Emilie Duvallet

Interleukin-23: A key cytokine in inflammatory diseases

Abstract Interleukin-23 (IL-23) is a pro-inflammatory cytokine composed of two subunits, p19 and p40. The p40 subunit is shared with IL-12. IL-23 and IL-12 have different receptors and different effects. Whereas IL-12 induces development of Th1 cells, which produce interferon-γ, IL-23 is involved in differentiation of Th17 cells in a pro-inflammatory context and especially in the presence of TGF-β and IL-6. Activated Th17 cells produce IL-17A, IL-17F, IL-6, IL-22, TNF-α, and GM-CSF. Inflammatory macrophages express IL-23R and are activated by IL-23 to produce IL-1, TNF-α, and IL-23 itself. These effects identify IL-23 as a central cytokine in autoimmunity and a highly promising treatment target for inflammatory diseases. IL-23 is found in the skin of patients with psoriasis, in the bowel wall of patients with chronic inflammatory bowel disease, and in synovial membrane of patients with rheumatoid arthritis. IL-23 is involved in osteoclastogenesis, independently from IL-17, via induction of RANKL expression. Debate continues to surround the role for IL-23 in the pathophysiology of inflammatory joint diseases (rheumatoid arthritis and spondyloarthritis). Ustekinumab, which inhibits IL-12 and IL-23 by blocking p40, has been found effective in cutaneous psoriasis and psoriatic arthritis, as well as in Crohns disease. Treatments that specifically target IL-23 (antibodies to p19) are being developed.

Active immunization against IL-23p19 improves experimental arthritis

INTRODUCTION IL-23 is a pro-inflammatory cytokine essential for the differentiation of Th17 lymphocytes, a subtype of T lymphocyte implied in auto-immunity. IL-23 shares a subunit with IL-12, IL-12/23p40, and comprises a specific subunit, IL-23p19. We previously demonstrated that active immunization against entire TNF-α and against peptides of IL-1β was protective in animal models of rheumatoid arthritis. The aim of this study was to evaluate the effect of peptide-based vaccines targeting the IL-23p19 subunit in collagen-induced arthritis (CIA). METHODS Using bioinformatics, the murine IL-23p19 subunit was modeled and two peptides were defined in the receptor interacting domain. Each peptide was coupled to keyhole limpet hemocyanin (KLH) to obtain two vaccines IL23-K1 and IL23-K2. Both vaccines were used for immunizations in incomplete Freund adjuvant (IFA) in groups of DBA/1 mice. Control groups received KLH or PBS at the same dates. CIA was induced by two subcutaneous injections of bovine type II collagen (CIIb), and the development of disease assessed during the next two months. Anti-CIIb and anti-IL-23 antibody levels were assessed by ELISA. Pro- and anti-inflammatory cytokines mRNA were quantified by qRT-PCR in the spleen and the synovium. T-cell populations in the spleen were evaluated by FACS analysis. RESULTS The clinical scores showed that mice treated with IL23-K1 developed less arthritis than negative controls (p<0.05). Mice immunized with IL23-K1 produced more anti-IL-23 antibodies than those immunized with IL23-K2 (p<0.001). mRNA quantification showed that the IL23-K1 immunization led to an increase of IL-10 in the spleen (p<0.05 vs. KLH), without any effect on IL-17 level. Histological examination showed that IL23-K1 strongly protected against joint destruction and inflammation (p<0.01 vs. KLH and p<0.001 vs. PBS). T-cell populations in the spleen were not modified by IL-23 modulation. CONCLUSION These data show that targeting IL-23p19 through a vaccination strategy is protective in CIA. This specific targeting of IL-23 might constitute a promising therapeutic approach to explore in rheumatoid arthritis.

Anti-cytokine vaccination in autoimmune diseases.

