Development of recombinant vaccines against IL-12/IL-23 p40 and in vivo evaluation of their effects in the downregulation of intestinal inflammation in murine colitis

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INTRODUCTIONStudies have provided evidence that

the pathogenesis of inflammatory boweldisease (IBD), which includes Crohn’sdisease and ulcerative colitis, is associ-ated with genetic factors, environmentalfactors, enteric flora and immunologicalabnormalities (1). It has been acceptedwidely that Crohn’s disease is caused byan overly aggressive Th1 immune re-sponse and, recently discovered, exces-sive IL-23/Th17 pathway activation tobacterial antigens in genetically predis-posed individuals (1–3). These Th1 cy-tokines, such as IL-12 and the IL-23/Th17 pathway, could be critical in gener-

ating and perpetuating the chronic intes-tinal inflammation of Crohn’s disease(3,4).

In active Crohn’s disease, the expres-sion of IL-12 receptors on mucosal cellsis increased (5). Various immunohisto-logical studies indicate that in situ IL-12is overproduced by macrophages inCrohn’s disease, and that macrophagesisolated from the inflammatory lesions ofpatients with Crohn’s disease produceincreased amounts of IL-12 ex vivo (6). InTNBS-induced colitis mice, administra-tion of an IL-12 antagonist (the IL-12p40-IgG2b fusion protein) (7) or anti-mouseIL-12 antibodies (8) abrogates mucosa in-

flammation, prevents body weight lossand restores a nearly normal histologicalappearance of the colon, through a mech-anism of induction of Fas-mediated apo-ptosis of Th1 cells (8). This treatment alsoreduces TNF levels and increases IL-10secretion in mice (7).

More recently, studies have high-lighted the roles of IL-23/Th17 pathwayin the pathogenesis of Crohn’s disease(9–13). Yen and his colleagues havedemonstrated that the development ofcolitis is suppressed by IL-23p19 (p19 isthe IL-23 specific subunit) deficiencybut not IL-12p35 (p35 is the IL-12 spe-cific subunit) deficiency in IL-10–/–

mice, and that administration of IL-23accelerates the onset of colitis and pro-motes inflammation through an IL-17-and IL-6-dependent mechanism (9).Anti-IL-23 mono clonal antibody canprevent and reverse active colitis in a T-cell-mediated colitis mouse model,

Targeting IL-12/IL-23 by Employing a p40 Peptide-BasedVaccine Ameliorates TNBS-Induced Acute and ChronicMurine Colitis

Qingdong Guan,1,2 Yanbing Ma,2 China-Li Hillman,2 Gefei Qing,3 Allan G Ma,1,2 Carolyn R Weiss,1,2

Gang Zhou,2 Aiping Bai,2 Richard J Warrington,4 Charles N Bernstein,4,5 and Zhikang Peng1,2

1Department of Immunology, 2Department of Pediatrics and Child Health, 3Department of Pathology, 4Department of InternalMedicine, and 5IBD Clinical and Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada

Interleukin (IL)-12 and IL-23 both share the p40 subunit and are key cytokines in the pathogenesis of Crohn’s disease. Previously,we have developed and identified three mouse p40 peptide-based and virus-like particle vaccines. Here, we evaluated the ef-fects and immune mechanisms of the optimal vaccine in downregulating intestinal inflammation in murine acute and chronic co-litis, induced by intrarectal administrations of trinitrobenzene sulfonic acid (TNBS). Mice were injected subcutaneously with vac-cine, vaccine carrier or saline three times, and then intrarectally administered TNBS weekly for 2 wks (acute colitis) or 7 wks (chroniccolitis). The severity of colitis was evaluated by body weight, histology and collagen and cytokine levels in colon tissue. Th1 andTh17 cells in mesenteric lymph nodes (MLN) were determined. Our results showed the vaccine induced high level and long- lastingspecific IgG antibodies to p40, IL-12 and IL-23. After administrations of TNBS, vaccinated mice had significantly less body weightloss and a significant decrease of inflammatory scores, collagen deposition and expression of p40, IL-12, IL-23, IL-17, TNF, iNOS andBcl-2 in colon tissues, compared with carrier and saline groups. Moreover, vaccinated mice exhibited a trend to lower percent-ages of Th1 cells in acute colitis and of Th17 cells in chronic colitis in MLN than in controls. In summary, administration of the vac-cine induced specific antibodies to IL-12 and IL-23, which was associated with improvement of intestinal inflammation and fibro-sis. This suggests that the vaccine may provide a potential approach for the long-term treatment of Crohn’s disease.© 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2010.00252

Address correspondence and reprint requests to Zhikang Peng, Department of Pediatrics

and Child Health, University of Manitoba, 532-715 McDermot Avenue, Winnipeg, Mani-

toba, Canada R3E 3P4. E-mail: [email protected].

Submitted December 6, 2010; Accepted for publication March 10, 2011; Epub

(www.molmed.org) ahead of print March 11, 2011.

