INFECTION AND IMMUNITY, Sept. 2009, p. 3909–3918 Vol. 77, No. 90019-9567/09/$08.00�0 doi:10.1128/IAI.00487-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Protection against Intestinal Amebiasis by a Recombinant Vaccine IsTransferable by T Cells and Mediated by Gamma Interferon�

Xiaoti Guo,1,2 Lisa Barroso,3 Steven M. Becker,1 David M. Lyerly,3 Thomas S. Vedvick,4Steven G. Reed,4 William A. Petri, Jr.,1,2*† and Eric R. Houpt1*†

Division of Infectious Diseases and International Health, Department of Internal Medicine1 and Department of Microbiology,2

University of Virginia, Charlottesville, Virginia 22908; TechLab Incorporated, Blacksburg, Virginia 240603; andInfectious Disease Research Institute, Seattle, Washington 981044

Received 3 May 2009/Returned for modification 16 June 2009/Accepted 22 June 2009

We have previously shown that vaccination with purified Entamoeba histolytica Gal/GalNAc lectin or recom-binant subunits can protect mice from intestinal amebiasis upon intracecal challenge. In this study, wedemonstrated with adoptive-transfer experiments that this lectin vaccine protection is mediated by T cells butnot serum. The cell-mediated immune (CMI) response was characterized by significant gamma interferon(IFN-�), interleukin 12 (IL-12), IL-2, IL-10, and IL-17 production. To move toward a human vaccine, weswitched to a recombinant protein and tested a range of adjuvants and routes appropriate for humans. Wefound that subcutaneous delivery of LecA with IDRI’s adjuvant system EM014 elicited a potent Th1-type CMIprofile and provided significant protection, as measured by culture negativity (79% efficacy); intranasalimmunization with cholera toxin provided 56% efficacy; and alum induced a Th2-type response that protected62 to 68% of mice. Several antibody and CMI cytokine responses were examined for correlates of protection,and prechallenge IFN-�� or IFN-�-, IL-2-, and tumor necrosis factor alpha-triple-positive CD4 cells in bloodwere statistically associated with protection. To test the role of IFN-� in LecA-mediated protection, weneutralized IFN-� in LecA-immunized mice and found that it abrogated the protection conferred by vaccina-tion. These data demonstrate that CMI is sufficient for vaccine protection from intestinal amebiasis and revealan important role for IFN-�, even in the setting of alum.

The enteric protozoan parasite Entamoeba histolytica is thecausative pathogen of amebic dysentery and liver abscess thataffects millions of people worldwide. Bangladeshi children ex-perience a 40% annual incidence of E. histolytica infection(24), and evidence of prior E. histolytica infection can be de-tected in 8.4% of the general population in Mexico (6). De-spite the availability of effective antibiotics, the World HealthOrganization estimates that up to 100,000 deaths occur annu-ally, highlighting the need for alternate approaches to controlamebiasis. One approach is to develop a vaccine to preventintestinal infection (26).

Several vaccine candidates for amebiasis have been proposed(48), including the serine-rich E. histolytica protein, peroxire-doxin, the EhCP112 molecule, and the galactose/N-acetyl-D-galactosamine-inhibitable lectin (Gal/GalNAc lectin). A signifi-cant body of work has focused on the latter: vaccination witheither parasite-purified Gal/GalNAc lectin (10, 29, 32, 38, 40)or recombinant lectin subunits has provided protection in ro-dent models against amebic liver abscess and amebic colitis(29, 37, 46, 47, 53). Although these results are encouraging,two limitations remain. First, in most of these vaccine studies,the adjuvants and delivery routes are not compatible with

eventual use in humans. Second, the mechanisms of amebiasisvaccine-mediated protection are still not fully understood. Forinstance, in the intestinal model, there was an association be-tween the presence of an antiparasite lectin fecal immunoglob-ulin A (IgA) response and subsequent protection, but theassociation did not extend to the recombinant antigen, andfecal IgA-negative mice remained statistically protected, sug-gesting that other immune mechanisms exist (29). Indeed,there is increasing evidence for a role of cell-mediated immu-nity (CMI) in protection from intestinal amebiasis (14, 20, 28).We have found that gamma interferon (IFN-�), the canonicalTh1 cytokine, can clear intestinal amebic infection in CBAmice (21). In this study, we demonstrate for the first time thatCMI plays a critical role in lectin-elicited protective immunityto intestinal amebic infection. A variety of vaccine adjuvantsand delivery routes were tested for their effectiveness in pro-tection and in eliciting CMI, and the tests demonstrated a clearrole for CMI and IFN-� in lectin-based vaccine protection.

MATERIALS AND METHODS

Antigens and immunizations. Groups of 3- to 4-week-old male CBA/J micewere obtained from the Jackson Laboratory to start immunization studies andwere maintained under specific-pathogen-free conditions at the University ofVirginia. The production of the two antigens used in trials 1 to 4, native E.histolytica Gal/GalNAc lectin and the recombinant subunit “LecA” (amino acids578 to 1154 of the lectin heavy chain), have been described previously (29). Fortrials 5 to 7, LecA was purified using immobilized metal affinity chromatography(IMAC). Briefly, the cells were lysed by sonication and treated with 0.7% NP-40and 0.7% sodium deoxycholate (Sigma-Aldrich). The isolated inclusion bodieswere washed with 0.05% Triton X-100 and denatured in 8 M urea containing 50mM dithiothreitol. To renature the proteins, the solution was diluted 10-fold andthe pH was gradually titrated from 11 to 7.5. IMAC purification was performed

* Corresponding author. Mailing address: Division of InfectiousDiseases and International Health, Department of Internal Medicine,University of Virginia, Charlottesville, VA 22908. Phone for Eric R.Houpt: (434) 243-9326. Fax: (434) 924-0075. E-mail: erh6k@virginia.edu. Phone for William A. Petri, Jr.: (434) 924-5621. Fax: (434) 924-0075.E-mail: wap3g@virginia.edu.

† W.A.P. and E.R.H. contributed equally to this study.� Published ahead of print on 29 June 2009.

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with a HisPrep 16/10 Fast Flow column (GE Healthcare), which eluted LecAwith 300 mM imidazole (Sigma-Aldrich).

