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`INFECTION AND IMMUNITY, June 2007, p. 2826–2832
`0019-9567/07/$08.00⫹0 doi:10.1128/IAI.00127-07
`Copyright © 2007, American Society for Microbiology. All Rights Reserved.
`
`Vol. 75, No. 6
`
`Transcutaneous Immunization with Clostridium difficile Toxoid A
`Induces Systemic and Mucosal Immune Responses and
`Toxin A-Neutralizing Antibodies in Mice䌤
`Chandrabali Ghose,1,2 Anuj Kalsy,1 Alaullah Sheikh,1 Julianne Rollenhagen,1,2 Manohar John,1,2
`John Young,1 Sean M. Rollins,1,2 Firdausi Qadri,3 Stephen B. Calderwood,1,2,4
`Ciaran P. Kelly,5 and Edward T. Ryan1,2,6*
`Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts1; Department of Medicine,
`Harvard Medical School, Boston, Massachusetts2; ICDDR,B Centre for Health and Populations Studies,
`Dhaka, Bangladesh3; Department of Microbiology and Molecular Genetics, Harvard Medical School,
`Boston, Massachusetts4; Division of Gastroenterology, Beth Israel Deaconess Medical Center,
`Harvard Medical School, Boston, Massachusetts5; and Department of Immunology and
`Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts6
`
`Received 25 January 2007/Returned for modification 2 March 2007/Accepted 13 March 2007
`
`Clostridium difficile is the leading cause of nosocomial infectious diarrhea. C. difficile produces two toxins (A
`and B), and systemic and mucosal anti-toxin A antibodies prevent or limit C. difficile-associated diarrhea. To
`evaluate whether transcutaneous immunization with formalin-treated C. difficile toxin A (CDA) induces sys-
`temic and mucosal anti-CDA immune responses, we transcutaneously immunized three cohorts of mice with
`CDA with or without immunoadjuvantative cholera toxin (CT) on days 0, 14, 28, and 42. Mice transcutaneously
`immunized with CDA and CT developed prominent anti-CDA and anti-CT immunoglobulin G (IgG) and IgA
`responses in serum and anti-CDA and anti-CT IgA responses in stool. Sera from immunized mice were able
`to neutralize C. difficile toxin A activity in an in vitro cell culture assay. CDA itself demonstrated adjuvant
`activity and enhanced both serum and stool anti-CT IgA responses. Our results suggest that transcutaneous
`immunization with CDA toxoid may be a feasible immunization strategy against C. difficile, an important cause
`of morbidity and mortality against which current preventative strategies are failing.
`
`Clostridium difficile is a spore-forming, gram-positive, anaer-
`obic bacillus and the leading cause of nosocomial diarrhea and
`colitis in the industrialized world. More than 300,000 cases of
`C. difficile-associated diarrhea are reported each year in the
`United States alone (3, 40, 57). Complications of C. difficile-
`associated diarrhea (CDAD)
`include pseudomembranous
`colitis, toxic megacolon, systemic inflammatory response syn-
`drome, and death. Broad-spectrum antibiotic usage, hospital-
`ization, advanced age, and comorbidities increase the risk of
`acquiring CDAD (32–34). Recently, a new, highly virulent
`strain of C. difficile, BI/NAP1/r027, has emerged and has been
`associated with outbreaks of severe nosocomial CDAD (4, 5,
`36, 38, 55). No vaccine effective at preventing C. difficile disease
`is currently commercially available, and measures to prevent C.
`difficile-associated diarrhea through patient isolation and im-
`plementation of hand hygiene and contact precautions have
`had variable and often limited success (2, 12, 24). The ongoing
`increase in the annual reported incidence of nosocomial
`CDAD in the United States may in large part reflect this
`failure of current disease control measures (39).
`C. difficile expresses two major virulence factors, toxin A and
`toxin B. These large toxins (toxin A, 308 kDa; toxin B, 270
`kDa) function as glucosyltransferases that inactivate Rho, Rac,
`
`* Corresponding author. Mailing address: Division of Infectious
`Diseases, Massachusetts General Hospital, Jackson 504, 55 Fruit
`Street, Boston, MA 02114. Phone: (617) 726-3812. Fax: (617) 726-
`7416. E-mail: etryan@partners.org.
