Research Article For reprint orders, please contact: [email protected]
Employing an IL‑23 p19 vaccine to block IL‑23 ameliorates chronic murine colitis Background: Overexpression of IL‑23 has been implicated in the pathogenesis of Crohn’s disease. Using vaccines to block overexpressed endogenous cytokines has emerged as a new therapeutic strategy for the long-term treatment of the disease. Aim: We sought to develop peptide-based vaccines specific to IL‑23 and evaluate their effects in colitis mice. Materials & methods: The vaccine was developed by inserting a peptide derived from mouse IL‑23 p19 into the carrier protein, hepatitis B core antigen, using molecular engineering methods. One vaccine against IL‑23 p19 was obtained that induced high-titered and longlasting antibodies to IL‑23 without the use of adjuvants. The inhibitory effect of vaccine-immunized serum was subsequently evaluated in vitro. To evaluate the in vivo effects, mice were subcutaneously injected with the vaccine, carrier or saline three times. Two weeks after the last injection, chronic colitis was induced in mice by seven weekly administrations with 2,4,6-trinitrobenzene sulfonic acid. Results: In vitro studies revealed that serum IL‑23 p19-specific IgG significantly suppressed IL‑23-induced IL‑17 production by splenocytes. In vivo evaluation of the effect of the vaccine in mice with chronic colitis indicated that vaccine-immunized mice exhibited a decrease in colon inflammation, collagen deposition and levels of IL‑23 and IL‑12 cytokines, compared with control groups. Conclusion: IL‑23 p19 vaccine is capable of downregulating inflammatory responses in chronic murine colitis, providing a novel therapeutic approach in Crohn’s disease. KEYWORDS: Crohn’s disease n IL‑23 n inflammatory bowel disease n TNBS-induced colitis n vaccine
Inflammatory bowel disease (IBD) refers to idiopathic intestinal inflammation, which is traditionally classified as Crohn’s disease or ulcerative colitis. Although the cause of IBD remains unknown, considerable progress has been made in recent years to unravel the patho‑ genesis of this disease. The pathogenesis of IBD is associated with genetic factors, environmental factors, enteric flora and immunological abnor‑ malities [1–3]. Evidence indicates that a dysregu‑ lation of mucosal immunity in the gut causes an overproduction of proinflammatory cytokines and aggregation of immune cells, especially T cells, in the intestinal mucosa, thus leading to uncontrolled mucosal inflammation. Crohn’s disease is caused by an overly aggressive Th1 immune response and, in particular, an exces‑ sive IL‑23/Th17 pathway activation to bacterial antigens in genetically predisposed individuals. These Th1 cytokines, such as IL‑12, and the IL‑23/Th17 pathway are critical in generat‑ ing and perpetuating the chronic intestinal inflammation of Crohn’s disease [4–6]. In the 1990s, IL‑12 was found to be increased in patients with active Crohn’s disease and it was shown that monoclonal antibodies against IL‑12 (IL‑12 p40) could ameliorate established
2,4,6-trinitrobenzene sulfonic acid (TNBS)induced murine colitis [7,8]. Since the discovery of IL‑23, many studies have highlighted the role of this cytokine in the pathogenesis of Crohn’s disease [9–13]. IL‑23 consists of two subunits: an IL‑23-specific subunit p19 and a subunit p40 that is shared with IL‑12. In IL‑10 knockout mice, a spontaneous IBD model, the develop‑ ment of colitis was suppressed by IL‑23 p19 deficiency, but not by IL‑12 p35 deficiency; administration of IL‑23 accelerated the onset of colitis and promoted colon inflammation through an IL‑17- and IL‑6-dependent mecha‑ nism [10]. By contrast, administration of antiIL‑23 monoclonal antibodies could prevent and reverse active colitis with the downregu‑ lation of a broad array of inflammatory cyto‑ kines and chemokines in the colon [14]. In a Helicobacter hepaticus-induced T cell-dependent colitis model, Kullberg et al. showed that adop‑ tive transfer of wild-type CD4+CD45RBhi cells into p40 and Rag double-deficient mice failed to develop typholocolitis; by contrast, adoptive transfer of the same T-cell populations into IL‑12 p35 and Rag double-deficient mice caused severe typhlocolitis, and adoptive transfer of the same T-cell populations into IL‑23 p19 and
10.2217/IMT.13.141 © 2013 Future Medicine Ltd
Immunotherapy (2013) 5(12), 1313–1322
Qingdong Guan1,2, Helen A Burtnick1, Gefei Qing3, Carolyn R Weiss1,2, Allan G Ma1,2, Yanbing Ma5, Richard J Warrington1,4 & Zhikang Peng*1,2 Department of Immunology, University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada 2 Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada 3 Department of Pathology, University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada 4 Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada 5 Institute of Medical Biology, Chinese Academy of Medical Science, Kunming, China *Author for correspondence: Tel.: +1 204 789 3815 Fax: +1 204 789 3937 [email protected] 1
part of
ISSN 1750-743X
1313
Research Article
Guan, Burtnick, Qing et al.