Mice were immunized with either native Gal/GalNAc lectin or recombinantLecA with the various regimens specified in Table 1. Briefly, our original proof-of-principle regimen, consisting of three intranasal immunizations and one in-traperitoneal immunization at 2-week intervals (29), was administered in trials 1to 5 (Table 1). In trial 5, LecA was formulated with the following adjuvant(s):IDRI adjuvant system EM014, which consisted of 1 mg/ml of synthetic lipid A,a TLR4 agonist, mixed with 1.25 mg/ml CpG of mouse origin in a stable emul-sion; complete (CFA) and incomplete (IFA) Freund adjuvant (Gibco); alum(Rehydragel LV; Reheis, Inc.); or cholera toxin (CT; Sigma-Aldrich). In trial 7,LecA was formulated in the IDRI adjuvant system Al001 (synthetic lipid Abound to the alum) or in LecA alone at 4-week intervals. Formulations withdifferent adjuvants were made as follows: for alum and Freund adjuvants, 1 partof adjuvant was mixed with 1 part of antigen (vol/vol) in a total volume of 100 �l.For EM014 and A1001, 20 �g of LecA was formulated with 20 �g of adjuvantand brought up to 100 �l with phosphate-buffered saline (PBS). For CT, 20 �gof LecA was mixed with 2 �g of adjuvant. Immunizations were administeredsubcutaneously (EM014, alum, A1001, CFA, and IFA), intranasally (CT) afterlight isofluorane anesthesia, or intrarectally (CT) after light isofluorane anesthe-sia, and animals were boosted by the same route used for the priming dose(Table 1). Endotoxin levels of purified proteins were monitored with the En-dosafe PTS instrument (Charles River Laboratory) and ranged from 0.021 en-dotoxin units (EU)/�g to 0.079 EU/�g. In all sham-immunized mice, the amountof lipopolysaccharide (Sigma-Aldrich) contained in 20 �g LecA was included asa control.

Measurement of immunogenicity. To assess the antigen-specific antibody re-sponses, tail vein sera and stool samples were obtained within 1 to 2 weeks afterthe final immunization (week 7 or 8). The preparation of stool suspensions wasdescribed previously (29). Both serum and fecal preparations were stored at�20°C prior to use. Antigen-specific fecal IgA and serum IgG to native lectin andLecA were assayed by enzyme-linked immunosorbent assay (ELISA). Briefly,ELISA plates were coated with 0.5 �g/well of native lectin or 0.2 �g/well of LecAin PBS at 4°C overnight and then blocked with 2% bovine serum albumin in PBSfor 1 h at 37°C. After three washes with PBS-Tween buffer, the plates wereincubated with fecal samples (100 �l per well) diluted 1:4 in PBS-Tween con-taining 2% bovine serum albumin. Horseradish peroxidase-conjugated goat anti-mouse IgA (Southern Biotechnology Associates) diluted 1:5,000 was added se-quentially. Antigen-specific antibodies were detected with 100 �l per well of

tetramethylbenzidine microwell peroxidase substrate (Kirkegaard & Perry Lab-oratories). The reaction was stopped with 100 �l per well of sulfuric acid stopsolution, and the absorbance was read at 450 nm. The IgG titer was determinedby end-point dilution. The serum IgG subtypes were determined by the ELISAdescribed above, except that 1:10,000-diluted horseradish peroxidase-conjugatedgoat anti-mouse IgG1 and IgG2a (Southern Biotechnology Associates) wereused as secondary antibodies. IgG1 and IgG2a units were determined by use ofa standard curve.

To assess antigen-specific CMI, spleens from three or four mice per groupwere harvested within 1 to 2 weeks after the final immunization and processedfor single-cell suspensions after lysis of red blood cells. Splenocytes (2 � 105)were cultured in 96-well plates (Costar) in RPMI 1640 medium (Invitrogen)supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 �g/ml strep-tomycin, 100 �g/ml gentamicin, and 50 �M beta-mercaptoethanol. Cells werestimulated with 2 �g/ml lectin or LecA for antigen-specific CMI. To analyze theperipheral blood mononuclear cell (PBMC) response, each individual mouse wasbled via the tail vein and about 250 �l whole blood was treated with 0.84%NH4Cl buffer to lyse the red blood cells. After two washes, 5 � 105 PBMCs werecultured in 24-well plates and stimulated with 2 �g/ml of LecA. One microgramof concanavalin A (ConA) was set up as a positive control, and medium alonewas used as a negative control in the CMI studies. Supernatants were collectedafter 48 h and analyzed by a multiplex suspension array system using Luminexbeads (Bio-Rad Laboratories), which included the following cytokines: Th1(IFN-�, interleukin-2 [IL-2], IL-12p35, and tumor necrosis factor alpha [TNF-�]), Th2 (IL-4, IL-5, and IL-13), Th17 (IL-17), and IL-10. Samples were runwithout dilution according to the manufacturer’s protocol and measured inpicograms per milliliter of supernatant.

Intracellular cytokine staining of PBMCs. A total of 5 � 105 PBMCs werecultured in complete Dulbecco’s modified Eagle medium (Invitrogen) and stim-ulated with 10 �g/ml of LecA for 22 h. Anti-CD28 and Golgiplug (BD Bio-sciences) were added in the last 12 h of stimulation. Phorbol myristoyl acetate (50ng/ml) and ionomycin (800 ng/ml) (Sigma-Aldrich) were used as positive controlsto assess the cytokine production capacity for each sample.

For intracellular staining, PBMCs were washed and incubated with CD16/CD32 monoclonal antibody (MAb; BD Biosciences) to block Fc binding, fol-lowed by surface staining with CD4-phycoerythrin (BD Biosciences). Cells werepermeabilized and stained with IFN-�–fluorescein isothiocyanate (BD Bio-sciences), IL-2–peridinin chlorophyll protein–Cy5.5 (eBioscience), and TNF-�–

TABLE 1. Vaccine regimens

Trial Antigena No. of micebSchemec

Wk 0 Wk 2 Wk 4 Wk 6–8d

1 Lectin 16 CT (i.n.) CT (i.n.) CFA (i.p.) CT (i.n.)

2 Lectin 20 CT (i.n.) CT (i.n.) CFA (i.p.) CT (i.n.)

3 LecA 16 CT (i.n.) CT (i.n.) CFA (i.p.) CT (i.n.)

4 LecA 16 CT (i.n.) CT (i.n.) CFA (i.p.) CT (i.n.)

5 LecA 20 CT (i.n.) CT (i.n.) CFA (i.p.) CT (i.n.)20 EM014 (s.c.) EM014 (s.c.) EM014 (s.c.) EM014 (s.c.)20 Alum (s.c.) Alum (s.c.) Alum (s.c.) Alum (s.c.)20 CFA (s.c.) IFA (s.c.) IFA (s.c.) IFA (s.c.)20 CT (i.n.) CT (i.n.) CT (i.n.) CT (i.n.)20 CT (i.r.) CT (i.r.) CT (i.r.) CT (i.r.)

6 LecA 60 EM014 (s.c.) EM014 (s.c.) EM014 (s.c.) EM014 (s.c.)60 Alum (s.c.) Alum (s.c.) Alum (s.c.) Alum (s.c.)

7 LecA 40 Al001 (s.c.) Al001 (s.c.) Al001 (s.c.)40 Alum (s.c.) Alum (s.c.) Alum (s.c.)40 No adjuvant No adjuvant No adjuvant

a In trials 1 to 3, 10 �g of antigen was delivered per dose; trials 4 to 7 used 20 �g of antigen per dose.b Sham-immunized mice were administered identical regimens of PBS with adjuvants in each trial.c Schemes are shown as adjuvant (route). EM014, synthetic lipid A and CpG mixed with stable emulsion; Al001, synthetic lipid A bound to the alum; i.n.,

intranasal; i.p., intraperitoneal; s.c., subcutaneous; i.r., intrarectal.d In trials 1 to 6, the last dose of vaccine was given in week 6; in trial 7, the last dose was given in week 8.

