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Immune-based treatment and prevention of Clostridium difficile infection ab

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a

a

Song Zhao , Chandrabali Ghose-Paul , Keshan Zhang , Saul Tzipori & Xingmin Sun

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Department of Infectious Diseases and Global Health; Tufts University Cummings School of Veterinary Medicine; North Grafton, MA USA b

Department of Gastroenterology; Jiangsu Province Hospital of TCM; Nanjing, China

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Aaron Diamond AIDS Research Center; New York, NY USA

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Clinical and Translational Science Institute; Tufts University; Boston, MA USA Published online: 10 Feb 2015.

Click for updates To cite this article: Song Zhao, Chandrabali Ghose-Paul, Keshan Zhang, Saul Tzipori & Xingmin Sun (2014) Immune-based treatment and prevention of Clostridium difficile infection, Human Vaccines & Immunotherapeutics, 10:12, 3522-3530, DOI: 10.4161/21645515.2014.980193 To link to this article: http://dx.doi.org/10.4161/21645515.2014.980193

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REVIEW Human Vaccines & Immunotherapeutics 10:12, 3522--3530; December 2014; Published with license by Taylor & Francis Group, LLC

Immune-based treatment and prevention of Clostridium difficile infection Song Zhao1,2, Chandrabali Ghose-Paul3, Keshan Zhang1, Saul Tzipori1, and Xingmin Sun1,4,* 1

Department of Infectious Diseases and Global Health; Tufts University Cummings School of Veterinary Medicine; North Grafton, MA USA; 2Department of Gastroenterology; Jiangsu Province Hospital of TCM; Nanjing, China; 3Aaron Diamond AIDS Research Center; New York, NY USA; 4Clinical and Translational Science Institute; Tufts University; Boston, MA USA

Keywords: bacterial toxins, clostridium difficile infection (CDI), immunotherapy, monoclonal antibody, vaccine

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Abbreviations: CDI, Clostridium difficile infection; AAD, antibiotic-associated diarrhea; GTD, glucosyltransferase domain; CPD, cysteine proteinase domain; TMD, transmembrane domain; RBD, receptor binding domain; IVIG, intravenous immunoglobulin; mAb, monoclonal antibody; HuMabs, human monoclonal antibodies; SLP, surface-layer protein.

Clostridium difficile (C. difficile) causes over 500,000 infections per year in the US, with an estimated 15,000 deaths and an estimated cost of $1–3 billion. Moreover, a continual rise in the incidence of severe C. difficile infection (CDI) has been observed worldwide. Currently, standard treatment for CDI is the administration of antibiotics. While effective, these treatments do not prevent and may contribute to a disease recurrence rate of 15–35%. Prevention of recurrence is one of the most challenging aspects in the field. A better knowledge of the molecular mechanisms of the disease, the host immune response and identification of key virulence factors of C. difficilenow permits the development of immune-based therapies. Antibodies specific for C. difficile toxins have been shown to effectively treat CDI and prevent disease relapse in animal models and in humans. Vaccination has been recognized as the most cost-effective treatment/prevention for CDI. This review will summarize CDI transmission, epidemiology, major virulent factors and highlights the rational and the development of immune-based approaches against this remerging threat.

Introduction C. difficile is a gram-positive, toxin-producing, spore-forming, anaerobic rod bacterium commonly associated with colitis and diarrhea in humans and other mammals.1,2 In 1935, it was first isolated in the stool of neonates and assumed to be part of the normal gut flora.3,4 It is now considered the leading cause of nosocomial antibiotic-associated diarrhea (AAD) in developed countries.5,6 The symptoms of C. difficile infection (CDI) range from mild diarrhea to fatal pseudomembranous colitis. Standard therapy depends on treatment with vancomycin, metronidazole or fidaxomicin. None of these are fully effective.7,8 Moreover, an © Song Zhao, Chandrabali Ghose-Paul, Keshan Zhang, Saul Tzipori, and Xingmin Sun *Correspondence to: Xingmin Sun; Email: [email protected] Submitted: 06/28/2014; Revised: 08/22/2014; Accepted: 09/03/2014 http://dx.doi.org/10.4161/21645515.2014.980193