`䌤 Published ahead of print on 19 March 2007.
`
`and Cdc42 within eukaryotic target cells, leading to actin po-
`lymerization, opening of tight junctions, and ultimately cell
`death (10, 54). Toxin A initiates intestinal epithelial damage
`and mucosal disruption that allows toxin B to gain access to
`underlying cells (37). A carboxyl-terminal 800-amino-acid por-
`tion of toxin A mediates binding of toxin A to receptors on
`epithelial cell surfaces (11, 30, 52). Monoclonal and polyclonal
`antibodies directed against this receptor-binding region of
`toxin A abrogate toxin activity and prevent clinical disease in
`animals (8, 13, 43). Antibodies against C. difficile are present in
`a majority of adults and older children, and serum immuno-
`globulin G (IgG) antibodies directed against toxin A are asso-
`ciated with protection against CDAD (34, 53). High mucosal
`antitoxin IgA antibody concentrations have also been associ-
`ated with protection against severe or recurrent CDAD (25–
`27, 51, 56).
`Transcutaneous immunization (TCI) involves the needle-
`free application of antigens directly to hydrated skin from
`which the stratum corneum has been gently removed (17, 18,
`23, 42). TCI usually requires the presence of an immunoadju-
`vant, and ADP-ribosylating proteins such as cholera toxin (CT)
`and heat-labile enterotoxin or their derivatives have most com-
`monly been used as immunoadjuvants during TCI (19, 23, 42,
`45, 46). During TCI, cutaneously applied antigens are taken up
`by Langerhans cells in the epidermis, and these cells then
`migrate to regional lymph nodes. Interestingly, TCI induces
`both systemic and mucosal immune responses (6, 22, 23, 28, 41,
`42, 48). TCI has been shown to be safe and effective in animals
`and humans (9, 21, 23, 42, 47, 58). In order to assess whether
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`TCI would induce immune responses against C. difficile toxin
`A, we therefore transcutaneously immunized mice with a tox-
`oid derivative of C. difficile toxin A (CDA), with or without the
`immunoadjuvant CT, and measured systemic and mucosal
`anti-CDA immune responses, including induction of toxin A-
`neutralizing antibodies in immunized mice.
`
`MATERIALS AND METHODS
`
`Preparation of CDA. We purified toxin A from C. difficile strain VPI 10463
`(American Type Culture Collection, VA) as previously described (35). Briefly,
`we fractionated culture supernatants by anion-exchange chromatography using a
`Sepharose column, precipitated toxin A with an acetate buffer, and further
`purified it by fast protein liquid chromatography using a MonoQ column (Phar-
`macia, Piscataway, NJ). We inactivated purified toxin A by formalin treatment,
`using 37% formaldehyde (Sigma Aldrich, St. Louis, MO) at 4°C for 6 days. We
`dialyzed inactivated CDA overnight at 4°C with regenerated cellulose dialysis
`tubing (Spectrum Laboratories, Rancho Dominguez, CA) against a 100-fold
`excess of 100 mM phosphate-buffered saline (PBS) with 0.016% formaldehyde
`and stored the product at 4°C. Prior to use, we concentrated CDA to a final
`concentration of 1 mg/ml by ultrafiltration through a 50-kDa membrane in a
`70-ml concentrator (Amicon, Beverly, MA). We calculated the CDA protein
`concentration using a bicinchoninic acid assay (Pierce Chemical Company,
`Rockford, IL), assessed purity by gel electrophoresis, and confirmed decreased
`toxicity using MRC-5 fibroblast cells in a toxicity assay as described below.
