Memory B Cells Encode Neutralizing Antibody Specific for Toxin Bfrom the Clostridium difficile Strains VPI 10463 and NAP1/BI/027 butwith Superior Neutralization of VPI 10463 Toxin B

T. Scott Devera,a Gillian A. Lang,a Jordi M. Lanis,a* Pragya Rampuria,a Casey L. Gilmore,a Judith A. James,b,c,d Jimmy D. Ballard,a

Mark L. Langa

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USAa; Arthritis and Clinical ImmunologyResearch Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USAb; Department of Medicine, University of Oklahoma Health Sciences Center,Oklahoma City, Oklahoma, USAc; Oklahoma Clinical and Translational Science Institute, Oklahoma City, Oklahoma, USAd

Secreted toxin B (TcdB) substantially contributes to the pathology observed during Clostridium difficile infection. To be success-fully incorporated into a vaccine, TcdB-based immunogens must stimulate the production of neutralizing antibody (Ab)-encod-ing memory B cells (Bmem cells). Despite numerous investigations, a clear analysis of Bmem cellular responses following vacci-nation against TcdB is lacking. B6 mice were therefore used to test the ability of a nontoxigenic C-terminal domain (CTD)fragment of TcdB to induce Bmem cells that encode TcdB-neutralizing antibody. CTD was produced from the historical VPI10463 strain (CTD1) and from the hypervirulent strain NAP1/BI/027 (CTD2). It was then demonstrated that CTD1 inducedstrong recall IgG antibody titers, and this led to the development of functional Bmem cells that could be adoptively transferredto naive recipients. Bmem cell-driven neutralizing Ab responses conferred protection against lethal challenge with TcdB1. Fur-ther experiments revealed that an experimental adjuvant (Imject) and a clinical adjuvant (Alhydrogel) were compatible withBmem cell induction. Reactivity of human Bmem cells to CTD1 was also evident in human peripheral blood mononuclear cells(PBMCs), suggesting that CTD1 could be a good vaccine immunogen. However, CTD2 induced strong Bmem cell-driven anti-body titers, and the CTD2 antibody was neutralizing in vitro, but its protection against lethal challenge with TcdB2 was limitedto delaying time to death. Therefore, CTD from different C. difficile strains may be a good immunogen for stimulating B cellmemory that encodes in vitro neutralizing Ab but may be limited by variable protection against intoxication in vivo.

The role of immune cell memory in Clostridium difficile infec-tion (CDI) remains poorly understood. CDI is complicated by

a high frequency of recurrence, often after disease has apparentlyresolved, and can be associated with progressively worsening pa-thology and ultimately death (1). However, patients that developantibodies (Ab) capable of neutralizing two toxins secreted by C.difficile (TcdA and TcdB) are less likely to experience recurrence(reviewed in reference 2). This suggests that memory B (Bmem)cells may contribute to resistance to reinfection by encodingtoxin-neutralizing Ab. Bmem cells have typically undergone affin-ity maturation and Ab class switch in the germinal centers of sec-ondary lymphoid organs (3). Bmem cells are therefore poised torespond rapidly to booster vaccinations or infection, by rapidlydifferentiating into plasma cells that secrete class-switched, high-affinity Ab (4). Such plasma cells may display a range of short-to-extreme longevity, be associated with transient or sustained Abtiters, or secrete neutralizing or nonneutralizing Ab (5–7).

In earlier studies, a correlation between bacterial load and ad-vanced age was observed during CDI, with older individuals lack-ing toxin-neutralizing Ab (8). In more recent work, the probabil-ity of HIV-positive patients experiencing CDI increased as theirCD4� T-cell counts declined (9), which could be partly attribut-able to altered CD4� T-cell-dependent B cell function (10). In-deed, there is growing concern about CDI in a variety of immuno-compromised individuals, including organ transplant recipients(reviewed in reference 11). These observations highlight the well-recognized importance of B cell responses and production of tox-in-neutralizing antibodies in resisting CDI. However, the under-lying characteristics of the Bmem cellular response and their

contributions to production of toxin-neutralizing Ab have notbeen described.

CDI is best known as a disease of the gastrointestinal tract,causing diarrhea, and this infection may progress to a severe path-ological condition in which pseudomembranous colitis and toxicmegacolon are evident (1, 2). However, CDI-associated mortalitymay be attributable to systemic sequelae of the disease (12). Al-though large-scale epidemiological studies are lacking, reportedsystemic complications include hepatic abscesses (13), ascites(14), pleural effusion with acute respiratory distress (15, 16), andsevere sepsis and multiorgan dysfunction (17).

TcdA and TcdB are large clostridial toxins that contribute sub-

Received 5 January 2015 Returned for modification 26 February 2015Accepted 19 October 2015

Accepted manuscript posted online 26 October 2015

Citation Devera TS, Lang GA, Lanis JM, Rampuria P, Gilmore CL, James JA, BallardJD, Lang ML. 2016. Memory B cells encode neutralizing antibody specific for toxinB from the Clostridium difficile strains VPI 10463 and NAP1/BI/027 but withsuperior neutralization of VPI 10463 toxin B. Infect Immun 84:194 –204.doi:10.1128/IAI.00011-15.

Editor: V. B. Young

Address correspondence to Mark L. Lang, mark-lang@ouhsc.edu.

* Present address: Jordi M. Lanis, School of Medicine—Gastroenterology,University of Colorado Denver, Aurora, Colorado, USA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00011-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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stantially to enteric and systemic pathology associated with CDI(18, 19). Blood-borne TcdB and TcdA can be detected in somepatients presenting with CDI, and diluted toxemic patient sera cankill target cells in vitro (20). C. difficile strains lacking TcdA andTcdB are less virulent than their toxin-expressing counterpartsand are associated with attenuated disease or asymptomatic colo-nization (21). However, TcdA-negative strains can still be highlyvirulent, resulting in severe CDI (22, 23). Systemic effects of TcdAand TcdB have been observed in mice and piglets, with toxemiacorrelating with disease severity and mortality (24, 25). TcdA andTcdB cause cardiac arrest in rabbits (26), and more recently, TcdBwas shown to be cardiotoxic in a zebrafish model (27).

While TcdA appears to be nearly identical among variousstrains of C. difficile, TcdB varies among these strains. TcdB1 (pre-viously termed TcdBHIST and TcdB003), derived from clinicalstrains such as CD630, shares 92% identity with TcdB2 (previ-ously termed TcdBHV and TcdB027) (27). TcdB2 is produced byrecently emerged hypervirulent strains of C. difficile such as strainsNAP1/027/BI (27). Toxemia is also a problem with TcdB2, whichis more potent than TcdB1 in in vitro assays (27, 28). Furthermore,TcdB2 is associated with a more aggressive form of CDI thanTcdB1 and represents a growing threat to public health (29–31).

TcdB1 and TcdB2 are single-chain 269-kDa proteins with atleast four functional domains (32). Unless administered in theformaldehyde-inactivated form, TcdB1 and TcdB2 are not suit-able vaccine candidates, due to their in vivo 100% lethal dose(LD100) of 5 �g/kg (or less) of body weight (27). Therefore, re-combinant fragments of TcdB or specific-domain containing fu-sion proteins may be better candidates than whole toxin (33, 34).This includes the 70-kDa C-terminal domains of TcdB1 andTcdB2 (CTD1 and CTD2) (28).

