Major pathogenic Clostridia in human and progress toward the clostridial vaccines

Document Type : Review Article


Department of Anaerobic Vaccine Research and Production, Specialized Clostridia Research Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran


The Clostridium genus is composed of a large spectrum of heterogeneous bacteria. They are Gram-positive, mostly mesophilic, and anaerobic spore-forming strains. Clostridia are widely distributed in oxygen-free habitats. They are found principally in the soil and intestines of ruminants as normal flora, but also are the cause of several infections in humans. The infections produced by important species in humans include botulism, tetanus, pseudomembranous colitis, antibiotics-associated diarrhea, and gas gangrene. Immunization with toxoid or bacterin-toxoid or genetically modified or other vaccines is a protective way against clostridial infection. Several experimental or commercial vaccines have been developed worldwide. Although conventional vaccines including toxoid vaccines are very important, the new generation of vaccines is an effective alternative to conventional vaccines. Recent advances have made it possible for new vaccines to increase immunogenicity. This review discusses briefly the important species of clostridia in humans, their toxins structure, and vaccine development and usage throughout the world.


1. Abdolmohammadi Khiav L, Zahmatkesh A. Vaccination against pathogenic clostridia in animals: A review. Trop Anim Health  Prod 2021; 53:1-12.
2. Abdolmohammadi Khiav L, Emadi A, Zahmatkesh A. A simple method for purification of epsilon toxin of Clostridium perfringens type D for serum neutralization assay. J Microbiol Meth 2022; 193:106395.
3. Guo P, Zhang K, Ma X, He P. Clostridium species as probiotics: Potentials and challenges. J Anim Sci Biotechnol 2020; 11:1-10.
4. Wilder-Kofie TD, Lúquez C, Adler M, Dykes JK, Coleman JD, Maslanka SE. An alternative in vivo method to refine the mouse bioassay for botulinum toxin detection. Comp Med 2011; 61:235-242.
5. Cruz-Morales P, Orellana CA, Moutafis G, Moonen G, Rincon G, Nielsen LK, et al. Revisiting the evolution and taxonomy of Clostridia, a phylogenomic update. Genome Biol Evol 2019; 11:2035-2044.
6. Lindstrom M, Korkeala H. Laboratory diagnostics of botulism. Clin Microbiol Rev 2006; 19:298-314.
7. Carter AT, Peck MW. Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II. Res Microbiol 2015; 166:303-317.
8. Zhang S, Masuyer G, Zhang J, Shen Y, Lundin D, Henriksson L, et al. Identification and characterization of a novel botulinum neurotoxin. Nat Commun 2017; 8:1-10.
9. Pirazzini M, Rossetto O, Eleopra R, Montecucco C. Botulinum neurotoxins: Biology, pharmacology, and toxicology. Pharmacol Rev 2017; 69:200-235.
10. Tighe AP, Schiavo G. Botulinum neurotoxins: Mechanism of action. Toxicon 2013; 67:87-93.
11. Dezfulian M, Dowell Jr V. Cultural and physiological characteristics and antimicrobial susceptibility of Clostridium botulinum isolates from foodborne and infant botulism cases. J Clin Microbiol 1980; 11:604-609.
12. Sundeen G, Barbieri JT. Vaccines against botulism. Toxins 2017; 9:268.
13. Peck MW. Biology and genomic analysis of Clostridium botulinum. Adv Microb Physiol 2009; 55:183-320.
14.Arnon SS, Schechter R, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, et al. Botulinum toxin as a biological weapon: Medical and public health management. JAMA 2001; 285:1059-1070.
15. Nejadrahim R, Delirrad M. Foodborne Botulism: A study of 57 cases in Northwest Iran. Iran J Toxicol 2016; 10:45-50.
16. Jeffery IA, Karim S. Botulism: Treasure Island (FL): StatPearls Publishing; 2017.
17. Brook I. Current concepts in the management of Clostridium tetani infection. Expert Rev Anti Infect Ther 2008; 6:327-336.
18. Ergonul O, Egeli D, Kahyaoglu B, Bahar M, Etienne M, Bleck T. An unexpected tetanus case. Lancet Infect Dis 2016; 16:746-752.