Rag double-deficient mice resulted in attenu‑ ated typholocolitis [13]. Using IL‑23 receptor (IL‑23R)-deficient mice, Powrie’s group dem‑ onstrated that through direct signaling in the intestine, IL‑23 promoted the proliferation and accumulation of Th17 cells, enhanced the gen‑ eration of IL‑17+IFN‑g+ T cells, and suppressed the differentiation of induced Treg (iTreg) and IL‑10 production from T cells [15]. Recently, genome-wide association scan has identified numerous single nucleotide polymorphisms in IL‑23R, with high association for Crohn’s disease and ulcerative colitis [16]. These results demonstrate that the IL‑23/IL‑23R pathway plays important roles in the pathogenesis of Crohn’s disease. Although immunotherapy using monoclonal antibodies, such as infliximab, has achieved great success, it has the disadvantages of a short half-life and side effects of infusion reactions (for infliximab), probably facilitated by the develop‑ ment of antibodies against infused monoclonal antibodies [17]. Recently, a new immunotherapy with cytokine vaccines is being investigated to ameliorate asthma and autoimmune diseases in animal models, as they induce long-lasting autoantibodies to the target cytokine [18–26]. An anti-human TNF vaccine has also been devel‑ oped and tested in mice transgenic for human TNF, resulting in protection against both acute and chronic human TNF-a exposure [20]. This vaccine strategy may be used as a maintenance treatment for the diseases. Our laboratory has successfully designed cytokine vaccines by inserting a small peptide derived from the target cytokine into a carrier protein, hepatitis B core antigen (HBcAg), using molecular engineer‑ ing methods. This type of vaccine presents as a virus-like particle, elicits sufficient autoantibod‑ ies to the target cytokine without the use of an adjuvant and results in the amelioration of the disease [23–25]. We have previously developed an IL‑12/IL‑23 p40 peptide-based vaccine, which induces longlasting and high-titered antibodies to IL‑12, IL‑23 and p40. Vaccine-induced antibodies can block IL‑12- or IL‑23-induced biological effects dose-dependently in vitro [18,26]. Administra‑ tion of this p40 vaccine significantly improves TNBS-induced murine acute and chronic colitis through downregulating Th1 and Th17 cyto‑ kines [23,26]. In the present study, using the same strategy, we developed a mouse IL‑23-specific peptide-based vaccine against IL‑23 p19 and explored the in vivo effects of this vaccine in the suppression of TNBS-induced chronic colitis. 1314
Immunotherapy (2013) 5(12)
Materials & methods Animals Female BALB/c mice (7–8 weeks old) purchased from Charles River Laboratories (QC, Canada) were maintained at the Central Animal Care Services, University of Manitoba (Canada). All protocols used were approved by the University Animal Ethics Committee. Selection of antigenic peptides Antigenic peptide prediction was performed based on the occurrence of amino acid residues in experimentally known segmental epitopes [101] and DNAstar software (DNASTAR, Inc., WI, USA). Utilizing the information from antigenic peptide prediction and the highresolution solution structures of human IL‑23 [27], priority was given to peptide sequences located in the possible receptor binding sites, terminal regions and/or regions with high antigenic index, hydrophilicity, surface prob‑ ability and flexibility (Figur e 1). Five peptides were selected from mouse IL‑23 p19 sub‑ unit, including peptide A (23PRSSSPD29), B ( 58 EEEDEETK NN V P 71), C ( 76 GCD‑ PQGLKDNSQ 88), D (139EDHPRETQQ147 ) and