The concept of therapeutic vaccination represents a novel strategy of active immunotherapy that can be applied to autoimmune disease. The principle is to design molecules which can trigger an immune response, targeting a cytokine that is pathogenic and over-expressed in a given disease. The mostly available vaccines are an application of vaccination using either the self-protein coupled to a carrier (type I A), or a modified form of the protein engineered to include neo-epitopes (type I B). These approaches have been developed in models of several autoimmune diseases, mainly in TNFα-dependent diseases such as rheumatoid arthritis and Crohns disease, but also in systemic lupus erythematosus, multiple sclerosis and myasthenia gravis. Clinical trials are in progress in rheumatoid arthritis, Crohns disease and diabetes. The benefit/risk ratio of anti-cytokine vaccination is currently under study to further develop the vaccination strategies.

Modulation of Anti-Tumor Necrosis Factor Alpha (TNF-α) Antibody Secretion in Mice Immunized with TNF-α Kinoid

ABSTRACT Tumor necrosis factor alpha (TNF-α) blockade is an effective treatment for patients with TNF-α-dependent chronic inflammatory diseases, such as rheumatoid arthritis, Crohns disease, and psoriasis. TNF-α kinoid, a heterocomplex of human TNF-α and keyhole limpet hemocyanin (KLH) (TNF-K), is an active immunotherapy targeting TNF-α. Since the TNF-K approach is an active immunization, and patients receiving this therapy also receive immunosuppressant treatment, we evaluated the effect of some immunosuppressive drugs on the generation of anti-TNF-α antibodies produced during TNF-K treatment. BALB/c mice were injected intramuscularly with TNF-K in ISA 51 adjuvant. Mice were also injected intraperitoneally with one of the following: phosphate-buffered saline, cyclophosphamide, methylprednisolone, or methotrexate. Anti-TNF-α and anti-KLH antibody levels were assessed by enzyme-linked immunosorbent assay and the anti-TNF-α neutralizing capacity of sera by L929 bioassay. Our results showed that current treatments used in rheumatoid arthritis, such as methylprednisolone and methotrexate, do not significantly alter anti-TNF-α antibody production after TNF-K immunization. In contrast, the administration of cyclophosphamide (200 mg/kg) after immunization significantly reduced anti-TNF-α antibody titers and their neutralizing capacity.

Targeting VEGF-A with a vaccine decreases inflammation and joint destruction in experimental arthritis

AbstractObjectivesInflammation and angiogenesis are two tightly linked processes in arthritis, and therapeutic targeting of pro-angiogenic factors may contribute to control joint inflammation and synovitis progression. In this work, we explored whether vaccination against vascular endothelial growth factor (VEGF) ameliorates collagen-induced arthritis (CIA).Methods Anti-VEGF vaccines were heterocomplexes consisting of the entire VEGF cytokine (or a VEGF-derived peptide) linked to the carrier protein keyhole limpet hemocyanin (KLH). Two kinds of vaccines were separately tested in two independent experiments of CIA. In the first, we tested a kinoid of the murine cytokine VEGF (VEGF-K), obtained by conjugating VEGF-A to KLH. For the second, we selected two VEGF-A-derived peptide sequences to produce heterocomplexes (Vpep1-K and Vpep2-K). DBA/1 mice were immunized with either VEGF-K, Vpep1-K, or Vpep2-K, before CIA induction. Clinical and histological scores of arthritis, anti-VEGF, anti-Vpep Ab titers, and anti-VEGF Abs neutralizing capacity were determined.ResultsBoth VEGF-K and Vpep1-K significantly ameliorated clinical arthritis scores and reduced synovial inflammation and joint destruction at histology. VEGF-K significantly reduced synovial vascularization. None of the vaccines reduced anti-collagen Ab response in mice. Both VEGF-K and Vpep1-K induced persistently high titers of anti-VEGF Abs capable of inhibiting VEGF-A bioactivity.ConclusionVaccination against the pro-angiogenic factor VEGF-A leads to the production of anti-VEGF polyclonal Abs and has a significant anti-inflammatory effect in CIA. Restraining Ab response to a single peptide sequence (Vpep1) with a peptide vaccine effectively protects immunized mice from joint inflammation and destruction.

