Author(s): Mario A Monteiro [*] 4 , Zuchao Ma 1 , Lisa Bertolo 1 , Yuening Jiao 1 , Luis Arroyo 1 , Douglas Hodgins 1 , Michael Mallozzi 2 , Gayatri Vedantam 2 , Martin Sagermann 3 , John Sundsmo 3 , Herbert Chow 3
antibiotic-associated diarrhea; Clostridium difficile; conjugate; polysaccharide; PSI; PSII; PSIII; vaccine
Clinical occurrence & carriers
Clostridium difficile is a Gram-positive, spore-forming and toxin-producing anaerobic gastrointestinal pathogen that is the major cause of antibiotic-associated colitis. C. difficile infection (CDI) with antibiotic-resistant hypervirulent strains now accounts for more than half of hospital and nursing home infections  with US$3.5 billion in patient costs  . Furthermore, CDI accounts for an estimated 8000-20,000 hospital deaths each year in the USA, mostly in elderly and infirm patients  .
Adult gastrointestinal infections in hospitals and nursing homes are ascribed to widespread environmental contamination at these sites combined with extensive use of a broad spectrum of antibiotics, immunosuppressive drugs and gastric acid proton-pump suppressants, which alter and/or eliminate commensal bacteria allowing colonization with resistant C. difficile . Recurrent CDI is treated with vancomycin and/or metronidazole, which tend to increase the risk for emergence of multidrug-resistant bacteria  . These treatments with metronidazole and vancomycin also carry an approximate 20% failure rate in the first treatment, 40% in the second and 60% in the third  , with time to recurrence being generally less than 4 weeks. The carrier rates in healthy adults is only 2-4%, but the rates among hospitalized patients have been reported to be 14-20% [4-6] and, during epidemics in hospitals, carrier rates >50% have been noted  .
C. difficile vaccine targets so far
C. difficile vaccines directed to toxins and surface proteins have been carefully reviewed very recently in this journal  . The vaccines in human clinical trials include TcdA (sponsored by Acambis/Sanofi, Paris, France) and recombinant hybrid TcdA/B (sponsored by Intercell/Novartis, Basel, Switzerland). TcdA, TcdB and binary CDT ( C. difficile ADP-ribosyltransferase) play predominant roles in symptomology, but vaccines targeting these exotoxins may inadvertently increase the number of healthy asymptomatic carriers. Siddiqui et al . reported that parenteral vaccination with TcdB protects hamsters against lethal challenge with TcdA- /TcdB+ strains, but as expected, did not prevent colonization  . Clinical trials of monoclonal-based antitoxin therapies are encouraging  (Medarex/Bristol-Myers Squibb, USA) and potentially life-saving, but raise the same community health issue. Targeting recurrent and residual disease will most likely require specific cellular (not secreted) antigens that are also not shared by commensal bacteria.
The C. difficile cell-wall polysaccharides PSI, PSII & PSIII
Fine structural investigations based on chemical and physical analyses have revealed that C. difficile vegetative cells can express three highly complex polysaccharides on their cell surface, named PSI, PSII and PSIII [11-14,101] . Most significantly, it has been established that PSII is a common C. difficile antigen abundantly expressed by all C. difficile ribotypes so far examined, including hypervirulent ribotype 027 [11-14] .
PSII is a polysaccharide composed of hexaglycosyl repeating blocks connected by a diester-phosphate (PO3 H)  . The variably linked monosaccharide constituents glucopyranose (Glc), mannopyranose (Man) and N -acetyl-galactopyranosamine (GalNAc)...