E (153SQQWQRPLL162). Preparation & identification of vaccines & carrier HBcAg Each peptide was inserted into the carrier protein HBcAg using molecular engineering methods as previously described [24]. The recombinant plas‑ mids pHBcAg, expressing carrier (i.e., truncated HBcAg; 1–149 amino acids), and pHBcAg-A13, expressing vaccine (i.e., a recombinant protein with a selected IL‑23 peptide inserted between 78–79 amino acids of HBcAg immunodominant epitope) were constructed. The recombinant plasmids were then identified by PCR and restriction endonucleases digestion. The expres‑ sion of recombinant protein was induced with isopropyl b-d-1-thiogalactopyranoside as rou‑ tine procedure and analyzed by SDS-PAGE and immunoblotting using a polyclonal biotinylated goat antimouse IL‑23 antibody (R&D Systems Inc., MN, USA). Among five IL‑23 p19 vaccines, only one vac‑ cine (peptide D) was expressed as a virus-like particle protein that could be used for immu‑ nization. This vaccine was purified by a combi‑ nation procedure consisting of ultrasonication lysis, 40% ammonium sulfate precipitation, 20% ammonium sulfate wash and size exclu‑ sion chromatography with Sepharose CL-4B (Sigma-Aldrich, Oakville, Canada). Endotoxin future science group
Blocking IL‑23 employing an IL‑23 p19 vaccine ameliorates chronic murine colitis
T C 4.5
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
Research Article
Scale Turn, regions – Chou Fasman Coil, regions – Garnier–Robson
0
Hydrophilicity plot – Kyte–Doolittle
-4.5 F 1.7
Flexible regions – Karplus–Schulz
0
Antigenic index – Jameson–Wolf
-1.7 6 Surface probability – Emini
1
Figure 1. Selection of peptides from mouse IL‑23 p19 subunit. (A) Peptide D predicted in mouse IL‑23 p19 subunit through DNAstar software (DNASTAR, Inc., WI, USA). (B) The corresponding peptide of human IL‑23 (peptide D) in the crystal structure of human IL‑23 (arrow indicates the position of the peptide selected). Please see color figure at www.futuremedicine.com/doi/full/10.2217/IMT.13.141
in the recombinant proteins was removed with Affinity-prep polymyxin matrix (Bio-Rad, Mississauga, Canada). The antigenicity of the vaccine was determined by immunization of the mice with the vaccine or the carrier protein four times without the use of an adjuvant, as we have previously reported [24]. Sera were collected at indicated times to detect IL‑23-specific IgG levels by ELISA. Measurements of IL‑23-specific IgG antibodies by ELISA Serum IL‑23-specific IgG levels were assayed by using ELISA techniques, as previously described [23,26], in which mouse IL‑23 was coated on the plates (0.25 µg/ml; eBioscience, CA, USA) followed by sequential incuba‑ tions with tested serum (1:200) and alkaline phosphatase conjugated goat antimouse IgG future science group
(1:5000). The results were expressed using optical density at 405 nm (OD 405). Serum IL‑23-specific IgG titers were also measured in the serum pooled from vaccinated mice or carrier-treated mice using ELISA. The titer of the test samples was determined as the recipro‑ cal of the highest dilution in which the OD405 was twice that of the corresponding control serum (mice receiving HBcAg) when its OD405 was 0.10. In vitro inhibition tests of vaccine‑induced IL‑23-specific IgG antibodies The bioactivity of IL‑23-specific IgG was assayed by its ability to inhibit IL‑23-dependent IL‑17 production from lymphocytes in vitro, as previ‑ ously described [18]. IgG antibodies were puri‑ fied from IL‑23 p19 vaccine-immunized sera or www.futuremedicine.com
1315
Research Article
Guan, Burtnick, Qing et al.