R E S E A R C H A R T I C L E

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with the downregulation of a broadarray of inflammatory cytokines andchemokines in the colon (14). Recently,a genome-wide association study hasreported IL-23R as an IBD gene that ishighly associated with Crohn’s disease(15). Even so, these findings do not ex-clude the role of an exaggerated Th1 re-sponse in Crohn’s disease, since IL-12/IFN-γ and IL-23/IL-17 may be parallelpathways involved in inflammatory responses (2).

These observations in mice are sup-ported by successful clinical trials. Mon-oclonal antibodies (mAb) against thoseoverproduced endogenous Th1 and Th17cytokines have emerged as a potentialtreatment in IBD (16–18). Administrationof a human mAb against p40, the sub-unit shared by IL-12 and IL-23 (4), in-duced significant clinical responses andremissions in patients with activeCrohn’s disease (19). Although the ther-apy with mAb may be useful clinically(3,16), these agents all act as passivelyadministered antagonists with short half-lives. Repeated intravenous (i.v.) or sub-cutaneous (s.c.) injections with largedoses of mAb are required to maintainthe effects. Side effects include high ratesof acute infusion reactions and the devel-opment of antibodies to the infusedmAb, which reduces the effectiveness ofthe treatment and occurs in 61% of thepatients receiving infliximab (an mAb toTNF) therapy (20,21) and also in patientsreceiving the mAb to IL-12/IL-23 p40treatment (19). Another highly relevantconcern is the extremely high cost associ-ated with such passively administeredtherapeutics.

To overcome these disadvantages, anew active immunization strategy, em-ploying vaccines that target overex-pressed endogenous molecules, is beinginvestigated. This new strategy may offerlong-term efficacy with fewer adverse ef-fects (22,23). To break immune tolerance,these vaccines generally are made by in-serting heterogeneous T cell epitopesinto the target self-cytokine (24), or link-ing the intact self cytokine to a foreigncarrier protein (25).

Previously, we have developed andidentified three recombinant mouseIL-12/ IL-23 p40 peptide-based virus-like particle vaccines (C, D and F),which induce high levels of specific an-tibodies against p40, IL-12 and IL-23(26). The vaccine is constructed by in-serting a peptide derived from mousep40 subunit into a carrier protein, hepa-titis B core antigen (HBcAg), using geneengineering methods. In this study, wesystemically evaluated the effectivenessof the best vaccine (F) in the attenuationof TNBS-induced both acute andchronic murine colitis. We further ex-plored the possible immune mecha-nisms of vaccine-mediated improve-ment, especially Th1 and IL-23/Th17responses.

MATERIALS AND METHODS

AnimalsFemale BALB/c mice (7–8 wks old),

purchased from Charles River Laborato-ries (Saint-Constant, Sherbrooke, QC,Canada), were maintained at the CentralAnimal Care Services, University ofManitoba. All protocols used were ap-proved by the University Animal EthicsCommittee.

Protocols of Vaccine Immunizationand Induction of Colitis

Vaccine F, which contains a peptide ofnine amino acids and a carrier proteinHBcAg, was prepared using the methodspreviously described (26) and used inTNBS-induced colitis, a widely used ani-mal model that exhibits characteristics ofCrohn’s disease (27).

The vaccine was evaluated firstly invivo in a TNBS-induced acute colitis inwhich mice were injected s.c. 3× at a2-wk interval with vaccine, vaccine car-rier HBcAg or saline (100 μg, 25 μg and25 μg in 200 μL, respectively) (n = 20).Two weeks later, mice were intrarectallychallenged with TNBS (Sigma-Aldrich,Oakville, ON, Canada) twice (1.5 mg and2.0 mg, respectively) at a 1-wk interval toinduce acute colitis (Figure 1A) accord-ing to the previous description (27).

The vaccine was further evaluated inTNBS-induced chronic colitis, in whichmice were immunized 4× with vaccine orcarrier (doses described in the legend ofFigure 2A) or saline (n = 12). Two weeksafter the third vaccination, chronic colitiswas induced by seven weekly adminis-trations of increasing doses of TNBS(1.0–2.5 mg) (Figure 2A).

Two days (for acute colitis) or 10 days(for chronic colitis) after the last TNBSdelivery, mice were euthanized. Colons,mesenteric lymph nodes and blood sam-ples were collected and processed ac-cording to different assays. The evalua-tions were repeated once, and similarresults were found.

Body WeightMouse body weight was monitored

daily in acute colitis (before the deliveryof TNBS on the delivery day and in themorning of remaining days) and weeklyin chronic colitis before each delivery ofTNBS.

Histological ExaminationTen percent buffered formalin fixed

and paraffin-embedded colon sectionswere cut and stained with H&E or Mas-son trichrome. Histological scoring wasevaluated by a pathologist blinded to thesource of treatment based on the methodpreviously described (28). During eachhistological examination, three differentparameters were estimated: severity ofinflammation, depth of injury and cryptdamage. All values were added to a sum,in which the maximum possible scorewas 10.

