Is Group A Strep the sole agent causing RHD?

Transcription

1 RHD Education Workshop, Brisbane, 23rd March 2016 Why do we require a laboratory model for ARF/RHD? Is Group A Strep the sole agent causing RHD? Natkunam Ketheesan Australian Institute of Tropical Health and Medicine James Cook University Australia

2 J Exp Med Mar 14;9(2):

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4 GAS infection triggers RF/RHD It is an immune-mediated complication of group A streptococcal (GAS) infection characterised by inflammation of the heart, joints, blood vessels and skin GAS Virulence Factors Environmental Factors Genetic Factors Streptococcus pyogenes Repeated GAS infections Repeated GAS infections destroys the heart tissue Susceptible child Skin sores Throat infection Acute Rheumatic Fever Rheumatic Heart Disease T & B cell responses against GAS & heart tissue Reversible damage Irreversible damage

5 M protein is a major virulence factor of GAS GAS virulence factors: Capsule, M protein, F protein, Erythrogenic exotoxin, Streptolysin O, Streptolysin S, Streptokinase, Peptidase, DNase M protein: inhibits phagocytosis, inhibits complement deposition, binding of host proteins, activates inflammation, adhesion, binding to immunoglobulins

6 Hypervariable Conserved GAS M protein as the heartbreaker A & B Highly variable C & D ~98% conserved between strains A B C D A, B & C Cross-reactive B & T cell epitopes could trigger autoimmune response Immune response against GAS generates crossrecognition of human tissue proteins -Antibodies cardiac myosin, vimentin, laminin, N- acetyl β-d-glucosamine -T cells cardiac myosin, valvular proteins (laminin, tropomyosin) Molecular mimicry and breaking of tolerance Heartbreaker by Debbie Taylor (2010 acrylic on canvas)

7 Immune cross-reactivity in RF/RHD Carapetis et al 2016 Nature Reviews Disease Primers Vol 2.1

8 Rat Autoimmune Valvulitis (RAV) Model Primary Immunisation Hock injection with 500µg GAS rm5 in CFA or PBS in CFA (Gorton et al 2010) Booster Immunisation Flank s.c. injection with 500µg GAS rm5 in IFA or PBS in IFA Day Pertussis Injection Euthanasia Blood - ELISA Heart - Immunohistology Spleen T cell proliferation assay (1) Histology (2) Antibody (3) T Cell Culture (4) ECG/ECHO Frontiers in Paediatrics Infection & Immunity (5): J Autoimmunity (3):

9 Repeat exposure to GAS M protein increases immune responses and heart damage Valve Tissue Myocardium Boost 1 Antibody responses to rm5 3 T cells Macrophages T cells Macrophages Antibody response to cardiac myosin T cell responses to rm5

10 Fractional shortening (%) Why are cardiac functional tests required? ECG ECHO MRI Reduction in LV fractional shortening ECHO 60 p< 0.01 p< PBS rm5 rm5 Cardiac (0.5mg) (1.0mg) Myosin Pre-treatment Post-treatment

11 Does this model have relevance to humans? Janelle Miles January 12, :00AM Quest for a RHD cure hits fever pitch Professor Good says the vaccine is based on a tiny molecule, known as J8, part of the M protein found on the surface of the germ. Before getting the approval for human trials of the Australian vaccine, the researchers had to show it did not cause heart disease in animals. They worked with Townsville-based James Cook University tested the product in rats. "It's the only model in the world where you can get rheumatic heart disease in an animal, other than humans," Good says. "We administered the vaccine in rats and it did not cause heart disease."

12 Volume 356, Issue 9236, 30 September 2000, Pages Cross-reactivity with Human Cardiac Myosin Cross-reactivity of antibodies to group C (16C, 64C, 79C) and group G (48G, 35G) streptococci with human cardiac myosin by ELISA. Antibodies to a streptococcal strain expressing M6 protein of group A streptococcus (M6) and five skin isolates (A153, A155, A175, A184, and A194) were used as controls. Values are means of triplicates.most studies linking ARF to group A streptococcus pharyngitis have come from more-developed countries and further epidemiological investigations are required in ARF-endemic regions where carriage of group C and group G is common. Our study indicates the rheumatogenic potential of these streptococcal isolates.