Recombinant mouse IL‑23 (final concentration 10 ng/ml; eBioscience) and purified vaccineinduced antibodies at different dilutions were added to the cultures. The cultures were then incubated at 37°C in a humidified atmosphere containing 5% CO2. After 72 h, IL‑17 concentra‑ tion in the culture supernatants was determined by ELISA. The inhibition percentage of mouse IgG antibodies was calculated as follows:
3
OD405
2 1 0 -1
0
2
4 6 Weeks
8
10 Inhibition(%) =
Carrier Vaccine Vaccine sc. immunization
IL -17 amount of carrier IgG IL-17 amount of IL -17 amount of carrier IgG vaccine IgG Inhibition(%) = # 100 IL -17 amount of carrier IgG
Figure 2. The levels of specific IgG antibodies to IL‑23 induced by the vaccine. Female BALB/c mice were primed sc. with 100 µg/200 µl of vaccine and carrier, and boosted at weeks 2, 4 and 8 with 25 µg/200 µl of the vaccine or the carrier (n = 8). Sera were obtained from the mice at the indicated weeks and diluted 1:200 for determination of specific IgG levels by an ELISA in which IL‑23 (0.25 µg/ml) were coated on the plates separately. Data are representative of two independent experiments. OD: Optical density; sc.: Subcutaneous.
carrier HBcAg-immunized sera using octanoic acid–ammonium sulfate (Sigma-Aldrich) [18]. Single-cell suspensions of normal mouse splenic mononuclear cells were cultured in 96-well plates (2 × 105 cells/well) containing plate-bound antiCD3e (5 µg/ml) and soluble anti-CD28 (1 µg/m; both from BD Biosciences, Mississauga, Canada).
Inhibition rate (%)
100 80 60 40 20 0
1:10
1:50 Dilution
Figure 3. In vitro inhibition of vaccineimmunized antisera on IL‑23-induced IL‑17 production from splenocytes. Splenocytes isolated from normal BALB/c mice were stimulated with anti-CD3e/CD28 for 3 days in the presence of IL‑6 (20 ng/ml), TGF-b (1 ng/ml) and IL‑23 (10 ng/ml) with various dilutions of the sera from mice receiving IL‑23 p19 vaccine or carrier. Supernatants were collected for measurement of expression levels of IL‑17 by ELISA. Data are representative of two independent experiments.
1316
IL-17 amount of IL -17 amount of carrier IgG vaccine IgG
Immunotherapy (2013) 5(12)
Protocols of vaccine immunization & induction of colitis The vaccine was in vivo evaluated in a model of TNBS-induced chronic colitis in which mice were subcutaneously injected three times at a 2-week interval with vaccine, vaccine carrier HBcAg (100, 25 and 25 µg in 200 µl, respec‑ tively) or saline (n = 12) [23]. Two weeks later, mice were intrarectally challenged with increas‑ ing doses of TNBS (1.0–2.5 mg) at a 1-week intervals for 7 consecutive weeks to induce chronic colitis. At 10 days after the last TNBS delivery, the mice were sacrificed. Colons and blood samples were collected and processed according to different assays. Histological examination Colons were fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded colon sections were cut and then stained with hematoxylin and eosin to examine inflammation or Masson’s trichrome to define collagen depo‑ sition. Histological scoring was evaluated by a pathologist blinded to the source of treatment based on the method previously described [28]. Briefly, for each histological examination, three different parameters were estimated: severity of inflammation, depth of injury and crypt dam‑ age. All values were added to a sum, in which the maximum possible score was 10. Preparation of colon tissue extracts & measurement of cytokines by ELISA Colon tissue extracts were prepared. Frozen colonic samples were mechanically homogenized in buffer containing Tris-HCl 1 M, NaCl 3 M, and 10% Triton supplemented with a prote‑ ase cocktail (Sigma-Aldrich). Samples were then frozen (-70°C) and thawed (37°C) three times and this was followed by centrifugation future science group
# 100
Blocking IL‑23 employing an IL‑23 p19 vaccine ameliorates chronic murine colitis
at 14,000 rpm for 30 min at 4°C. Supernatants were frozen at -70°C until assay. The amounts of IL‑12 and IL‑23 in colon tissue extracts were measured by ELISA according to the manufacturer’s instructions (BD Biosciences).