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estimated 15–35% of those infected with C. difficile relapse following treatment.9,10 Treatment of recurrent CDI is one of the major challenges in the field,11-13 and is attributed to the direct impact of antimicrobial agents on the integrity of the gut microflora, which in turns help promote bacterial colonization by C. difficile of the large bowel. With the continued rise of antibiotic resistance and concern about high rate of recurrence/relapse related to such treatment, the search for non-antibiotic and immune-based therapies against CDI has been renewed.14 C. difficile exists as inactive spores or vegetative cells.15-17 It can infect both humans and animals, and is transmitted by the fecal-oral route.18,19 Host microbiota in the gut prevents colonization, expansion and persistence of C. difficile in the intestine. Antibiotic treatment disrupts microbiota-host homeostasis and creates an environment within the gut that promotes C. difficile spore germination, followed by vegetative growth.20,21 C. difficile can adhere to the mucus layer carpeting the enterocytes and penetrate the mucus layer with the help of proteases and flagella. Virulence factors that play important role during intestinal colonization and adherence include cysteine protease Cwp84,22 S-layer P36, P47,23 Cwp66,24 GroEL,25 Flagellin, and flagellar cap protein.26 Following spore germination and vegetative cell colonization, the vegetative cells secrete 2 toxins: toxin A (TcdA) and toxin B (TcdB), which are C. difficile’s two major virulence factors.27-29 TcdA and TcdB share similar domain structures that include the N-terminal catalytic glucosyltransferase domain (GTD), the autolytic cysteine proteinase domain (CPD), the central transmembrane domain (TMD) and the C-terminal receptor-binding domain (RBD).30,31 The TMD facilitates the insertion of the Nterminus into and through the endosomal membrane.32 The CPD cleaves and releases GTD into the cytosol of host cell.33 The RBD is responsible for toxin binding to the cell surface possibly via multivalent interactions, leading to endocytosis.34 The GT domain is capable of transferring glucose residues from UDP-glucose to small GTPases including RhoA, Rac1 and Cdc42,35 which leads to the deregulation of actin cytoskeleton and tight junction integrity, and ultimately cell death.29,36 In addition to TcdA and TcdB, up to 35% of C. difficile strains express binary toxin which enhance virulence of C. difficile

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through stimulation of the host cells to form microtubule protrusions facilitating bacterial attachment.37,38 Binary toxin has 2 subunits (CDTa and CDTb) which can catalyze ADP-ribosylation of G-actin, resulting in the depolymerization of F-actin filaments.39,40 The incidence of CDI has increased dramatically over the last decade, and new “low risk” patient groups have been affected. Increased incidence and morbidity of the disease correlates with the emergence of new hypervirulent strains known as BI/NAP1/ 027.41 These strains have recently been associated with community-based outbreaks of CDI.42 Some evidence suggests that these strains may produce more TcdA and TcdB, exhibit a higher rate of sporulation, produce binary toxin and exhibit high-level fluoroquinolone resistance allowing for easier dissemination of these strains.43-46 Recurrence is one of the major challenges in managing CDI, either due to relapse (i.e., endogenous persistence of the same strain of C. difficile) or reinfection (i.e., acquisition of a new strain of C. difficile from an exogenous source). Up to 33% of patients experience recurrence after an initial episode47-50 and recurrences can reach 45% after a second episode.51 Recurrence of disease correlates well with a failure to mount effective neutralizing anti-toxin antibodies. Re-colonization of the gut by normal intestinal microbiota as well as the magnitude of the antibody response to the first episode together determines the probability of recurrence.