`Toxicity assay. To confirm reduced toxicity of CDA, we grew freshly
`trypsinized MRC-5 cells to confluence in 96-well plates (4 ⫻ 104 cells/well) in
`minimal essential medium (Gibco, Grand Island, NY) containing 10% fetal
`bovine serum for 5 days at 37°C in a 5% CO2 atmosphere. We added the CDA
`preparation to MRC-5 cells starting at 45 ␮g/well and serially diluted threefold
`to 0.9 pg/well. We used toxin A as a control. We incubated cells and CDA or
`wild-type toxin A dilutions at 37°C in a 5% CO2 atmosphere for 48 h, determin-
`ing the proportion of cell rounding every 3 h.
`Serum neutralization assay. To measure the neutralizing activity of sera, we
`used MRC-5 cells in a manner similar to that used in the cytotoxicity assay. We
`incubated twofold dilutions of sera from mice, starting at a 1:50 dilution in
`minimal essential medium containing 10% fetal bovine serum, at 37°C for 1 h
`with C. difficile toxin A at 60 ng/well. We used four times the minimal dosage of
`toxin A in the absence of serum required to cause 100% cell rounding after 48 h
`(0.6-␮g/ml final concentration or 60 ng/well). We used commercially available
`goat anti-C. difficile toxin A (List Biological Laboratories, Campbell, CA), toxin
`A alone, and medium alone as controls. We added toxin-serum mixtures to
`MRC-5 cells, incubated the plates for 24 h, and determined the proportion of cell
`rounding. We defined the neutralization antibody titer as the reciprocal of
`the highest serum dilution that inhibited cell rounding ⬎50%.
`Immunization regimen. We immunized female, 3- to 5-week-old, Swiss Web-
`ster mice (Taconic, Germantown, NY). Animal work was approved by the In-
`stitutional Animal Care and Use Committee. We transcutaneously immunized
`three cohorts of 15 mice each with either 25 ␮g of CT (List Biological Labora-
`tories) or 100 ␮g of CDA or a combination of 25 ␮g of CT and 100 ␮g of CDA.
`We transcutaneously immunized mice on days 0, 14, 28, and 42, as previously
`described (42). Briefly, we shaved a 3- by 5-cm2 area on the dorsa of mice by
`using a clipper with a no. 40 blade (Wahl Clipper Corp, Sterling, IL) and then
`rested the mice for 24 h. Prior to application of antigen, we anesthetized the mice
`with 2,2,2-tribromoethanol (Avertin; Sigma Aldrich) administered intraperito-
`neally at 0.4 mg/g of body weight. We then hydrated the previously shaved area
`of skin with warm water for 5 min. We then removed the stratum corneum by
`gently stroking the hydrated area with 10 strokes of an emery board. We then
`rehydrated the prepared area, applied vaccine antigens, and covered the vacci-
`nation site with hydrated gauze and porous Kendall Curity tape (Fisher Scien-
`tific, Pittsburgh, PA). The following day, we removed the tape and washed the
`dorsa of mice with 1 liter of warm water to remove residual antigen. We also
`immunized a cohort of 15 mice subcutaneously with 25 ␮g of CDA and 2.5 ␮g of
`CT on days 0, 14, 28, and 42.
`Immunological sampling. We collected, processed, and stored blood and stool
`samples from mice on day 0, 12, 26, 40, and 63 as previously described (44). In
`preparing stool specimens, we placed each stool pellet in 1 ml of a 3:1 mixture of
`PBS–0.1 M EDTA containing soybean trypsin inhibitor (type II-S; Sigma Al-
`drich) at a concentration of 0.1 mg/ml and vortexed until the pellet was broken.
`We centrifuged the mixture twice, added 20 ␮l of 100 mM phenylmethylsulfonyl
`fluoride (Sigma) to each 1 ml of final recovered supernatant, and stored samples
`at ⫺70°C for further analysis.