19. Roper MH, Wassilak SG, Tiwari T, Orenstein WA. In: Plotkin S, Orenstein W, Offit P, editors. Vaccines. 6th ed: Elsevier-Saunders; 2013. p. 746-772.
20. Popoff MR. Tetanus in animals. J Vet Diagn Invest 2020; 32:184-191.
21. Pearce J. Notes on tetanus (lockjaw). J Neurol Neurosurg  Psychiatry 1996; 60:332.
22. Khan AA, Zahidie A, Rabbani F. Interventions to reduce neonatal mortality from neonatal tetanus in low and middle income countries-a systematic review. BMC Public Health 2013; 13:1-7.
23. Brüggemann H, Brzuszkiewicz E, Chapeton-Montes D, Plourde L, Speck D, Popoff MR. Genomics of Clostridium tetani. Res Microbiol 2015; 166:326-331.
24. Herreros J, Lalli G, Schiavo G. C-terminal half of tetanus toxin fragment C is sufficient for neuronal binding and interaction with a putative protein receptor. Biochem J 2000; 347:199-204.
25. Fitzsimmons SP, Clark KC, Wilkerson R, Shapiro MA. Inhibition of tetanus toxin fragment C binding to ganglioside GT1b by monoclonal antibodies recognizing different epitopes. Vaccine 2000; 19:114-121.
26. Berkowitz AL. Tetanus, botulism, and diphtheria. Continuum 2018; 24:1459-1488.
27. Enany S. Structural and functional analysis of hypothetical and conserved proteins of Clostridium tetani. J Infect Public Health 2014; 7:296-307.
28. Brüggemann H, Bäumer S, Fricke WF, Wiezer A, Liesegang H, Decker I, et al. The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad Sci U S A 2003; 100:1316-1321.
29. Radjou A, Hanifah M, Govindaraj V. Tetanus following dog bite. Indian J Community Med 2012; 37:200-201.
30. World Health Organization (2017). Tetanus vaccines: Wkly Epidemiol Rec 92 :53-76. 
31. Moghaddam HM, Mohammad Zadeh A, Bagheri S, Moosafarkhani M. Neonatal Tetanus in Mashhad (North East of Iran) over a 17 Year period. Iranian J Neonatol 2014; 5:31-33.
32. Springer DA, Phillippi-Falkenstein K, Smith G. Retrospective analysis of wound characteristics and tetanus development in captive macaques. J Zoo Wildl Med 2009; 40:95-102.
33. Popoff M. Ecology of neurotoxigenic strains of clostridia. Curr Top Microbiol Immunol 1995;195:1-29. .
34. Ribeiro MG, Nardi Júnior Gd, Megid J, Franco MM, Guerra ST, Portilho FV, et al. Tetanus in horses: An overview of 70 cases. Pesquisa Veterinária Brasileira 2018; 38:285-293.
35. Beheshti S, Khajehdehi A, Rezaian GR, Khajehdehi P. Current status of tetanus in Iran. Arch Iran Med 2002; 5:216-218.
36. Modaber I. Clostridium difficile. Acta Med Iran 1975; 18:111-128.
37. Chandrasekaran R, Lacy DB. The role of toxins in Clostridium difficile infection. FEMS Microbiol Rev 2017; 41:723-750.
38.Carter GP, Lyras D, Allen DL, Mackin KE, Howarth PM, O’connor JR, et al. Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator. J Bacteriol 2007; 189:7290-7301.
39. Awad MM, Johanesen PA, Carter GP, Rose E, Lyras D. Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen. Gut Microbes 2014; 5:579-593.
40. Tijerina-Rodríguez L, Villarreal-Treviño L, Morfin-Otero R, Camacho-Ortíz A, Garza-González E. Virulence factors of Clostridioides (Clostridium) difficile linked to recurrent infections. Can J Infect Dis Med Microbiol 2019; 2019: 7127850.
41. Montgomery VA, Makuch RS, Brown JE, Hack DC. The immunogenicity in humans of a botulinum type F vaccine. Vaccine 1999; 18:728-735.
42. Edelman R, Wasserman SS, Bodison SA, Perry JG, O’Donnoghue M, DeTolla Jr LJ. Phase II safety and immunogenicity study of type F botulinum toxoid in adult volunteers. Vaccine 2003; 21:4335-4347.