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allophycocyanin (eBioscience) and run on a BD FACSCalibur (BD Biosciences).The data were analyzed with FlowJo software.

Parasites and intracecal inoculation. Immunized CBA mice were challengedintracecally with E. histolytica trophozoites 4 weeks after the final boost. Tro-phozoites were originally derived from laboratory strain HM1:IMSS (ATCC)that were sequentially passed in vivo through the mouse cecum. Cecal contentswere cultured in trypsin-yeast-iron (TYI-S-33) medium supplemented with 2%Diamond vitamins (JRH Biosciences), 13% heat-inactivated bovine serum (Sig-ma-Aldrich), and 100 U/ml penicillin plus 100 �g/ml streptomycin. For all in-tracecal inoculations, trophozoites were grown to the log phase and 2 � 106

trophozoites in 150 �l were injected intracecally after laparotomy as describedpreviously (30).

Evaluation of amebic infection. Mice were sacrificed 7 to 11 days after chal-lenge. Half of the cecum was placed in Bouin’s fixative (Sigma-Aldrich) andprocessed for hematoxylin and eosin staining. Pathology was scored in a blindmanner as described before (30), based on the number of visible ameba anddegree of inflammation (scale of 0 to 5). The other half of each cecum was rinsedwith PBS and stored in RNAlater (Ambion) for quantitative reverse transcrip-tion-PCR analysis. Three hundred microliters of cecal rinse was cultured incomplete TYI-S-33 medium with supplemental antibiotics for 5 days at 37°C.

Adoptive-transfer experiments. Mice were immunized with native Gal/GalNAclectin per trial 2 (Table 1). Two weeks after the final immunization, lectin-immunized and PBS-treated mice were bled for sera and sacrificed to obtainsplenocytes and mesenteric lymph node cells. Pooled single-cell suspensionsfrom either lectin- or PBS-immunized donors were enriched for CD3� T-cellpopulations by negative selection (EasySep mouse T-cell enrichment kit; Stem-Cell Technologies). To determine the efficiency of T-cell enrichment, the donorlymphocytes were stained with phycoerythrin-conjugated anti-B220 (BD Bio-sciences) and fluorescein isothiocyanate-conjugated anti-CD3 (BD Biosciences)antibodies by flow cytometry before and after T-cell enrichment. For T-celltransfer, 4 � 107 enriched donor cells were injected intravenously into naiveCBA mouse recipients. For serum transfer, recipients received 400 �l serumintraperitoneally. Both T-cell and serum recipients were challenged 3 days aftertransfer and sacrificed 10 days postchallenge. The lectin-specific CMI in recipi-ents was assessed 3 days after transfer to confirm successful reconstitution. Insome MAb experiments, monoclonal IgG or IgA was purified from cell bags bythe UVA Hybridoma Center and administered intraperitoneally or intracecallyat the doses indicated in Results. Some of the serum and MAb transfer exper-iments utilized C3H/HeJ mice purchased from the Jackson Laboratory.

Cytokine depletion in vivo. For CBA mice that had completed four doses ofvaccination, 1 mg of anti-IL-4 (11B11) or anti-IFN-� (XMG1.2) MAb or acontrol rat IgG (Lampire Bio lab) was administered intraperitoneally every 3days, from 3 days before to 6 days after challenge (four doses total). The micewere sacrificed at day 8 for evaluation of the infection and vaccine efficacy.

Statistical analysis. All statistical analyses were performed using GraphPadPrism for Windows, version 5.0 (GraphPad Software). Student’s t tests were usedto compare values for vaccinated and control groups or protected and unpro-tected individuals. Two-tailed P values of �0.05 were considered significant.

RESULTS

Gal/GalNAc lectin-mediated protection is transferable by Tcells but not by serum. We have previously reported thatpurified Gal/GalNAc lectin protects the C3H mouse from in-testinal amebiasis (29). In this study, we first confirmed theeffectiveness of this vaccine in male CBA mice, which offer amore-efficient model since they exhibit a slightly greater sus-ceptibility to infection (3). As expected, native lectin protectedCBA mice from amebic infection, as measured by cecal cul-ture, with 71% efficacy (P of �0.001 in comparison with con-trols) (Fig. 1A). As in previous work, histopathology mirroredthe culture results, and there were no significant differences ininflammation scores between infected mice from the shamgroup and those from the vaccinated group for this or any ofthe trials (data not shown).

To determine the mechanisms underlying Gal/GalNAc lec-tin-mediated protection, adoptive-transfer experiments wereperformed to discern whether Gal/GalNAc lectin-induced pro-

tection could be transferred by T cells or serum antibodies.Lymphocytes and sera were obtained from lectin-immunizedor PBS-treated mice. The donor lymphocytes were enrichedfor CD3� T cells from 33% to 95% per the methods describedin the legend to Fig. 1B. Purified T cells or sera from lectin-vaccinated or PBS-treated donors were intravenously trans-ferred to naive recipients. Successful transfer was confirmed bydocumenting the transfer of lectin-specific CMI and serumresponses (e.g., IFN-� production, 298 � 88 pg/ml in spleno-cytes of vaccine T-cell recipients versus 7 � 2 pg/ml in spleno-cytes from sham T-cell recipients; P � 0.05). Three days aftertransfer, recipients of either T cells or sera were challengedwith E. histolytica and sacrificed at day 10 to assess protection.Mice that received lectin-immunized donor T cells exhibited alower culture positivity rate than the sham-immunized donorT-cell recipients (Fig. 1C). In contrast, we did not observeprotection by passive immunization with serum (Fig. 1C). Ofnote, recipients of sera from sham-immunized mice exhibited asurprisingly low infection rate, which appeared to be real sincewe obtained an identical result previously in C3H/HeJ mice(44% culture positivity in immune serum recipients versus 21%in sham serum recipients [n - 16 and 14 mice, respectively]; theP value was not significant), and may reflect a protective effectof nonspecific immunoglobulin or other serum components. Insupport of the lack of protection by immune sera, we were alsounable to confer protection using antilectin MAbs: transfer ofclones 7F4, 1G7, and H85, with 0.33 mg of each per mouse,yielded protection for 8/15 culture-positive mice versus 8/20mice treated with control IgG (data not shown). Similarly,administration of 500 �g (intraperitoneally) plus 340 �g (in-tracecally) of a multimeric antilectin monoclonal IgA did notconfer protection (observed for 9/13 culture-positive mice thatreceived antilectin IgA versus 6/9 control IgA recipients; datanot shown). These adoptive-transfer results indicate that lec-tin-specific T cells were sufficient for vaccine protection.

Antibody and CMI induced by Gal/GalNAc lectin with theCT-CFA regimen. Lectin vaccination (trial 1) elicited high lev-els of antigen-specific fecal IgA (Fig. 2A) and serum IgG (datanot shown), yet there was no correlation of fecal IgA level withprotection (Fig. 2A) (29). Since protection was transferredfrom an immunized to a naïve mouse by T cells, we focused onthe cell-mediated response to vaccination. Splenocytes fromvaccinated mice produced higher levels of IFN-�, IL-12, IL-2,IL-10, and IL-17, but not IL-4, than sham-immunized controlsin response to Gal/GalNAc lectin (Fig. 2B). Furthermore, cellsfrom lectin-immunized mice exhibited greater IFN-�, IL-12,and IL-17 production with ConA stimulation than those fromsham-immunized mice, suggesting some intrinsic immuno-stimulatory properties of native lectin itself. Mesenteric lymphnode cells were also assayed and exhibited a CMI patternsimilar to that of splenocytes (data not shown).

