TABLE 1

Antibodies used in the study of Clostridium difficile infection interventions

Antibody typeAntibody no.Antibody name/typeMethodSummaryNotesReference(s)
Polyclonal antibodies/exptl vaccines1PolyclonalToxoid immunizationImmunization of hamsters with toxoid A and/or toxoid B resulted in a protective antitoxin polyclonal response100% protection with toxoid A+B; ∼23% protection with toxoid A or B alone. In animals with serological response, reciprocal reduction in cecal TcdA or TcdB concentration or both in the presence of antisera.59
2Polyclonal, subdomain specificPeptide vaccinationHamsters were vaccinated with the majority of the C-terminal domain of TcdA and challenged with a TcdA+B+ strain. Hamsters with higher serum titers survived.Immunized sequence is (presumed to be) derived from VPI10463; the infection challenge strain was VPI7698, which produces moderate levels of toxin. Notable that sera against TcdA alone (presumed) protected against a strain expressing both TcdA and TcdB regarding survival but not onset of diarrhea.81
3Polyclonal IgG, bovineOrogastric dosingHyperimmune IgG fraction from bovine colostrum protected ∼66% of hamsters when given alone or protected 100% hamsters when given with infant formula milk.900 mg/day of IgG was given daily starting 48 h prior to infection. >10-fold-higher neutralization of TcdB than of TcdA.38
4Immunization of hamsters by various routesHamsters were immunized with toxoid, inactivated culture filtrates, or formalin-killed cells and immunized with cholera toxin or RIBI adjuvants by one of eight different combinations of route of administration.Data suggested that C. difficile may have some ability to “evade” host immune response when encountered in the gastrointestinal (GI) tract and that TcdB in particular may be difficult to raise antibody responses against.82
5Polyclonal, bovine colostrumOrogastric dosingCows were immunized with inactivated TcdA or culture filtrate. Anti-filtrate colostrum IgG neutralized the in vitro effects of TcdA or culture filtrate (TcdA and TcdB).39
6PolyclonalVaccineOral immunization of rabbits with attenuated Vibrio cholerae strains secreting TcdA subdomainsResulted in serum IgG that protected against TcdA challenge in a ligated ileal loop model.35
7PolyclonalToxoid immunizationImmunization with TcdA or TcdA and TcdB, but not TcdB alone, protected hamsters against infection. Neutralizing antibodies could be transferred to infants by immunized biological and foster mothers.Purified TcdA or TcdB alone could be lethal when dosed orogastically (o.g.). Hamster antitoxin IgG was detected in cecal homogenates.36, 60
8Polyclonal, subdomain specificOrogastric dosing of IgYIgY was raised against subdomains spanning full-length toxin sequence. Antibodies against the C-terminal domain were the most effective. Anti-TcdA alone conferred ∼70% protection to hamsters, but anti-TcdA and anti-TcdB conferred 100% protection.Weight loss was measured as marker of morbidity. Protection of hamsters was dose dependent but required large doses, up to 320 mg/day. Anti-TcdA was sufficient for prophylactic protection, but both anti-TcdA and anti-TcdB were required for therapeutic protection. Protected animals survived rechallenge.40
9PolyclonalToxoid immunization. Passive transfer of MAbsVarious sites of immunization and adjuvant conferred 20% to 100% protection of hamsters due to neutralizing anti-TcdA and anti-TcdB. Hamsters were protected by passively transferred hyperimmune hamster sera or mouse ascitic fluid but not goat polyclonal sera in a dose-dependent manner.Demonstration of hamster weight loss after challenge. Demonstration that high serum titer or large parenteral serum doses were required for protection of hamsters. Protection was due to the IgG fraction, and autologous Fc domains may be required. Antitoxin IgG was not detected in feces.37