Antibody Responses and Development of Clinical Symptoms Upon C. difficile Infection CDI symptoms range from asymptomatic carriage to lifethreatening pseudomembranous colitis. The main risk factor for the development of CDI is use of antibiotics. In addition, age, underlying and co-morbid disease, possibly immunosuppression, drug therapy and immune responses also influence the onset, progression and severity of CDI. Antibodies against C. difficile are present in a majority of adults and older children (»60%), although less than 3% of adults and older children are colonized. Environmental exposure to poorly pathogenic C. difficile strains or other clostridial species, such as Clostridium sordellii, which possesses cross-reacting antigens, can induce antibody production. Individuals may be transiently exposed to C. difficile at infancy and then throughout life, via repeated exposure from the environment, food, as well as domestic animals.52-55 The development of antibodies may allow a person to become an asymptomatic carrier. Carriage of C. difficile varies throughout lifetime, with up to 60%–70% of newborns colonized at birth, decreasing to about 2% in healthy adults. Increasing evidence suggests that these individuals serve as a reservoir of C. difficile.56 The level of serum IgG antibodies against TcdA and TcdB is related with protection against CDI. It was demonstrated that asymptomatic patients have increased serum anti-toxin IgG compared to patients who develop symptomatic disease.57 Clinical studies have also shown that acquired immunity after an initial

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episode, featured with increased serum anti-toxin IgG, protects against recurrent CDI.58,59 The role of anti-toxin antibody, especially anti-TcdA response, in the prevention of primary disease and disease recurrence is undeniable. Asymptomatic carriers and patients with a single episode of C. difficile diarrhea also show greater immune responses to TcdB although the differences in TcdB responses between protected and vulnerable subjects were not as dramatically statistically different as for TcdA.58,60,61 TcdB is 10 times more potent than TcdA in inducing epithelial injury and electrophysiological changes in human colonic strips in vitro.61 Recent studies using TcdA-negative C. difficile mutants demonstrate the importance of TcdB in a CDI animal model.62 There have also been numerous reports of TcdA-negative/TcdBpositive strains of C. difficile that are able to cause CDI.63 Taken together, these findings suggest that both toxins are central to disease pathogenesis and therefore both must be targeted for effective protection. The correlation of antibody response with protection against CDI provides a theoretical basis for the development of immune-based therapies. In addition, both passive and active immunization are generally accepted as effective approaches for the prevention or treatment of CDI.65

Passive Immunization Against CDI Passive immunization against C. difficile toxins was originally studied in animals. Anti-C. difficile bovine immunoglobulin concentrate (BIC), prepared from the colostrum of cows immunized with culture filtrate toxoid, protected against diarrhea and death in hamsters infected with C.difficile.71 Several years later, researchers produced 2 different bovine IgG preparations by immunizing cattle with C. difficile culture filtrates or with formalin-inactivated TcdA. Both preparations could neutralize TcdAinduced cytotoxicity in vitro and inhibit the enterotoxic effects of TcdA on rat gut loops.66,67 Avian immunoglobulins also protected hamster models from diarrhea and death. Meanwhile, neutralizing antibodies to both TcdA and TcdB provided complete protection from diarrhea and death in this model.68 In contrast to animal studies where antibodies were delivered orally or systemically, the majority of human studies thus far have used the systemic delivery route. The results of animal studies and the prevalence of serum antibodies against TcdA and TcdB in healthy populations have prompted investigators to evaluate the therapeutic activity of Intravenous immunoglobulin (IVIG) preparations in individuals experiencing severe or recurrent CDI.69-72 However, the limited availability of IVIG hinders its general use as a therapeutic for severe or recurrent CDI. An alternative to standard IVIG preparations, which rely on antitoxin antibodies raised in response to natural exposure to the organism, is the production of hyper-immune globulin derived from volunteers immunized with C. difficile toxoid vaccine. This strategy aims to produce an immunoglobulin preparation which has a higher specific activity than IVIG developed from source plasma.