Soluble Collagen AssayFor quantitative measurement of colla-

gen, a marker of chronic inflammation,colons were homogenized in 0.5 mol/Lacetic acid containing 1 mg of pepsin (ata concentration of 10 mg of tissue/5 mLof acetic acid solution). The resultingmixture was then incubated and stirredfor 24 h at 4°C. Total soluble collagencontent of the mixture was determinedwith a Sircol Collagen Assay Kit (BiocolorLtd, Westbury, NY, USA) (27). Acid solu-


I L - 1 2 / I L - 2 3 P 4 0 V A C C I N E I M P R O V E S M U R I N E C O L I T I S

ble type I collagen supplied with the kitwas used to generate a standard curve.

Myleroperoxidase (MPO) ActivityAssay

MPO activity, an index of neutrophilicinfiltration, was assayed using themethod described previously (29). The

colon tissue (100 mg tissue/mL) was ho-mogenized in potassium phosphatebuffer (50 mmol/L, pH 6.0,) containing5% hexadecyltrimethylammpnium bro-mide, and then subjected to three cyclesof freezing and thawing. After centrifug-ing for 15 min at 18,300g at 4°C, the su-pernatant was transferred to a 96-well

plate (7 μL per well, triplicate each sam-ple). The enzyme reaction was carriedout by adding 200 μL of phosphate buffer(50 mmol/L, pH 6.0) containing 0.167

Figure 1. Vaccine ameliorates TNBS-induced acute colitis. (A) Protocol. (B) Body weight.(C) Colon tissue sections (H&E staining). (D) Semiquantitative analysis of histological in-flammation. (E) MPO activity. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Immunization with the p40 vac-cine induces specific IgG antibodies top40, IL-12 and IL-23. (A) Protocol. Micewere subcutaneously primed with 100 μg/200 μL of vaccine or carrier or 200 μL ofsaline, and boosted at wks 2, 4 and 9 with25 μg/200 μL of vaccine or carrier or 200 μLsaline. Chronic colitis was induced byseven weekly administrations of TNBS. (B)Serum specific IgG antibodies. Sera wereobtained at the indicated weeks and di-luted 1:200 for determination of specificIgG levels by ELISA, in which p40, IL-12, orIL-23 (0.25 μg/mL) were coated on theplates, separately. Data represent one ofat least two repeated experiments.



mg/mL o-dianisodine hydrochloride (Sigma-Aldrich) and 0.0005% H2O2. The kineticsof absorbance changes at 460 nm wasmeasured at 0, 30 and 60 min in a mi-crotiter reader. The MPO activity is pre-sented as MPO units per gram of tissue.

Measurements of Antibodies andCytokines by ELISA

Frozen colonic samples were homog-enized mechanically in buffer contain-ing 1 mol/L Tris-HCl, 3 mol/L NaCl,and 10% Triton supplemented with pro-tease cocktail (Sigma-Aldrich). Sampleswere then frozen (–70°C) and thawed(37°C) 3×, followed by centrifugation at18,300g for 30 min at 4°C. Supernatantswere frozen at –70°C until assay (13).

Serum p40-, IL-12- and IL-23-specificIgG levels were assayed by using ELISAtechniques established in our laboratory(30,31). The results obtained from eachsample were expressed using optical den-sity at 405 nm (OD405). The results ob-tained from sera pooled from each groupwere expressed using “titer,” the recipro-cal of the highest dilution in which theOD405 was twice that of the correspon-ding control serum when its OD405 was0.10. Cytokine concentrations in colon tis-sues were measured by ELISA accordingto the manufacturer’s instructions. IL-12p40, IL-17, IFN-γ, IL-13 and TNF-βwere purchased from BD Biosciences(Mississauga, ON, Canada); IL-23 and IL-10 were from eBioscience (San Diego, CA,USA); TGFβ1 was from R&D Systems(Burlington, ON, Canada).

Real-Time Reverse-TranscriptionPolymerase Chain Reaction (RT-PCR)

Briefly, total RNA was extracted fromcolons by Trizol (Invitrogen, Burlington,ON, Canada) according to the manufac-turer’s instruction. Complementary DNAwas synthesized using iScript cDNA syn-thesis kit (Bio-Rad, Mississauga, ON, Can-ada). Primer sequence was as follows: 5′-GGCAAATTCAACGGCACAGT-3′ and5′-AGATGGTGATGGGCTTCCC-3′ forGAPDH; 5′-ACTTCGCAGAGATGTCCAGTCA-3′ and 5′-TGGCA AAGCG

TCCCCTC-3′ for Bcl-2; 5′-AACATCTGGAACCACGGGCACTAT-3′ and 5′-GCATTGCTTGAGGCT GCGTATGAT-3′ for Foxp3;5′-GTGACGGCAAACATG ACTTC AG-3′and 5′-GCCATCGGGCATCTGGTA-3′ foriNOS; 5′-TTCCGAATTCACTGGAGCCTCGAA-3′ and 5′-TGCACCTCAGGGAAGAATCTGGAA-3′ for TNF; 5′-CTCACCTGTG ACACGCCTGA-3′ and 5′-CAGGACACTGAATACTTCTC-3′ for IL-12/IL-23p40; 5′-TGAATTCCCTGGGTGAGAAGCTGA-3′ and 5′-TGGCCTTGTAGACACCTTGGTCTT-3′ for IL-10. Real-timequantitative reverse transcription poly-merase chain reaction (RT-PCR) was as-sayed in special optical tubes of 96-wellmicrotiter plates with ABI PRISM 7700Sequence Detector Systems (AppliedBiosystems, Foster City, CA, USA). Thereaction mixture consisted of SYBR GreenPCR master mix (Applied Biosystems).PCR retain conditions were 50°C for2 min, 95°C for 10 min and then 40 cyclesat 95°C for 15 s, 60°C for 15 s and 72°Cfor 35 s, followed by a dissociation stage.Gene expression was calculated relativeto the housekeeping gene GAPDH.