10PolyclonalMouse immunization with part of TcdA C terminusHigh binding titer and some neutralizing serum, but no mucosal antibody titer, was seen against TcdA.83
11Polyclonal, bovine WPC-40 “MucoMilk”Bovine colostrumA preparation (prepn) of 40% whey protein from cows immunized with inactivated bacteria and culture filtrate tested as an oral prepn in a human uncontrolled cohort study for the prevention of recurrence.The immunoglobulin fraction comprised 10% of the whey prepn, and the ratio of sIgA to IgG was 100:1. Whey neutralized TcdA with a titer of ∼3,100 and neutralized TcdB with a titer of ∼330. Protection of hamsters was 80 to 90% when given orally for 3 days. None of the 16 patients had a recurrence; median follow-up period was 333 (range, 35 to 365) days.84
12PolyclonalTranscutaneous TcdA immunizationTranscutaneous immunization of mice with toxoid A elicited potent IgG and IgA responses. Sera were potently neutralizing (in vitro), and IgA was shown in feces.85
13PolyclonalDNA immunization with part of TcdA C terminusHigh binding titer and protection against lethal challenge with TcdA86
14PolyclonalIVIGReview of reports of use of intravenous (i.v.) pooled human immunoglobulin to treat protracted, recurrent or severe CDI.Uncontrolled data suggest efficacy of pooled human immunoglobulin, particularly to treat patients with multiple recurrences of CDI.47
15PolyclonalChimeric inactive toxinMice and hamsters were immunized with a glucosyl transferase inactive version of TcdB, which had its C-terminal cell binding domain switched to that of TcdA.Mice and hamsters were protected against infection challenge. Antibody (Ab) responses were neither cross-neutralizing nor cross-protective. Ab responses were mostly IgG1 and IgG2a. TcdA was more immunogenic than TcdB.67
16PolyclonalImmunization with a hybrid C terminus comprised of approx half of the TcdA and most of the TcdB cell binding domains.Mice, hamsters, and monkeys were immunized with hybrid C-terminal domain.TcdA domains were more immunogenic (higher neutralizing titer and sooner) than TcdB domains in the presence and absence of adjuvant. Noninfected hamsters had antitoxin IgG in stool. Hamsters were protected 100% against lethality but not against symptoms.87
17Polyclonal seraAlpaca-derived seraMice were protected against bacterial challenge by either immunization with toxoid or passive immunization with alpaca-derived polyclonal sera.Mice challenged with spores produced lower levels of antitoxin IgG response than those immunized with toxoid. Regardless, IgG responses were stronger than those of IgA, and anti-TcdA was stronger than anti-TcdB. Antitoxin antibodies, but not vancomycin, were required to protect mice against recurrent disease.20
18Polyclonal, sheep IgGPolyclonal IgG (pIgG) intraperitoneally (i.p.)Generation of neutralizing titers (1/16,000 to 1/20,000) required four immunizations over 14 weeks.Anti-TcdA and anti-TcdB mixtures conferred ∼50 to 90% protection after a total of 75 mg of each antisera (25 mg per dose, “top-up” dosing) but poorly protective at 7.5 mg total dose. Administration of anti-TcdA sera alone slowed symptoms but ultimately offered no protection against death, while anti-TcdB alone had no protective effects.61
19Polyclonal, alpaca IgGPassive immunization of neutralizing polyclonal antibodies (PAbs) were shown to prevent serum toxemia caused by gut infections with C. difficile.Both TcdA and TcdB can be found in serum of infected animals and cause elevation of serum proinflammatory cytokines. pIgG against TcdB alone was found to protect piglets from infection-related GI symptoms and toxemia, but pIgG against TcdA alone did not protect against disease (actually appeared to worsen outcomes).76, 88