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The oral administration of anti-C.difficile antibodies has been explored for the treatment of severe or recurrent CDI. The orally delivered anti-toxin therapy was first reported in 1993,73 which involved the treatment of CDI in a child with C. difficile- specific IgA. The bovine IgG preparation found to be effective in animal models, as mentioned above, was also evaluated in a clinical study aimed at determining the survival of bovine IgG following passage through the GI tract.74 The degradation of bovine IgG during transit by intestinal proteases was found to substantially reduce the activity of anti-C. difficile antibodies recovered in stool. Recently, some studies demonstrated the effectiveness of orally delivered bovine immune whey to treat CDI patients.75,76 Whey containing bovine immunoglobulins was obtained from immunization of cattle with inactivated C. difficile culture. In addition to immunoglobulin-based therapies which rely on the activity of polyclonal anti-toxin antibodies. Monoclonal antibody (mAb)-based passive immunotherapy is also effective in animals. The earliest animal study involving mAbs specific for TcdA and TcdB was performed by Lyerly et al.77 This group demonstrated that oral administration of the premixed antiTcdA mAb (PCG-4) with TcdA completely protected hamsters from fatal doses of TcdA. Another report demonstrated that mAbs directed against the repeating units of TcdA protected axenic mice from C. difficile disease.78 Recently, Babcock et al. described the first fully human monoclonal antibodies (HuMAbs) generated by immunizing mice transgenic for human immunoglobulin genes with inactivated full-length toxins.79 This group found that the combined peritoneal injection of the anti-TcdA HuMAb (CDA1) with the anti-TcdB HuMAb (MDX-1388) resulted in a statistically significant reduction of mortality in both primary and relapse hamster models of CDI, relative to either mAb alone. Similar to the most efficacious antibodies reported before, both HuMAbs recognized the RBDs of TcdA and TcdB, respectively. Passive immunotherapy for the treatment of CDI in human has been also studied using CDA1 and CDB1.80,81 Medarex performed a randomized, double-blind, placebo-controlled study of these 2 neutralizing mAbs, and found a significant reduction in CDI recurrence compared to controls, with only 7% of those receiving the mAb therapy relapsing compared to a 25% relapse

rate among patients receiving placebo.80 Merck is completing a study to investigate the efficacy, safety and tolerability of infusion of 3 HuMAbs in patients receiving antibiotic therapy for CDI (Table 1). Although a reduced recurrence of C. difficile diarrhea was reported, this antibody therapy did not improve the severity of the diarrheal illness, the duration of hospitalization, or the time to resolution of the diarrhea.82

Development of Vaccines Against CDI Because of the importance of toxins in the outcome of CDI, most vaccine efforts have been focused on the toxins. Given the success of toxoid based vaccines, the first candidate vaccine against C. difficile to be tested in humans is a toxoid-based vaccine containing formalin-inactivated purified TcdA and TcdB, adjuvanted with alum, licensed by Sanofi Pasteur. This candidate vaccine has successfully completed 6 phase-I clinical trials to evaluate its safety and immunogenicity. In more than 200 young healthy adults, 18 to 55 y of age, and healthy adults greater than 65 y of age, this vaccine was found to be safe and immunogenic with no vaccine related adverse events reported.83,84 Sanofi Pasteur also initiated the 2 Phase-II studies using an adjuvanted vaccine dose of 50 ug or higher in 600 volunteers in USA and UK. The efficacy of this vaccine in successfully treating recurrent CDI was studied in patients following an initial episode of CDI. This vaccine was able to successfully treat a small number of patients with recurrent CDI.85 The randomized, placebo-controlled, double-blind, dose-finding study was accomplished to assess the efficacy of the vaccine for the prevention of CDI in at-risk volunteers. Sanofi Pasteur has initiated a Phase III study in 2013 with plans to include up to 200 sites in 17 countries, involving 15.000 volunteers to evaluate the efficacy of the toxoid vaccine to prevent primary CDI in elderly patients with comorbidities who are at a high- risk for CDI. The requirement of 3 parenteral administrations of this vaccine to reach serum antitoxin antibody levels significantly higher than those associated with natural infection, makes this vaccine somewhat impractical in a public health setting.86,87 Residual toxicity associated with formalin inactivation also reduces the safety of this vaccine.