43. Torii Y, Tokumaru Y, Kawaguchi S, Izumi N, Maruyama S, Mukamoto M, et al. Production and immunogenic efficacy of botulinum tetravalent (A, B, E, F) toxoid. Vaccine 2002; 20:2556-2561.
44. Rusnak JM, Smith LA. Botulinum neurotoxin vaccines: Past history and recent developments. Hum Vaccin 2009; 5:794-805.
45. Krüger M, Skau M, Shehata AA, Schrödl W. Efficacy of Clostridium botulinum types C and D toxoid vaccination in Danish cows. Anaerobe 2013; 23:97-101.
46. Anniballi F, Fiore A, Löfström C, Skarin H, Auricchio B, Woudstra C, et al. Management of animal botulism outbreaks: From clinical suspicion to practical countermeasures to prevent or minimize outbreaks. Biosecur Bioterror 2013; 11:S191-S199.
47. Kobayashi R, Kohda T, Kataoka K, Ihara H, Kozaki S, Pascual DW, et al. A novel neurotoxoid vaccine prevents mucosal botulism. J Immunol 2005; 174:2190-2195.
48. Smith J. Diphtheria and tetanus toxoids. Br Med Bull 1969; 25:177-182.
49. Brgles M, Prebeg P, Kurtović T, Ranić J, Marchetti-Deschmann M, Allmaier G, et al. Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology. Prep Biochem Biotechnol 2016; 46:695-703.
50. Hughes M, Thomson R, Knight P, Stephen J. The immunopurification of tetanus toxoid. J Appl Bacteriol 1974; 37:603-621.
51. Muni C, Mani KR, Subashkumar R. Large scale recovery of tetanus toxin and toxoid from fermentation broth by microporous tangential flow filtration. Int J Biotechnol Mol Biol Res 2013; 4:28-37.
52. Stojićević I, Dimitrijević L, Dovezenski N, Živković I, Petrušić V, Marinković E, et al. Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation. J Chromatograph B 2011; 879:2213-2219.
53. Lyerly DM, Saum KE, Macdonald DK, Wilkins TD. Effects of Clostridium difficile toxins given intragastrically to animals. Infect Immun 1985; 47:349-352.
54. Siddiqui F, O’Connor JR, Nagaro K, Cheknis A, Sambol SP, Vedantam G, et al. Vaccination with parenteral toxoid B protects hamsters against lethal challenge with toxin A–negative, toxin B–positive Clostridium difficile but does not prevent colonization. J Infect Dis 2012; 205:128-133.
55. Kim P-H, Iaconis JP, Rolfe RD. Immunization of adult hamsters against Clostridium difficile-associated ileocecitis and transfer of protection to infant hamsters. Infect Immun 1987; 55:2984-2992.
56. Kotloff KL, Wasserman SS, Losonsky GA, Thomas Jr W, Nichols R, Edelman R, et al. Safety and immunogenicity of increasing doses of a Clostridium difficile toxoid vaccine administered to healthy adults. Infect Immun 2001; 69:988-995.
57. Ghose C, Kalsy A, Sheikh A, Rollenhagen J, John M, Young J, et al. Transcutaneous immunization with Clostridium difficile toxoid A induces systemic and mucosal immune responses and toxin A-neutralizing antibodies in mice. Infect Immun 2007; 75:2826-2832.
58. Anosova NG, Brown AM, Li L, Liu N, Cole LE, Zhang J, et al. Systemic antibody responses induced by a two-component Clostridium difficile toxoid vaccine protect against C. difficile-associated disease in hamsters. J Med Microbiol 2013; 62:1394-1404.
59. Greenberg RN, Marbury TC, Foglia G, Warny M. Phase I dose finding studies of an adjuvanted Clostridium difficile toxoid vaccine. Vaccine 2012; 30:2245-2249.
60. Foglia G, Shah S, Luxemburger C, Pietrobon PJF. Clostridium difficile: development of a novel candidate vaccine. Vaccine 2012; 30:4307-4309.
61. Donald RG, Flint M, Kalyan N, Johnson E, Witko SE, Kotash C, et al. A novel approach to generate a recombinant toxoid vaccine against Clostridium difficile. Microbiology 2013; 159:1254.