LecA recapitulated the protection and immunogenicity oflectin with a CT-CFA regimen. Although native Gal/GalNAclectin has proven effective, its usefulness as a vaccine antigen islimited and therefore we moved our vaccine work toward abacterially expressed subunit of the Gal/GalNAc lectin, termed“LecA.” LecA was expressed and purified from E. coli usingIMAC purification and contained not only the major LecA75-kDa band, but additional proteins of approximately 250,150, 60, and 40 kDa (Fig. 3A, Coomassie). Western blot anal-

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ysis using native lectin-specific MAbs (3F4 and 7F4) reactedstrongly with the major band and also with each minor band,suggesting that these proteins are derived from LecA (Fig. 3A,3F4 MAb and 7F4 MAb). Based on densitometry scans, theimmunoreactive bands comprise 95% of the total protein.Negative-control lysates did not react with any of the bands.Silver stain analysis showed the same protein banding patterndemonstrated by Coomassie blue staining (data not shown),indicating the absence of significant contamination of nonre-active bands. Endotoxin levels of purified LecA ranged from0.021 EU/�g to 0.079 EU/�g. In all sham-immunized mice, thesame amount of lipopolysaccharide was included as a control.

CBA mice were immunized with LecA with the original CT-

CFA regimen and challenged intracecally with E. histolytica tro-phozoites. As shown in Fig. 3B, LecA provided protection inCBA mice (P � 0.05 in comparison with controls) with 57%efficacy. LecA also elicited high levels of antigen-specific fecal IgA(Fig. 3C) and serum IgG (with a predominance of IgG2a overIgG1) (data not shown), the levels of which did not correlate withprotection. The splenocyte CMI response in LecA-vaccinatedmice was similar to that seen with lectin, with significantly higherlevels of IFN-�, IL-12, IL-2, IL-10, and IL-17 than in the sham-immunized controls. The production of the Th2 cytokine IL-4 byvaccinated splenocytes was comparable to that of control cells,without significant elevation (Fig. 3D). Altogether, our data indi-cated that the LecA vaccination with the original CT-CFA regi-

FIG. 1. Gal/GalNAc lectin-mediated protection is adoptively transferred by T cells, not serum antibodies. Purified Gal/GalNAc lectin-immunized (closed bars) and PBS sham-immunized (open bars) CBA mice were challenged with E. histolytica trophozoites 4 weeks after the finalboost. (A) The infection rate was determined by culture positivity and is indicated in the graph as the number of infected mice/total number ofmice. Vaccine efficacy was calculated as 100 � [1 � (percentage of vaccinated mice with infection/percentage of sham mice with infection)].(B) Splenocytes and mesenteric lymph node cells were isolated from lectin-immunized or PBS sham-immunized mice and enriched for CD3� Tcells. The purity of enriched cells was determined by fluorescence-activated cell sorter staining for CD3 and B220 before and after enrichment.(C) T cells or sera from vaccinated or control donor mice were transferred to naive recipients, which were challenged with E. histolyticatrophozoites 3 days after transfer. The recipients were sacrificed 10 days postchallenge to assess protection, as determined by culture of the cecalcontents. **, P � 0.01; ***, P � 0.001. n.s., not significant.

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men recapitulated the protection and immunogenicity profiles ofnative lectin.

LecA-mediated protection by different formulations. LecAadministered with the combination of mucosal (CT) and sys-temic (CFA) adjuvants effectively protected the mice fromamebic colitis, but the contribution of mucosal versus systemicroutes to protection was not clear. To address the roles ofadjuvants and route of immunization in protection, a variety ofimmunization schemes were evaluated (trial 5) (Table 1).Based on the standard measure of culture positivity upon sac-rifice, several schemes were protective, including LecA admin-istered via the original intranasal CT/intraperitoneal CFA reg-imen, intranasal CT, and subcutaneous immunization with theadjuvants EM014, alum, and Al001 (Fig. 4). There were dif-ferences in the calculated efficacy rates (79% for EM014, 62 to68% for alum), but these differences were not statistically sig-nificant at these sample sizes. CFA/IFA administered subcu-taneously failed to provide statistically significant protectioncompared to the level for sham-immunized controls, suggest-ing that the effect of CT used intranasally in the original pro-tective intranasal CT/intraperitoneal CFA regimen was addi-

tive if not critical. As for CT adjuvant alone, the intranasallyadministered vaccine reduced the infection rate by 56% (P �0.05), while the intrarectally administered vaccine failed toprotect, which could reflect the immunologic superiority of thenasal mucosa for this vaccine or simply technical difficulties inkeeping antigen in the lumen of the rectum. Interestingly,though not statistically significant, subcutaneous delivery ofLecA alone, without adjuvant, lowered the infection rate (29%efficacy relative to the level for alum/PBS control mice; P 0.10). Finally, simplification of the immunization scheme tothree monthly doses did not diminish the efficacy of LecA plusalum (Fig. 4). Of note, for all experiments illustrated in Fig. 4,the histological ameba scores and inflammation scores ob-tained were consonant with the culture positivity data andrevealed no significant differences in infected mice from thesham and vaccinated groups (data not shown).

Immunogenicity of different regimens and surrogate mark-ers for protection. Prechallenge sera, stool, splenocytes, andPBMCs were obtained from mice at 2 to 3 weeks after the finalimmunization to assess the LecA-specific immunogenicity ofthese different schemes. For LecA-specific CMI, cytokine re-

FIG. 2. Fecal IgA and splenocyte cytokine response induced by Gal/GalNAc lectin with the CT-CFA regimen. (A) Gal/GalNAc lectin-immunized and PBS sham-immunized CBA mice were assessed for prechallenge antilectin fecal IgA. The vaccinated and infected mice (numberof vaccine failures, 4) are represented by filled circles, whereas vaccinated and uninfected mice (number of protected mice, 16) are representedby open circles. (B) For lectin-specific CMI, three or four mice per group were sacrificed prior to challenge and splenocytes were in vitro stimulatedwith lectin for production of IFN-�, IL-12, IL-2, IL-10, IL-17, and IL-4. ConA-stimulated cells were used as positive controls, while medium aloneserved as a negative control. Filled bars, cells from lectin-immunized animals; open bars, cells from PBS control animals. *, P � 0.05; **, P � 0.01;***, P � 0.001.

