IMM-529 for the prevention and treatment of Clostridium difficile infections
Clostridium difficile http://www.nlm.nih.gov/medlineplus/images/clostridiumdifficile.jpg • Gram positive, spore forming anaerobe • Major nosocomial pathogen • Pathogenic C. difficile strains produce toxins that cause diarrhoea and mediate gut damage • Resistant to most clinical antibiotics
Clostridium difficile • Leading cause of infectious antibiotic-associated diarrhoea in hospitals worldwide • C . difficile only colonises the gut if the normal microbiota is disrupted • People at greatest risk include those on antibiotics, the elderly and immunocompromised patients • High rate of relapse, >25%
C. difficile infection (CDI) • Causesa spectrum of diseasescollectively knownas CDI: - Mild self-limiting diarrhoea - Pseudomembranouscolitis(PMC) • May progressto toxic megacolon, sepsis, death normal gut PMC toxic megacolon S. Kirov, U Tas
C. difficile infection (CDI) Centersfor Disease Control andPrevention(CDC):ANTIBIOTIC RESIST ANCETHREATS inthe UnitedStates, 2013 C . difficile is listed as the number one antibiotic resistance threat to the US healthcare system.
C. difficile spores • Spore-forming ability and spore persistence are major problems • Infectious particle • Survival mechanism • Heat, ethanol and UV resistant – allowsfor persistence in hospitals
The infectious cycle of C. difficile Disruption to the gut microbiota by antibiotics T oxin production by 1 Ingestion of spores 4 vegetative cells Germination of spores 2 • T oxin A in the colon • T oxin B Toxin-mediated damage 5 Colonisation by 3 vegetative cells in the colon
C. difficile toxins • T oxin A (308 kDa) & T oxin B (269.6 kDa) • Monoglucosyltransferasesencoded on PaLoc (19.6 kb) tcdC tcdE tcdA tcdR tcdB PaLoc 1 kb T cdR = alternativesigmafactor T cdE = putative holin-like protein T cdC = anti-sigma factor (negativeregulator of toxin production) • T oxin B shown to be essential for disease (Lyras et al ., 2009) • Some strains also produce Binary toxin – may be involved in colonisationand adherence
Pathogenesis of C. difficile toxins InactivationofRho GTPases Depolymerisationofactin filaments Cytoskeleton disruption Cellroundingand death Jank et al ., 2007
The hypervirulent strains • Emergence of hypervirulent strains associated with an increase in disease incidence, severity and mortality • Epidemicstrains (ribotype 027) have been isolated worldwide • Higher relapse rates • Increased virulence may be due to: – A deletion in tcdC – loss of negative regulation of toxin production – More toxin produced = more bowel damage – Resistance to fluoroquinolones – Presenceof binary toxin
C. difficile in hospitals • 3% of healthy people and 20-40% hospitalised patients colonised with C . difficile • associated with short-stay hospitals • poor hospital practices • antibiotic stewardship • ability to form spores – major problem C.difficile spore image:JoanneWee – resistantto many cleaning agents (notsporocidal) – persistin environmentfor6 months+
Current treatment of CDI 1) Mild disease - Discontinue use of antibiotics 2) Moderate/severe disease - Treat with metronidazole, vancomycin or fidaxomicin 3) A range of non-antibiotic treatments have also been tested: • Intravenous IgG antibodies • Monoclonal antibodies • Probiotics • Non-toxigenic strains of C. difficile http://choices.studentlife.wfu.edu/files/2012/08/prescription-drug-addictions.jpg • Faecal transplant therapy
Faecal transplant therapy • Prepared by blending and filtering a fresh donor stool • Is administered via the upper or lower gastrointestinaltract by nasogastric/duodenaltube, colonoscopyor enema. • Restore the diversity of the gut microbiota and reverse the dysbiosisof CDI • Concernsover the long-term unknown risks of the therapy: • transmissionof unrecognised infectious agents • potential associationof the gut microbiota with conditions such as irritable bowel syndrome, metabolic syndromes, obesity and chronic fatigue • Donorscreening protocols have not been established
New methods for the prevention and/or treatment of CDI are urgently required
IMM-529 Production • IMM-529 is a natural product which is intended to prevent and treat C. difficile infections • IMM-529 contains high concentrations of specific antibodies that are predominantly IgG (86%), IgA (7%) and IgM (7%) • IMM-529 does not destroy the gut microbiota like antibiotictreatment
Advantages of IMM-529 • Inexpensive • Antibodiessurvive transit through the stomachand remain functional in the large intestine (site of C. difficile infection and toxin production) • Technology platform already used for the prevention and treatment of other gastrointestinaldiseases ( Cryptosporidium ,Rotavirus,Enterotoxigenic Escherichiacoli )
Evaluation of IMM-529 for passive immunotherapy in the prevention and treatment of C. difficile infections
Different markets for IMM-529 • Preventionof initial disease • Treatment • Preventionof disease relapse
Vaccine targets Disruptionto the gut microbiotaby antibiotics Ingestionof spores T oxinproduction • T oxinA Germinationofspores • T oxinB Infection Colonisationby vegetativecells
IMM-529 Products • IMM-529B contains high levels of specific antibodies which have been generated against recombinant Toxin B • IMM-529S contains high levels of specific antibodies which target the very infectious C. difficle spores • IMM-529V contains high levels of specific antibodies which target cell surface antigens present on vegetative cells • These are produced by vaccinating pregnant cows with specific antigens and harvesting colostrum upon calving.