Statistical analyses Values were expressed as mean ± standard error of the mean. Differences between experimen‑ tal groups were assessed by one-way analysis of variance followed by Newman–Keuls multiple comparison test (GraphPad Prism; GraphPad‑ Software, Inc., CA, USA). A p-value
Employing an IL‑23 p19 vaccine to block IL‑23 ameliorates chronic murine colitis Background: Overexpression of IL‑23 has been implicated in the pathogenesis of Crohn’s disease. Using vaccines to block overexpressed endogenous cytokines has emerged as a new therapeutic strategy for the long-term treatment of the disease. Aim: We sought to develop peptide-based vaccines specific to IL‑23 and evaluate their effects in colitis mice. Materials & methods: The vaccine was developed by inserting a peptide derived from mouse IL‑23 p19 into the carrier protein, hepatitis B core antigen, using molecular engineering methods. One vaccine against IL‑23 p19 was obtained that induced high-titered and longlasting antibodies to IL‑23 without the use of adjuvants. The inhibitory effect of vaccine-immunized serum was subsequently evaluated in vitro. To evaluate the in vivo effects, mice were subcutaneously injected with the vaccine, carrier or saline three times. Two weeks after the last injection, chronic colitis was induced in mice by seven weekly administrations with 2,4,6-trinitrobenzene sulfonic acid. Results: In vitro studies revealed that serum IL‑23 p19-specific IgG significantly suppressed IL‑23-induced IL‑17 production by splenocytes. In vivo evaluation of the effect of the vaccine in mice with chronic colitis indicated that vaccine-immunized mice exhibited a decrease in colon inflammation, collagen deposition and levels of IL‑23 and IL‑12 cytokines, compared with control groups. Conclusion: IL‑23 p19 vaccine is capable of downregulating inflammatory responses in chronic murine colitis, providing a novel therapeutic approach in Crohn’s disease. KEYWORDS: Crohn’s disease n IL‑23 n inflammatory bowel disease n TNBS-induced colitis n vaccine
Inflammatory bowel disease (IBD) refers to idiopathic intestinal inflammation, which is traditionally classified as Crohn’s disease or ulcerative colitis. Although the cause of IBD remains unknown, considerable progress has been made in recent years to unravel the patho‑ genesis of this disease. The pathogenesis of IBD is associated with genetic factors, environmental factors, enteric flora and immunological abnor‑ malities [1–3]. Evidence indicates that a dysregu‑ lation of mucosal immunity in the gut causes an overproduction of proinflammatory cytokines and aggregation of immune cells, especially T cells, in the intestinal mucosa, thus leading to uncontrolled mucosal inflammation. Crohn’s disease is caused by an overly aggressive Th1 immune response and, in particular, an exces‑ sive IL‑23/Th17 pathway activation to bacterial antigens in genetically predisposed individuals. These Th1 cytokines, such as IL‑12, and the IL‑23/Th17 pathway are critical in generat‑ ing and perpetuating the chronic intestinal inflammation of Crohn’s disease [4–6]. In the 1990s, IL‑12 was found to be increased in patients with active Crohn’s disease and it was shown that monoclonal antibodies against IL‑12 (IL‑12 p40) could ameliorate established
2,4,6-trinitrobenzene sulfonic acid (TNBS)induced murine colitis [7,8]. Since the discovery of IL‑23, many studies have highlighted the role of this cytokine in the pathogenesis of Crohn’s disease [9–13]. IL‑23 consists of two subunits: an IL‑23-specific subunit p19 and a subunit p40 that is shared with IL‑12. In IL‑10 knockout mice, a spontaneous IBD model, the develop‑ ment of colitis was suppressed by IL‑23 p19 deficiency, but not by IL‑12 p35 deficiency; administration of IL‑23 accelerated the onset of colitis and promoted colon inflammation through an IL‑17- and IL‑6-dependent mecha‑ nism [10]. By contrast, administration of antiIL‑23 monoclonal antibodies could prevent and reverse active colitis with the downregu‑ lation of a broad array of inflammatory cyto‑ kines and chemokines in the colon [14]. In a Helicobacter hepaticus-induced T cell-dependent colitis model, Kullberg et al. showed that adop‑ tive transfer of wild-type CD4+CD45RBhi cells into p40 and Rag double-deficient mice failed to develop typholocolitis; by contrast, adoptive transfer of the same T-cell populations into IL‑12 p35 and Rag double-deficient mice caused severe typhlocolitis, and adoptive transfer of the same T-cell populations into IL‑23 p19 and
10.2217/IMT.13.141 © 2013 Future Medicine Ltd
Immunotherapy (2013) 5(12), 1313–1322
Qingdong Guan1,2, Helen A Burtnick1, Gefei Qing3, Carolyn R Weiss1,2, Allan G Ma1,2, Yanbing Ma5, Richard J Warrington1,4 & Zhikang Peng*1,2 Department of Immunology, University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada 2 Department of Pediatrics & Child Health, University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada 3 Department of Pathology, University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada 4 Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada 5 Institute of Medical Biology, Chinese Academy of Medical Science, Kunming, China *Author for correspondence: Tel.: +1 204 789 3815 Fax: +1 204 789 3937 [email protected] 1
part of
ISSN 1750-743X
1313
Research Article
Guan, Burtnick, Qing et al.