Intracellular Staining for Identificationof Th1 and Th17 Cells

To examine the percentages of Th1 andTh17 cells, single-cell suspensions con-taining 1 × 106 mesenteric lymph node(MLN) cells were cultured and stimu-lated for 7 h with 50 ng/mL phorbolmyristate acetate and 1 μg/mL iono-mycin (Sigma-Aldrich), with brefeldin Aadded for the last 4 h of culture. Cellswere harvested, washed and stained withanti-CD4 (eBioscience). Surface-stainedcells were fixed (2% parafor maldehyde)and resuspended in permeabilizationbuffer (0.5% saponin). This was followedby staining for intracellular IL-17 or IFN-γ(eBioscience). Cells were analyzed usinga FACSCalibur (BD Biosciences).

Statistical AnalysisValues were expressed as mean ± SD.

Differences between experimentalgroups were assessed by one-way analy-sis of variance (ANOVA) followed byNewman-Keuls multiple comparison test

(GraphPad Software, San Diego, CA,USA). P values <0.05 were consideredstatistically significant.

RESULTS

Vaccine Induces High Levels ofSpecific IgG Antibodies Against p40,IL-12, and IL-23, and ImprovesIntestinal Inflammation in Acute Colitis

After three immunizations, the p40peptide-based vaccine induced signifi-cantly high levels of specific antibodiesagainst p40, IL-12 and IL-23. The titers ofthe serum pooled from vaccinated micewere 150,000 for p40-specific IgG, 80,000for IL-12-specific IgG and 10,000 forIL-23-specific IgG. This was similar toour previous results (26).

After mice developed high levels ofantibodies against p40, IL-12 and IL-23,they were intrarectally challenged withTNBS twice to induce acute colitis (seeFigure 1A). As shown in Figure 1B, aftereach TNBS administration, mice of salineand carrier groups show obvious bodyweight loss, but vaccine-treated micehave significantly improved body weightloss (P < 0.05) and their body weight isquickly recovered to the level of normalmice on days 3–7.

Histological analysis revealed that un-treated mice (saline and carrier groups)exhibited severe thickening of the mus-cularis, a marked mucosal inflammatorycell infiltrate, many focal lesions charac-terized by epithelial cell sloughing, in-creased neutrophilic infiltrate, and com-plete loss of crypt architecture, resultingin high scores (Figure 1C, D). In contrast,colons from vaccine-treated mice showedmuch less inflammation than controls(P < 0.05), indicating that vaccine treat-ment could ameliorate intestinal inflam-mation significantly.

The effectiveness of vaccination, seen inhistological analysis, was further con-firmed quantitatively by the results ob-tained from the measurement of MPO ac-tivity, an enzyme specific to granulocytelysosomes, and, therefore, directly corre-lated with the number of granulocytes.MPO activity was increased about four-



fold in carrier and saline groups com-pared with normal mice. In contrast, therewas a significant decrease in the MPO ac-tivity in vaccine-treated mice (P < 0.05)that was close to normal mice (Figure 1E).

We examined cytokine expression lev-els in colon tissue by ELISA. The resultsshowed that vaccine treatment sup-pressed the protein levels of p40, IL-12,IFN-γ, IL-17 and TNF, but increasedIL-10 production compared with salineand carrier groups (P < 0.05) (Figure 3A).IL-23 levels were not elevated signifi-cantly in any colitis group (data notshown). We further examined cytokineand proinflammatory molecules byusing real time RT-PCR. Vaccine treat-ment also resulted in a decrease in themRNA expression levels of p40, TNF,proinflammatory molecules induciblenitric oxide synthase (iNOS) and anti-apoptotic molecules Bcl-2 (Figure 3B).Consistent with the increased proteinlevels of IL-10 measured by ELISA, themRNA levels of IL-10 and Foxp3 wereincreased in the vaccine-treated group.Taken together, these data indicate thatthe p40 vaccine ameliorates intestinalinflammation in TNBS-induced acutecolitis by inhibiting Th1 cytokines andinflammatory molecule expression andincreasing IL-10 production.