Table 1. Summary of passive immunizations against CDI in clinical development Antibody

Sponsor

Composition

Status of development

Patient groups

Anti-TcdA and anti-TcdB mAbs

Medarex

CDA1 plus CDB1

Phase 2

Merck

MK-3415, MK-6072 or MK3415A

Phase 3

IVIGs

None

Small-scale studies

Bovine immune whey

None

Extracted from the plasma of blood donors Whey protein concentrate contains sIgA

200 patients with symptomatic CDI 1600 patients with confirmed diagnosis of CDI and under antibiotics therapy Patients with recurrent CDI or severe CDI Patients with recurrent CDI

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Preclinical

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References/study (clinicalTrials.gov) 80/NCT00350298 NCT01241552 NCT01513239

69–72 75, 76

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Historically, holotoxins have been difficult to purify and produce, require either chemical or molecular inactivation, are unstable and degrade over time and contain some contaminating antigens. To overcome these limitations, newer commercial bioprocess technologies have been developed by leading pharmaceutical companies. Pfizer’s novel expression platform for overexpressing genetically inactivated native toxins in a non-toxigenic non-sporulating strain of C. difficile leads to the production of highly stable toxoids.88 This stability is due to mutations preventing autocatalytic cleavage. Furthermore, expression of the toxoids is uncoupled from counterproductive sporulation and metabolic transcriptional regulation due to the use of a constitutive promoter, resulting in much higher antigen yields. Pfizer’s toxoidbased vaccine is first rendered 10,000 fold less toxic than the wild-type toxins by in vitro cell-based toxicity assays by the introduction of targeted mutations in the N-terminal glucosyltransferase domains, followed by chemical treatment to remove any residual toxicity. Furthermore, Pfizer has invented irreversible 1ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and NHydroxysuccinimide (NHS) modifications to remove minor residual toxicity of its genetic toxoids without affecting the neutralizing activity of functional antibodies (patent US201330371A1). A Phase-I vaccine study currently underway by Pfizer is a placebo-controlled, randomized, observer-blinded study of an adjuvanted vaccine containing TcdA and TcdB requiring at least 3 parenterally administered doses, in healthy volunteers, 50 to 85 y of age to evaluate the vaccine’s safety and immunogenicity (ClinicalTrials.gov: NCT02052726). Pfizer is using a live cell neutralization assay to monitor functional immunogenicity as its

secondary readout which is found to be more accurate than the conventional toxin neutralizing assay.88 Pfizer is also enrolling subjects for a Phase II non-adjuvanted “proof-of-concept” trial (ClinicalTrials.gov: NCT02117570). A summary of C. difficile vaccines in clinical studies is shown in Table 2. Recombinant protein and peptide vaccines targeting the smaller toxin domains are being developed as potential vaccines against CDI. Multiple laboratories and pharmaceutical companies are working on various domains of TcdA and TcdB, especially the RBD of both toxins to identify epitopes that have potent toxin neutralizing ability in murine and hamster models of CDI. Recombinant protein-based vaccine targeting the RBDs of the C. difficile toxins adjuvanted with S. typhimurium flagellin can induce rapid, high level protection in a mouse model of CDI that closely represents the human disease, using a simple primeboost regimen. Parenteral immunization with a recombinant protein expressing 33 of the 38 C-terminal repeats of TcdA can generate a TcdA-neutralizing systemic antibody response that partially protect against toxin challenge. Another study has reported the use of an attenuated S. typhimurium aroA aroD vaccine strain, BRD509, expressing 14 C-terminal repeats of TcdA as a fusion to the immunogenic, nontoxic fragment C of tetanus toxin to target the mucosal immune system.89 Both intragastric and intranasal immunizations were shown to be efficient at generating anti-TcdA antibodies that could neutralize the cytotoxicity of the intact TcdA. Importantly, mucosal local anti-TcdA IgA responses were also induced by both immunization routes. Using such an approach, Valneva (Intercell) has completed a phase 1a/ Ib open-label dose-escalation study in 60 healthy adults, aged 18 to 65 y, as well as in 81 healthy at-risk volunteers greater than 65