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sponses were measured in splenocytes, PBMCs, and mesen-teric lymph node cells. Across all the trials, the Th1 adjuvantEM014 induced the highest level of IFN-� release in spleno-cytes (Fig. 5A) or PBMCs (Fig. 5C); other Th1 cytokines,including IL-12 and TNF-�, were also detected at the highestlevel in the EM014 group (data not shown). A modest increasein splenocyte-produced IL-4 was observed in the EM014 andalum groups (Fig. 5B); however, alum appeared to induce themost IL-4 secretion by PBMCs (Fig. 5D). We also measuredIL-17 production by splenocytes, mesenteric lymph node cells,and PBMCs and found that vaccination with CT via mucosalroutes (CT-CFA and CT intranasally and CT intrarectally)induced strong IL-17 production by all three cell populations.CT administered intranasally elicited the highest level of fecalIgA (Fig. 5E). Serum IgG subtype was evaluated as a surrogate

marker of Th1/Th2 responses. A predominance of IgG2a overIgG1 was observed in the EM014, CT-CFA, and CT (intrana-sal) groups, with EM014 exhibiting the most pronouncedIgG2a bias. In contrast, alum induced anti-LecA IgG1 withoutdetectable IgG2a, consistent with its Th2 adjuvant nature (4, 5,45, 52). CFA/IFA elicited comparable levels of the IgG sub-types, and the CT (intrarectal) route failed to induce any de-tectable serum IgG response (Fig. 5F). When the immunoge-nicity data were compared between protected and infectedmice within each group, none of these measures appeared tooffer a clear correlate for protection (Fig. 5C to F). In contrast,the frequency of postvaccination but prechallenge IFN-�� cy-tokine-positive CD4 T cells, and IFN-��, IL-2�, and TNF-��

CD4 T cells in blood (Fig. 5G and H), correlated with vaccineprotection (P � 0.05).

FIG. 3. LecA recapitulated the protection and immunogenicity of lectin with the CT-CFA regimen. CBA mice were immunized withrecombinant LecA using the CT-CFA regimen and challenged with E. histolytica trophozoites 4 weeks after the final boost. (A) Bacteriallyexpressed LecA was purified and run in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel stained with Coomassie blue. Westernblot analysis showed reactivity of anti-native lectin MAbs (3F4 and 7F4) to the LecA bands. Based on densitometry scans, the immunoreactivebands comprise 95% of the total protein. (B) The infection rate was determined by culture positivity. (C) The prechallenge anti-LecA fecal IgAwas assessed, and vaccinated and infected mice (vaccine failures) are represented by filled circles, whereas vaccinated and uninfected (protected)mice are represented by open circles. (D) For LecA-specific CMI, three or four mice per group were sacrificed prior to challenge and splenocyteswere in vitro restimulated with LecA for production of IFN-�, IL-12, IL-2, IL-10, IL-17, and IL-4. ConA-stimulated cells were used as positivecontrols, while medium alone served as a negative control. *, P � 0.05; **, P � 0.01; ***, P � 0.001. Filled bars, cells from lectin-immunizedanimals; open bars, cells from PBS control animals.

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IFN-� is important in LecA vaccine-mediated protection.We hypothesized that IFN-� was playing a crucial role invaccine-mediated protection, based on vaccine protection be-ing transferred by T cells, the high IFN-� responses with thehigh-efficacy EM014 vaccine, and the correlation with IFN-��

CD4 T-cell frequency. We tested the role of IFN-� in protec-tion conferred by the different vaccine formulations (Fig. 6).Interestingly, anti-IFN-� abrogated protection for all of thevaccines. Specifically, EM014- or CT-CFA-immunized miceadministered anti-IFN-� lost the statistically significant protec-tion that the immunized/control IgG-treated mice exhibited. Itwas not possible to make this conclusion for alum, since theprotection in immunized/control IgG-treated mice was notquite statistically significant (as was seen for the non-IgG-treated controls). Yet by a simpler comparison, the culturepositivity rate of the immunized/anti-IFN-�-treated mice washigher than that of the immunized/IgG-treated mice for eachof the EM014, CT-CFA, and alum groups, defining an impor-tant role for IFN-� across these three protective vaccine regi-mens. Not surprisingly, as we previously showed, anti-IL-4cleared infection in sham-immunized mice (21) and had noadditional impact on the vaccinated animals (Fig. 6, anti-IL-4columns).

DISCUSSION

There are two main findings from this work: T cells aresufficient to provide vaccine protection against intestinal ame-biasis, and IFN-� is required for this protection.

The finding that CMI is protective in this model was some-what surprising, since there was substantial anticipation thatthe presence of secretory IgA would be critical. In severalhuman studies, fecal antilectin IgA to lectin or its subunits wasassociated with a decreased risk of amebic reinfection (1, 24,25, 27, 42). In vitro, antibodies to lectin diminish adherence tocolonic mucins (9), providing a putative mechanism for anti-body-based protection. However, the vaccines tested here pro-vided protection with no clear correlation between levels ofantigen-specific fecal IgA or serum IgG and subsequent pro-

tection. Furthermore, passive transfer experiments using eitherimmune serum or monoclonal antilectin IgA/IgG did not dem-onstrate protection.

The mouse model has increasingly shown that CMI re-sponses can contribute to both protection and disease, in thatCD4 cells can promote the severity of colitis in C3H mice (30)and, conversely, can mediate clearance, in that anti-IL-4 treat-ment clears infection in CBA mice via an IFN-�-dependentmechanism (21). Human data also suggest that high parasite-specific IFN-� production by PBMCs is associated with pro-tection against invasive amebiasis (28). In this context, it is notsurprising that CMI is protective. Whether the mechanism ofCMI protection is through outright prevention of the estab-lishment of infection, or through rapid clearance of establishedinfection, is unknown.

Though we have not established the cytokine requirementsof the T cells for protection, all evidence points to IFN-�. Thisis best argued from the IFN-� depletion studies (Fig. 6) andthe correlation of protection with vaccine-induced IFN-��

CD4 T cells (Fig. 5). Furthermore, several protective vaccines(EM014 plus LecA, CT-CFA plus lectin, and LecA alone),including the CT-CFA plus lectin vaccine that was documentedto be CMI transferable, exhibited strong antigen-specificIFN-� production. The Th1 polarity of synthetic lipid A-basedadjuvant systems is well appreciated (34, 39, 50, 51). As for thedownstream mechanism of IFN-�-based protection, there areseveral possibilities. The simplest explanation is that IFN-�activates macrophages and neutrophils for E. histolytica killing,which has been shown in vitro (13, 36). Alternately, it is pos-sible that IFN-� exerts protective effects (e.g., inducing pro-duction of chemokines [15, 18] and mucosal defense molecules[16]) on the epithelium, which bone marrow chimera experi-ments indicate is a critical cell type in the mouse strain-specificresistance to intestinal E. histolytica infection (23).

It is surprising that IFN-� was required for vaccine protec-tion with alum-based LecA vaccines, since alum is a Th2 typeadjuvant and only a marginal level of IFN-� was detected inour alum-immunized PBMCs or splenocyte supernatants.However, there was a significant frequency of LecA-specific

FIG. 4. Protection by LecA formulated with various regimens. CBA mice were immunized at either weeks 0, 2, 4, and 6 (four doses) or weeks0, 4, and 8 (three doses) with LecA formulated with EM014, alum, CFA and IFA, CT, Al001, or LecA alone as indicated on the x axis. Vaccinatedand control mice were challenged 4 weeks after the final immunization and sacrificed at day 7 postchallenge. Levels of protection (infection rates)were determined by culture of cecal contents. *, P � 0.05; ***, P � 0.001. i.n., intranasal; i.r., intrarectal.