Vaccine Induces Specific IgGAntibodies and Improves Body WeightLoss in Chronic Colitis

Since IBD is a chronic disease, itwould be more important and relevantto evaluate this vaccine strategy in achronic colitis model, so we further ex-plored the effects of the vaccine inTNBS-induced chronic model. As shownin Figure 2B, after two immunizations,the vaccine induces significantly highlevels of specific antibodies against p40,IL-12 and IL-23, which reach a plateauafter three immunizations and levels re-main high in the entire experiment.These antibodies were undetectable incarrier and saline groups (P < 0.001).The titers of the serum pooled from vac-cinated mice were 200,000 for p40- specific IgG, 150,000 for IL-12-specific

IgG and 20,000 for IL-23-specific IgG,which were slightly higher than those inacute colitis. This may be owing to theuse of different batches of mice in thetwo experiments as immune responsesare individually different. After mice de-veloped high levels of antibodies, theywere intrarectally challenged with TNBSseven times to induce a chronic colitis(see Figure 2A). Consistent with thefindings in acute colitis, in this chroniccolitis model mice in saline and carriergroups showed obvious body weightloss after the first administration ofTNBS that lasted from wk 6 to wk 9, andthen gradually regained weight and re-covered from other signs of chronic ill-ness despite continued TNBS adminis-trations after wk 10 (Figure 4). Incontrast, vaccine-immunized miceshowed no obvious body weight loss

compared with saline and carrier groups(P < 0.01), and their body weightchanges were similar to those of normalhealthy mice and even higher than nor-mal mice between wk 12 and wk 13.These results indicated that the vaccine

Figure 3. Vaccine downregulates the levels of proinflammatory cytokines and other medi-ators involved in acute colitis, but upregulates IL-10 production. (A) Colon cytokine levelsmeasured by ELISA. (B) Colon mRNA levels evaluated by real-time RT-PCR. *P < 0.05; **P <0.01; ***P < 0.001.

Figure 4. Vaccine improves body weightloss in chronic colitis. Body weight was mon-itored weekly on the day after TNBS admin-istration. *P < 0.05; **P < 0.01; ***P < 0.001.



could lessen body weight loss in micewith TNBS-induced chronic colitis.

Vaccine Suppresses IntestinalInflammation in Chronic Colitis

In addition to the improvement ofbody weight loss, vaccine treatment alsoameliorated other manifestations ofchronic colitis. Histological analysis re-vealed that colons from saline and carriergroups exhibited obvious inflammation,including inflammatory cell infiltration,goblet cell reduction and distorted archi-tecture (Figure 5A). In contrast, colon tis-sues of vaccine-treated mice showedmuch less inflammation than controls.The vaccine-induced improvement ofcolon inflammation was further con-firmed by semiquantitative analysis inwhich vaccine-treated mice had signifi-cantly lower inflammatory scores thansaline and carrier groups (P < 0.001) (Figure 5B).

Vaccine Inhibits Fibrosis in ChronicColitis

As fibrosis is an important indicatorfor chronic inflammation, colon collagendeposition was evaluated. As shown inFigure 6A, compared with the normalgroup, the amount of collagen is in-creased in the subepithelium, in deeperlayers of the colonic lamina propria, andin the muscular layer in saline and car-rier groups. No significant increase incollagen deposition was observed in vac-cine-immunized mice. The reduction incollagen deposition was further corrobo-rated by the Sircol collagen assay thatdetects soluble collagen quantitatively.The amount of soluble collagen in vac-cine-treated mice was significantly lowerthan that in saline and carrier groups,and was reduced almost to the normallevel (P < 0.01) (Figure 6B), confirmingthat vaccine immunization could de-crease fibrosis in chronic colitis.

Expression Levels of Cytokines inChronic Colitis

Studies have provided evidence thatthe production of some proinflammatorycytokines (such as IL-23, IL-17 and TNF)

and a profibrotic cytokine (TGFβ1), areincreased in colon tissue of chronic coli-tis. As indicated in Figure 7, after admin-istrations of TNBS, the protein levels ofp40, IL-23, IL-17 and TGFβ1 in colon tis-sue of saline and carrier groups were sig-nificantly higher than those in normalcontrol (Figure 7A). As expected, vaccine-treated mice had a decrease in p40, IL-23,IL-17 and TGFβ1 levels compared withcarrier and saline groups (P < 0.05). Thedecreased p40 and TNF levels in the vac-cine-treated group were further con-firmed by the measurement of mRNA ex-pression (Figure 7B). However, incontrast to the finding in acute colitis thatIL-10 was increased in vaccinated mice,IL-10 levels were decreased in vaccinatedmice in chronic colitis. The decreasedIL-10 was supported by reduced IL-10mRNA expression in colon tissue, com-pared with control groups. The mRNAexpression levels of Bcl-2 and Foxp3 alsowere downregulated in the vaccine-treated group. The IFN-γ levels in the

normal control group were comparativewith other groups (data not shown),which was similar to the results reportedby Fichtner-Feigl et al. (27). We also de-tected the expression of IL-13, a typicalTh2 cytokine, in the colon tissue. Our re-sults showed that after TNBS challenge,IL-13 expression was increased signifi-cantly in saline, carrier and vaccinegroups when compared with normal con-trol; however, no significant differenceswere found between these three groups.