Given the dearth of available information on memory re-sponses to TcdB1 and TcdB2, we analyzed whether CTD1- andCTD2-specific Bmem cells encoded TcdB1- and TcdB2-neutral-izing IgG. Using a tractable mouse model, we explored the funda-mental characteristics of Bmem cell-driven responses to the twoforms of TcdB. We report that recombinant CTD1 stimulatedBmem cell-driven TcdB1-neutralizing Ab responses that also pro-tected in vivo. In contrast, CTD2 induced high Bmem cell-drivenTcdB2-specific Ab titers that neutralized TcdB2 in vitro but dem-onstrated limited protection in vivo by delaying time to death.These findings provide new and important insights into theBmem cellular response to a critical C. difficile virulence factor andsuggest key differences that could impact neutralizing Ab re-sponses due to vaccination or infection.

MATERIALS AND METHODSEthics. This study was carried out in strict accordance with the recom-mendations in the Guide for the Care and Use of Laboratory Animals ofthe National Institutes of Health. All animal procedures were approved bythe OUHSC Institutional Animal Care and Use Committee (Protocol12-120). The human subject studies were approved by the OUHSC Insti-tutional Review Board (Protocol 2275). Written informed consent wasgiven by study participants.

Expression and purification of TcdB and CTD. C. difficile was cul-tured and TcdB purified as previously described (27, 35). The CTD-en-coding region of tcdb gene (YP_001087135.1, nucleotides 4961 to 7111)from strains VPI-10463 and NAP1/BI/027 were codon optimized andcloned into pET15b (Genscript). The VPI-10463 and NAP1/BI/027 CTDgenes were amplified using primers 5=-GATCATATGCTGTATGTGGGTAACCG-3= and 5=-AACGGATCCTTATTCGCTAATAACCA-3= con-

taining BamHI and NdeI sites for cloning into pET15b. VPI-10463 CTDand NAP1/BI/027 CTD (representing VPI-10463 TcdB1651–2366 andNAP1/BI/027 TcdB1651–2366) were expressed in Escherichia coli BL21 starDE3 (Invitrogen) and purified by Ni2� affinity chromatography(HisTrap; GE Life Sciences). VPI-10463 CTD and TcdB are referred to as“CTD1” and “TcdB1,” respectively, while the designations “CTD2” and“TcdB2” are used for NAP1/BI/027.

Immunizations, blood collection, and experimental schedule. Fe-male C57BL/6 mice were purchased from the National Cancer Institute(Bethesda, MD) and housed in a specific-pathogen-free facility. Mice wereanesthetized with a vaporized 4% isoflurane–96% oxygen mixture forprocedures. Mice were immunized subcutaneously (s.c.) at 6 to 10 weeksof age with the dosage divided evenly over both flanks. Vaccines consistedof 25, 50, or 100 �g of CTD in 200 �l sterile phosphate-buffered saline(PBS) adsorbed to Imject Alum (Thermo Scientific, Rockford, IL) or Al-hydrogel Alum (Invivogen, San Diego, CA). Two vaccination scheduleswere employed. (i) The initial vaccine was administered on day 0, and thebooster consisting of one-half of the original dose of CTD in PBS (noadjuvant) was administered on day 60. Mice were bled retro-orbitallyusing heparinized capillary tubes on days 0, 28, 60, 67, 74, and 180. (ii) Theinitial vaccine was administered on day 0, and the booster was adminis-tered on day 180. Blood samples were collected on days 180, 187, and 194.

Splenocyte isolation. Spleens were harvested into RPMI medium andmechanically disrupted before ammonium chloride lysis of erythrocytes.Cells were enumerated using a Nexcelom Cellometer (Lawrence, MA).Viability was �98% by trypan blue exclusion.

B cell isolation and adoptive transfer. B-lineage cells were enrichedfrom preimmunized IgHb B6 donors by incubating splenocytes on icewith 10 �g of anti-Thy1.2, anti-CD4 monoclonal antibody (MAb) perspleen. The splenocytes were washed, and then T-lineage cells were re-moved by complement-mediated lysis. Cells were incubated with 66�g/ml of LoTox rabbit complement (Cedarlane Laboratories, Burlington,NC) for 45 min at 37°C. The cells were then washed in sterile PBS andcounted. This method was reported by us previously and gives B cellenrichment comparable to that of magnetic sorting (36, 37). B-cell-en-riched splenocytes contained �95% B cells by flow cytometry of CD19�

cells (data not shown). Twenty-five million cells in 100 �l sterile PBS wereinjected paraorbitally into IgHa congenic recipient mice. The recipientmice received a boost of 50 �g/ml CTD 24 h after the adoptive transfer.Sera were collected at 14 days following administration of the boostervaccine. This is a standard method for confirming establishment of anti-gen (Ag)-specific B cell memory (38).

ELISA. Immulon 4 enzyme-linked immunosorbent assay (ELISA)plates (Dynex, Chantilly, VA) were coated with CTD1 or CTD2, and spe-cific endpoint IgG1, IgG2b, IgG2c, and IgG3 titers were determined asdescribed previously (39). In adoptive transfers, IgHb was also detected byELISA as described previously (37).

In vitro neutralization. CHO cells (5 � 104 cells in 100 �l of mediumconsisting of F-12K, 4 �M L-glutamine, and 10% fetal calf serum [FCS])were seeded into 96-well plates and cultured overnight (5% CO2, 37°C).Sera were diluted 1/500 in medium, mixed with TcdB1 or TcdB2 (finalconcentration, 2.5 nM unless indicated otherwise), and incubated for 1 hat room temperature. Concentrations of toxins were previously calibratedto cause 85 to 95% CHO cell death. Medium was removed from cellcultures and replaced with the serum-toxin-medium mixture. Plates werecultured for 24 h before addition of Cell Counting kit-8 (CCK-8) reagent(Dojindo Technologies, Rockville, MD) and an additional 2 h of incuba-tion. A450 was then measured, and percent survival was calculated as fol-lows: (A450 of sample � A450 of toxin-treated cells)/(A450 of untreatedcells � A450 of toxin-treated cells � 100).

Toxin challenge. Mice were tail vein injected with PBS containing 500ng TcdB1, 75 ng TcdB2, or 200 ng TcdB2. Survival was monitored at 1- to4-h intervals. Terminally moribund animals were euthanized, and time todeath was recorded. Passive serum transfer was achieved by intraperito-

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neal (i.p.) injection of 400 �l of serum from naive or CTD1-immunizedmice 16 h prior to TcdB1 challenge.

Peripheral blood mononuclear cell preparation. Peripheral venousblood was drawn from healthy volunteers into heparinized vacuum tubesand layered onto 20 ml of lymphocyte separation medium (Lonza, Allen-dale, NJ) and centrifuged (700 relative centrifugal force [RCF], 35 min,room temperature). PBMCs were removed, washed with PBS, and resus-pended in medium (RPMI, 10% FCS, 40 �g/ml gentamicin). Cell viabilitywas routinely 80 to 90%.

Polyclonal stimulation of Bmem cells. The standard method forstimulating human Bmem cells to differentiate into Ab-secreting plasmacells was employed by culturing PBMCs with polyclonal stimuli (40). Inbrief, CpG 2006 (Invivogen), Pokeweed Mitogen (a kind gift from EmoryUniversity Vaccine Center), and Staphylococcus aureus protein A (Sigma,St. Louis, MO) were added at 6 �g/ml, 1/100,000 dilution of stock, and1/10,000 dilution of stock and cultured for 6 days.