62. Yari K, Fatemi SS-A, Tavallaei M. High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli. Bioprocess Biosyst Eng 2012; 35:407-414.
63. Smith LA, Jensen MJ, Montgomery VA, Brown DR, Ahmed SA, Smith TJ. Roads from vaccines to therapies. Mov Disord 2004; 19:S48-S52.
64. Sayadmanesh A, Ebrahimi F, Hajizade A, Rostamian M, Keshavarz H. Expression and purification of neurotoxin-associated protein HA-33/A from Clostridium botulinum and evaluation of its antigenicity. Iran Biomed J 2013; 17:165.
65. Doosti A. Cloning of the gene encoding neurotoxin heavy chain of Clostridium botulinum in E. coli. J Microb World 2013; 5:77-84.
66. Mousavi Gargari SL, Rasooli I, Valipour E, Basiri M, Nazarian S, Amani J, et al. Immunogenic and protective potentials of recombinant receptor binding domain and a C-terminal fragment of Clostridium botulinum neurotoxin type E. Iran J Biotechnol 2011; 9:181-187.
67. Baghban R, Gargari SLM, Rajabibazl M, Nazarian S, Bakherad H. Camelid‐derived heavy‐chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris. Biotechnol Appl Biochem 2016; 63:200-205.
68. Webb RP, Smith TJ, Wright PM, Montgomery VA, Meagher MM, Smith LA. Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins. Vaccine 2007; 25:4273-4282.
69. Hamidi B, Ebrahimi F, Hajizadeh A, Hajizadeh A, Keshavarz Alikhani H. Fusion and cloning of the binding domains of botulinum neurotoxin type A and B in E. coli DH5α. Eur J Exp Biol 2012; 2:1154-1160.
70. Ebrahimi F, Rasaee MJ, Mousavi SL, Babaeipour V. Production and characterization of a recombinant chimeric antigen consisting botulinum neurotoxin serotypes A, B and E binding subdomains. J Toxicol Sci 2010; 35:9-19.
71. Arimitsu H, Lee J-C, Sakaguchi Y, Hayakawa Y, Hayashi M, Nakaura M, et al. Vaccination with recombinant whole heavy chain fragments of Clostridium botulinum Type C and D neurotoxins.  Clin Diagn Lab Immunol. 2004;11:496-502.
72. Gil LA, da Cunha CEP, Moreira GM, Salvarani FM, Assis RA, Lobato FCF, et al. Production and evaluation of a recombinant chimeric vaccine against Clostridium botulinum neurotoxin types C and D. PloS one 2013; 8:e69692.
73. Moosavi SJ, Rashidiani J, Farasat A, Ebrahimi F. Recombinant expression of light chain of botulinum neurotoxin type-a in E. coli and evaluation of its enzymatic activity. Mol Biol Res Commun 2014; 3:58.
74. Kiyatkin N, Maksymowych AB, Simpson LL. Induction of an immune response by oral administration of recombinant botulinum toxin. Infect Immun 1997; 65:4586-4591.
75. Webb RP, Smith TJ, Wright P, Brown J, Smith LA. Production of catalytically inactive BoNT/A1 holoprotein and comparison with BoNT/A1 subunit vaccines against toxin subtypes A1, A2, and A3. Vaccine 2009; 27:4490-4497.
76. Przedpelski A, Tepp WH, Kroken AR, Fu Z, Kim J-JP, Johnson EA, et al. Enhancing the protective immune response against botulism. Infect Immun 2013; 81:2638-2644.
77. Shone C, Agostini H, Clancy J, Gu M, Yang H-H, Chu Y, et al. Bivalent recombinant vaccine for botulinum neurotoxin types A and B based on a polypeptide comprising their effector and translocation domains that is protective against the predominant A and B subtypes. Infect Immun 2009; 77:2795-2801.
78. Agarwal R, Binz T, Swaminathan S. Analysis of active site residues of botulinum neurotoxin E by mutational, functional, and structural studies: Glu335Gln is an apoenzyme. Biochemistry 2005; 44:8291-8302.