Table 2. Summary of vaccines evaluated in clinical studies Antigens

Sponsor

Regimen

Status of development

Sanofi

3 doses i.m

Phase 1

50 healthy adults (aged 18–55 y)

110/NCT00127803

Phase 1

48 elderly volunteers ( 65 y of age) 116 patients with first event of CDI (aged 18–85 y) 661 risk patients without CDI (aged 40–75 y) 15,000 risk patients ( 50 y of age) 102 healthy adults in Japan (aged 40–75 y) 82 healthy adults (18 y of age) 192 healthy adults (aged 50 to 85 y)

110/NCT00214461

Estimated 344 healthy adults (aged 50 to 85 y) Estimated 749 healthy adults (aged 50 to 85 y)

88/NCT02052726

Toxoid A and B (ACAMCDIFFTM )

3 doses i.m § adjuvant

Phase 2

Phase 2

Fusion protein (IC84) Genetically modified TcdA and B

Valneva

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Pfizer

3 doses i.m

Phase 3

4 doses i.m

Phase 1, 2

4 doses i.m § adjuvant 3 doses i.m § adjuvant

Phase 1

3 doses i.m with adjuvant 3 doses i.m

Phase 1

Phase 1

Phase 2

Patient groups

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References/study (clinicalTrials.gov)

83/NCT00772343

83/NCT01230957 NCT01887912 NCT01896830 NCT01296386 NCT01706367

NCT02117570

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y of age to test the safety of a recombinant protein-based parenteral vaccine consisting of truncated TcdA and TcdB, with or without aluminum hydroxide as adjuvant.90 This vaccine consists of 4 parenterally delivered immunizations, thus bringing into question the feasibility of such a vaccine. In addition to parenteral immunizations that induce systemic anti-toxin immune responses, several routes of vaccination targeting the mucosal immune system have also been studied, such as transcutaneous, intranasal and oral etc using a formalin detoxified toxoid vaccine in the presence of several mucosal adjuvants and are found to be immunogenic in the murine model. Given the importance of mucosal immunity in CDI, delivery of a vaccine by a mucosal route using a mucosal adjuvant is highly desirable. A novel approach using Bacillus subtilis spores to deliver antigens orally is currently being developed for clinical use by Royal Holloway, University of London, commonly called CDVAX.91 CDVAX is a novel B. subtilis vaccine expressing a fragment of TcdA fused to surface proteins of B. subtilis, CotB and CotC. The vaccine is able to induce serum and fecal antibodies that neutralize TcdB and is protective in hamsters following oral administration of B. subtilis PP108 (CotB-A26–39 CotC A26–39). Hypervirulent strains of C. difficile BI/NAP1/027 express binary toxin CDT. CDT, an iota-like toxin such as Clostridium botulinum C2 toxin and Clostridium perfringens E toxin, is a 2component toxin encoded by 2 genes, cdtA (enzymatic domain) and cdtB (binding domain).92 CDT leads to the disruption of the actin cytoskeleton, leading to the formation of microtubulebased cell protrusions allowing enhanced colonization of the gut epithelium by C. difficile. The role of CDT in CDI is additive to TcdA and TcdB, although recent studies using an isogenic mutant (A¡B¡CC) producing only CDT suggests an independent role of CDT in causing disease. Patients with CDI caused by toxigenic strains producing CDT are at a higher risk for recurrent disease.93 Therefore, a vaccine approach targeting all 3 toxins is reasonable in stemming the spread of hypervirulent strains of C. difficile BI/NAP1/027. Merck Research Laboratories are developing a multivalent 4-component vaccine that targets TcdA, TcdB and CDT domains. The vaccine consists of recombinant proteins with mutations in the active enzymatic sites of TcdA, TcdB and CDT that leads to full protection against lethal challenge with epidemic C. difficile NAP1/B1/027 strain in animal models of CDI. These antigens are over-expressed using Baculovirus insect cells (patent WO2013112867). This approach and the one used by Pfizer (patent US201330371A1) highlight the drawbacks to the formalin inactivation of toxins used by Sanofi Pasteur. Colonization of the host by C. difficile is an important step in the disease pathogenesis of CDI. While a strong immune response against the toxins can prevent symptomatic disease, it does not prevent colonization or persistence of infection in asymptomatic carriers who, in turn, can produce environmental contamination by C. difficile spores and transmit the organism to susceptible individuals.54,61,94-96 Thus, an ideal vaccine, in addition to targeting the toxins, would eliminate carriage and dissemination of C. difficile spores.