ELISPOT assay. Enzyme-linked immunospot (ELISPOT) assay plates(Millipore, Bedford, MA) were coated with CTD1, and Ab-secreting cellswere detected as described previously (39), with the exception that horse-radish peroxidase (HRP)-conjugated anti-human IgG was used for detec-tion (Southern Biotech, Birmingham, AL).

Statistics. A two-tailed Mann-Whitney U test and an analysis of vari-ance (ANOVA) with Bonferroni’s posttest were used to compare two andmultiple experimental groups, respectively. For challenge experiments,survival data were subjected to Kaplan-Meier analysis with the log ranktest.

RESULTSThe C-terminal domain of VPI 10463 TcdB (CTD1) inducesTcdB-neutralizing protective Ab recall responses. C57BL/6 micewere immunized with 25, 50, or 100 �g of CTD1 adsorbed toImject Alum adjuvant (Fig. 1). Primary serum CTD1-specificIgG1 titers were dependent on the amount of protein adminis-tered. A booster administered 60 days after the initial vaccine ledto an approximate 10-fold increase in IgG1 titer regardless of pri-mary IgG1 titer. IgG1 titers were sustained for at least 180 days, atwhich time a nonsignificant decline in titer was observed. IgG2b,IgG2c, and IgG3 titers were determined for samples obtained be-fore and after the booster (days 60 and 67, respectively) (Fig. 2).IgG2b and IgG2c titers were enhanced at least 10-fold by thebooster. IgG3 was not induced. IgG2a was not measured becauseC57BL/6 mice have a �2A deletion in the Ig locus (41). Therefore,CTD1 induced significant Ab recall responses that were stimu-lated 60 days after the initial immunization. This is consistent withinduction of CTD1-specific Bmem cells.

CHO cells were incubated with TcdB1 in the absence or pres-ence of sera obtained before and after the booster (Fig. 3). Immu-nization with 25 �g of CTD1 did not induce measurable neutral-ization of TcdB1 by the day 60 sera, because CHO cells died underthose conditions (Fig. 3A). The day 67 sera variably neutralizedTcdB1 as evidenced by CHO cell survival. As the immunizationdose increased to 50 �g and 100 �g, neutralization conferred byday 60 sera also increased. Neutralization was further enhanced byday 67 sera from mice immunized and boosted with higher dosesof CTD1. The results were confirmed by repeating the assays witha range of serum dilutions (Fig. 3B; see also Fig. S1 in the supple-mental material). Under these conditions, the stark differencesbetween TcdB1 neutralization by the day 60 and 67 sera were stillobserved.

To confirm the results, CHO TcdB1-induced cell rounding wasanalyzed (Fig. 3C). The day 60 sera did not substantially alterTcdB1-induced cell rounding, but the day 67 sera prevented cell

rounding, such that the flattened adherent morphology observedin untreated cells was observed. Therefore, Ab recall responses ledto enhanced neutralization of TcdB1 and were consistent withBmem cells encoding TcdB1-neutralizing Ab.

CTD1-specific bone marrow plasma cells were detected byELISPOT assay in immunized mice, and the response was signif-icantly increased by the booster vaccine (data not shown). Partialneutralization of TcdB1 by primary sera was therefore attributableto serum IgG secreted by a small number of existing plasma cells.

FIG 1 Imject Alum-adsorbed CTD1 stimulates primary and recall IgG1 re-sponses. C57BL/6 mice were immunized s.c. with Imject-adsorbed CTD1.Mice received a booster vaccine consisting of CTD1 in PBS on day 60. Serawere collected at the times indicated, and endpoint CTD1-specific IgG1 titerswere determined by ELISA for mice immunized with 25 �g (top), 50 �g (mid-dle), or 100 �g (bottom) of CTD1. Each data point represents an individualmouse. Statistical significance was determined by ANOVA and is indicated byasterisks: **, P � 0.01 to 0.001; ***, P 0.001. The day 60 titers were comparedfor each immunization by ANOVA, and titers were significantly higher for the100-�g dose than the 50-�g or 25-�g dose (P 0.001).

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However, stimulation of Bmem cells was necessary to generatesufficient plasma cell-derived Ab to efficiently neutralize TcdB1.

Sustained TcdB1 neutralization was observed in immunizedmice because sera obtained 180 days after immunization neutral-ized TcdB1 (Fig. 4A). Indeed, the neutralization observed after180 days was comparable to that observed for sera obtained after67 days. The CTD1-immunized mice were then challenged in vivoby the intravenous (i.v.) route with a 2� LD100 dose of TcdB1 (Fig.4B). Naive mice all succumbed to TcdB1 and died within 20 h.Immunized mice were protected. Mice immunized with the low-est dose of CTD1 did not succumb to TcdB1 challenge despitevariable in vitro neutralization. This suggests that the in vitro as-says underestimate the amount of protection afforded in vivo.Mice immunized with higher doses were also protected, with 80%survival in the 50 �g group and 100% survival in the 100 �g group.Immunization with Alum alone followed by challenge was notperformed due to the lack of Ab or neutralization in the absence ofCTD1 (data not shown) and because it is well documented that

Alum does not confer Ag-specific immune responses unless thatAg is adsorbed to it (reviewed in reference 42).

To show that Ab-mediated mechanisms contributed to in vivoprotection, naive mice received a passive transfer of serum fromnaive or immunized mice and then were challenged with TcdB1.Mice receiving serum from naive donors succumbed rapidly toTcdB1, displaying 0% survival after 16 h (Fig. 4C). Mice receivingserum from immunized mice succumbed over a period spanning44 h, consistent with the temporary protection conferred by pas-sive transfer (Fig. 4C).

The data presented in Fig. 1 through 4 therefore show that

FIG 2 Imject Alum-adsorbed CTD1 stimulates primary and recall IgG2 re-sponses. Endpoint CTD1-specific IgG2b (top), IgG2c (middle), and IgG3(bottom) titers in the day 60 and 67 sera from the experiment whose results areshown in Fig. 1 were measured by ELISA (note that the booster was adminis-tered on day 60). Each data point represents an individual mouse. Statisticalsignificance was determined by Mann-Whitney U-test, and P values areshown.

FIG 3 Bmem-encoded Ab neutralizes TcdB1 in vitro. Sera from the experi-ment whose results are shown in Fig. 1 were incubated with active TcdB1before adding in triplicate to CHO cell cultures. The final concentration of serawas 1/500, and the final concentration of TcdB1 was 2.5 nM (sufficient to cause85 to 95% cell death in the culture). After 24 h, the CCK-8 cell viability reagentwas added and A405 was measured after a further 3 h. (A) Percent neutraliza-tion when comparing sera obtained before and after the booster vaccine (day60 versus day 67). Black-filled symbols indicate serum number 487, used togenerate the data in panels B and C. Statistical significance between neutral-ization in the day 60 and day 67 groups was determined by Mann-Whitney Utest, and P values are shown. Administration of Alum alone did not induce Abtiters or neutralization responses (data not shown). (B) Effects of dilution ofsera on CHO cell viability for selected sera (mouse ID number 487, immunizedwith 50 �g CTD1). Green symbols depict day 67 sera, and orange symbolsdepict day 60 sera. Serum titrations for every mouse that received the 50-�gdose of CTD1 are shown in Fig. S1 in the supplemental material. (C) Repre-sentative images from a cell-rounding assay whereby cells were treated as de-scribed for panel A and visualized by light microscopy 2 h after application ofTcdB1 and/or sera (from mouse ID number 487).

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stimulation of CTD1-specific Ab recall responses led to durableand sustained in vivo protection against TcdB1. This is consis-tent with CTD1-specific Bmem cells having the capacity to dif-ferentiate into long-lived plasma cells that secrete TcdB1-neu-tralizing Ab.