Vaccine Decreases the Percentagesof Th1 and Th17 in MLNs in ChronicColitis

Studies have shown that IL-12 pro-motes the differentiation of Th1 cells andthat IL-23 stabilizes the proliferation ofTh17 cells. Vaccination, by blocking IL-12and IL-23, may inhibit the differentiationof Th1 and Th17 cells. As shown in Fig-ure 8, in both acute (A) and chronic (B)colitis, the percentages of CD4+IFN-γ +Th1and CD4+IL-17+Th17 cells in MLNs, de-

Figure 5. Vaccine ameliorates intestinal inflammation in chronic colitis. (A) RepresentativeH&E staining histological images of colon samples (original magnification, 200×). (B) Semi-quantitative analysis of histological inflammation. *P < 0.05; **P < 0.01; ***P < 0.001.



tected by flow cytometry analysis, tend tobe higher in the saline and carrier groupscompared with normal mice. In contrast,the vaccine-treated group had lower per-centages (close to that of normal mice)than saline and carrier groups. Althoughthe differences didn’t reach statistical sig-nificance, the trends were supported bythe results obtained from ELISA and RT-PCR (see Figures 3, 7). If the numbers oftest samples were to be increased, a statis-tically significant difference may bereached. Furthermore, the mean percent-age of Th1 cells was higher and that ofTh17 cells was lower in acute colitis thanthose in chronic colitis. These data indi-cate that predominant Th1 cell responsesmay initiate the acute intestinal inflamma-tion, while Th17 cell responses perpetuatethe chronic intestinal inflammation, whichare in agreement with previous findings(27). Taken together, vaccine treatmenttends to decrease the percentage of Th1and Th17 cells in MLN.

DISCUSSIONWe have previously developed a

mouse IL-12/IL-23 p40 peptide-basedvirus-like particle vaccine that inducedhigh titered and relative long-lasting an-tibodies to p40, IL-12 and IL-23, and theantisera could inhibit IL-12-induced interferon-γ (26) and IL-23-induced IL-17production of splenocytes in vitro (datanot shown). In this study, we systemi-cally evaluated the in vivo effectivenessof the vaccine and the immune mecha-nisms involved in both TNBS-inducedacute and chronic colitis models.

To date, to overcome B cell tolerance,vaccines against excessive endogenouscytokines are developed mainly by eitherinserting a foreign peptide into the intactcytokine molecule (24) or linking the in-tact cytokine to a heterogeneous carriersuch as ovalbumin (25). Antibodiesraised by these vaccines act against mul-tiple antigen determinants of the targetcytokine. As such, their use may be hin-dered by undesirable crossreactions with

other self-proteins containing similar epi-topes. This is of particular concern whenthis strategy is used in humans. Cytokinepeptide-based vaccines are currentlyunder development; however, these vac-cines use inappropriate carrier proteinssuch as keyhole limpet hemocyanin (32).As these vaccines are not virus-like parti-cles, their antigenicity is low, requiringstrong adjuvants, such as complete Fre-und adjuvant, to elicit high titers of anti-bodies to the target cytokine. Virus-likeparticles induce potent B-cell responseseven in the absence of adjuvants (30,31),and as carrier proteins, they are prefer-able to other proteins. More recently, cy-tokine peptide-based virus-like particlevaccines are being investigated by chem-ically conjugating synthesized cytokinepeptides to bacteriophage Qβ virus-likeparticles (33). Compared with the vac-cines that are currently being investi-gated, the p40 vaccine used in the pres-ent study has unique advantages: (1) ituses a small peptide, avoiding possiblecross-reaction with other self proteins;(2) it is highly antigenic, eliciting long-lasting specific antibodies without theuse of an adjuvant owing to its virus-likeparticle feature; (3) it is a recombinantprotein, making it easier to prepare andto control the quality of the vaccine thanthose by chemical coupling (33); (4) thesafety of the vaccine carrier HBcAg hasbeen confirmed in a phase I clinical trialfor a malaria vaccine used in humans(34), making this vaccine even more at-tractive for human use. For these rea-sons, the vaccine developed in the pres-ent study exhibits great advantages overother cytokine vaccine strategies.

In the TNBS-induced chronic colitismodel, the initial Th1 response subsidesafter three weekly TNBS challenges and,then, is supplanted by a Th17 responseafter 4 to 5 weeks (27). This pattern of im-mune response is confirmed in the pres-ent study in which CD4+IFN-γ+ T cells,not CD4+IL-17+ T cells, tended to increasein the acute colitis, while CD4+IL-17+

T cells, not CD4+IFN-γ+ T cells, were in-creased in chronic colitis (see Figure 8).Although the differences didn’t reach sta-

Figure 6. Vaccine reduces colon fibrosis in chronic colitis. (A) Representative colon sec-tions stained with Masson trichrome showing collagen deposition (the blue color) (originalmagnification, 100×). (B) Quantitation of soluble collagens assayed by a Sircol assay. *P <0.05; **P < 0.01; ***P < 0.001.