`
`Measurement of immune responses. To detect antibody responses to CDA, we
`coated plates with 100 ng/well of purified C. difficile toxin A in 50 mM carbonate
`buffer, pH 9.6. To detect antibody responses to CT, we coated plates sequentially
`with 1 ␮g of type III ganglioside (Sigma Aldrich) in 50 mM carbonate buffer
`(pH 9.6) and then with 100 ng/well of CT in PBS. We blocked plates with
`PBS–1% bovine serum albumin (BSA) (Sigma Aldrich). To detect anti-CDA
`and anti-CT IgG and IgA responses in serum, we diluted sera 1:1,000 or 1:50
`in PBS containing 0.05% Tween 20 (PBS-T) (Sigma Aldrich), respectively,
`and incubated the plates at 37°C for 1 h. We detected bound antibodies using
`a 1:1,000 dilution in PBS-T of either goat anti-mouse IgG conjugated with
`horseradish peroxidase (HRP) (Southern Biotech, Birmingham, AL) or goat
`anti-mouse IgA conjugated with HRP (Southern Biotech), incubating plates
`for 1 h at37°C. We developed the plates with 2, 2⬘ -azino-bis (3-ethylbenz-
`thiazoline-6-sulfonic acid) (ABTS) (Sigma Aldrich) and 0.03% H2O2 (Sigma
`Aldrich) and determined optical density using a Vmax microplate reader
`(Molecular Devices Corp, Sunnyvale, CA) at 405 nm kinetically for 5 min at
`14-second intervals as previously reported (44). To equilibrate, we divided
`readings of milliunits of optical density per minute for samples by those for
`plate controls comprised of pooled blood or stool standards from unrelated
`experimental cohorts and reported the results as enzyme-linked immunosor-
`bent assay (ELISA) units.
`To detect anti-CDA and anti-CT specific antibodies in stool, we first measured
`total stool IgA. We coated plates with 100 ␮l/well of rat anti-mouse IgA (South-
`ern Biotech) at a dilution of 1:1,000 in 50 mM carbonate buffer, pH 9.6. Fol-
`lowing blocking and washing of plates, we added 100 ␮l/well of a 1:1,000 PBS-
`BSA dilution of the previously prepared mouse stool samples and incubated the
`plates overnight at room temperature. We detected bound antibody using goat
`anti-mouse IgA-HRP conjugate at a dilution of 1:1,000 in PBS-T–0.1% BSA,
`incubating plates for 1 h at37°C. We developed the plates and measured optical
`density as described above. We calculated total stool IgA using a mouse IgA
`standard (Kappa TEPC 15; Sigma). To detect specific anti-CDA or anti-CT
`antibodies in stool, we added 725 ␮g of total stool IgA in PBS-T to wells in
`ELISAs as described above.
`Statistical analysis. For normally distributed data, we used an unpaired
`Student t test analysis for comparison of means; for nonparametric data, we
`used the Mann-Whitney U test. We performed statistical analyses using
`Microsoft Excel 2002 and Statistical Package for Social Sciences (SPSS)
`version 12.0 and plotted graphs using GraphPad Prism (GraphPad Software,
`San Diego, CA).
`
`RESULTS
`
`Preparation of CDA. CDA was 46,000 times less toxic than
`toxin A in a cell-rounding MRC-5 cell assay: after 48 h, toxin
`A was able to cause cell rounding in a cell-rounding MRC-5
`cell assay at a concentration of 0.192 ng/well; after 48 h, for-
`malin-inactivated CDA required 9 ␮g/well to cause similar cell
`rounding. Residual formalin was present in the final CDA
`preparation at 0.016% by volume.
`Systemic and mucosal anti-CDA and anti-CT antibody re-
`sponses in mice transcutaneously immunized with CDA
`and/or CT. TCI of mice with CDA and CT resulted in a
`significant anti-CDA IgG response following the second TCI
`(P ⬍ 0.01) (Fig. 1A). Mice that were transcutaneously immu-
`nized with CDA alone developed a significant serum anti-CDA
`IgG response following the third immunization (P ⬍ 0.01).
`Coadministration of CDA with immunoadjuvantative CT re-
`sulted in a significant increase in the serum anti-CDA IgG
`response by day 63 (following the fourth TCI) in comparison to
`mice that were transcutaneously immunized with CDA alone
`(P ⬍ 0.01). All cohorts of mice that received TCI with CT
`developed prominent serum anti-CT IgG responses following
`the first TCI (P ⬍ 0.001) (Fig. 1B).