CTD1 induces specific and functional Bmem cells. Mice wereimmunized with Imject-adsorbed CTD1 and rested for 2 months.This was followed by isolating splenocytes from naive and immu-nized IgHb-congenic mice and subjecting them to depletion of allT-lineage cells before adoptive transfer into IgHa congenic recip-ients (Fig. 5, right panel). After immunization of recipient mice

with a mock booster (CTD1 in PBS), serum IgHb Ab titers wereobserved only in those mice receiving cells from immunized do-nors. IgHa titers were not detected, showing that the Ab responsewas donor derived. This demonstrates that CTD immunizationgenerated transferable Bmem cells that were stimulated with Ag todifferentiate into productive Ab-secreting plasma cells. Neutral-ization and challenge experiments were not performed becausethis method does not generate sufficient Ab titers.

Alhydrogel-adsorbed CTD1 induces long-term B cell mem-ory. To determine if long-term B cell memory could be stimulatedby CTD1 adsorbed to a clinically relevant adjuvant, mice wereimmunized with CTD1 alone, CTD1 adsorbed to Imject Alum, orCTD1 adsorbed to Alhydrogel Alum. Mice were rested for 6months before administration of a booster vaccine (Fig. 6). Imjector Alhydrogel was necessary to stimulate high CTD1-specific IgG1titers (Fig. 6A) as well as IgG2b and IgG2c titers (Fig. 6B). After a6-month resting period, administration of a booster led to Abtiters that were at least 10-fold higher than preboost titers. Thiswas observed for Alhydrogel, which stimulated larger IgG1,IgG2b, and IgG2c titers than did Imject.

In vitro assays revealed that CTD1 immunization alone led tominimal neutralization of TcdB (Fig. 6C). Neutralization in theCTD1/Imject group was variable and did not differ significantlybetween pre- and postbooster sera (Fig. 6C). In contrast, nearlycomplete and sustained TcdB1 neutralization was observed inpostbooster sera in the CTD1/Alhydrogel group (Fig. 6C). Due tothe apparent absence of neutralization in the CTD1 and CTD1/Imject groups, the assay was repeated using a higher concentra-tion of serum, revealing the presence of neutralizing Ab in thepostbooster sera from the CTD1/Imject group (Fig. 6D). The ob-servation that neutralization was significantly higher in sera ob-tained after the boosters demonstrates that Bmem cells could bestimulated to differentiate into plasma cells and secrete TcdB1-neutralizing Ab 6 months after the initial immunization.

Following in vivo challenge with TcdB1, all naive mice died

FIG 4 Bmem-encoded neutralizing Ab is durable and protects against in vivochallenge with TcdB. (A) Six months after the initial immunization (4 monthsafter booster), mice from the experiment whose results are shown in Fig. 1were bled again, and neutralization assays were repeated. Statistical signifi-cance was determined by ANOVA and is indicated by asterisks: **, P � 0.01 to0.001; ***, P 0.001. (B) Mice were then challenged i.v. with TcdB1 (20 ng/gof body weight). The graph shows survival of naive mice (n � 5) and thoseimmunized with 25 �g (n � 9), 50 �g (n � 10), or 100 �g (n � 10) ofImject-adsorbed CTD1. (C) Naive B6 mice were transferred (i.p. route) with400 �l of serum from naive or immunized donor mice. After 24 h, mice werechallenged with TcdB1 (20 ng/g of body weight). The graph depicts percentsurvival over time. Three mice per group were used. Statistical significance inpanels B and C was determined by Kaplan-Meier analysis with log rank test,and P values comparing immunized or serum-transferred groups to naivecontrols are shown.

FIG 5 CTD1 induces functional Bmem IgHb C57BL/6 mice were immunizedwith 100 �g Imject-adsorbed CTD1 and rested for 2 months. Splenocytes werethen subjected to T-cell depletion before adoptive transfer of cells to naive IgHa

C57BL/6 congenic recipient mice. Recipient mice were then immunized with50 �g CTD1 in PBS. All recipient mice were immunized because transferredcells die if not stimulated with Ag. IgHb-specific Abs were then used in anELISA to detect donor Bmem-derived anti-CTD1 Ab. The graph showsmeans standard deviations (SD) of A405 at a 1/200 dilution of sera (n �2 for naive cell transfer, n � 5 for immune cell transfer). Statistical signif-icance between the two groups was determined by Mann-Whitney U test,and the P value is shown.

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within 16 h. Of the CTD1 group, 50% survived. Of the CTD1/Imject and CTD1/Alhydrogel groups, 100% survived (Fig. 6E). Ina further experiment, B6 mice were immunized with CTD/Alhy-drogel, rested for 4 months, and then “immunized/challenged”

with a sublethal dose of TcdB1 (Fig. 6F). CTD1-specific IgG1 titerswere boosted by the toxin, suggesting that during natural infec-tion, exposure to the active toxin may result in stimulation ofCTD1-specific Bmem and production of more neutralizing Ab.

FIG 6 Imject- and Alhydrogel Alum-adsorbed CTD1 stimulates long-term Bmem responses. Mice were not immunized (naive) or were immunized with 50 �gCTD1 in PBS, CTD1 adsorbed to Imject Alum, or CTD1 adsorbed to Alhydrogel. Mice were rested for 180 days before boosting with 25 �g CTD1 in PBS. Serawere collected before the booster and 7 and 14 days after the booster (days 187 and 194). (A) Endpoint CTD1-specific IgG1 titers. (B) IgG2b and IgG2c titers. (C)Neutralization of TcdB1 in vitro. (D) Results of neutralization assay in which sera were diluted 1/100 instead of 1/500. (E) Survival of mice following challengewith TcdB1 in vivo. (F) Mice were immunized with 50 �g CTD1 adsorbed to Alhydrogel and rested for 4 months. Mice were immunized i.p. with 100 ng of TcdB1.Sera were collected before and after TcdB1 administration as indicated. The graph shows CTD1-specific IgG1 titers, and each symbol represents an individualmouse. Statistical significance was determined in panels A, C, D, and F by ANOVA and is indicated by asterisks: *, P � 0.05 to 0.01; **, P � 0.01 to 0.001; ***, P 0.001. Statistical significance in panel B was determined by Mann-Whitney U test, and P values are shown. Statistical significance in panel F was determined byKaplan-Meier analysis with log rank test, and P values comparing CTD/adjuvant groups to naive controls and CTD1 only are shown.

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Therefore, CTD1 adsorbed to a clinically relevant adjuvant stim-ulated long-term B cell memory against C. difficile toxin B.

Detection of CTD1-specific Bmem cells in human subjects.PBMCs were obtained from healthy volunteers over age 55 thatwere unaware of previous C. difficile infection or from a cohortwho had a positive diagnosis of CDI within the 5 years beforesample collection. PBMCs were cultured with polyclonal stimulithat drive differentiation of Bmem cells into Ab-secreting plasmacells (Fig. 7). ELISPOT analysis showed that 2/19 individuals withno known history of C. difficile infection had peripheral bloodBmem that encoded CTD1-specific IgG. CTD1-specific Bmemcells were also observed in 4/8 individuals with a previous C. dif-ficile infection. The number of CTD1-specific spots detectedranged from 0.01 to 0.1% of total IgG-secreting cells (of all spec-ificities) and is consistent with recent observations by anotherlaboratory (43). Therefore, Bmem cells in some human subjectsencoded CTD1-specific IgG.