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IFN-�-positive CD4 cells in peripheral blood postvaccination,and this response correlated with vaccine-induced protection.Moreover there is precedent for IFN-� in alum-based protec-tion. For instance in an alum-based respiratory syncytial viruspeptide vaccine, loss of protection was seen in mice treatedwith IFN-� neutralizing antibody, and purified CD4 T cellsfrom vaccinated IFN-� knockout donors failed to transfer pro-tection (41). Other vaccine studies employing alum as an ad-juvant against Toxoplasma gondii (11) and Streptococcus pneu-moniae (17) infection also suggested a possible role of IFN-�response in protection.

As for how the IFN-� was elicited during alum vaccination,this response may be due to the LecA antigen itself. First,IFN-� production seemed to be promoted by LecA, in that

ConA-stimulated cells from our LecA-immunized mice pro-duced more IFN-� than cells from sham-immunized mice. Fur-thermore, the frequency of IFN-�-secreting CD4 T cells in theLecA-only group was significantly higher than that observedfor the control. There is a long precedent for the potential Th1induction of Gal/GalNAc lectin, with several laboratories (8,31, 43, 44) reporting that native lectin contains epitopes thatstimulate IL-12 and TNF-� in macrophages and dendritic cells,particularly amino acids 596 to 1082, which are located withinLecA. Such a combination of Th1 stimulus and alum has en-hanced the efficacy of vaccines against human immunodefi-ciency virus (33), Leishmania major (22, 35), malaria (49), andSchistosoma mansoni (2). Perhaps the LecA afforded a fre-quency of IFN-�-secreting CD4 cells in the LecA-alum vaccine

FIG. 5. Immunogenicity across different adjuvants and the correlate markers for protection. Mice were immunized with LecA formulated withEM014, alum, CFA and IFA, CT, or original CT-CFA adjuvants via different routes. The prechallenge immunogenicities of six regimens wereassessed for LecA-specific IFN-� and IL-4 produced by splenocytes (A and B) and PBMCs (C and D). Anti-LecA fecal IgA (E) and the serumIgG2a/G1 ratio (F) were also measured. For LecA-specific IFN-�-producing CD4 (G) and IFN-�� IL-2� TNF-�� multifunctional CD4 T cells (H),PBMCs were stimulated with LecA for 22 h. The frequency of cytokine-producing cells was determined by flow cytometry on CD4-gated cells. Inpanel A, the only statistically significant difference in IFN-� production levels was between EM014 and the control. In panels C to H, vaccinatedand infected mice (unprotected) in each group are represented by filled circles, whereas vaccinated and uninfected (protected) mice arerepresented by open circles. In panels G and H, protected mice in each group were compared to unprotected mice for statistical analysis. *, P �0.05; **, P � 0.01; ***, P � 0.001. i.n., intranasal; i.r., intrarectal.

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that was above a certain threshold for protection (�20%),even though the total quantity detected in the LecA-alumPBMC supernatants was marginal (and not predictive).

The IFN-�-producing, or triple IFN-�-, TNF-�-, and IL-2-producing, CD4 T-cell frequency served as the one meaningfulmarker for the protection observed. The so-called “multifunc-tional CD4” T cells that secrete IFN-�, TNF-�, and IL-2 con-cordantly have been shown to correlate with protection fromLeishmania major (12) and Mycobacterium tuberculosis (19)better than a single marker alone. We examined them in E.histolytica because TNF-� and IFN-� synergistically activatemacrophages for trophozoite killing (36) and IL-2 is associatedwith resistance to reinfection in amebic liver abscess models(7). The additive value of TNF-� and IL-2 to IFN-� is unclear,however, since the frequency of triple cytokine production wasno more correlative than IFN-� production alone.

In conclusion, we have demonstrated that protection by Gal/GalNAc lectin-derived vaccines was transferred to naive ani-mals by the transfer of immune T cells and that IFN-� wasessential for protection. The frequency of IFN-�-producingCD4 T cells in blood stands as a useful surrogate marker foralum-based LecA-mediated protection. The demonstration ofefficacy by a vaccine utilizing alum and the delineation of theprotective role of IFN-� represent important steps toward ahuman amebiasis vaccine.

ACKNOWLEDGMENTS

This research was supported by U.S. National Institutes of Healthgrants U01 AI070384 (to W.A.P.) and R01 AI071373 (to E.R.H.).

We thank members of the Advisory Committee for grant U01 (AlanShaw, Brian Kelsall, Myron Levine, Steven Reed, and Annie Mo) forvaluable input. We thank Suzanne Stroup for assistance in amebatrophozoite culture and Caitlin Foley for IMAC purification and eval-uation of LecA.

REFERENCES

1. Abd-Alla, M. D., T. F. Jackson, T. Rogers, S. Reddy, and J. I. Ravdin. 2006.Mucosal immunity to asymptomatic Entamoeba histolytica and Entamoebadispar infection is associated with a peak intestinal anti-lectin immunoglob-ulin A antibody response. Infect. Immun. 74:3897–3903.

2. Argiro, L., S. Henri, H. Dessein, A. J. Dessein, and A. Bourgois. 1999.Induction of a protective immunity against Schistosoma mansoni withovalbumin-coupled Sm37-5 coadsorbed with granulocyte-macrophage col-ony stimulating factor (GM-CSF) or IL-12 on alum. Vaccine 17:13–18.

3. Asgharpour, A., C. Gilchrist, D. Baba, S. Hamano, and E. Houpt. 2005.Resistance to intestinal Entamoeba histolytica infection is conferred by in-nate immunity and Gr-1� cells. Infect. Immun. 73:4522–4529.

4. Brewer, J. M., M. Conacher, C. A. Hunter, M. Mohrs, F. Brombacher, andJ. Alexander. 1999. Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling.J. Immunol. 163:6448–6454.

5. Brewer, J. M., M. Conacher, A. Satoskar, H. Bluethmann, and J. Alexander.1996. In interleukin-4-deficient mice, alum not only generates T helper 1responses equivalent to Freund’s complete adjuvant, but continues to induceT helper 2 cytokine production. Eur. J. Immunol. 26:2062–2066.

6. Caballero-Salcedo, A., M. Viveros-Rogel, B. Salvatierra, R. Tapia-Conyer, J.Sepulveda-Amor, G. Gutierrez, and L. Ortiz-Ortiz. 1994. Seroepidemiologyof amebiasis in Mexico. Am. J. Trop. Med. Hyg. 50:412–419.

7. Campbell, D., and K. Chadee. 1997. Interleukin (IL)-2, IL-4, and tumornecrosis factor-alpha responses during Entamoeba histolytica liver abscessdevelopment in gerbils. J. Infect. Dis. 175:1176–1183.

8. Campbell, D., B. J. Mann, and K. Chadee. 2000. A subunit vaccine candidateregion of the Entamoeba histolytica galactose-adherence lectin promotesinterleukin-12 gene transcription and protein production in human macro-phages. Eur. J. Immunol. 30:423–430.