Rag double-deficient mice resulted in attenu‑ ated typholocolitis [13]. Using IL‑23 receptor (IL‑23R)-deficient mice, Powrie’s group dem‑ onstrated that through direct signaling in the intestine, IL‑23 promoted the proliferation and accumulation of Th17 cells, enhanced the gen‑ eration of IL‑17+IFN‑g+ T cells, and suppressed the differentiation of induced Treg (iTreg) and IL‑10 production from T cells [15]. Recently, genome-wide association scan has identified numerous single nucleotide polymorphisms in IL‑23R, with high association for Crohn’s disease and ulcerative colitis [16]. These results demonstrate that the IL‑23/IL‑23R pathway plays important roles in the pathogenesis of Crohn’s disease. Although immunotherapy using monoclonal antibodies, such as infliximab, has achieved great success, it has the disadvantages of a short half-life and side effects of infusion reactions (for infliximab), probably facilitated by the develop‑ ment of antibodies against infused monoclonal antibodies [17]. Recently, a new immunotherapy with cytokine vaccines is being investigated to ameliorate asthma and autoimmune diseases in animal models, as they induce long-lasting autoantibodies to the target cytokine [18–26]. An anti-human TNF vaccine has also been devel‑ oped and tested in mice transgenic for human TNF, resulting in protection against both acute and chronic human TNF-a exposure [20]. This vaccine strategy may be used as a maintenance treatment for the diseases. Our laboratory has successfully designed cytokine vaccines by inserting a small peptide derived from the target cytokine into a carrier protein, hepatitis B core antigen (HBcAg), using molecular engineer‑ ing methods. This type of vaccine presents as a virus-like particle, elicits sufficient autoantibod‑ ies to the target cytokine without the use of an adjuvant and results in the amelioration of the disease [23–25]. We have previously developed an IL‑12/IL‑23 p40 peptide-based vaccine, which induces longlasting and high-titered antibodies to IL‑12, IL‑23 and p40. Vaccine-induced antibodies can block IL‑12- or IL‑23-induced biological effects dose-dependently in vitro [18,26]. Administra‑ tion of this p40 vaccine significantly improves TNBS-induced murine acute and chronic colitis through downregulating Th1 and Th17 cyto‑ kines [23,26]. In the present study, using the same strategy, we developed a mouse IL‑23-specific peptide-based vaccine against IL‑23 p19 and explored the in vivo effects of this vaccine in the suppression of TNBS-induced chronic colitis. 1314
Immunotherapy (2013) 5(12)
Materials & methods Animals Female BALB/c mice (7–8 weeks old) purchased from Charles River Laboratories (QC, Canada) were maintained at the Central Animal Care Services, University of Manitoba (Canada). All protocols used were approved by the University Animal Ethics Committee. Selection of antigenic peptides Antigenic peptide prediction was performed based on the occurrence of amino acid residues in experimentally known segmental epitopes [101] and DNAstar software (DNASTAR, Inc., WI, USA). Utilizing the information from antigenic peptide prediction and the highresolution solution structures of human IL‑23 [27], priority was given to peptide sequences located in the possible receptor binding sites, terminal regions and/or regions with high antigenic index, hydrophilicity, surface prob‑ ability and flexibility (Figur e 1). Five peptides were selected from mouse IL‑23 p19 sub‑ unit, including peptide A (23PRSSSPD29), B ( 58 EEEDEETK NN V P 71), C ( 76 GCD‑ PQGLKDNSQ 88), D (139EDHPRETQQ147 ) and