`Mice that were transcutaneously immunized with CDA and
`CT developed a significant anti-CDA serum IgA response fol-
`lowing the third TCI (P ⬍ 0.05; Fig. 1C). The concomitant
`administration of CT during TCI with CDA also resulted in a
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`FIG. 1. Serum anti-CDA IgG (A) and anti-CDA IgA (C) responses and serum anti-CT IgG (B) and anti-CT IgA (D) responses in mice
`transcutaneously immunized on days 0, 14, 28, and 42 with CDA alone (CDA TCI), CT alone (CT TCI), or CT and CDA (CT⫹CDA TCI). Results
`were determined by kinetic ELISA and are reported as ELISA units; the geometric mean plus standard error of the mean for each immunization
`cohort is shown.
`
`significant increase in the anti-CDA IgA serum response in
`day 63 samples in comparison to the responses in mice that
`received TCI with CDA alone (P ⬍ 0.05). Anti-CT serum
`IgA responses were present in all cohorts of mice transcu-
`taneously immunized with CT following the second TCI
`(P ⬍ 0.01) (Fig. 1D).
`Comparison of immune responses in mice that were immu-
`nized transcutaneously versus responses in mice that were
`immunized subcutaneously. Comparing responses in day 63
`samples by cohorts of animals grouped by route of immuniza-
`tion, mice that were subcutaneously immunized with CDA and
`CT had a significantly increased serum anti-CDA IgG response
`in comparison to mice that were transcutaneously immunized
`with CDA and CT (P ⬍ 0.01) (Fig. 2A), although anti-CT
`serum IgG responses were comparable in all mice that were
`immunized with CT, either transcutaneously or subcutaneously
`(Fig. 2B). In comparison, mice that were transcutaneously im-
`munized with CDA and CT had a significantly increased day 63
`serum anti-CDA IgA response in comparison to mice that
`were subcutaneously immunized with CDA and CT (P ⬍ 0.05)
`(Fig. 2C). Anti-CT IgA serum responses were also significantly
`increased in mice that were transcutaneously immunized with
`CDA and CT versus the response in mice that were subcuta-
`neously immunized with CDA and CT (P ⬍ 0.001) (Fig. 2D).
`
`We also measured immune responses in stool samples. TCI
`of mice with CDA and CT resulted in a significant anti-CDA
`IgA response in stool (P ⬍ 0.01) (Fig. 3A). Interestingly, mice
`that were transcutaneously immunized with CDA and CT had
`a significantly increased stool anti-CDA IgA response in com-
`parison to mice that were subcutaneously immunized with
`CDA and CT (P ⬍ 0.01). Anti-CT IgA responses in stool were
`also more prominent in mice that were transcutaneously im-
`munized with CDA and CT than in mice that were transcuta-
`neously immunized with CDA alone (P ⬍ 0.001) (Fig. 3B) or
`with CT alone (P ⬍ 0.01). TCI with CDA and CT also resulted
`in more prominent stool anti-CT IgA responses than those
`observed in mice subcutaneously immunized with CDA and
`CT (P ⬍ 0.001).
`Induction of C. difficile toxin A-neutralizing responses. TCI
`with CDA alone resulted in induction of C. difficile toxin A-
`neutralizing serum antibodies (P ⬍ 0.001) (Fig. 4). TCI with
`CDA and immunoadjuvantative CT resulted in an increased
`toxin A-neutralizing response in comparison to the response
`seen in mice transcutaneously immunized with CDA alone
`(P ⬍ 0.001). Subcutaneous immunization with CDA and CT
`resulted in the most prominent toxin A-neutralizing re-
`sponse (P ⬍ 0.001).
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`FIG. 2. Day 63 serum anti-CDA IgG (A), anti-CDA IgA (C), anti-CT IgG (B), and anti-CT IgA (D) responses in mice either transcutaneously
`immunized on days 0, 14, 28, and 42 with CDA alone (CDA TCI), CT alone (CT TCI), or CT and CDA (CT⫹CDA TCI) or subcutaneously
`immunized on days 0, 14, 28, and 42 with CT and CDA (CT⫹CDA SQ). Results were determined by kinetic ELISA and are reported as ELISA
`units; the geometric mean plus standard error of the mean for each immunization cohort is shown.