79. Bagheripour M, Ebrahimi F, Hajizadeh A, Nazarian S, Arefpour M. Preparation of chitosan based botulinum neurotoxin e recombinant nanovaccine and evaluation of its immunogenicity as oral & intradermal route in mice. J Rafsanjan Univ Med Sci 2016; 14:923-938.
80. Eisel U, Jarausch W, Goretzki K, Henschen A, Engels J, Weller U, et al. Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins. The EMBO J 1986; 5:2495-2502.
81. Villarreal-Ramos B, Manser JM, Collins RA, Dougan G, Howard CJ. Cattle immune responses to tetanus toxoid elicited by recombinant S. typhimurium vaccines or tetanus toxoid in alum or Freund’s adjuvant. Vaccine 2000; 18:1515-1521.
82. Yu R, Hou L, Yu C, Liu S, Ren J, Fang T, et al. Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate. Immunobiology 2011; 216:485-490.
83. Yu R, Fang T, Liu S, Song X, Yu C, Li J, et al. Comparative immunogenicity of the tetanus toxoid and recombinant tetanus vaccines in mice, rats, and cynomolgus monkeys. Toxins 2016; 8:194.
84. Tierney R, Nakai T, Parkins CJ, Caposio P, Fairweather NF, Sesardic D, et al. A single-dose cytomegalovirus-based vaccine encoding tetanus toxin fragment C induces sustained levels of protective tetanus toxin antibodies in mice. Vaccine 2012; 30:3047-3052.
85. Jank T, Giesemann T, Aktories K. Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function. Glycobiology 2007; 17:15R-22R.
86. Lyerly DM, Johnson JL, Frey SM, Wilkins TD. Vaccination against lethal Clostridium difficile enterocolitis with a nontoxic recombinant peptide of toxin A. Curr Microbiol 1990; 21:29-32.
87. Ryan ET, Butterton JR, Smith RN, Carroll PA, Crean TI, Calderwood SB. Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain. Infect Immun 1997; 65:2941-2949.
88. Ward SJ, Douce G, Figueiredo D, Dougan G, Wren BW. Immunogenicity of a Salmonella typhimurium aroA aroD vaccine expressing a nontoxic domain of Clostridium difficile toxin A. Infect Immun 1999; 67:2145-2152.
89. Ward SJ, Douce G, Dougan G, Wren BW. Local and systemic neutralizing antibody responses induced by intranasal immunization with the nontoxic binding domain of toxin A from Clostridium difficile. Infect Immun 1999; 67:5124-5132.
90. Permpoonpattana P, Hong HA, Phetcharaburanin J, Huang J-M, Cook J, Fairweather NF, et al. Immunization with Bacillus spores expressing toxin A peptide repeats protects against infection with Clostridium difficile strains producing toxins A and B. Infect Immun 2011; 79:2295-2302.
91. Leuzzi R, Spencer J, Buckley A, Brettoni C, Martinelli M, Tulli L, et al. Protective efficacy induced by recombinant Clostridium difficile toxin fragments. Infect Immun 2013; 81:2851-2860.
92. Castagliuolo I, Sardina M, Brun P, DeRos C, Mastrotto C, Lovato L, et al. Clostridium difficile toxin A carboxyl-terminus peptide lacking ADP-ribosyltransferase activity acts as a mucosal adjuvant. Infect Immun 2004; 72:2827-2836.
93. Gardiner DF, Rosenberg T, Zaharatos J, Franco D, Ho DD. A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A. Vaccine 2009; 27:3598-3604.
94. Seregin SS, Aldhamen YA, Rastall DP, Godbehere S, Amalfitano A. Adenovirus-based vaccination against Clostridium difficile toxin A allows for rapid humoral immunity and complete protection from toxin A lethal challenge in mice. Vaccine 2012; 30:1492-1501.
95. Tian J-H, Fuhrmann SR, Kluepfel-Stahl S, Carman RJ, Ellingsworth L, Flyer DC. A novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models. Vaccine 2012; 30:4249-4258.
96. Ghose C, Verhagen JM, Chen X, Yu J, Huang Y, Chenesseau O, et al. Toll-like receptor 5-dependent immunogenicity and protective efficacy of a recombinant fusion protein vaccine containing the nontoxic domains of Clostridium difficile toxins A and B and Salmonella enterica serovar typhimurium flagellin in a mouse model of Clostridium difficile disease. Infect Immun 2013; 81:2190-2196.