tistical significance, the trends were sup-ported by the results obtained fromELISA and RT-PCR. In acute colitis, Th1cytokine expression (IFN-γ, IL-12, TNF)was increased significantly after TNBSchallenge (see Figure 3); in chronic colitis,Th17 cytokine expression (IL-17, IL-23,TGFβ1) was increased significantly (seeFigure 7). As the p40 vaccine effectivelytargets both IL-12 and IL-23, it exerts itsantiinflammatory effects during the entirecourse of chronic colitis. We speculatethat this vaccine, by downregulatingIL-12, is able to halt the differentiation ofnaïve T cells into Th1 cells, leading to areduction in IFN-γ and TNF. As IL-12 alsohas been shown to inhibit Fas-mediatedcell death of CD4+ T cells through the in-hibition of caspase-3 activity and the up-regulation of the anti-apoptotic molecule

Bcl-2 (35), blocking IL-12 resulted in a de-creased mRNA expression of Bcl-2 incolon tissue (see Figures 3, 7), which indi-cated an increase of T-cell apoptosis indi-rectly. In addition, the vaccine, by block-ing IL-23, may hinder the differentiationof naïve T cells into Th17 cells. Throughthese immune mechanisms, vaccine treat-ment indeed decreases the percentages ofTh1 and Th17 cells in the MLN afterTNBS challenges, returning them almostto the levels in normal mice. Althoughthe decrease is a trend, the significantlyreduced Th1 and Th17 cytokine levels incolon tissue support the trend of the de-creased percentages of Th1 and Th17cells. In the future, we will use flow cy-tometry analysis or immunohistologicalstaining to quantitate the frequency and

apoptosis of Th1/Th17 in the colon tissue.

In the acute colitis model, the IL-17level in colon tissue was elevated signifi-cantly (see Figure 3) which is in agree-ment with previous reports (36–38).However, the percentage of Th17 inMLN cells was not increased signifi-cantly (Figure 8A right). Although IL-17is described as a Th17 cell-secreted cy-tokine, much of the IL-17 released duringan inflammatory response is producedby innate immune cells which include γδT cells, CD11b+ cells, macrophages andneutrophils, etc. (39). Therefore, the sig-nificantly increased IL-17 levels in colontissue might be produced by local innateimmune cells, not the T cells in themesenteric lymph nodes. Indeed, Fitz-patrick et al. has reported that TNBS ad-ministration resulted in increased stain-ing cells for IL-17 and that the IL-17+

stained cells at the top of the muscularismucosa appeared to be macro phages(37). In the future, we will use immuno-histological staining to identify IL-17producing cells in colon tissue, whichmay be more relevant than the analysisof Th17 cells in MLN cells.

Studies have demonstrated that themain sources of IL-10 production are reg-ulatory T cells (Treg) and CD11b+

myeloid cells (40,41) and that IL-10 actson Treg cells to maintain Foxp3 expres-sion (41). In the present study, colon IL-10 levels were measured by both pro-tein and mRNA expression. As Treg areone of the important IL-10 producingcells, IL-10 levels may reflect the functionof Treg. Our results showed that in acutecolitis, IL-10 levels, measured two daysafter TNBS challenge, were higher in thevaccine-treated group than in the controlgroups (see Figure 3), which coincideswith results reported by Stallmach etal.(7). Recently, IL-23 has been found torestrain the activity of Treg to driveT cell-dependent colitis (42), and the ho-modimer of p40 is found to suppress theexpression of CD25 and Foxp3 (43). Wespeculate that, by blocking p40 andIL-23, Treg might be activated or re-cruited rapidly into the local colon tissue

Figure 7. Vaccine inhibits colon proinflammatory cytokines production in chronic colitis.(A) Protein levels of p40, IL-23, IL-17, TGFβ1, IL-13 and IL-10, measured by ELISA. (B) mRNAexpression levels of p40, TNF IL-10, Bcl-2 and Foxp3 detected by real-time RT-PCR. *P < 0.05;**P < 0.01; ***P < 0.001.



to control inflammation in the acutephase of colitis. Therefore, p40 vaccinetreatment might ameliorate colitisthrough improving the function and/ornumbers of Treg in colon tissue in acutecolitis. This is supported by the increasedmRNA expression in vaccinated mice. Inchronic colitis, levels of IL-10 and Foxp3,measured 10 days after the last TNBSchallenge, were significantly lower invaccine-treated mice than those in salineand carrier controls (see Figure 7). Be-cause vaccine-treated mice had signifi-cantly reduced colon inflammation atthis time point, accompanied by signifi-cantly lower inflammatory cytokinessuch as p40, IL-23 and IL-17, Tregs mightnot be required to exert their antiinflam-matory effects. This is supported by clin-ical observations that IL-10 expression inlamina propria mononuclear cells of pa-tients with Crohn’s disease had a ten-dency to decrease after treatment withmonoclonal antibodies against IL-12/IL-23p40 (19). In future studies, the fre-quency of CD4+CD25+Foxp3+ Treg incolon tissue will be investigated to con-firm this finding.

Vaccine treatment reduced histologicalinflammation and MPO activity. This

may be through the inhibition of IL-12.IL-12 has been demonstrated to bechemotactic for neutrophils and NK cellsthrough induction of synthesis ofplatelet-activating factor (44,45). It alsohas been reported that IL-12 is critical toneutrophil activation and resistance topolymicrobial sepsis induced by cecalligation and puncture (46). Therefore,vaccine-induced specific antibodiesblocked the biophysical functions ofIL-12, leading to less migration and acti-vation of neutrophils to the local colontissue and, thus, ameliorating intestinalinflammation.