13 Do streptococci other than GAS cause RHD? Streptococcus dysgalactiae subspecies equisimilis (SDSE) have been known under β-hemolytic groups C and G streptococci GGS colonises the pharynx, skin, large intestines and GU tract Causes pharyngitis, respiratory tract infection, cellulitis, sepsis, meningitis and endocarditis Complications include arthritis and glomerulonephritis (NOT RHD?) Shares sequence homology with GAS M protein and cross-reactivity with cardiac myosin High carriage rates of GGS in RHD endemic regions of Australia and India GGS M Cardiac Myosin GGS M Cardiac Myosin GGS M Cardiac Myosin

14 Rat Autoimmune Valvulitis (RAV) Model Primary Immunisation Hock injection with 500µg GAS rm5 in CFA or PBS in CFA (Gorton et al 2010) Booster Immunisation Flank s.c. injection with 500µg GAS rm5 in IFA or PBS in IFA Day Pertussis Injection Euthanasia Blood - ELISA Heart - Immunohistology Spleen T cell proliferation assay (1) Histology (2) Antibody (3) T Cell Culture (4) ECG/ECHO Frontiers in Paediatrics Infection & Immunity (5): J Autoimmunity (3):

15 Histological changes in cardiac tissue Score Valve Myocardium 0 No inflammatory cells Diffuse, individual cells 1 <5 isolated cells in/on valvular tissue 1-2 small foci ( 50 cells) 2 >5 cells on valve surface only >2 small foci 3 1 focal lesion in valve Large focal lesion (>50 cells) 4 >1 lesion Aschoff-type lesion Score 0 Score 2 Score 4 Mitral valve Score 0 Score 4 Myocardium

16 Histological changes in cardiac tissue Whole killed GAS injected Whole killed GGS injected **** **** **** **** PBS (SC4; <70d) (SC5; >200d) (SC4; <70d) (SC5; >200d) GAS GGS GAS rm GGS rm **** **** (SC4; <70d) (SC4; <70d)

17 Antibodies from killed GAS & GGS injected rats Respond to whole killed GAS *** *** *** *** PBS (SC4; <70d) (SC5; >200d) (SC4: <70d) (SC5; >200d) GAS Respond to whole killed GGS GGS *** *** *** *** (SC4; <70d) (SC5; >200d) (SC4; <70d) (SC5; >200d)

18 Antibodies cross-react with cardiac myosin Whole killed GAS injected Whole killed GGS injected *** ** ** ** PBS (SC4; <70d) (SC5; >200d) (SC4; <70d) (SC5; >200d) GAS GGS GAS rm injected GGS rm injected ** * (SC4; <70d) (SC4; <70d)

19 Antibodies cross-react with collagen type 1 Whole killed GAS injected Whole killed GGS injected ** ns ns ns PBS (SC4; <70d) GAS rm injected (SC5; >200d) (SC4; <70d) GGS rm injected (SC5; >100d)) GAS GGS ** * (SC4; <70d) (SC4; <70d)

20 T cell responses to GAS rm GAS rm Killed GAS GGS rm ** *** ** Killed GGS * (SC4; <70d) (SC5; >200d) T cell responses to GGS rm (SC4; <70d) (SC5; >200d) GAS rm Killed GAS GGS rm Killed GGS ** ** ** ** PBS GAS GGS (SC4; <70d) (SC5; >200d) (SC4; <70d) (SC5; >200d)

21 Prolongation of P-R interval in hyper-immunised rats ECG PBS Immunised rm5 Immunised

22 Prolongation of P-R following GAS & GGS injection Whole killed GAS Whole killed GGS **** *** *** **** PBS (SC4; <70d) GAS rm (SC5; >200d) (SC4; <70d) GGS rm **** **** (SC5; >200d) GAS GGS (SC4; <70d) (SC4; <70d)

23 Echo changes following GAS & GGS injection Valvular thickening 0 - Normal 1 - Moderate 2 - Distinct Score 0 Score 2 Valvular nodules Score 0 Score nodules nodules 2 > 2 nodules Whole killed GAS GAS rm **** **** Whole killed GGS GGS rm **** **** (5SC; >200d) (5SC; >200d) (5SC; >200d) (5SC; >200d)

24 Antibodies from patients with ARF cross-reacts with both GAS & GGS Whole killed GAS Whole killed GGS ** ** Control serum Patient serum Control serum Patient serum

