Table 1.

Advantages and disadvantages of potential C. difficile mucosal vaccine candidates.

Target Antigen	Advantages	Disadvantages
TcdA and TcdB	Direct presentation of antigens at mucosal sites by engineered commensal bacteria [38,40,97] TcdA/TcdB fragment-expressing commensals provide strong protection from death in animal models [38,40,97] TcdA C-terminal-expressing C. difficile spores provided colonization resistance and cross-reactivity to TcdB [98,99]	Rectally administered, formalin-deactivated TcdA and TcdB generated poor antibody responses and poor protection [36] No protection from colonization [34,100] May promote asymptomatic carriage of CDI [101,102] Long-term binding effectiveness of vaccine induced anti-TcdB antibodies is unclear [103]. May not protect against hypervirulent C. difficile [105]
C. difficile membrane preparation	Rectal vaccination of mice with a C. difficile membrane fraction reduced colonization [39] Intrarectal vaccination of mice with ntCDMF reduced fecal bacterial load and decreased death [107,108]	Which antigens were protective in these studies has not been fully determined [39,107,108]
SlpA	Intra-rectal vaccination of mice reduced colonization [113]	Immunogenicity varies widely based on adjuvant [35,113] Protection from death was not significant in hamsters [113]
Cwp84	Immunogenic in CDI patients [115] Rectal vaccination reduced colonization in mice [39] Rectal vaccination reduced hamster deaths [39] Encapsulated Cwp84 is stable in the GI tract [39]	Rectally vaccinated hamsters are still colonized [39] Susceptible to degradation in the gut [39] Anti-Cwp84 antibody levels did not correlate with survival in a hamster vaccine model [32]
Cwp66	Cwp66 C-terminal region is surface-exposed [117] and is more immunogenic than TcdA or TcdB [106]	Cwp66 has yet to be tested as a mucosal vaccine
CD0873	Encapsulated CD0873 stimulated strong SIgA and IgG responses in hamsters [121] Anti-CD0873 antibodies blocked C. difficile adherence and protected from death [121] Liposome delivery of CD0873 produced even greater antibody responses than encapsulated antigen [122]	The liposome delivery mechanism needs further characterization [122]
GroEL	Intranasal vaccination reduced colonization in mice [124]	Intrarectal vaccination of hamsters prolonged survival, but it did not offer strong protection [124]
Fbp68	Immunogenic in CDI patients [106] More immunogenic than TcdA or TcdB [106]	Untested as mucosal vaccine
FliC and FliD	CDI patients produce strong antibody responses to FliC and FliD [106] Rectal administration of FliD generated significant IgA and IgG levels in mice [39] May be able to reduce colonization if used with other surface antigens [39]	Intraperitoneal FliC injections were protective in mice [126], but mucosal administration has not been tested Intranasal and intragastric FliD vaccination was not strongly immunogenic [39] While B. subtilis spores expressing FliD fragments have been developed, their effectiveness has not been evaluated [127]
CdeC and CdeM	Abundant in exosporium and unique to C. difficile [131,133] Both proteins are immunogenic in mice [131] Intraperitoneal vaccination offered strong protection in both mice and hamsters [131] Intraperitoneal vaccination with either protein reduced spore shedding in mice [131] Other vaccine designs related to B. subtilis spores, expressing the TcdA C-terminal [98,99], generated antibody responses against CdeC	Not tested as mucosal vaccine Other vaccine designs, such as B. subtilis spores expressing the TcdA C-terminal [98,99], generated anti-CdeC antibodies without intentionally including CdeC as an antigen
BclA2	An intranasally delivered BclA2 fragment (BclA2CTD) protected mice from death [134] Free and spore-adsorbed BclA2CTD triggered significant murine IgG responses [134]	Neither free nor spore-adsorbed BclA2CTD blocked colonization or mitigated CDI symptoms in vivo [134]
Bcla3	An intranasally delivered BclA3 fragment (BclA3CTD) was immunogenic in mice [135] Intranasal delivery of the C-terminal domain of the spore surface protein BclA3 (BclA3CTD) produced IgG responses in mice Vaccination with free BclA3CTD prevented weight loss in mice [135].	Spore-displayed BclA3CTD was less immunogenic than free BclA3CTD and does not appear to improve vaccine performance [135,136] BclA3CTD vaccination in mice was unable to reduce diarrhea prevalence, diarrhea severity, spore load in the gut, or toxin levels in feces [135,136] Recombinant BclA3 glycosylation is not representative of the dominant glycan structure on the spore coat, possibly contributing to poor vaccine performance [136]
Bcla1		Intraperitoneal vaccination was not protective in mice [131] RT027 strains express truncated BclA1 [131], possibly requiring a separate BclA1 vaccine for these strains Not evaluated as a mucosal vaccine
SleC	Intraperitoneal vaccination reduced spore shedding in mice [131]	Intraperitoneal vaccination did not protect mice from death [131] SleC-mutant spores can still germinate [138] Not evaluated as a mucosal vaccine
CotA	Intraperitoneal vaccination with CotA protected mice from death [131]	Intraperitoneal CotA vaccination did not reduce spore shedding [128] CotA is not expressed on all spores [131] Not evaluated as a mucosal vaccine
Non-toxigenic C. difficile (NTCD)	NTCD may compete with toxigenic strains for the same niche in the gut [139] NTCD can be modified to express fragments of C. difficile toxins and colonization factors [30,142,143] Oral vaccination with modified NTCD strains ellicited antibody responses against toxins, surface components, and colonization factors [30,142,143] Vaccination with modified NTCD strains offers strong, sometimes complete, protection from death in mice in hamsters [30,142]	Concerns over toxigenic conversion of administered NTCD strains [145] Long-term protection may wane if NTCD is not retained after completing therapy [146]