20PolyclonalSubdomain-specific immunization of mice and hamsters.Hamsters were fully protected when immunized with at least one subdomain from TcdA and one from TcdB, but neither alone was protective.Antibody titer (in mice) was not predictive of neutralizing titer. TcdB-derived domains were generally less immunoreactive than TcdA-derived domains. The most effective domains for protection were found to be the C-terminal domain of TcdA and the N-terminal domain of TcdB. Sera were found to be weakly cross-reactive for toxin binding, but not cross-neutralizing. IgG antibodies were found in the gut lumen.62
21PolyclonalSubdomain-specific DNA immunization of mice and rabbits.C-terminal domain of TcdA but not TcdB is immunogenic in mice. Central domains of TcdA and TcdB are not immunogenic. N-terminal domain of TcdB is immunogenic in mice.TcdA antisera was fully protective against an i.p. challenge with purified TcdA. Protection against diluted culture filtrate require administration of both anti-TcdA (C-terminal) and anti-TcdB (N-terminal) sera.63
Monoclonal antibodies22MAb, murine PCG-4 G-2MAb + purified toxinPCG-4 is a TcdA-specific neutralizer. G-2 is a nonneutralizing cross-reactive MAb against TcdA and TcdBPCG-4 protected hamsters against the effects of purified o.g. TcdA. Shown to bind to the C terminus of TcdA at ≥2 epitopes. PCG-4 binds minimally to positions 2097 to 2193 (2078 to 2193) and 2279 to 2414, which map to CROP3 and CROP4. PCG-4 blocks the binding of TcdA to Caco-2 cells.49, 78, 89, 90
23MAb, murine G2 various 5288; 1339, 1134, 1142MAbFirst reports of cross-reactive MAbs, specifically those which bind to both TcdA and TcdB; however, these do not neutralize either toxin.49, 50, 51, 91
24MAb, murine 37B5Mouse IgG2b MAb37B5 neutralized enterotoxicity of TcdA in a rabbit ligated ileal loop assay but did not neutralize hemagglutination, mouse lethality, or cytotoxicity.The apparent separation of enterotoxicity and hemagglutinin (HA) functionalities was in contrast to PCG-4, which neutralized both, and was suggestive of distinct epitopes for both. Seven (nonneutralizing) IgM MAbs cross-reacted to TcdA and TcdB and remained nonneutralizing when combined together.92
25MAb, murine TTC8 2CVMAbTTC8 is a mouse MAb specific for the C terminus of TcdA and mapped to a minimal 30-amino-acid (aa) peptide that encodes a predicted epitope repeated eight times in the toxin. 2CV is a mouse MAb specific for the C terminus of TcdB and mapped to a minimal 140-aa peptide that contains a predicted unique epitope. TTC8 precipitates and neutralizes TcdA, 2CV neither precipitates nor neutralizes TcdB.Suggestion that MAbs that bind oligoclonally are discoverable and might be more strongly neutralizing than those which bind at one epitope79
26MAb, human CDA1MAb, hIgG1Human phase II clinical trial of anti-TcdA alone.No effect on recurrence rate but a trend toward a delay in recurrence was observed. Low levels of patient anti-TcdB and infection with strain 027 were associated with recurrence.29
27MAb, human MK-3415 (anti-TcdA also CDA1, Actoxumab, 3D8). MK-6072 (anti-TcdB also MDX-1388, CDB-1, 124–152, Bezlotoxumab)2 doses of hIgG1 human-mouse MAb mixture, i.p. (hamsters), i.v. (humans)A mixture of two MAbs both directed against the toxin C-terminal domains. Most advanced of all C. difficile antitoxin antibodies; partway through phase 3 clinical trials (MODIFY I and II). Anti-TcdA was first tested alone in humans without clinical effect before the addition of anti-TcdB. Phase II clinical trials showed a 72% reduction in recurrence rate, but no effect on the duration or severity of diarrhea.Hamsters were dosed with a total of 200 mg of anti-TcdA or 200 mg of anti-TcdB or 200 mg of each prior to infection (“prophylactic dosing”) with strain BI. Protection levels at day 2 postinfection were approximately 55%, 17%, and 94%, respectively; these rates declined to 5%, 0%, and 55%, respectively. MAbs subsequently shown by others to lack neutralizing capacity for toxins produced by strains of ribotype 027 and 078. Shown to reduce the production of inflammatory markers tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) in peripheral blood mononuclear cells (PBMCs) in human colonic explants. MDX-1388 alone was found to protect piglets from infection related GI symptoms and toxemia, but CDA1 alone did not protect against disease (there was trend toward worse outcomes).29, 34, 52, 55, 56, 58, 88, 93; clinical trials registered at ClinicalTrials.gov under registration nos. NCT01241552 and NCT01512239