Immunization with CTD2 leads to Bmem cell-driven pro-duction of Ab that neutralizes CTD2 in vitro but provides lim-ited protection in vivo. Since CTD1 appeared to be a good vaccine

candidate, we then examined CTD2 from the “hyper-virulent”strain NAP1/BI/027. B6 mice were immunized with Alhydrogel-adsorbed CTD2 and boosted with CTD2 alone after 60 days (Fig.8). Sera were collected before (day 60) and after (days 67 and 74)the booster vaccine and examined by ELISA. It was evident thatCTD2 caused a significant IgG1, IgG2b, and IgG2c recall responseconsistent with Bmem cell restimulation by the booster vaccine(Fig. 8A). Sera obtained after the booster vaccine were able toneutralize TcdB2 in vitro, while sera obtained before the boosterwere not (Fig. 8B). However, a modest, but statistically significantdrop in neutralization was observed between the day 67 and day74 sera, showing that neutralizing titers were not sustained. Titra-tion of sera was also performed and showed that neutralizationcould not be detected when the sera were further diluted (Fig. 8C).Titrations of sera for all mice in the study are shown in Fig. S2 inthe supplemental material. A cell-rounding assay was also per-formed, in which cell morphology was analyzed 2 h after treat-ment of CHO cells with TcdB2 (Fig. 8D). The TcdB2 treatmentresulted in previously flattened cells adopting a shrunken roundedmorphology. Sera from mice obtained before the CTD2 boosterdid not change the shrunken appearance, but sera obtained afterthe booster limited the changes induced by TcdB2. The cellstreated with TcdB2 plus postbooster sera had a rounded appear-ance but were not shrunken, indicating partial protection fromtoxicity. Mice were also challenged with TcdB2, and despite thegood in vitro neutralization observed, 100% of mice ultimatelysuccumbed to TcdB2, although time to death was significantlydelayed (Fig. 8E). These data show the CTD2-specific Bmem cell-encoded Ab that could partially neutralize TcdB2 in vitro but notafford sustained protection in vivo.

DISCUSSION

Here, we demonstrate that the C-terminal domain of TcdB fromC. difficile strain VPI 10463 (CTD1) stimulated long-term Ag-specific B cell memory that encoded toxin-neutralizing Ab. Ag-specific IgG1, IgG2b, and IgG2c titers were boosted at least 10- to40-fold by administration of CTD1 alone to B6 mice that hadpreviously been immunized with CTD1 adsorbed to Imject Alum.The ability of CTD1 to stimulate an enhanced Ab response 60 daysafter a primary immunization was consistent with induction ofBmem cells (44). Importantly, Bmem cell-encoded IgG1, IgG2b,and IgG2c have the potential to invoke a diverse range of effectorfunctions in vivo. Future experiments to determine whether IgG1,IgG2b, and IgG2c are singly or collectively protective in vivo arewarranted. The Bmem cell-driven IgG titers were sustained andconferred protection against an in vivo toxin challenge some 4months later. It is unlikely that the IgG detected by ELISA wasderived from newly differentiated plasma cells at this later timepoint (6 months after immunization); it is more likely that it wasderived from bone marrow-resident long-lived Ab-secretingplasma cells that were established at an earlier time (5–7). Inter-estingly, immunization of B6 mice with higher doses of CTD1 ledto variable but measurable in vitro neutralization in serum ob-tained prior to administering a booster vaccine. This suggests thatin the vaccine setting, a single immunization may afford limitedprotection but at least one booster would be necessary to achievegood protection.

TcdB-specific serum Ab are well documented to protectagainst CDI in patients and animal models by reducing the sever-ity of enteric disease, recurrence of disease, and mortality (re-

FIG 7 Detection of CTD1-specific Bmem in human subjects. PBMCs were ob-tained from healthy individuals age 55 years and older that had previously beendiagnosed with C. difficile infection (gray bars) or were not aware of previousinfection (black bars). PBMCs were cultured with polyclonal stimuli as describedin Materials and Methods. Cells were then cultured on ELISPOT assay platescoated with CTD1. IgG reactive to CTD1 was then detected using HRP-conju-gated anti-human IgG. The graph shows the number of Ab-secreting cells (ASC)per million total cells for each individual in the cohort. Data are represented asmeans standard errors of the means (SEM) for triplicate samples. The dottedarea depicts background (from unstimulated samples) plus 2 standard deviations.The inset image shows raw ELISPOT assay data from donor 880.

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viewed in reference 2). Furthermore, circulating TcdB-specificIgG is a correlate of protection against recurrent CDI (45). Themechanism by which serum IgG can protect against enteric CDI isnot entirely clear, but mucosal TcdB-specific IgG can be detectedin human intestines (43). The neonatal IgG Fc receptor (FcRn)mediates bidirectional transport of IgG across intestinal epithelia(46). Indeed, such transport of Citrobacter rodentium-specific IgGprotects the gut of infected mice (47). The epithelial transportpathway therefore represents a strong possibility for a mechanism

by which TcdB-specific IgG could limit the pathology associatedwith CDI.

The role of mucosal IgA in protection against CDI is lessclear, but fecal IgA concentrations were reported to be lower inindividuals with recurrent CDI than in healthy controls or inpatients with a single occurrence (48, 49). Studies in mice havealso shown that mucosal IgA was induced during infection andwas protective in the absence of serum IgG responses, but mu-cosal IgA was also dispensable since mice lacking the poly-Ig

FIG 8 Immunization with CTD2 leads to Bmem-driven production of Ab that neutralizes CTD2 in vitro but does not protect in vivo. Mice were immunized with50 �g CTD2 adsorbed to Alhydrogel and rested for 60 days before boosting with 25 �g CTD2 in PBS. Sera were collected before the booster and 7 and 14 daysafter the booster (days 67 and 74). (A) Endpoint CTD2-specific IgG1 titers, IgG2b, and IgG2c titers. Geometric mean titers are indicated for the 15 mice in eachgroup, and statistical significance was determined by one-way ANOVA followed by Bonferroni’s posttest (*, P � 0.05 to 0.01; ***, P 0.001). (B) Neutralizationof TcdB2 in vitro using sera obtained before and after booster vaccine administration. Statistical significance was determined by ANOVA and Bonferroni’sposttest (**, P � 0.01 to 0.001; ***, P 0.001). (C) Representative titration of serum in the neutralization assay (from mouse number 273). Results from all othersera are shown in Fig. S2 in the supplemental material. (D) Images from cell-rounding assay, in which TcdB2 was applied to CHO cells in the absence or presenceof sera. Images depict sera from mouse number 264, where in vitro neutralization was most representative of the average for the group. (E) Survival of micefollowing challenge with 200 ng (left) or 75 ng (right) of TcdB2/mouse. Kaplan-Meier analysis with log rank test was used to determine significant differencesbetween naive and immunized mice (**, P � 0.01 to 0.001). Data are representative of two similar experiments.

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receptor had outcomes similar to those of wild-type controls(50).

Passive transfers in this study confirmed that IgG-mediatedmechanisms contributed to protection in vivo but did not conferfull protection. Passive transfer is unlikely to provide the recipientmouse with as much IgG as that produced by the endogenoushumoral immune response and may be one reason why it did notafford complete protection. Another reason may be that otherIgG-independent mechanisms could contribute to protection,and this could also explain why the in vitro neutralization assaywas better able to discriminate between experimental groups thanthe in vivo challenge assay. Indeed, �-defensins were recentlyshown to contribute to neutralization of TcdB1 in a manner de-pendent on binding to the N terminus of the protein, thus sug-gesting that neutralization could be complementary to that pro-vided by C terminus-targeting IgG (51, 52).