25 Conclusions GAS & GGS induce myocarditis and valvulitis in RAV model GAS & GGS activate antibodies and T cells that cross-react with each other Antibodies against GAS & GGS cross-react with Cardiac Myosin and Collagen GAS & GGS cause similar ECG and echocardiographic changes in RAV model Experimental evidence for the role of GGS in initiating and or potentiating RF/RHD? Clinical and epidemiological implications

26 Acknowledgements (Rheumatic Fever and Rheumatic Heart Disease Programme) James Cook University (IDIRG) & Townsville Hospital Current: Brenda Govan, Robert Norton, Cathy Rush, Suchandan Sikder, Tom O'Donohoe, Ryan Schrale, Donna Rudd, Scott Simpson Past: Davina Gorton, Anne Fane, Saffron Bryant, Lisa Chilton, Natasha Williams, Robyn Lymbury, Alan Sive Menzies School of Health Research Bart Currie Griffith University Michael Batzloff, Michael Good QIMR & USC Kadaba Sriprakash, David McMillan RHD Queensland Louise Axford-Haines & Co University of Auckland Nikki Moreland OUHSC Madeleine Cunningham $

27 Immune mechanisms leading to RHD GAS ACMA Initiation Infection with GAS Generation of anti- GAS antibodies and T cells Deposition of immune complexes Binding of crossreactive Ab???? Activation Initial tissue injury and release of heart muscle proteins Development of heart muscle reactive antibodies???? Amplification Inflammation leading to irreversible damage Lymphocyte infiltration Degeneration Functional impairment & RHD Valve stenosis Heart failure

28 Anti cardiac myosin antibodies as possible markers to detect RHD activity S1 S2 LMM Diagram of cardiac myosin fragment The Paediatric Infectious Disease Journal 30,11, 2011

29 Prolongation of P-R interval in hyper-immunised rats

30 The Canadian Medical Journal 1929 Jan; 20(1): Arnold Branch

31 Immune cross-reactivity in RF/RHD Carapetis et al 2016 Nature Reviews Disease Primers Vol 2.1

32 Immune cross-reactivity in heart tissue in RF

33 Immune cross-reactivity in joints tissue in RF

34 Immune cross-reactivity in Syndenham s Chorea

35 Immune cross-reactivity and Erythema marginatum

36 Cardiac Myosin M5-B.1 M5-B.2 M5-B.3 M5-B.4 M5-B.5 M5-B.6 M5-C.9 Stimulation Index T cells are involved in pathology Infiltration of T cells (CD3+) on day 21 following rm5 immunisation T cells from immunised rats crossreact with cardiac myosin M5-B.6 peptide-immunised rats Controls Stimulating antigen Amino Acid sequence alignments M5-B.4 IAKEQENKETIGTLKKILDE M5-B.5 IGTLKKILDETVKDKIAKEQ M5-B.6 TVKDKIAKEQENKETIGTLK M5-B. ----TVKDKIAKEQENKETIG---TKL-- 20 HCM KGTLEDQIIQANPALEAFGNAKTVRND 240 *::*:* : : *::* *:: rm5-immunised (* =identities, : =conserved substitutions)

37 ECG data Whole killed GAS exp. Whole killed GGS exp. **** *** *** **** PBS GAS GGS (4/70) (5/240) (4/70) (5/240) GAS rm5 exp. GGS rm exp. **** **** (4/35) (4/35) One way ANOVA with multiple comparison

38 >2 foci/nodules Scores 0 Echocardiography on rats Valvular thickness Scores 0 Nodules on the leaflets 1 No inflammatory thickening 1 No nodules 2 Moderately thick foci/nodule(s) Distinctly thick

39 Echocardiography on rats Whole killed GAS exp. GAS rm5 exp. Whole killed GGS exp. GGS rm exp. **** **** **** **** (5/240) (5/210) (5/240) (5/210) One way ANOVA with multiple comparison

40 Antibody response to whole killed GAS Whole killed GAS exp. Whole killed GGS exp. *** *** *** *** (4/70) (5/240) (4/70) (5/240) Antibody response to whole killed GGS *** *** *** *** (4/70) (5/240) (4/70) (5/240) Mann-Whitney t test

41 Antibody response to GAS rm5 Whole killed GAS exp. Whole killed GGS exp. **** *** **** ** (4/70) (5/240) (4/70) (5/240) GAS rm5 exp. GGS rm exp. *** ** (4/70) (4/70) Mann-Whitney t test

