FOR PROTECTIVE ANTIGENS

Produce the antigens

Vaccinate 1 mouse to produce the Abs

Select protective antigen

In vitro screening for protection

DEVELOP TWO ASSAYS

Produce a green Chlamydia bacteria (visible in a flow cytometer)

Show that an antibody binding to it

can mediate uptake into neutrophil or macrophage

Induction of phagocytosis Neutralization

FS

C-H

SS

C-A

FSC-A

Gating strategy for stained bacteria

BacteriaSinglets

APC-A

FIT

C-A

unstained CSFE-labeled a-LPS + 2nd Ab only 2nd Aba-LPS + 2nd AbCSFE-labeled &

STAINING THE BACTERIA

Green bacteria Staining LPS

1. Permeabilize the cells, stain the bacteria, and FACS

2. FACS (because the bacteria are green)

Two ways to measure uptake :

UPTAKE INTO PLB-985 CELLS

CFSE-labeled SvD bacteria were preincubated with no serum, serum

from naıve rabbits, or serum from rabbits vaccinated with Hirep1 for

40 min at 37C and incubated for 4 h with DMF-stimulated PLB-985

cells

100

20

40

60

80

MOI

% p

hag

ocyto

sin

g C

FS

E+

cells

MOMP _1:10

MOMP _1:100

MOMP _1:1000

MOMP _1:10000

naive_1:10

naive_1:100

naive_1:1000

naive_1:10000

% Phagocytosis

1:10 1:100 1:1000 1:100000

20

40

60

80

Serum dilution

% p

ha

go

cy

tos

ing

CF

SE

+ c

ells MOMP

CTO43

TB

naive

FSC-A

FIT

C-A

6.8 %7.2 %

9.3 %65.4 %

naiveTB

CTO43Hirep1

TESTING TWO SURFACE ANTIGENS – MOMP AND CT043

Being surface exposed does not

automatically lead to phagocytosis.

n o c yto c h D 0 .1 µ g 1 µ g 1 0 µ g 1 0 0 µ g

0

2 0

4 0

6 0

8 0

g C y to c h a la s in D

% p

ha

go

cy

tos

ing

CF

SE

+ c

ell

s

H ire p 1

n a iv e ra b b it se ru m

n o fc -b lo c k 1 :1 0 0 1 :1 0 1 :4

0

2 0

4 0

6 0

8 0

F c b lo c k d ilu t io n

% p

ha

go

cy

tos

ing

CF

SE

+ c

ell

s

H ire p 1

n a iv e ra b b it se ru m

Fcγ receptor blocking

Actin-polymerization inhibition

INHIBITING FC RECEPTORS OR ACTIN POLYMERIZATION

OTHER USED FOR THE ASSAY

Another obvious use for a FACS based

phagocytosis assay is the high-throughput

testing of serum samples from human

donors.

It was, therefore, important to show that

the assay is also suitable for human and murine serum

1 :1 0 0 1 :1 0 0 0 1 :1 0 0 0 0

0

2 0

4 0

6 0

8 0

S e ru m d ilu t io n

% p

ha

go

cy

tos

ing

CF

SE

+ c

ell

s

P o s itiv e H u m a n d o n o r A b

N e g a tiv e H u m a n d o n o r A b

Phagocytosis induced by human serum

NEUTRALIZATION ASSAY – PREVENTION OF UPTAKE

Induction of phagocytosis Neutralization

The standard assay includes incubation of C. trachomatis with serum

followed by infection of a HaK cell line (20h) and microscopy counting of

inclusions

Labour intensive

The assay

COMPARISON OF NEUTRALISATION METHODS

Sample

1

Sample

2

Sample

3

Sample

4

Sample

5

Sample

6

10

100

1000

10000

Titers determined by 2 different methods

Neu

trali

sati

on

tit

er

Flow cytometry

Microscopy

1 10 100 1000 100001000000

20

40

60

80

100 Serum titration

Dilution of sera

% N

eu

tralisati

on

MOMP

CT043

1:10 1:100 1:10001:100000

20

40

60

80

Serum dilution

% p

ha

go

cy

tos

ing

CF

SE

+ c

ells MOMP

CTO43

Phagocytosis

Conclusion: Flow cytometry is an alternative to

the standard microscopy method

Mouse serum Human serum

Antibodies against surface exp. proteins does not automatically

lead to phagocytosis or neutralization

CONCLUSIONS

Developing two in vitro assays to measure antibody mediate protection

against intracellular bacteria

Phagocytosis

assayNeutralization

assay

We can use these assays in the screening

part of the antigen discovery process