9. Chadee, K., W. A. Petri, Jr., D. J. Innes, and J. I. Ravdin. 1987. Rat andhuman colonic mucins bind to and inhibit adherence lectin of Entamoebahistolytica. J. Clin. Investig. 80:1245–1254.

10. Cheng, X. J., and H. Tachibana. 2001. Protection of hamsters from amebicliver abscess formation by immunization with the 150- and 170-kDa surfaceantigens of Entamoeba histolytica. Parasitol. Res. 87:126–130.

11. Cuppari, A. F., V. Sanchez, B. Ledesma, F. M. Frank, A. Goldman, S. O. Angel,and V. Martin. 2008. Toxoplasma gondii protease inhibitor-1 (TgPI-1) is a novelvaccine candidate against toxoplasmosis. Vaccine 26:5040–5045.

12. Darrah, P. A., D. T. Patel, P. M. De Luca, R. W. Lindsay, D. F. Davey, B. J.Flynn, S. T. Hoff, P. Andersen, S. G. Reed, S. L. Morris, M. Roederer, andR. A. Seder. 2007. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat. Med. 13:843–850.

13. Denis, M., and K. Chadee. 1989. Cytokine activation of murine macrophagesfor in vitro killing of Entamoeba histolytica trophozoites. Infect. Immun.57:1750–1756.

14. Denis, M., and K. Chadee. 1989. Human neutrophils activated by interferon-gamma and tumour necrosis factor-alpha kill Entamoeba histolytica tropho-zoites in vitro. J. Leukoc. Biol. 46:270–274.

15. Egesten, A., M. Eliasson, H. M. Johansson, A. I. Olin, M. Morgelin, A.Mueller, J. E. Pease, I. M. Frick, and L. Bjorck. 2007. The CXC chemokineMIG/CXCL9 is important in innate immunity against Streptococcus pyo-genes. J. Infect. Dis. 195:684–693.

16. Fahlgren, A., V. Baranov, L. Frangsmyr, F. Zoubir, M. L. Hammarstrom,and S. Hammarstrom. 2003. Interferon-gamma tempers the expression ofcarcinoembryonic antigen family molecules in human colon cells: a possiblerole in innate mucosal defence. Scand. J. Immunol. 58:628–641.

17. Ferreira, D. M., M. Darrieux, M. L. Oliveira, L. C. Leite, and E. N. Miyaji.2008. Optimized immune response elicited by a DNA vaccine expressingpneumococcal surface protein a is characterized by a balanced immunoglob-ulin G1 (IgG1)/IgG2a ratio and proinflammatory cytokine production. Clin.Vaccine Immunol. 15:499–505.

18. Flanagan, K., Z. Modrusan, J. Cornelius, A. Chavali, I. Kasman, L.Komuves, L. Mo, and L. Diehl. 2008. Intestinal epithelial cell up-regulation

FIG. 6. Role of IFN-� in LecA-mediated protection. Mice immunized with LecA (filled bars) or PBS (open bars) in the EM014 and alumregimens were administered anti-IFN-�, anti-IL-4, or control IgG from 3 days before to 6 days after challenge (four doses in total). Similarly, inanother trial, mice immunized with LecA in the CT-CFA regimen were treated with anti-IFN-� or control IgG. Mice were sacrificed at 8 dayspostchallenge. *, P � 0.05; **, P � 0.01; ***, P � 0.001.

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nloaded from

of LY6 molecules during colitis results in enhanced chemokine secretion.J. Immunol. 180:3874–3881.

19. Forbes, E. K., C. Sander, E. O. Ronan, H. McShane, A. V. Hill, P. C.Beverley, and E. Z. Tchilian. 2008. Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protectionagainst Mycobacterium tuberculosis aerosol challenge in mice. J. Immunol.181:4955–4964.

20. Guo, X., E. Houpt, and W. A. Petri, Jr. 2007. Crosstalk at the initial encoun-ter: interplay between host defense and ameba survival strategies. Curr.Opin. Immunol. 19:376–384.

21. Guo, X., S. E. Stroup, and E. R. Houpt. 2008. Persistence of Entamoebahistolytica infection in CBA mice owes to intestinal IL-4 production andinhibition of protective IFN-�. Mucos. Immunol. 1:139–146.

22. Gurunathan, S., D. L. Sacks, D. R. Brown, S. L. Reiner, H. Charest, N.Glaichenhaus, and R. A. Seder. 1997. Vaccination with DNA encoding theimmunodominant LACK parasite antigen confers protective immunity tomice infected with Leishmania major. J. Exp. Med. 186:1137–1147.

23. Hamano, S., A. Asgharpour, S. E. Stroup, T. A. Wynn, E. H. Leiter, and E.Houpt. 2006. Resistance of C57BL/6 mice to amoebiasis is mediated bynonhemopoietic cells but requires hemopoietic IL-10 production. J. Immu-nol. 177:1208–1213.

24. Haque, R., I. M. Ali, R. B. Sack, B. M. Farr, G. Ramakrishnan, and W. A.Petri, Jr. 2001. Amebiasis and mucosal IgA antibody against the Entamoebahistolytica adherence lectin in Bangladeshi children. J. Infect. Dis. 183:1787–1793.

25. Haque, R., P. Duggal, I. M. Ali, M. B. Hossain, D. Mondal, R. B. Sack, B. M.Farr, T. H. Beaty, and W. A. Petri, Jr. 2002. Innate and acquired resistanceto amebiasis in Bangladeshi children. J. Infect. Dis. 186:547–552.

26. Haque, R., C. D. Huston, M. Hughes, E. Houpt, and W. A. Petri, Jr. 2003.Amebiasis. N. Engl. J. Med. 348:1565–1573.

27. Haque, R., D. Mondal, P. Duggal, M. Kabir, S. Roy, B. M. Farr, R. B. Sack,and W. A. Petri, Jr. 2006. Entamoeba histolytica infection in children andprotection from subsequent amebiasis. Infect. Immun. 74:904–909.

28. Haque, R., D. Mondal, J. Shu, S. Roy, M. Kabir, A. N. Davis, P. Duggal, andW. A. Petri, Jr. 2007. Correlation of interferon-gamma production by pe-ripheral blood mononuclear cells with childhood malnutrition and suscepti-bility to amebiasis. Am. J. Trop. Med. Hyg. 76:340–344.

29. Houpt, E., L. Barroso, L. Lockhart, R. Wright, C. Cramer, D. Lyerly, andW. A. Petri. 2004. Prevention of intestinal amebiasis by vaccination with theEntamoeba histolytica Gal/GalNac lectin. Vaccine 22:611–617.

30. Houpt, E. R., D. J. Glembocki, T. G. Obrig, C. A. Moskaluk, L. A. Lockhart,R. L. Wright, R. M. Seaner, T. R. Keepers, T. D. Wilkins, and W. A. Petri,Jr. 2002. The mouse model of amebic colitis reveals mouse strain suscepti-bility to infection and exacerbation of disease by CD4� T cells. J. Immunol.169:4496–4503.