97. Yoshino Y, Kitazawa T, Ikeda M, Tatsuno K, Yanagimoto S, Okugawa S, et al. Clostridium difficile flagellin stimulates toll-like receptor 5, and toxin B promotes flagellin-induced chemokine production via TLR5. Life Sci 2013; 92:211-217.
98. Clayton J, Middlebrook JL. Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A. Vaccine 2000; 18:1855-1862.
99. Shyu R-H, Shaio M-F, Tang S-S, Shyu H-F, Lee C-F, Tsai M-H, et al. DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice. J Biomed Sci 2000; 7:51-57.
100. Trollet C, Pereira Y, Burgain A, Litzler E, Mezrahi M, Seguin J, et al. Generation of high-titer neutralizing antibodies against botulinum toxins A, B, and E by DNA electrotransfer. Infect Immun 2009; 77:2221-2229.
101. Jathoul AP, Holley JL, Garmory HS. Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters. Vaccine 2004; 22:3942-3946.
102. Scott VL, Villarreal DO, Hutnick NA, Walters JN, Ragwan E, Bdeir K, et al. DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge. Hum Vaccin Immunother 2015; 11:1961-1971.
103. Chen S, Xu Q, Zeng M. Oral vaccination with an adenovirus-vectored vaccine protects against botulism. Vaccine 2013; 31:1009-1011.
104. Li J, Diaz-Arévalo D, Chen Y, Zeng M. Intranasal vaccination with an engineered influenza virus expressing the receptor binding subdomain of botulinum neurotoxin provides protective immunity against botulism and influenza. Front Immunol 2015; 6:170.
105. Hudacek AW, Al-Saleem FH, Willet M, Eisemann T, Mattis JA, Simpson LL, et al. Recombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo. Mol Ther Methods Clin Dev 2014; 1:14046.
106. Yu Y, Liu S, Ma Y, Gong Z-W, Wang S, Sun Z-W. Pentavalent replicon vaccines against botulinum neurotoxins and tetanus toxin using DNA-based Semliki Forest virus replicon vectors. Hum Vaccin Immunother 2014; 10:1874-1879.
107. Lee JS, Pushko P, Parker MD, Dertzbaugh MT, Smith LA, Smith JF. Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system. Infect Immun 2001; 69:5709-5715.
108. Wright A, Drudy D, Kyne L, Brown K, Fairweather NF. Immunoreactive cell wall proteins of Clostridium difficile identified by human sera. J Med Microbiol 2008; 57:750-756.
109. Ní Eidhin DB, O’Brien JB, McCabe MS, Athié-Morales V, Kelleher DP. Active immunization of hamsters against Clostridium difficile infection using surface-layer protein. FEMS Immunol  Med Microbiol 2008; 52:207-218.
110. Péchiné S, Denève C, Le Monnier A, Hoys S, Janoir C, Collignon A. Immunization of hamsters against Clostridium difficile infection using the Cwp84 protease as an antigen. FEMS Immunol Med Microbiol 2011; 63:73-81.
111. Sandolo C, Péchiné S, Le Monnier A, Hoys S, Janoir C, Coviello T, et al. Encapsulation of Cwp84 into pectin beads for oral vaccination against Clostridium difficile. Eur J Pharm  Biopharm 2011; 79:566-573.
112. Adamo R, Romano MR, Berti F, Leuzzi R, Tontini M, Danieli E, et al. Phosphorylation of the synthetic hexasaccharide repeating unit is essential for the induction of antibodies to Clostridium difficile PSII cell wall polysaccharide. ACS Chem Biol 2012; 7:1420-1428.
113. Bertolo L, Boncheff AG, Ma Z, Chen Y-H, Wakeford T, Friendship RM, et al. Clostridium difficile carbohydrates: glucan in spores, PSII common antigen in cells, immunogenicity of PSII in swine and synthesis of a dual C. difficile–ETEC conjugate vaccine. Carbohydr Res 2012; 354:79-86.
114. Cox AD, Michael FS, Aubry A, Cairns CM, Strong PC, Hayes AC, et al. Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection. Glycoconj J 2013; 30:843-855.