28MAb, murine A1H3mIgG2aMAb that enhances the activity of TcdA on cells by ∼1,000 timesA1H3 (de facto a nonneutralizing MAb) recruits TcdA to the cell surface via FcγRI and has been used to develop an extremely sensitive assay capable of demonstrating the presence of serum-borne toxin in a piglet model.76, 94, 95
29MAb, murine 3358 and 3359mIgGMurine MAbs against the C-terminal domain of TcdA, given i.p. in hamster model.Modestly neutralizing MAbs, which became more complete neutralizers as a combination. One neutralizing MAb (3358) increased binding of TcdA to cells, while another (3359) blocked TcdA binding to cells, suggestive of different modes of neutralization. Both MAbs were shown to bind to TcdA multiple times (14 and 8 times, respectively). The combination offered some delay in disease onset in the hamster model, but zero protection at ca. day 6 when infected with an A+B+ strain. Patent engineered enhanced stability into MAbs in order to facilitate o.g. dosing.42, 80
30MAb, llamaLlama VHHImmunized llamas used for “phage display discovery of VHH domains”Anti-TcdA VHH were partially neutralizing in vitro, anti-TcdB VHH were nonneutralizing in vitro. Combinations of 2 or 3 VHH conferred more-complete neutralization. No data for infection models. Molecular basis for multiple binding events shown with X-ray crystallography77, 96
31MAb, humanized PA-50 (anti-TcdA), PA-41 (anti-TcdB)2 doses of hIgG1 humanized mouse MAb mixture, i.p. (hamsters)A mixture of two MAbs, anti-TcdA (PA-50) directed against the toxin C-terminal domains and anti-TcdB (PA-41) directed against the catalytic N-terminal domain.Both MAbs had low pM activities against their respective toxins in vitro and shown to neutralize toxin from nine different ribotypes (20 strains). Levels of protection in hamsters were 90 to 100% at day 39 postinfection, but MAb was dosed both pre- and postinfection (“top-up/therapeutic” dosing), making direct comparison with the data of Babcock et al. (55) not possible.58, 66
32MAb, humanized CA997 (anti-TcdA) CA1125 (anti-TcdB) CA1151 (anti-TcdB)3 doses of hIgG1A mixture of three MAbs, one anti-TcdA (CA997) and two anti-TcdB (CA1125, CA1151), all targeting the C-terminal domainsMAbs were high-affinity toxin binders (pM) and high-potency (low ng/ml) neutralizers of R0003, R027, and R078 toxins in vitro. MAbs demonstrated multiple binding events to toxin “oligoclonality”. CA997 protected against TEER loss in vitro. Hamster challenge with R012 showed 100% protection to day 11, 82% protection to day 28 and were shown to be superior to the CDA1/MDX-1388 mix.52
MAb, murine 4A4 (anti-TcdA) 2C7 (anti-TcdA)2 doses of IgGA mixture of two MAbs for detection of and protection against TcdA.The most potent MAb (4A4) conferred 50% protection to mice challenged i.p. with purified TcdA. Combination with 2C7 resulted in ∼90% protection.97
33MAb, humanEpstein-Barr virus (EBV) immortalized human B cellEV029105a (anti-TcdA) and EV029104 (anti-TcdB) are both human IgG1 directed against the C-terminal domains.EV029105a (0.15 nM affinity) was a more potent neutralizer than CDA1 (3G8) comparator, while EV029104 was less potent than MDX1388. The MAb pair was 100% protective of hamsters to day 10 against R012 challenge when given as three 50-mg/kg doses on days −1, 0, and 1 and were shown to be superior to the CDA1/MDX-1388 mix.98