42 Antibody response to GGS rm Whole killed GAS exp. Whole killed GGS exp. **** *** **** *** (4/70) (5/240) (4/70) (5/240) GAS rm5 exp. ** GGS rm exp. ** (4/70) (4/70) Mann-Whitney t test

43 Antibody response to cardiac myosin Whole killed GAS exp. Whole killed GGS exp. *** ** ** ** (4/70) (5/240) (4/70) (5/240) GAS rm5 exp. GGS rm exp. ** * (4/70) (4/70) Mann-Whitney t test

44 Antibody response to collagen I Whole killed GAS exp. Whole killed GGS exp. ** ns ns ns (4/70) (5/240) (4/70) (5/240) GAS rm5 exp. GGS rm exp. ** * (4/70) (4/70) Mann-Whitney t test

45 Spleen T cell response to GAS rm5 at 96 hrs incubation GAS rm5 exp. Whole killed GAS exp. GGS rm exp. ** *** ** Whole killed GGS exp. * (4/35) (5/240) (4/35) (5/240) T cell response to GGS rm GAS rm5 exp. Whole killed GAS exp. ** ** GGS rm exp. ** Whole killed GGS exp. ** (4/35) (5/240) Mann-Whitney t test (4/35) (5/240)

46 Histological exam. of cardiac tissues by H&E staining Scores Valves Myocardium 0 No inflammatory cells assoc. with valves Diffuse, individual cells throughout tissue 1 <5 isolated cells in/on valves 1-2 small foci ( 50 cells) 2 >5 cells on valve surface only >2 small foci 3 Focal lesion in valve Large focal lesion (>50 cells) 4 >1 lesion Aschoff-type lesion Mitral valves 3 4

47 Myocardium, a: Aschoff type body, b: Aschoff type cell, c: Anitschkow type cells a b c 3 4

48 Carditis scores comparison Whole killed GAS exp. Whole killed GGS exp. **** **** **** **** (4/70) (5/240) (4/70) (5/240) GAS rm5 exp. GGS rm exp. **** **** (4/70) (4/70) One way ANOVA with multiple comparison

49 Human RHD sera antibody response to GAS antigen Human RHD sera antibody response to GGS antigen Whole killed GAS exp. GAS rm5 exp. Whole killed GGS exp. GGS rm exp. ** ** * * Mann-Whitney t test

50 Recombinant GAS/GGS protein alternative immunisation exp. (short term) PBS rm5 rggsm rggsm rm5 ECG at day 35 analysis ** **** T cell response to GAS rm5 and GGS rm ** ** * * (4/35) (4/35) One way ANOVA with multiple comparison

51 Recombinant GAS/GGS protein alternative immunisation exp. (short term) Antibody response to GAS rm5 and GGS rm Antibody response to myosin and collagen I ** ** * ** * ** ns ns Histology: Carditis scores comparison **** **** One way ANOVA with multiple comparison

52 S tim u la tio n in d e x S tim u la tio n in d e x S tim u la tio n in d e x S tim u la tio n in d e x Whole killed GAS/GGS exp. long term Spleen T cell response to cardiac myosin at 96 hrs incubation ns ns PBS GAS GGS (5/240) (5/240) 9 6 P Spleen T cell response to collagen I at 96 hrs incubation ns 9 6 A P ns 9 6 G (5/240) (5/240) 9 6 P 9 6 A P 9 6 G Mann-Whitney t test