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Several adherence factors have been characterized, although the mechanisms of adhesion are still poorly understood. Putative adherence factors include the surface-layer proteins (SLPs), flagella, Cwp66 adhesin, Fbp68 fibronectin binding protein, GroEL heat-shock protein, and certain hydrolytic enzymes.97,98 Limited human studies in small number of patients indicate that these proteins are antigenic. Whether host humoral immune responses to these surface proteins of C. difficile can determine the clinical outcome of CDI by influencing bacterial colonization, persistence and toxin effects is unknown. Active vaccination using colonization factors as antigens are also being studied in animal models of CDI for potential development as vaccine candidates, as discussed below. C. difficile is surrounded by a paracrystalline S-layer that consists of a high molecular weight (HMW-SLP) and a low molecular weight SLP (LMW-SLP).99 Although S-layer proteins can modulate the host’s innate and adaptive immune systems via TLR4 activation active immunization with SLPs with various adjuvants have only afforded partial protection against C. difficile challenge in hamsters.100 C. difficile flagellar proteins play an important role in the pathogenesis of CDI by influencing adherence, toxin production, and biofilm formation; although this role is strain specific. C. difficile flagellum is made up of 2 components, the 39-kDa FliC (flagellin) and the 56-kDa flagellar cap protein FliD.101,102 Several studies have reported that flagella proteins are highly immunogenic and that natural anti-flagella immune responses may play a role in protection against colonization.103 Furthermore, FliC-FliD immunized mice showed reduced intestinal colonization by C. difficile.104 C. difficile expresses 3 highly complex cell-surface polysaccharides named PSI, PSII and PSIII, on the cell surface.105 PSII is abundantly expressed by historical and hypervirulent C. difficile BI/NAP1/027 ribotypes.106 PSII is being developed as an oligosaccharide-conjugate vaccine. PSII fused with various carrier proteins such as TcdA and TcdB is found to be immunogenic and protective in animal model of CDI.107 Germination of spores into vegetative cells is a critical step during C. difficile’s life cycle because stopping their function would prevent downstream events including the production of toxins, and thus, the development of CDI. The C. difficile spore proteome consists of more than 300 proteins that probably contribute to the resistance phenotype of the spore, as well sensor proteins that lead to germination and colonization of host tissue. C. difficile expresses 3 collagen-like exosporium proteins (BclA1, BclA2 and BclA3) on the outer most surface of the spore, the exosporium layer. The role of C. difficile BclA proteins in spore-host interactions and pathogenesis is unknown. An ortholog of C. difficile BclA1 in Bacillus anthracis protects immunized mice from B. anthracis spore colonization. BclA1 is not immunogenic in mice and affords partial protection in hamsters orally immunized with B. subtilis spores to express BclA1 on CotB from lethal challenge. The utility of BclA2 and BclA3 as potential vaccine candidates hasn’t been studied yet.