`
`DISCUSSION
`
`C. difficile is the leading cause of nosocomial infectious di-
`arrhea, with more than 30% of patients admitted to high-risk
`hospital wards acquiring C. difficile in their intestines and 10%
`developing CDAD during hospitalization (31). Recently, the
`emergence of C. difficile strain BI/NAP1/r027 has been associ-
`ated with disease outbreaks, increased severity of CDAD, and
`CDAD that may be less responsive to treatment (4, 5, 36, 38).
`Strain BI/NAP1/027 has also been associated with cases of
`community-acquired CDAD,
`including cases in individuals
`who have not recently received treatment with antimicrobial
`agents. The emergence of BI/NAP1/r027 has been linked to
`the widespread use of fluoroquinolone antibiotics (14), and
`increased virulence of strain BI/NAP1/r027 has been attributed
`to a greater-than-20-fold-increased toxin production compared
`to that of historical strains (55). Strain BI/NAP1/r027 also
`expresses a binary toxin whose contribution to virulence is
`currently unclear (15). Although the spread of C. difficile dis-
`ease can be reduced or prevented by careful adherence to hand
`hygiene and contact precautions among medical personnel and
`by isolation of individuals with CDAD, such control practices
`are costly and have had variable and less-than-optimal results
`
`(2, 12, 24), indicating that evaluation of alternative preventa-
`tive strategies is warranted.
`Studies with humans have shown that protection against
`disease and relapse with C. difficile correlates predominantly
`with the presence of serum antibodies directed against C. dif-
`ficile toxin A and less strongly with anti-toxin B antibody levels
`(25). Individuals with low anti-toxin A antibody levels are at
`increased risk of C. difficile-associated disease and relapse (33,
`34). Studies with humans have also detected anti-toxin A an-
`tibodies in intestinal secretions (26), and mucosal anti-toxin A
`IgA responses contribute to protection against CDAD in ani-
`mal models (16, 51, 56). Currently, no anti-C. difficile vaccine is
`commercially available, although a candidate vaccine has been
`evaluated in phase I and IIa studies with humans (1, 29, 49).
`This vaccine consists of formalin-detoxified C. difficile toxins A
`and B, and parenteral immunizations with this vaccine induce
`anti-C. difficile toxin IgG and toxin-neutralizing antibody re-
`sponses (1, 29). Since TCI is a noninvasive immunization strat-
`egy that induces both systemic and mucosal immune responses,
`we were interested in evaluating whether TCI with CDA could
`induce both systemic and mucosal anti-C. difficile responses.
`We found that TCI with CDA and immunoadjuvantative CT
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`FIG. 3. Day 63 stool anti-CDA IgA (A) and anti-CT IgA (B) responses in mice either transcutaneously immunized on days 0, 14, 28, and 42
`with CDA alone (CDA TCI), CT alone (CT TCI), or CT and CDA (CT⫹CDA TCI) or subcutaneously immunized on days 0, 14, 28s and 42 with
`CT and CDA (CT⫹CDA SQ). Results were determined by kinetic ELISA and are reported as ELISA units; the geometric mean plus standard
`error of the mean each immunization cohort is shown.
`
`induces serum anti-C. difficile toxin A IgG responses following
`two immunizations and induces anti-CT IgG responses follow-
`ing one application. Serum responses against CDA continued
`to increase following subsequent TCIs, although anti-CT re-
`sponses were prominent and plateaued following three TCIs.
`The most prominent serum IgG anti-CDA response occurred
`in mice that were immunized subcutaneously, although serum
`anti-CT IgG responses in mice immunized transcutaneously
`were comparable to responses observed in mice immunized
`subcutaneously.