In our in vivo challenge experiments, mice received 500 ng ofCTD1 from the VPI 10463 strain. Directly relating this dosage tohuman disease is not straightforward, especially as toxemia pres-ents only in a subset of patients, methods to measure toxin con-centrations in serum vary in sensitivity, and serum Ab in infectedindividuals mask toxin detection (20). However, a 1/10 dilution ofserum from toxemic patients was reported to be sufficient to kill,or cause 100% rounding up in, cultured Vero cells, similar to whatwas observed by adding TcdB1 at a 25-pg/ml concentration (20).Arguably, sera from toxemic patients could contain TcdB1 at aconcentration exceeding the estimated 250-pg/ml concentrationbecause neutralizing anti-TcdB1 IgG causes a 125-fold loss of sen-sitivity of detection and perhaps intoxication capacity (20). Thetrue concentration of TcdB1 in toxemic sera could therefore behigher than 31 ng/ml (125 � 250 pg/ml). Our challenge dose of500 ng/mouse equates to approximately 20 ng/ml if distributedevenly throughout the mouse, or 250 ng/ml if confined to a closed2-ml circulatory system. Our challenge assays therefore reason-ably recapitulate the likely concentrations observed during tox-emia in patients.

As Imject is not used clinically, we also measured Bmem cellresponses with Alhydrogel, which is used in the clinic. Longer-term experiments were also performed to determine if the Bmemcell response was durable. Alhydrogel led to higher Bmem cell-driven neutralizing Ab titers than Imject. CTD1 alone led to poorAb titers and poor neutralization but was associated with partial(50%) protection in the in vivo challenge assays. Protection in vivowas the same for the Imject and Alhydrogel groups (100%). Thisfurther suggests that in addition to Ab-mediated protection, thereis an Ab-independent component, which should be pursued infollow-up studies.

Polyclonal stimulation of human PBMCs showed that CTD1-specific Bmem cells were present in the blood of healthy individ-uals with no awareness of previous C. difficile infection or with aconfirmed previous infection. This was consistent with a recentstudy in which reactivity to TcdB was detected in human PBMCs(43). Therefore, human Bmem cells can encode CTD1-specificIgG and indicate that the protein used in our mouse studies couldbe a good candidate for stimulating TcdB1-neutralizing Bmemcells in patients. There are several reasons why some individualswith no knowledge of prior infection had TcdB1-specific Bmemcells. Approximately 60% of individuals are colonized with C. dif-ficile as infants, and it is maintained as a gut commensal (2). Someindividuals may have had a mild infection and not be aware of it,

or natural infection may not adequately immunize some individ-uals. Alternatively, the Bmem cells generated by infection may notbe long-lived. The cohort in this study was diagnosed up to 5 yearsbefore obtaining the blood sample. Larger-scale studies will there-fore be necessary to better understand Bmem cell responses toinfection and immunization. However, we propose that CTD1 hasthe potential to be exploited as a vaccine Ag that stimulates long-lived Bmem cell responses that encode TcdB-specific Ab.

Due to the emergence of C. difficile strain NAP1/B/027, whosehypervirulence is attributable in part to secretion of a TcdB variant(referred to as TcdB2 here), we tested the ability of the CTD fromTcdB2 (CTD2) to stimulate B cell memory. We observed thatCTD2 induced a somewhat different response from that of CTD1.Both CTD1 and CTD2 induced Bmem restimulation, but the lat-ter appeared to stimulate a slower recall response, reaching max-imal IgG1 and IgG2b titers after 2 weeks rather than 1 week. CTD1resulted in production of neutralizing Ab with responses that wereobserved up to 4 months after administration of the booster vac-cine. In contrast, CTD2 resulted in Bmem cell-driven neutralizingAb that was not sustained and diminished within 2 weeks of thebooster vaccine. In vitro neutralization of CTD1 was able to with-stand a 4,000-fold dilution of the sera. When anti-CTD2 sera weresimilarly titrated, in vitro neutralization was lost with one furtherdilution of sera. In vivo challenge experiments revealed that moreor less complete protection against TcdB1 could be achieved, butwith TcdB2, all mice died, albeit with time to death significantlydelayed.

Ab titers showed that CTD2 was at least as immunogenic asCTD1, so differences in in vitro neutralization and in vivo protec-tion were attributable to differences in Bmem cells encoding neu-tralizing Ab. We also immunized mice with a mutant CTD2 inwhich an amino acid sequence, SINKVIST, corresponding to res-idues 1791 to 1798 was substituted for an AAAAAIAA sequence.Following immunization with mutant CTD2, mice were chal-lenged with wild-type TcdB2. We observed that survival was sim-ilar whether mice were immunized with CTD2 or mutant CTD2(data not shown). The mutant CTD2 has a conformation thatresults in exposure of neutralizing epitopes (53), suggestingthat immunogenicity of CTD2 is not a limitation in our exper-iments.

It should be noted that Ab raised to TcdB from one strain of C.difficile may not be effective at cross-neutralization of TcdB fromother strains. CTD1- and CTD2-specific Ab raised in rabbits andin mice did not cross-neutralize TcdB1 and TcdB2 (27, 53). Abraised against CTD1 neutralized TcdB1 but not TcdB2. Surpris-ingly, Ab raised in rabbit against CTD2 did not neutralize TcdB1or TcdB2. Differences in the overall make-up of neutralizingepitopes in the CTD1 and CTD2 may in part explain the absenceof cross-neutralization. This does not, however, explain whyCTD2 did not stimulate a neutralizing response to TcdB2 itself. Inthe present study, we observed some CTD2-induced neutraliza-tion of TcdB2, suggesting that there are some species-related dif-ferences in response but that CTD1-specific Bmem cells encodeneutralizing Ab to a greater degree than do CTD2-specific Bmemcells. Therefore, the two forms of the toxin may be fundamentallydifferent in how they stimulate an effective Bmem cell-driven neu-tralizing Ab response. Collectively, our data demonstrate thatCTD1 may be a useful immunogen, but engineering of CTD2 toimprove establishment of Bmem cells that better encode neutral-izing IgG is necessary. Further research to understand why such

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closely related immunogens result in such disparate immunolog-ical outcomes is also warranted.

ACKNOWLEDGMENTS

We thank Virginia Roberts, OMRF Clinical Coordinator, for recruitingand enrolling volunteers and procuring volunteer blood samples.

We have no conflicts of interest to declare.

FUNDING INFORMATIONOklahoma Center for Advancement of Science and Technology (OCAST)provided funding to Mark L. Lang under grant number HR12-005.HHS | National Institutes of Health (NIH) provided funding to Judith A.James, Jimmy D. Ballard, and Mark L. Lang under grant numbersAI078993, AI062629, GM103510, GM104938, and AI119048.

The funders had no role in study design, data collection and interpreta-tion, or the decision to submit the work for publication.

REFERENCES1. Bartlett JG. 2010. Clostridium difficile: progress and challenges. Ann N Y

Acad Sci 1213:62– 69. http://dx.doi.org/10.1111/j.1749-6632.2010.05863.x.

2. Kaslow DC, Shiver JW. 2011. Clostridium difficile and methicillin-resistant Staphylococcus aureus: emerging concepts in vaccine develop-ment. Annu Rev Med 62:201–215. http://dx.doi.org/10.1146/annurev-med-051109-101544.