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Future Perspectives for CDI Immunotherapy In 1974, pseudomembranous colitis was diagnosed in patients who had been treated with clindamycin.108 A few years later, Bartlett et al. identified toxigenic C. difficile as the cause of the symptoms.109 Adaptive immune responses of hostdetermine the outcomes of CDI; therefore, immune-based therapies are bound to play a key role in stopping the spread of this epidemic. Many cases documenting the successful treatment of relapsing CDI with antibody-based reagents have relied on systemicallydelivery antibody administration. As mentioned above, some antibodies likely need to reach the GI tract to work effectively. To enhance the efficacy of orally delivered antibodies, the exploration of protective antibody formulations and engineered antibodies with robust biophysical properties may be warranted. Meanwhile, recombinant antibody (rAb) fragments offer some advantages over conventional antibodies. They are amenable to in vitro display selection and can be engineered for greater efficacy. In addition, single-domain antibodies (sdAbs) are recombinant, in vitro selected fragments and include the VH and VL domains of conventional immunoglobulins. rAbs and sdAbs might provide new approaches in passive administration of antibody products. A large number of studies mentioned above have confirmed the efficiency of protective immunity induced by C. difficile vaccine, in animal models of CDI as well as in humans enrolled in clinical trials. Both toxoid-based and recombinant vaccines have proven to be highly immunogenic in healthy and at-risk volunteers. Three experimental vaccines against C. difficile are currently under clinical evaluation, all of them aim to prevent CDI in adults and elderly.83,85,110 These vaccines will be challenged by their own ability to induce a rapid, long lasting, and protective immunity in elderly and immunocompromised population. As key colonization factors, C. difficile surface layer proteins (SLPs) have been evaluated in animal models of CDI.103,110 Among the most common C. difficile strains, PSII is a conservative surface antigen and represents a promising target for the development of a carbohydrate-based vaccine.107 Conjugation of C. difficile carbohydrate antigens to toxin fragments is a promising approach for the design of a multivalent vaccine targeting References 1. Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 1996; 9:216-34; PMID:8964036 2. Keel MK, Songer JG. The comparative pathology of Clostridium difficile-associated disease. Vet Pathol 2006; 43:225-40; PMID:16672570; http://dx.doi. org/10.1354/vp.43-3-225 3. Richardson SA, Alcock PA, Gray J. Clostridium difficile and its toxin in healthy neonates. Brit Med J 1983; 287:878; PMID:6412868; http://dx.doi.org/ 10.1136/bmj.287.6396.878 4. INTESTINAL flora of infants. Nutr Rev 1952; 10:198-9; PMID:14957293 5. Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 2009; 7:526-36; PMID:19528959; http://dx.doi. org/10.1038/nrmicro2164

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both colonization and toxin-induced symptoms simultaneously. More studies are needed to fully understand protection efficacy of such constructs against CDI. A number of vaccines against CDI are currently under development. The toxoid vaccine developed by Sanofi is currently undergoing a Phase III trial although the requirement for multiple doses decreases the viability of the vaccines. Additionally the protocol of making the toxoid is cumbersome. To overcome these issues, Pfizer, currently in Phase II, has used a recombinant approach to inactivate the toxins without the use of formalin to reduce cytotoxicity, although its use is also hindered by multiple required doses. Due to the new epidemic of caused by hypervirulent strains expressing binary toxin, Merck is developing a 4-component vaccine targeting TcdA, TcdB and binary toxin. The feasibility of this vaccine has not progressed to clinical trials yet. Other novel approaches such as targeting colonization factors and spore proteins by utilizing B. subtilis spores as an oral vaccine is an exciting new concept that will be tested in clinical trials in the near future. For a vaccine to warrant clinical development and be clinically and commercially feasible for patients susceptible to C. difficile in a public health setting, the vaccine must induce a rapid, high level protection against autologous and heterologous strains, requiring the fewest immunizations with or without the added help of an adjuvant. With the emergence of new hypervirulent strains and spread of the infection in a younger, community-based population, there is an urgent need for developing novel non-antimicrobial approaches against C. difficile. Immune-based therapies represent logic and cost-effective methods to deal with the CDI epidemic.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

Financial support to XS from NIDDK (grant: K01DK092352) and Tufts Collaborates 2013! (grant: V330421) is gratefully acknowledged.

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