antibodies 39 me Runion Interdisciplinaire de Chimiothrapie - PowerPoint PPT Presentation

Local humoral response during M. ulcerans infection production of mycolactone neutralizing antibodies 39 ème Réunion Interdisciplinaire de Chimiothérapie Anti-Infectieuse Lundi 16 et mardi 17 décembre 2019 Palais des Congrès de Paris Mélanie FOULON – phD student Team ATOMycA – U1232, eq. 06

CONTEXT Buruli ulcer, a neglected tropical disease 90 % of cases Outbreak in Australia ➢ Third most common mycobacterial disease ➢ Neglected tropical disease (WHO) 2

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 Tissue destruction is associated to a toxin secret ed by Mycobacterium ulcerans Slow-growing bacteria (30°C) Generation time estimated to be 3,5 day → 2 months to obtain colonies… Acid Fast Bacilli Cytotoxic effect Immunomodulatory effect Analgesic effect MYCOLACTONE

CONTEXT What about the involvement of the immune response ? NO MEDICAL TREATMENT Cytotoxic effect Immunomodulatory effect Analgesic effect WHO N ECROSIS AMPUTATION

CONTEXT What about the involvement of the immune response ? NO MEDICAL TREATMENT Cytotoxic effect Immunomodulatory effect Analgesic effect ~5% of cases WHO N ECROSIS AMPUTATION WHO S PONTANEOUS HEALING Marion et al. 2016, OFID Host’s strategy to counteract the effect of O’Brien et al. 2019, M. ulcerans and the mycolactone PLoS Negl Trop Dis

CONTEXT What about the involvement of the immune response ? C LUSTERS OF B- CELLS SURROUNDING INFECTION SITE Ruf et al. 2017, Virulence Local B-cells Local humoral response enhance local defense and immunity in chronic inflammatory skin diseases

AIM OF OUR STUDY Our aim ------------------------------------ ------------------------------------ Characterize the local humoral response during spontaneous healing process Novel diagnostic and therapeutic strategies

EXPERIMENTAL MODEL In our lab : original mice model of spontaneous healing C57Bl/6 model FVB/N model Acute inflammation Inflammation resolution No infection control Infection control Marion et al . 2016, Journal of Immunology

RESULTS Is the local humoral response involved in the spontaneous healing process ? F LOW C YTOMETRY H ISTOLOGY Plasma cells = CD45 + , CD19 - , B220 int , CD138 + HPS staining of inflammatory infiltrate Immunostaining of CD138 + cells Rise of local antibodies-producing cells during first steps of spontaneous healing process

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

RESULTS Global cutaneous immunoglobulins production during the infection course in mice SERUM SKIN 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 4 0 0 0 C 5 7 B l/6 Ig G T is s u e T itra tio n (n g /m L ) ** F V B /N * 3 0 0 0 ** 2 0 0 0 ** 1 0 0 0 SERUM 0 SKIN ) ) ) ) ) 5 5 0 0 0 - 1 3 4 6 D - - D D D ( D ( ( ( y ( r g h s a e n t s m c i l l e l a a e U n e d e d H h E e s R v s i s o r c e N 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 Recognition of the mycolactone by local immunoglobulins purified from skin 0.4 IgG Tissue (OD 450 nm) 0.3 0.2 0.1 Redness Edema Ulcers Healthy Healing 0 .6 Ig G 2 a T is s u e A b s o rb a n c e IgG2a 0 .5 ELISA against the 0 .4 mycolactone * 0 .3 * ✓ Immunoglobulins M 0 .2 ✓ Immunoglobulins A ✓ Immunoglobulins G 0 .1 ▪ IgG1 C 5 7 B l/6 F V B /N ▪ IgG2a Identification of a signature of the spontaneous healing ▪ IgG2b ▪ IgG3 process in mycolactone recognition

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 What about anti-mycolactone antibodies in Buruli ulcer patients ? * * 0 .8 0 .7 0 .6 0 .5 O D 0 .4 0 .3 0 .2 0 .1 0 .0 C o n tro l B u ru li u lc e r Human organism is able to produce anti-mycolactone antibodies

CONCLUSION Local approache : a key in the study of Buruli ulcer ? Novel diagnosis tools ? monoclonal antibodies Development of a new simple diagnosis tool adapted for endemic regions Patent application

ACKNOWLEDGMENTS U1232 CRCINA, team ATOMycA (06) - Angers U1163, Laboratory of Human Genetics of Infectious • Estelle MARION Diseases, Necker Branch - Paris • Laurent MARSOLLIER • Jérémy MANRY • Fida KHATER • Alexandre ALCAÏS • Marie KEMPF • Laurent ABEL • Vincent DUBEE • Mathieu EVEILLARD Pathology Department, CHU - Angers • Marie ROBBE-SAULE • Jean-Paul SAINT-ANDRE • Amandine DURAND • Anne CROUE • Lucille ESNAULT • Alban BESNARD • Amélie POUCHIN • Mélanie FOULON

RESULTS What about anti-mycolactone antibodies in Buruli ulcer patients ? Comparison of RNA from whole blood of patients at the time of diagnosis and after the antibiotic treatment The human host triggering a humoral immune response at the time of Buruli ulcer diagnosis

RESULTS Is the local humoral response involved in the spontaneous healing process ? B-cell populations characterization P1 P2

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

CONTEXT 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 Demangel & High 2018, Biol Cell