3. McHeyzer-Williams LJ, McHeyzer-Williams MG. 2005. Antigen-specific memory B cell development. Annu Rev Immunol 23:487–513.http://dx.doi.org/10.1146/annurev.immunol.23.021704.115732.

4. Amanna IJ, Slifka MK. 2010. Mechanisms that determine plasma celllifespan and the duration of humoral immunity. Immunol Rev 236:125–138. http://dx.doi.org/10.1111/j.1600-065X.2010.00912.x.

5. Slifka MK, Ahmed R. 1998. Long-lived plasma cells: a mechanism formaintaining persistent antibody production. Curr Opin Immunol 10:252–258. http://dx.doi.org/10.1016/S0952-7915(98)80162-3.

6. Slifka MK, Antia R, Whitmire JK, Ahmed R. 1998. Humoral immunitydue to long-lived plasma cells. Immunity 8:363–372. http://dx.doi.org/10.1016/S1074-7613(00)80541-5.

7. McHeyzer-Williams MG, Ahmed R. 1999. B cell memory and the long-lived plasma cell. Curr Opin Immunol 11:172–179. http://dx.doi.org/10.1016/S0952-7915(99)80029-6.

8. Nakamura S, Mikawa M, Nakashio S, Takabatake M, Okado I, Ya-makawa K, Serikawa T, Okumura S, Nishida S. 1981. Isolation ofClostridium difficile from the feces and the antibody in sera of young andelderly adults. Microbiol Immunol 25:345–351. http://dx.doi.org/10.1111/j.1348-0421.1981.tb00036.x.

9. Haines CF, Moore RD, Bartlett JG, Sears CL, Cosgrove SE, Carroll K,Gebo KA. 2013. Clostridium difficile in a HIV-infected cohort: incidence,risk factors, and clinical outcomes. AIDS 27:2799 –2807. http://dx.doi.org/10.1097/01.aids.0000432450.37863.e9.

10. Moir S, Fauci AS. 2013. Insights into B cells and HIV-specific B-cellresponses in HIV-infected individuals. Immunol Rev 254:207–224. http://dx.doi.org/10.1111/imr.12067.

11. Collini PJ, Bauer M, Kuijper E, Dockrell DH. 2012. Clostridium difficileinfection in HIV-seropositive individuals and transplant recipients. J In-fect 64:131–147. http://dx.doi.org/10.1016/j.jinf.2011.12.003.

12. Karas JA, Enoch DA, Aliyu SH. 2010. A review of mortality due toClostridium difficile infection. J Infect 61:1– 8. http://dx.doi.org/10.1016/j.jinf.2010.03.025.

13. Sakurai T, Hajiro K, Takakuwa H, Nishi A, Aihara M, Chiba T. 2001.Liver abscess caused by Clostridium difficile. Scand J Infect Dis 33:69 –70.http://dx.doi.org/10.1080/003655401750064112.

14. Tsourous GI, Raftopoulos LG, Kafe EE, Manoleris EK, Makaritsis KP,Pinis SG. 2007. A case of pseudomembranous colitis presenting withmassive ascites. Eur J Intern Med 18:328 –330. http://dx.doi.org/10.1016/j.ejim.2006.09.034.

15. Boaz A, Dan M, Charuzi I, Landau O, Aloni Y, Kyzer S. 2000. Pseu-domembranous colitis: report of a severe case with unusual clinical signsin a young nurse. Dis Colon Rectum 43:264 –266. http://dx.doi.org/10.1007/BF02236993.

16. Jacob SS, Sebastian JC, Hiorns D, Jacob S, Mukerjee PK. 2004. Clos-

tridium difficile and acute respiratory distress syndrome. Heart Lung 33:265–268. http://dx.doi.org/10.1016/j.hrtlng.2004.04.003.

17. Dobson G, Hickey C, Trinder J. 2003. Clostridium difficile colitis causingtoxic megacolon, severe sepsis and multiple organ dysfunction syndrome.Intensive Care Med 29:1030.

18. Ballard JD. 2010. Medical microbiology: a toxin contest. Nature 467:665–666. http://dx.doi.org/10.1038/467665a.

19. Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP.2010. The role of toxin A and toxin B in Clostridium difficile infection.Nature 467:711–713. http://dx.doi.org/10.1038/nature09397.

20. Yu H, Chen K, Wu J, Yang Z, Shi L, Barlow LL, Aronoff DM, GareyKW, Savidge TC, von Rosenvinge EC, Kelly CP, Feng H. 2015. Identi-fication of toxemia in patients with Clostridium difficile infection. PLoSOne 10:e0124235. http://dx.doi.org/10.1371/journal.pone.0124235.

21. Shim JK, Johnson S, Samore MH, Bliss DZ, Gerding DN. 1998. Primarysymptomless colonisation by Clostridium difficile and decreased risk ofsubsequent diarrhoea. Lancet 351:633– 636. http://dx.doi.org/10.1016/S0140-6736(97)08062-8.

22. Drudy D, Fanning S, Kyne L. 2007. Toxin A-negative, toxin B-positiveClostridium difficile. Int J Infect Dis 11:5–10. http://dx.doi.org/10.1016/j.ijid.2006.04.003.

23. Lyras D, O’Connor JR, Howarth PM, Sambol SP, Carter GP, Phu-moonna T, Poon R, Adams V, Vedantam G, Johnson S, Gerding DN,Rood JI. 2009. Toxin B is essential for virulence of Clostridium difficile.Nature 458:1176 –1179. http://dx.doi.org/10.1038/nature07822.

24. Steele J, Chen K, Sun X, Zhang Y, Wang H, Tzipori S, Feng H. 2012.Systemic dissemination of Clostridium difficile toxins A and B is associatedwith severe, fatal disease in animal models. J Infect Dis 205:384 –391. http://dx.doi.org/10.1093/infdis/jir748.

25. Steele J, Feng H, Parry N, Tzipori S. 2010. Piglet models of acute orchronic Clostridium difficile illness. J Infect Dis 201:428 – 434. http://dx.doi.org/10.1086/649799.

26. Siarakas S, Damas E, Murrell WG. 1995. Is cardiorespiratory failureinduced by bacterial toxins the cause of sudden infant death syndrome?Studies with an animal model (the rabbit). Toxicon 33:635– 649.

27. Lanis JM, Barua S, Ballard JD. 2010. Variations in TcdB activity and thehypervirulence of emerging strains of Clostridium difficile. PLoS Pathog6:e1001061. http://dx.doi.org/10.1371/journal.ppat.1001061.

28. Lanis JM, Heinlen LD, James JA, Ballard JD. 2013. Clostridium difficile027/BI/NAP1 encodes a hypertoxic and antigenically variable form ofTcdB. PLoS Pathog 9:e1003523. http://dx.doi.org/10.1371/journal.ppat.1003523.

29. Muto CA, Pokrywka M, Shutt K, Mendelsohn AB, Nouri K, Posey K,Roberts T, Croyle K, Krystofiak S, Patel-Brown S, Pasculle AW, Pater-son DL, Saul M, Harrison LH. 2005. A large outbreak of Clostridiumdifficile-associated disease with an unexpected proportion of deaths andcolectomies at a teaching hospital following increased fluoroquinoloneuse. Infect Control Hosp Epidemiol 26:273–280. http://dx.doi.org/10.1086/502539.

30. Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S,Bourgault AM, Nguyen T, Frenette C, Kelly M, Vibien A, Brassard P,Fenn S, Dewar K, Hudson TJ, Horn R, Rene P, Monczak Y, Dascal A.2005. A predominantly clonal multi-institutional outbreak of Clostridiumdifficile-associated diarrhea with high morbidity and mortality. N Engl JMed 353:2442–2449. http://dx.doi.org/10.1056/NEJMoa051639.