Our results showed that the colon in-flammatory scores in the chronic colitiswere slightly lower than those in theacute colitis. This is different from otherreports where colon inflammatoryscores are significantly higher in thechronic phase than those in the acutephase (36,37). The reason may be due tothe following factors: 1) Different detec-tion times. In our acute colitis, micewere euthanized 2 days after the secondTNBS delivery; while in the chronic co-litis, mice were euthanized 10 days afterthe last TNBS delivery. In our experi-ence, after a TNBS challenge, the body

weight shows an obvious loss in thefirst 2 days, and then gradually recoversduring the next 4 days. At day 7, thebody weight is close to that of baseline,accompanied with a decreased colon in-flammation. 2) Different TNBS doses. Inour acute colitis, mice were intrarectallychallenged with TNBS twice (1.5 mg and2.0 mg, respectively), and in the chroniccolitis, the mice were challenged withincreasing doses of TNBS (1.0–2.5 mg)over 7 weeks. 3) Different experimentaldesigns. In our study, the acute andchronic experiments were performedseparately at different times with differ-ent mouse batches. This makes the dataless compatible between acute andchronic colitis, when compared withother reports (36,37). In future studies,we will combine the acute and chronicexperiments as one experiment for ki-netic observations.

Our present study also indicated thatvaccine treatment could ameliorate fi-brosis observed in chronic colitis. Thisinhibition may be through the downreg-ulation of p40, TGFβ1 and IL-17 levels.Studies have shown that IL-12/IL-23p40is overproduced during the establish-ment of an experimental fibrotic process(47,48), TGFβ1, involved in most of theprocesses of wound repair, is identifiedas a major profibrotic factor, and IL-17has profibrotic functions in multipleforms of fibrosis (49–51). In the presentstudy, vaccine treatment decreased theexpression of IL-12/IL-23p40, IL-17 andTGFβ1 in colon tissue (see Figure 7),and, therefore, ameliorated intestinal fibrosis.

A potential concern with cytokine vac-cines is that the level of the target cy-tokine may be reduced to a level belowbaseline, thus impeding the normalfunctions of the cytokine. First, as seenin our previous studies, the target cy-tokine levels are downregulated only toa level that is lower than the diseasecondition but still above the normallevel (30,31). Second, vaccine-inducedantibodies are capable of neutralizingthe excessive target cytokine within theextracellular compartment, not the cy-

Figure 8. Vaccine treatment tends to reduce the percentages of Th1 and Th17 cells inMLNs. After stimulation with PMA and inomycin, lymphocytes from MLNs were stained withanti-CD4, anti-IFN-γ and anti-IL-17 to detect the percentages of CD4+IFN-γ+Th1 andCD4+IL-17+Th17 cells by flow cytometry. (A) Acute colitis. (B) Chronic colitis. *P < 0.05; **P <0.01; ***P < 0.001.



tokines inside of the immunologicalsynapse where normal cytokineprocesses occur (22). Third, the antibodylevels that persist for about 4 months arereversible and can be adjusted by thefrequency of immunization as the im-munogenicity of cytokine vaccines ismuch less than that of microbe vaccines(22). Therefore, the vaccine strategy maybe safe when used with careful monitor-ing. The influence of this vaccine on thesusceptibility to intracellular bacteria in-fections in mice is currently under inves-tigation in our group.

In summary, we have demonstratedthat the IL-12/IL-23p40 peptide-basedvaccine that induces specific antibodiesto IL-12 and IL-23 is capable of amelio-rating experimental colitis significantly,as seen by improved body weight lossand a decrease of colon inflammationand collagen deposition, accompaniedwith reduced expressions of Th1 andTh17 cytokines and other inflammatorymediators in colon tissues. By using anactive immunization strategy, the p40vaccine, we offer an innovative and long-lasting immune inhibitory approach forthe treatment of IBD. In addition, thisstrategy can serve as a research tool inour understanding of the pathogenesis ofIBD, as it provides a persistent blockadeof a pathogenic cytokine rather than thetemporary blockage using mAbs or thepermanent deletion using the geneknockout method.

ACKNOWLEDGMENTSThis research was supported by grants

from the Canadian Institutes of HealthResearch (ROP-92387 to Z Peng) and con-ducted using facilities of the Manitoba In-stitute of Child Health, Inc. Q Guan is therecipient of the CIHR Frederick Bantingand Charles Best Canada Graduate Schol-arship. CN Bernstein is supported in partby a Research Scientist Award of theCrohn’s and Colitis Foundation of Canada and the Bingham Chair in Gastroenterology.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molecu-lar Medicine, or other interests that mightbe perceived to influence the results anddiscussion reported in this paper.

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Development of recombinant vaccines against IL-12/IL-23 p40 and in vivo evaluation of their effects in the downregulation of intestinal inflammation in murine colitis