antibodies 39 me Runion Interdisciplinaire de Chimiothrapie - - PowerPoint PPT Presentation

Local humoral response during M. ulcerans infection

production of mycolactone neutralizing antibodies

Mélanie FOULON – phD student

Team ATOMycA – U1232, eq. 06

39ème Réunion Interdisciplinaire de Chimiothérapie Anti-Infectieuse

Lundi 16 et mardi 17 décembre 2019 Palais des Congrès de Paris

Outbreak in Australia 90 % of cases

➢ Third most common mycobacterial disease ➢ Neglected tropical disease (WHO)

2

Buruli ulcer, a neglected tropical disease

CONTEXT

Buruli ulcer, a neglected tropical disease characterized by large ulcerative lesions

CURRENT TREATMENT

+

A combination of two antibiotics for 8 weeks (+ surgery for severe cases)

3

CONTEXT

Cytotoxic effect Immunomodulatory effect Analgesic effect

MYCOLACTONE

Tissue destruction is associated to a toxin secreted by Mycobacterium ulcerans

Acid Fast Bacilli Slow-growing bacteria (30°C) Generation time estimated to be 3,5 day → 2 months to obtain colonies…

CONTEXT

What about the involvement of the immune response ?

Cytotoxic effect Immunomodulatory effect Analgesic effect

WHO

CONTEXT

NO MEDICAL TREATMENT

NECROSIS

AMPUTATION

What about the involvement of the immune response ?

Cytotoxic effect Immunomodulatory effect Analgesic effect

SPONTANEOUS HEALING Host’s strategy to counteract the effect of

• M. ulcerans and the mycolactone

O’Brien et al. 2019, PLoS Negl Trop Dis Marion et al. 2016, OFID

WHO WHO

CONTEXT

NO MEDICAL TREATMENT

NECROSIS

AMPUTATION

~5% of cases

What about the involvement of the immune response ?

CLUSTERS OF B-CELLS SURROUNDING INFECTION SITE Local B-cells Local humoral response enhance local defense and immunity in chronic inflammatory skin diseases

Ruf et al. 2017, Virulence

CONTEXT

AIM OF OUR STUDY

Our aim

Characterize the local humoral response during spontaneous healing process

• Novel diagnostic and therapeutic strategies

In our lab : original mice model of spontaneous healing

EXPERIMENTAL MODEL

C57Bl/6 model FVB/N model Acute inflammation No infection control Inflammation resolution Infection control

Marion et al. 2016, Journal of Immunology

FLOW CYTOMETRY

Plasma cells = CD45+, CD19-, B220int, CD138+

HISTOLOGY

Immunostaining

• f CD138+ cells

HPS staining of inflammatory infiltrate

Is the local humoral response involved in the spontaneous healing process ?

RESULTS

Rise of local antibodies-producing cells during first steps of spontaneous healing process

IDENTIFICATION OF SKIN-SPECIFIC ANTIBODIES DURING M. ulcerans INFECTION PROCESS

RESULTS

Global cutaneous immunoglobulins production during the infection course in mice

SKIN

SERUM

The course of M. ulcerans infection is associated with a local humoral response

RESULTS

Global cutaneous immunoglobulins production during the infection course in mice

SKIN

SERUM

H e a l t h y ( D

• )

R e d n e s s ( D

• 1

5 ) E d e m a ( D

• 3

) U l c e r ( D 4 5 ) N e c r

• s

i s v s h e a l i n g ( D 6 ) 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 F V B /N C 5 7 B l/6

Ig G T is s u e T itra tio n (n g /m L )

** ** * **

SKIN

Do these IgGs recognize the mycolactone ?

RESULTS

Recognition of the mycolactone by local immunoglobulins purified from skin ELISA against the mycolactone

✓ Immunoglobulins M ✓ Immunoglobulins A ✓ Immunoglobulins G ▪ IgG1 ▪ IgG2a ▪ IgG2b ▪ IgG3

RESULTS

Healthy Redness Edema Ulcers Healing 0.1 0.2 0.3 0.4

IgG Tissue (OD 450 nm) C 5 7 B l/6 F V B /N 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6

Ig G 2 a T is s u e A b s o rb a n c e

* *

IgG2a

Identification of a signature of the spontaneous healing process in mycolactone recognition

ELISA against the mycolactone

✓ Immunoglobulins M ✓ Immunoglobulins A ✓ Immunoglobulins G ▪ IgG1

▪ IgG2a

▪ IgG2b ▪ IgG3

Recognition of the mycolactone by local immunoglobulins purified from skin

RESULTS

Are these immunoglobulins able to neutralize the mycolactone ?

Neutralization test

RESULTS

Are these immunoglobulins able to neutralize the mycolactone ?

Neutralization test

RESULTS

Are these immunoglobulins able to neutralize the mycolactone ?

Neutralization test

IgG from FVB/N skins are able to neutralize the mycolactone

RESULTS

WHAT ABOUT THE HUMORAL RESPONSE IN BURULI ULCER LESIONS ?

RESULTS

B u ru li u lc e r C o n tro l 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 0 .7 0 .8

O D

* *

Human organism is able to produce anti-mycolactone antibodies

RESULTS

What about anti-mycolactone antibodies in Buruli ulcer patients ?

CONCLUSION

Novel diagnosis tools ?

monoclonal antibodies

Local approache : a key in the study of Buruli ulcer ?

Development of a new simple diagnosis tool adapted for endemic regions

Patent application

ACKNOWLEDGMENTS

U1232 CRCINA, team ATOMycA (06) - Angers

• Estelle MARION
• Laurent MARSOLLIER
• Fida KHATER
• Marie KEMPF
• Vincent DUBEE
• Mathieu EVEILLARD
• Marie ROBBE-SAULE
• Amandine DURAND
• Lucille ESNAULT
• Alban BESNARD
• Amélie POUCHIN
• Mélanie FOULON

U1163, Laboratory of Human Genetics of Infectious Diseases, Necker Branch - Paris

• Jérémy MANRY
• Alexandre ALCAÏS
• Laurent ABEL

Pathology Department, CHU - Angers

• Jean-Paul SAINT-ANDRE
• Anne CROUE

What about anti-mycolactone antibodies in Buruli ulcer patients ?

RESULTS

The human host triggering a humoral immune response at the time

• f Buruli ulcer diagnosis

Comparison of RNA from whole blood of patients at the time of diagnosis and after the antibiotic treatment

B-cell populations characterization

P1 P2

Is the local humoral response involved in the spontaneous healing process ?

RESULTS

Global cutaneous immunoglobulins expression during the infection course in mice

mRNA encoding for IgM, IgA and IgG are significantly higher in the spontaneous healing model

RESULTS

What about the involvement of the immune response ?

Cytotoxic effect Immunomodulatory effect Analgesic effect

Mycolactone is targeting Sec61channel during the early stages of the infection

→inhibition of maturation of cytokines

CONTEXT

Demangel & High 2018, Biol Cell