E (153SQQWQRPLL162). Preparation & identification of vaccines & carrier HBcAg Each peptide was inserted into the carrier protein HBcAg using molecular engineering methods as previously described [24]. The recombinant plas‑ mids pHBcAg, expressing carrier (i.e., truncated HBcAg; 1–149 amino acids), and pHBcAg-A13, expressing vaccine (i.e., a recombinant protein with a selected IL‑23 peptide inserted between 78–79 amino acids of HBcAg immunodominant epitope) were constructed. The recombinant plasmids were then identified by PCR and restriction endonucleases digestion. The expres‑ sion of recombinant protein was induced with isopropyl b-d-1-thiogalactopyranoside as rou‑ tine procedure and analyzed by SDS-PAGE and immunoblotting using a polyclonal biotinylated goat antimouse IL‑23 antibody (R&D Systems Inc., MN, USA). Among five IL‑23 p19 vaccines, only one vac‑ cine (peptide D) was expressed as a virus-like particle protein that could be used for immu‑ nization. This vaccine was purified by a combi‑ nation procedure consisting of ultrasonication lysis, 40% ammonium sulfate precipitation, 20% ammonium sulfate wash and size exclu‑ sion chromatography with Sepharose CL-4B (Sigma-Aldrich, Oakville, Canada). Endotoxin future science group
Blocking IL‑23 employing an IL‑23 p19 vaccine ameliorates chronic murine colitis
T C 4.5
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
Research Article
Scale Turn, regions – Chou Fasman Coil, regions – Garnier–Robson
0
Hydrophilicity plot – Kyte–Doolittle
-4.5 F 1.7
Flexible regions – Karplus–Schulz
0
Antigenic index – Jameson–Wolf
-1.7 6 Surface probability – Emini
1
Figure 1. Selection of peptides from mouse IL‑23 p19 subunit. (A) Peptide D predicted in mouse IL‑23 p19 subunit through DNAstar software (DNASTAR, Inc., WI, USA). (B) The corresponding peptide of human IL‑23 (peptide D) in the crystal structure of human IL‑23 (arrow indicates the position of the peptide selected). Please see color figure at www.futuremedicine.com/doi/full/10.2217/IMT.13.141
in the recombinant proteins was removed with Affinity-prep polymyxin matrix (Bio-Rad, Mississauga, Canada). The antigenicity of the vaccine was determined by immunization of the mice with the vaccine or the carrier protein four times without the use of an adjuvant, as we have previously reported [24]. Sera were collected at indicated times to detect IL‑23-specific IgG levels by ELISA. Measurements of IL‑23-specific IgG antibodies by ELISA Serum IL‑23-specific IgG levels were assayed by using ELISA techniques, as previously described [23,26], in which mouse IL‑23 was coated on the plates (0.25 µg/ml; eBioscience, CA, USA) followed by sequential incuba‑ tions with tested serum (1:200) and alkaline phosphatase conjugated goat antimouse IgG future science group
(1:5000). The results were expressed using optical density at 405 nm (OD 405). Serum IL‑23-specific IgG titers were also measured in the serum pooled from vaccinated mice or carrier-treated mice using ELISA. The titer of the test samples was determined as the recipro‑ cal of the highest dilution in which the OD405 was twice that of the corresponding control serum (mice receiving HBcAg) when its OD405 was 0.10. In vitro inhibition tests of vaccine‑induced IL‑23-specific IgG antibodies The bioactivity of IL‑23-specific IgG was assayed by its ability to inhibit IL‑23-dependent IL‑17 production from lymphocytes in vitro, as previ‑ ously described [18]. IgG antibodies were puri‑ fied from IL‑23 p19 vaccine-immunized sera or www.futuremedicine.com
1315
Research Article
Guan, Burtnick, Qing et al.