In vitro characterisation of C. difficile IMM-529 antibodies (spore, vegetative cell and toxin B)
IMM-529S1 antibodiesare cross-reactive with the exosporiumlayer from C. difficile spores exosporium 1 2 3 4 5 6 7 8 250 kDa Non-immune 1-KI (A + B + ) 250 kDa 2-M7404 (A + B + ) 3-VPI10463 (A + B + ) IMM-529S1 #1 4-GE (A + B + ) 5-MDU2992 (A - B + ) 6-JGS6133 (A + B + ) 250 kDa 7-AI35 (A - B + ) 8-1470 (A - B + ) IMM-529S1 #2 IMM-529S1 contains antibodies that are cross-reactive with the exosporium layer of spores from a variety of isolates (human and animal) Strain used to generate product
IMM-529V1 antibodiesare cross-reactive with cell lysates from C. difficile vegetative cells Animal Human 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 75 kDa 50 kDa 37 kDa 25 kDa IMM-529V1 contains antibodies that are cross-reactive with vegetative cell whole cell lysates from a variety of human and animal C. difficile isolates Strain used to generate product
IMM-529B1 antibodiesare cross-reactive with Toxin B from different C. difficile strains 1 2 3 4 5 6 7 8 9 10 11 1-KI (A + B + ) 2-M7404 (A + B + ) 250 kDa 3-VPI10463 (A + B + ) 4-GE (A + B + ) Non-immune 5-MDU2992 (A - B + ) 6-JGS6133 (A + B + ) 7-AI35 (A - B + ) 250 kDa 8-1470 (A - B + ) IMM-529B1 #1 9-CD37 (A - B - ) 10-Purified Toxin B (commercial) 250 kDa IMM-529B1 #2 IMM-529B1 contains antibodies specific to Toxin B from a variety of human and animal isolates
IMM-529B1 antibodies neutralise T oxin B from historical and hypervirulent strains C o m m e rcia l p u rifie d T o xin B H isto rica l T o xin B H y p e rv iru le n t T o xin B (stra in V P I1 0 4 6 3 ) (strain K I) (stra in 6 3 0 ) *** **** **** *** **** **** 1 0 0 1 0 0 1 0 0 7 5 7 5 % c e ll d e a th % c e ll d e a th 7 5 % c e ll d e a th 5 0 5 0 5 0 2 5 2 5 2 5 0 0 0 N o N o n -im m u n e IM M -5 2 9 B 1 N o N o n -im m u n e IM M -5 2 9 B 1 N o N o n -im m u n e IM M -5 2 9 B 1 A n tib o d y A n tib o d y A n tib o d y
IMM-529B1 antibodies neutralise T oxin B from a historical strain T oxin neutralisationassays 50 µm 50 µm 50 µm Vero cells only Vero + Toxin B-Hist Vero + Toxin B-Hist + anti-Toxin B antibody 50 µm 50 µm Vero + Toxin B-Hist + Non-immune Vero + Toxin B-Hist + IMM-529B1 • Antibodies in IMM-529B1 neutralise historicalT oxinB from the historical (Hist) strain 630
IMM-529B antibodies neutralise T oxin B from a hypervirulent strain T oxin neutralisationassays 50 µm 50 µm 50 µm Vero cells only Vero + Toxin B-HV Vero + Toxin B-HV + Toxin B antibody 50 µm 50 µm Vero + Toxin B-HV + IMM-529B1 Vero + Toxin B-HV + Non-immune • Antibodies in IMM-529B1 cross-neutraliseT oxin B from a hypervirulent(HV) strain
In vivo characterisation of IMM-529
The C. difficile mouse model C57BL/6 mice 6 – 7 weeks IMM-529 administration Day -10 -3 -2 -1 0 C. difficile challenge Antibiotics in drinking water to (10 3 spores) induce susceptibility to C. difficile Monitor: • Weight loss • Physiological appearance • Activity • Diarrhoea
IMM-529S1 protects 40% of mice from C. difficile disease % survival % weight loss *** x Non-immunecontrol IgG N = 10 mice/group *** p = 0.0005 IMM-529S1 SEM x = mice culled
IMM-529B1 protects 80% of mice from C. difficile disease % W e igh t (re lative to d a y 0 ) % survival % weight loss 11 0 *** 10 0 90 80 70 0 12 2 4 3 6 4 8 6 0 7 2 84 9 6 T im e (h o u rs ) *** p < 0.0001 Uninfected (N=11) SEM No IgG(N=10) Non-immune IgG(N=20) IMM-529B1 (N=20)
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