31. McDonald LC, Killgore GE, Thompson A, Owens RC, Jr, Kazakova SV,Sambol SP, Johnson S, Gerding DN. 2005. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 353:2433–2441. http://dx.doi.org/10.1056/NEJMoa051590.

32. Pruitt RN, Chambers MG, Ng KK, Ohi MD, Lacy DB. 2010. Structuralorganization of the functional domains of Clostridium difficile toxins Aand B. Proc Natl Acad Sci U S A 107:13467–13472. http://dx.doi.org/10.1073/pnas.1002199107.

33. Tian JH, Fuhrmann SR, Kluepfel-Stahl S, Carman RJ, Ellingsworth L,Flyer DC. 2012. A novel fusion protein containing the receptor bindingdomains of C. difficile toxin A and toxin B elicits protective immunityagainst lethal toxin and spore challenge in preclinical efficacy models.Vaccine 30:4249 – 4258. http://dx.doi.org/10.1016/j.vaccine.2012.04.045.

34. Permpoonpattana P, Hong HA, Phetcharaburanin J, Huang JM, CookJ, Fairweather NF, Cutting SM. 2011. Immunization with Bacillus sporesexpressing toxin A peptide repeats protects against infection with Clostrid-ium difficile strains producing toxins A and B. Infect Immun 79:2295–2302. http://dx.doi.org/10.1128/IAI.00130-11.

C. difficile Toxin-Specific Memory B Cells

January 2016 Volume 84 Number 1 iai.asm.org 203Infection and Immunity

on January 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

35. Krivan HC, Wilkins TD. 1987. Purification of Clostridium difficile toxin Aby affinity chromatography on immobilized thyroglobulin. Infect Immun55:1873–1877.

36. Lang GA, Devera TS, Lang ML. 2008. Requirement for CD1d expressionby B cells to stimulate NKT cell-enhanced antibody production. Blood111:2158 –2162. http://dx.doi.org/10.1182/blood-2007-10-117309.

37. Lang GA, Johnson AM, Devera TS, Joshi SK, Lang ML. 2011. Reductionof CD1d expression in vivo minimally affects NKT-enhanced antibodyproduction but boosts B-cell memory. Int Immunol 23:251–260. http://dx.doi.org/10.1093/intimm/dxq477.

38. Foy TM, Laman JD, Ledbetter JA, Aruffo A, Claassen E, Noelle RJ.1994. gp39-CD40 interactions are essential for germinal center formationand the development of B cell memory. J Exp Med 180:157–163. http://dx.doi.org/10.1084/jem.180.1.157.

39. Devera TS, Shah HB, Lang GA, Lang ML. 2008. Glycolipid-activatedNKT cells support the induction of persistent plasma cell responses andantibody titers. Eur J Immunol 38:1001–1011. http://dx.doi.org/10.1002/eji.200738000.

40. Crotty S, Aubert RD, Glidewell J, Ahmed R. 2004. Tracking humanantigen-specific memory B cells: a sensitive and generalized ELISPOT sys-tem. J Immunol Methods 286:111–122. http://dx.doi.org/10.1016/j.jim.2003.12.015.

41. Martin RM, Brady JL, Lew AM. 1998. The need for IgG2c specificantiserum when isotyping antibodies from C57BL/6 and NOD mice. JImmunol Methods 212:187–192. http://dx.doi.org/10.1016/S0022-1759(98)00015-5.

42. Lindblad EB. 2004. Aluminium compounds for use in vaccines. Im-munol Cell Biol 82:497–505. http://dx.doi.org/10.1111/j.0818-9641.2004.01286.x.

43. Monaghan TM, Robins A, Knox A, Sewell HF, Mahida YR. 2013.Circulating antibody and memory B-cell responses to C. difficile toxins Aand B in patients with C. difficile-associated diarrhoea, inflammatorybowel disease and cystic fibrosis. PLoS One 8:e74452. http://dx.doi.org/10.1371/journal.pone.0074452.

44. Benson MJ, Elgueta R, Schpero W, Molloy M, Zhang W, Usherwood E,Noelle RJ. 2009. Distinction of the memory B cell response to cognateantigen versus bystander inflammatory signals. J Exp Med 206:2013–2025.http://dx.doi.org/10.1084/jem.20090667.

45. Leav BA, Blair B, Leney M, Knauber M, Reilly C, Lowy I, Gerding DN,Kelly CP, Katchar K, Baxter R, Ambrosino D, Molrine D. 2010. Serumanti-toxin B antibody correlates with protection from recurrent Clostrid-ium difficile infection (CDI). Vaccine 28:965–969. http://dx.doi.org/10.1016/j.vaccine.2009.10.144.

46. Yoshida M, Claypool SM, Wagner JS, Mizoguchi E, Mizoguchi A,Roopenian DC, Lencer WI, Blumberg RS. 2004. Human neonatal Fcreceptor mediates transport of IgG into luminal secretions for delivery ofantigens to mucosal dendritic cells. Immunity 20:769 –783. http://dx.doi.org/10.1016/j.immuni.2004.05.007.

47. Yoshida M, Kobayashi K, Kuo TT, Bry L, Glickman JN, Claypool SM,Kaser A, Nagaishi T, Higgins DE, Mizoguchi E, Wakatsuki Y, Roope-nian DC, Mizoguchi A, Lencer WI, Blumberg RS. 2006. Neonatal Fcreceptor for IgG regulates mucosal immune responses to luminal bacteria.J Clinical Invest 116:2142–2151. http://dx.doi.org/10.1172/JCI27821.

48. Warny M, Vaerman JP, Avesani V, Delmee M. 1994. Human antibodyresponse to Clostridium difficile toxin A in relation to clinical course ofinfection. Infect Immun 62:384 –389.

49. Johal SS, Lambert CP, Hammond J, James PD, Borriello SP, MahidaYR. 2004. Colonic IgA producing cells and macrophages are reduced inrecurrent and non-recurrent Clostridium difficile associated diarrhoea. JClin Pathol 57:973–979. http://dx.doi.org/10.1136/jcp.2003.015875.

50. Johnston PF, Gerding DN, Knight KL. 2014. Protection from Clostrid-ium difficile infection in CD4 T cell- and polymeric immunoglobulin re-ceptor-deficient mice. Infect Immun 82:522–531. http://dx.doi.org/10.1128/IAI.01273-13.

51. Furci L, Baldan R, Bianchini V, Trovato A, Ossi C, Cichero P, CirilloDM. 2015. New role for human alpha-defensin 5 in the fight againsthypervirulent Clostridium difficile strains. Infect Immun 83:986 –995.http://dx.doi.org/10.1128/IAI.02955-14.

52. Kudryashova E, Quintyn R, Seveau S, Lu W, Wysocki VH, KudryashovDS. 2014. Human defensins facilitate local unfolding of thermodynami-cally unstable regions of bacterial protein toxins. Immunity 41:709 –721.http://dx.doi.org/10.1016/j.immuni.2014.10.018.

53. Larabee JL, Krumholz A, Hunt JJ, Lanis JM, Ballard JD. 2015. Exposureof neutralizing epitopes in the carboxyl-terminal domain of TcdB is al-tered by a proximal hypervariable region. J Biol Chem 290:6975– 6985.http://dx.doi.org/10.1074/jbc.M114.612184.

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