53 GAS 1 MARENTNKHYSLRKLKKGTASVAVALSVLGAGLVV NT 37 MAR+NTNKHYSLRKLK GTASVAVAL+V+GAGLV ++ GGS 1 MARKNTNKHYSLRKLKTGTASVAVALTVVGAGLVAGQTVKANNSEDITSMMPILSGVGSS 60 GAS 38 NEVSAAVTRGTI----NDPQRAKEALD---KYELENHDLKTKNEGLKTENEGLKTENEGL 90 N V + T + N+P Y+ +D +N+ L+ + + GGS 61 NAVDSNFTADQLAKRMNNPKAIEKTIQVGREYKQGLYDQLVRNKKLEEDLRVKNLLADAA 120 GAS 91 KTENEGLKTEKKEHEAENDKLKQQRDTLSTQKETLEREVQNTQYNN--ETLKIKNGDLTK 148 E + E +E E L +++ LE +Q + ++ KIK + K GGS 121 SLRAESAERELQELEDKYKQVETEKTALKSRQAELESALQGAMNFSAEDSAKIKALEAEK 180 GAS 149 ELNKTRQELANKQQESKENEKALNELLEKTVK-DKIAKEQENKETIGTLKKILDETVKD- 206 K+RQ + N A + K ++ +K A + E L D GGS 181 TALKSRQAELESALQGAMNFSAEDSAKIKALEAEKTALKSRQAELESALQGAMNFSAEDS 240 GAS 207 -KIAKEQENKETIGTLKKILDETVKDKLAKEQKSKQNIGA LKQELAKKDEANK 258 KI + K L+ + A Q ++++ A L+ E K +E + GGS 241 AKIKALEAEKTALESRQAELEHQTQVLNANRQSLRRDLDASREAKKQLEAEYQKLEEEKQ 300 GAS 259 ISDASRKGLRRDLDASREAKKQLEAEHQKLEEQNKISEASRKGLRRDLDASREAKKQLE- 317 ISDASR+GLRRDL+ASREAKKQLEAEHQKLEEQNKISEASRKGLRRDL+ASR AKKQ+E GGS 301 ISDASRQGLRRDLNASREAKKQLEAEHQKLEEQNKISEASRKGLRRDLNASRAAKKQVEK 360 GAS AEHQKLEEQNKISEASRKGLRRDLDASREAKKQVEKALEEANSKLAALEKLNKE 371 AE K++E+ +IS+ASR+GLRRDLDASREAKKQVEKALEEANSKLAALEKLNKE GGS 361 DLANLTAELDKVKEEKQISDASRQGLRRDLDASREAKKQVEKALEEANSKLAALEKLNKE 420 GAS 372 LEESKKLTEKEKAELQAKLEAEAKALKEQLAKQAEELAKLRAGKASDSQTPDTKPGNKAV 431 LEESKKLTEKEKAELQAKLEAEAKALKE+LAKQAEELAKLRAGKASDSQTPD KPGNK V GGS 421 LEESKKLTEKEKAELQAKLEAEAKALKEKLAKQAEELAKLRAGKASDSQTPDAKPGNKVV 480 GAS 432 PGKGQAPQAGTKPNQNKAPMKETKRQLPSTGETANPFFTAAALTVMATAGVAAVVKRKEE 491 PGKGQAPQAGTKPNQNK PMKETKRQLPSTGE NPFFTAAAL VMATAGVAAV KRKEE GGS 481 PGKGQAPQAGTKPNQNKEPMKETKRQLPSTGEATNPFFTAAALAVMATAGVAAVAKRKEE 540 GAS 492 N 492 N GGS 541 N 541 GAS and GGS M protein sequence alignment Sequence alignment GAS M5 v GGS M (stg480.0) Myosin reactive CD4 T cell epitope (GAS) M5B.6 TVKDKIAKEQENKETIGTLK Recognised by heart infiltrating T cell clones (Human) Myocardium and aortic valve derived heart tissue proteins (RHD)

54 Diseases caused by GAS DISEASE CATEGORY DISEASE Superficial Diseases Deep Infections Toxin Mediated Immune Mediated pharyngitis, skin and soft tissue infection, pyoderma, erysipelas bacteremia, necrotising fasciitis, cellulitis, meningitis, puerperal sepsis, septic arthritis toxic shock, scarletina RF/RHD, PSGN, Reactive Arthritis Slide provided by Professor Kadaba Sriprakash

55 Absorbance Antibody reactivity to rm5 peptides Hypervariable Conserved PBS immunised rats (n=4) rm5 immunised rats (0.5 mg; n=4) A B Patients with ARF & confirmed carditis (n=6) C D 0 A B C

56 Cardiac Myosin M5-B.1 M5-B.2 M5-B.3 M5-B.4 M5-B.5 M5-B.6 M5-C.9 Stimulation Index T cells are involved in pathology Infiltration of T cells (CD3+) on day 21 following rm5 immunisation T cells from immunised rats crossreact with cardiac myosin M5-B.6 peptide-immunised rats Controls Stimulating antigen Amino Acid sequence alignments M5-B.4 IAKEQENKETIGTLKKILDE M5-B.5 IGTLKKILDETVKDKIAKEQ M5-B.6 TVKDKIAKEQENKETIGTLK M5-B. ----TVKDKIAKEQENKETIG---TKL-- 20 HCM KGTLEDQIIQANPALEAFGNAKTVRND 240 *::*:* : : *::* *:: rm5-immunised (* =identities, : =conserved substitutions)