Recombinant mouse IL‑23 (final concentration 10 ng/ml; eBioscience) and purified vaccineinduced antibodies at different dilutions were added to the cultures. The cultures were then incubated at 37°C in a humidified atmosphere containing 5% CO2. After 72 h, IL‑17 concentra‑ tion in the culture supernatants was determined by ELISA. The inhibition percentage of mouse IgG antibodies was calculated as follows:
3
OD405
2 1 0 -1
0
2
4 6 Weeks
8
10 Inhibition(%) =
Carrier Vaccine Vaccine sc. immunization
IL -17 amount of carrier IgG IL-17 amount of IL -17 amount of carrier IgG vaccine IgG Inhibition(%) = # 100 IL -17 amount of carrier IgG
Figure 2. The levels of specific IgG antibodies to IL‑23 induced by the vaccine. Female BALB/c mice were primed sc. with 100 µg/200 µl of vaccine and carrier, and boosted at weeks 2, 4 and 8 with 25 µg/200 µl of the vaccine or the carrier (n = 8). Sera were obtained from the mice at the indicated weeks and diluted 1:200 for determination of specific IgG levels by an ELISA in which IL‑23 (0.25 µg/ml) were coated on the plates separately. Data are representative of two independent experiments. OD: Optical density; sc.: Subcutaneous.
carrier HBcAg-immunized sera using octanoic acid–ammonium sulfate (Sigma-Aldrich) [18]. Single-cell suspensions of normal mouse splenic mononuclear cells were cultured in 96-well plates (2 × 105 cells/well) containing plate-bound antiCD3e (5 µg/ml) and soluble anti-CD28 (1 µg/m; both from BD Biosciences, Mississauga, Canada).
Inhibition rate (%)
100 80 60 40 20 0
1:10
1:50 Dilution
Figure 3. In vitro inhibition of vaccineimmunized antisera on IL‑23-induced IL‑17 production from splenocytes. Splenocytes isolated from normal BALB/c mice were stimulated with anti-CD3e/CD28 for 3 days in the presence of IL‑6 (20 ng/ml), TGF-b (1 ng/ml) and IL‑23 (10 ng/ml) with various dilutions of the sera from mice receiving IL‑23 p19 vaccine or carrier. Supernatants were collected for measurement of expression levels of IL‑17 by ELISA. Data are representative of two independent experiments.
1316
IL-17 amount of IL -17 amount of carrier IgG vaccine IgG
Immunotherapy (2013) 5(12)
Protocols of vaccine immunization & induction of colitis The vaccine was in vivo evaluated in a model of TNBS-induced chronic colitis in which mice were subcutaneously injected three times at a 2-week interval with vaccine, vaccine carrier HBcAg (100, 25 and 25 µg in 200 µl, respec‑ tively) or saline (n = 12) [23]. Two weeks later, mice were intrarectally challenged with increas‑ ing doses of TNBS (1.0–2.5 mg) at a 1-week intervals for 7 consecutive weeks to induce chronic colitis. At 10 days after the last TNBS delivery, the mice were sacrificed. Colons and blood samples were collected and processed according to different assays. Histological examination Colons were fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded colon sections were cut and then stained with hematoxylin and eosin to examine inflammation or Masson’s trichrome to define collagen depo‑ sition. Histological scoring was evaluated by a pathologist blinded to the source of treatment based on the method previously described [28]. Briefly, for each histological examination, three different parameters were estimated: severity of inflammation, depth of injury and crypt dam‑ age. All values were added to a sum, in which the maximum possible score was 10. Preparation of colon tissue extracts & measurement of cytokines by ELISA Colon tissue extracts were prepared. Frozen colonic samples were mechanically homogenized in buffer containing Tris-HCl 1 M, NaCl 3 M, and 10% Triton supplemented with a prote‑ ase cocktail (Sigma-Aldrich). Samples were then frozen (-70°C) and thawed (37°C) three times and this was followed by centrifugation future science group
# 100
Blocking IL‑23 employing an IL‑23 p19 vaccine ameliorates chronic murine colitis
at 14,000 rpm for 30 min at 4°C. Supernatants were frozen at -70°C until assay. The amounts of IL‑12 and IL‑23 in colon tissue extracts were measured by ELISA according to the manufacturer’s instructions (BD Biosciences).
Statistical analyses Values were expressed as mean ± standard error of the mean. Differences between experimen‑ tal groups were assessed by one-way analysis of variance followed by Newman–Keuls multiple comparison test (GraphPad Prism; GraphPad‑ Software, Inc., CA, USA). A p-value