31. Ivory, C. P., and K. Chadee. 2007. Activation of dendritic cells by theGal-lectin of Entamoeba histolytica drives Th1 responses in vitro and in vivo.Eur. J. Immunol. 37:385–394.

32. Ivory, C. P., K. Keller, and K. Chadee. 2006. CpG-oligodeoxynucleotide is apotent adjuvant with an Entamoeba histolytica Gal-inhibitable lectin vaccineagainst amoebic liver abscess in gerbils. Infect. Immun. 74:528–536.

33. Jankovic, D., P. Caspar, M. Zweig, M. Garcia-Moll, S. D. Showalter, F. R.Vogel, and A. Sher. 1997. Adsorption to aluminum hydroxide promotes theactivity of IL-12 as an adjuvant for antibody as well as type 1 cytokineresponses to HIV-1 gp120. J. Immunol. 159:2409–2417.

34. Johnson, D. A. 2008. Synthetic TLR4-active glycolipids as vaccine adjuvantsand stand-alone immunotherapeutics. Curr. Top. Med. Chem. 8:64–79.

35. Kenney, R. T., D. L. Sacks, J. P. Sypek, L. Vilela, A. A. Gam, and K.Evans-Davis. 1999. Protective immunity using recombinant human IL-12and alum as adjuvants in a primate model of cutaneous leishmaniasis. J. Im-munol. 163:4481–4488.

36. Lin, J. Y., R. Seguin, K. Keller, and K. Chadee. 1994. Tumor necrosis factoralpha augments nitric oxide-dependent macrophage cytotoxicity against Ent-amoeba histolytica by enhanced expression of the nitric oxide synthase gene.Infect. Immun. 62:1534–1541.

37. Lotter, H., F. Khajawa, S. L. Stanley, Jr., and E. Tannich. 2000. Protectionof gerbils from amebic liver abscess by vaccination with a 25-mer peptide

derived from the cysteine-rich region of Entamoeba histolytica galactose-specific adherence lectin. Infect. Immun. 68:4416–4421.

38. Lotter, H., T. Zhang, K. B. Seydel, S. L. Stanley, Jr., and E. Tannich. 1997.Identification of an epitope on the Entamoeba histolytica 170-kD lectinconferring antibody-mediated protection against invasive amebiasis. J. Exp.Med. 185:1793–1801.

39. Mosca, F., E. Tritto, A. Muzzi, E. Monaci, F. Bagnoli, C. Iavarone, D.O’Hagan, R. Rappuoli, and G. E. De. 2008. Molecular and cellular signaturesof human vaccine adjuvants. Proc. Natl. Acad. Sci. USA 105:10501–10506.

40. Petri, W. A., Jr., and J. I. Ravdin. 1991. Protection of gerbils from amebicliver abscess by immunization with the galactose-specific adherence lectin ofEntamoeba histolytica. Infect. Immun. 59:97–101.

41. Plotnicky-Gilquin, H., D. Cyblat-Chanal, J. P. Aubry, T. Champion, A. Beck,T. Nguyen, J. Y. Bonnefoy, and N. Corvaia. 2002. Gamma interferon-depen-dent protection of the mouse upper respiratory tract following parenteralimmunization with a respiratory syncytial virus G protein fragment. J. Virol.76:10203–10210.

42. Ravdin, J. I., M. D. bd-Alla, S. L. Welles, S. Reddy, and T. F. Jackson. 2003.Intestinal antilectin immunoglobulin A antibody response and immunity toEntamoeba dispar infection following cure of amebic liver abscess. Infect.Immun. 71:6899–6905.

43. Schain, D. C., R. A. Salata, and J. I. Ravdin. 1992. Human T-lymphocyteproliferation, lymphokine production, and amebicidal activity elicited by thegalactose-inhibitable adherence protein of Entamoeba histolytica. Infect. Im-mun. 60:2143–2146.

44. Seguin, R., B. J. Mann, K. Keller, and K. Chadee. 1997. The tumor necrosisfactor alpha-stimulating region of galactose-inhibitable lectin of Entamoebahistolytica activates gamma interferon-primed macrophages for amebicidalactivity mediated by nitric oxide. Infect. Immun. 65:2522–2527.

45. Siegrist, C. A., H. Plotnicky-Gilquin, M. Cordova, M. Berney, J. Y. Bonnefoy,T. N. Nguyen, P. H. Lambert, and U. F. Power. 1999. Protective efficacyagainst respiratory syncytial virus following murine neonatal immunizationwith BBG2Na vaccine: influence of adjuvants and maternal antibodies. J. In-fect. Dis. 179:1326–1333.

46. Soong, C. J., K. C. Kain, M. bd-Alla, T. F. Jackson, and J. I. Ravdin. 1995.A recombinant cysteine-rich section of the Entamoeba histolytica galactose-inhibitable lectin is efficacious as a subunit vaccine in the gerbil model ofamebic liver abscess. J. Infect. Dis. 171:645–651.

47. Soong, C. J., B. E. Torian, M. D. Abd-Alla, T. F. Jackson, V. Gatharim, andJ. I. Ravdin. 1995. Protection of gerbils from amebic liver abscess by immu-nization with recombinant Entamoeba histolytica 29-kilodalton antigen. In-fect. Immun. 63:472–477.

48. Stanley, S. L., Jr. 2006. Vaccines for amoebiasis: barriers and opportunities.Parasitology 133(Suppl.):S81–S86.

49. Su, Z., M. F. Tam, D. Jankovic, and M. M. Stevenson. 2003. Vaccination withnovel immunostimulatory adjuvants against blood-stage malaria in mice.Infect. Immun. 71:5178–5187.

50. Vandepapeliere, P., Y. Horsmans, P. Moris, M. M. Van, M. Janssens, M.Koutsoukos, B. P. Van, F. Clement, E. Hanon, M. Wettendorff, N. Garcon,and G. Leroux-Roels. 2008. Vaccine adjuvant systems containing monophos-phoryl lipid A and QS21 induce strong and persistent humoral and T cellresponses against hepatitis B surface antigen in healthy adult volunteers.Vaccine 26:1375–1386.

51. Wack, A., B. C. Baudner, A. K. Hilbert, I. Manini, S. Nuti, S. Tavarini, H.Scheffczik, M. Ugozzoli, M. Singh, J. Kazzaz, E. Montomoli, G. G. Del, R.Rappuoli, and D. T. O’Hagan. 2008. Combination adjuvants for the induc-tion of potent, long-lasting antibody and T-cell responses to influenza vac-cine in mice. Vaccine 26:552–561.

52. Yip, H. C., A. Y. Karulin, M. Tary-Lehmann, M. D. Hesse, H. Radeke, P. S.Heeger, R. P. Trezza, F. P. Heinzel, T. Forsthuber, and P. V. Lehmann. 1999.Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious di-chotomy defines the class of response. J. Immunol. 162:3942–3949.

53. Zhang, T., and S. L. Stanley, Jr. 1994. Protection of gerbils from amebic liverabscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica. Infect. Immun. 62:2605–2608.

Editor: J. F. Urban, Jr.

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