`Interestingly, parenteral immunization with CDA did not
`induce serum or stool anti-C. difficile toxin A responses, de-
`
`FIG. 4. C. difficile toxin A-neutralizing antibody titers in day 63 sera
`collected from mice transcutaneously immunized with CDA alone
`(CDA TCI), CT alone (CT TCI), or CT and CDA (CT⫹CDA TCI) or
`subcutaneously immunized with CT and CDA (CT⫹CDA SQ). The
`neutralizing titer against toxin A was determined by a cell toxicity assay
`in MRC-5 cells. Results are reported as the geometric mean plus
`standard error of the mean of the reciprocal titer for each immuniza-
`tion cohort.
`
`spite repetitive immunization. In comparison, transcutaneous
`application of CDA with immunoadjuvantative CT resulted in
`anti-C. difficile toxin A in both serum and stool. TCI has pre-
`viously been shown to induce both mucosal and systemic im-
`mune responses (6, 22, 28, 41, 42), including induction of IgA
`antibody-secreting cell (ASC) responses (20). ASC responses
`measure transient migration of activated lymphocytes in pe-
`ripheral circulation prior to lymphocyte homing to mucosal
`surfaces, and ASC responses correlate with development of
`subsequent mucosal immune responses at mucosal surfaces
`(20, 21). The mechanism by which TCI induces mucosal im-
`mune responses is currently unclear.
`Induction of immune responses to antigens applied transcu-
`taneously usually requires coapplication of an immunoadju-
`vant (19). We found induction of anti-C. difficile toxin A IgG
`and neutralizing antibody responses following TCI with CDA
`alone, although coadministration of CDA and immunoadju-
`vantative CT increased the magnitude of the anti-C. difficile
`toxin A IgG and toxin-neutralizing antibody responses. In ad-
`dition, coadministration of antigen and CT resulted in induc-
`tion of anti-C. difficile toxin A IgA responses in both serum and
`stool, and such responses were not induced when CDA alone
`was applied transcutaneously. Mice that were transcutaneously
`immunized with CT and CDA developed more prominent
`anti-CT IgA responses in serum and stool than mice that were
`transcutaneously immunized with CT alone. These observa-
`tions and our detection of anti-C. difficile toxin A responses
`following TCI with CDA alone may reflect immunoadjuvanta-
`tive properties of the carboxyl terminus of C. difficile toxin A
`itself (7).
`We found that TCI with CDA alone or CDA and CT in-
`duced C. difficile toxin A-neutralizing antibody responses in
`serum. Serum C. difficile toxin A-neutralizing responses have
`previously been associated with protection from C. difficile-
`associated disease (16, 29), suggesting that TCI can result in
`protective anti-C. difficile immune responses. The new epi-
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`demic strain of C. difficile, BI/NAP1/r027, has a deletion in a
`regulatory tcdC gene, resulting in increased expression of both
`toxins A and B (38), and antitoxin immune responses would be
`predicted to protect against this newly emergent strain just as
`with other toxigenic strains. BI/NAP1/r027 also expresses a
`binary toxin, an iota-like toxin similar to one produced by
`Clostridium perfringens type E (50); however, the contribution
`of binary toxin to pathogenesis is unclear, since strains of C.
`difficile expressing binary toxin but deficient in toxins A and B
`fail to cause disease in animal models (15).
`In summary, our results suggest that TCI with CDA and
`immunoadjuvantative CT induces not only serum IgG and
`toxin-neutralizing antibody responses but also mucosal anti-C.
`difficile toxin A IgA responses in serum and stool. Our results
`suggest that TCI with CDA may be a feasible immunization
`strategy against C. difficile, an important cause of morbidity
`and mortality against which current preventative strategies are
`inadequate.
`
`ACKNOWLEDGMENTS
`
`This work was supported by funding from NIH grants AI40725 (to
`E.T.R.) and AI53069 (to C.P.K.), by New England Regional Center of
`Excellence/Biodefense and Emerging Infectious Disease Career De-
`velopment Award U54 AI057159 (to S.M.R.), and by Fogarty Inter-
`national Center Global Infectious Disease Training Fellowship Award
`D43 TW05572 (to A.S.).
`We thank Wendy Kallas for assistance with cell culture assays.
`
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