Sıra | DOSYA ADI | Format | Bağlantı |
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01. | Difficile Toxins Difficile Infection | ppt | Sunumu İndir |
Transkript
Xingmin Sun, PhDAssistant ProfessorDepartment of Molecular MedicineMorsani College of MedicineUniversity of South Florida Novel chimeric vaccines against Clostridium difficile infection
Clostridium difficile Gram positive, toxin-producing, spore-forming anaerobic bacterium. One of the three urgent antibiotic resistance threats in US. Leading cause of nosocomial antibiotic-associated diarrhea (AAD) in developed countries. The most common organism to cause healthcare-associated infections in US, leading to 14,000 deaths per year. A continual rise in the incidence of severe C. difficile infection (CDI) has been observed worldwide. Emergence of more virulent strains like NAP1/BI/027: increased toxin production and sporulation; altered antibiotic resistance pattern; secretion of additional toxin.
Toxin A (TcdA) and Toxin B (TcdB): potent cytotoxic enzymes that damage the human colonic mucosa.Binary toxin (CDT): present ~6% of C. difficile isolates, but found in all hypervirulent strains.Major virulence factorsTMD RBDCPDGTD
Major colonization factors Two C. difficile flagellar proteins FliC and FliD are involved in the attachment of the organism to host cells and mucus layer. Serum antibody responses against both FliC and FliD are detected in patients with C. difficile infection. C. difficile Cwp84 is a cysteine protease, and plays a critical role in the maturation of surface-layer proteins. Immunization with Cwp84 provides significant protection in hamsters by delaying C. difficile colonization. Cwp84 is highly immunogenic and conserved.
Step 1- Ingestion of sporesStep 2- Germination into vegetative cellsStep 3 - Altered intestine flora allows proliferation of C. difficile in colonStep 4 . Toxin production leads to colon damage +/- pseudomembraneCDI: transmission through spores
Antibiotics: Standard treatment Vancomycin (only FDA-approved treatment) Metronidazole (most commonly used treatment) Fidaxomicin (lower recurrence rates)Recurrence is common ~20% after first CDI episode ~40% after first recurrence ~60% after 2 or more recurrencesCDI: Standard treatment and major challenges
Lee et al., Vaccine, 2010 Jul 19;28(32):5245-53The potential value of Clostridium difficile vaccine: An economic computer simulation model.No vaccine against CDI is currently licensed “C. difficile vaccination could be cost-effective over a wide range of C. difficile risk, especially when being used post-CDI treatment to prevent recurrent disease.”
GT RBD TMDCPD GT RBD TMDCPDW DXDNNCC27102366185118481 544543 769767TcdATcdBW102A D288N GT CPDN CmTcd138TcdB TcdA RBDNovel vaccine candidate targeting both toxins: mTcd138
• The RBD is the immunodominant region of TcdA and has potent adjuvant activity. • The neutralizing epitopes in TcdB are primarily confined to the N terminus since preincubation of polysera from the atoxic TcdB-immunized mice with recombinant TcdB-RBD did not significantly reduce the serum neutralizing activity. • The N terminus in TcdB is more highly conserved between historical and hypervirulent strains than its RBD. • Neutralizing antibodies to the TcdA-RBD were shown to be cross-reactive with TcdB-RBD. Construction of mTcd138: fusion of the N terminus of TcdB with the receptor binding domain (RBD) of TcdA
75 kDa100 kDa150 kDa250 kDamTcd138 mTcd138(A) (B) (C)Expression and purification of mTcd138 Analysis of purified 138 kDa fusion protein by SDS-PAGE (A), and Western blot analysis with anti-TcdA antibody (B) and anti-TcdB antibody (C)
(A) (B)mTcd138 is atoxic
mTcd138 immunization via intraperitoneal (i.p.), intramuscular (i.m.) or intradermal (i.d.) routes induces similar levels of antibody response
(A) (B)mTcd138 immunization induced potent neutralizing antibodies against both toxins
mTcd138 immunization protected mice against systemic toxin challenge
(A)(B)(C)mTcd138 immunization of mice provided full protection against infection with a hypervirulent C. difficile strain
Development of a vaccine which targets C. difficile colonization and reduces C. difficile excretion Two C. difficile flagellar proteins FliC and FliD are involved in the attachment of the organism to host cells and mucus layer.Serum antibody responses against both FliC and FliD are detected in patients with C. difficile infection.
FliCFliD immunization of mice provides significant protection against infection with a hypervirulent C. difficile strain
10 510 710 910 1 1D ay 1D ay 3D ay 5D ay 7C. difficile (CFU/g)******FliCFliD immunization significantly reduces C. difficile excretion from mice
Summary of vaccine development We have constructed a novel vaccine targeting both C. difficile toxins. Immunization with mTcd138 induced potent antibody and protective responses against both TcdA and TcdB. We have generated a novel vaccine (FliCFliD) which can significantly reduce C. difficile colonization and excretion.
AcknowledgementsDr. Sun LabYuanguo WangYing CaiXianghong JuPrevious members:Keshan ZhangSong Zhao Xianghong JuYuankai WangDr. Abraham L. Sonenshein(Tufts University) Laurent BouillautDr. Qiaobing Xu(Tufts University)Ming WangNIDDK (K01DK092352); NIAID (R21 AI113470) ; Tufts Collaborates! 2013-2014; Tufts Institute Innovation Award 2014-2015.