Protection from articular damage by passive or active anti‐tumour necrosis factor (TNF)‐α immunotherapy in human TNF‐α transgenic mice depends on anti‐TNF‐α antibody levels

Active anti‐tumour necrosis factor (TNF)‐α immunization with the kinoid of TNF‐α (TNF‐K) induces polyclonal anti‐TNF‐α antibodies and ameliorates arthritis in human TNF‐α (hTNF‐α) transgenic mice (TTg). We compared the efficacy of TNF‐K to that of infliximab (IFX) and of TNF‐K and IFX co‐administration, and evaluated whether the titres of anti‐hTNF‐α antibodies induced by immunization were a determinant of TNF‐K efficacy. Forty‐eight TTg mice received one of the following treatments: TNF‐K immunization (TNF‐K group); weekly IFX throughout the study duration (IFXw0–15); TNF‐K plus weekly IFX for 4 weeks (TNF‐K + IFX); and weekly IFX for 4 weeks (IFXw0–4); PBS. Animals were killed at week 16. Anti‐hTNF‐α antibody titres and clinical and histological scores were compared. All TNF‐K immunized mice (TNF‐K and TNF‐K + IFX) produced anti‐hTNF‐α antibodies. Titres were higher in TNF‐K versus TNF‐K + IFX (P < 0·001) and correlated inversely with histological inflammation (R = −0·78; P = 0·0001) and destruction (R = −0·67; P = 0·001). TNF‐K + IFX had higher histological inflammation and destruction versus TNF‐K (P < 0·05). A receiver operating characteristic (ROC) analysis of anti‐hTNF‐α antibody titres identified the criterion cut‐off value to discriminate most effectively between the TNF‐K and TNF‐K + IFX groups. Mice with high versus low titres had less histological inflammation and destruction (P < 0·05). In a model of TNF‐α‐dependent arthritis, protection from articular damage by TNF‐K correlates with the titres of induced anti‐hTNF‐α antibodies. The co‐administration of TNF‐K and a short course of infliximab does not result in less articular damage versus solely TNF‐K, due probably to lower anti‐hTNF‐α antibody production. These results are relevant for future development of active anti‐TNF‐α immunization in human disease.

A1.36 Active immunisation against peptides of vegf improves joint inflammation and destruction in collagen-induced arthritis

Background Angiogenesis is a key pathophysiological process in rheumatoid arthritis (RA). VEGF-A is considered the major player in both physiological and pathological angiogenesis. VEGF–VEGFR system blockade delays synovitis onset and ameliorates collagen-induced arthritis (CIA). We previously demonstrated the effectiveness of active anti-cytokine immunisation in RA models, with a class of therapeutics called kinoids. The kinoids (heterocomplexes consisting of a cytokine conjugated to a carrier protein, the keyhole limpet hemocyanin, KLH) induce the endogenous production of polyclonal anti-cytokine Abs. Here we tested the effectiveness in CIA of two distinct peptidic vaccines obtained by conjugation of one of two different VEGF-derived peptides to KLH. Objectives To demonstrate the inhibitory effect on CIA of sustained VEGF blockade by vaccines based on VEGF-derived peptides linked to KLH (Vpep-K). Materials and Methods Two peptides were chosen in the sequence of the VEGF murine isoformVEGF-A164 (90% identity and 93% homology with the human VEGF-A165): Vpep1 (16 aminoacids (aa), position 98-113) localised in the region cleaved by the plasmin between the exons 4 and 5, and Vpep2 (77 carboxy-terminal aa) which comprises the Arginine 164, essential for binding of VEGF to Neuropilin-1 (NRP1). Each peptide was linked to keyhole limpet hemocyanin (KLH) to form Vpep1-kinoid (Vpep1-K) and Vpep2-kinoid (Vpep2-K), respectively. CIA was induced in 48 DBA/1 male mice with two injections of 100µg of bovine collagen type II (day 0 and 21). Mice were divided in 4 groups to receive one of the following: Vpep1-K, Vpep2-K, KLH or PBS emulsified in Incomplete Freund Adjuvant (IFA, 5 intra muscular injections at days -36, -22, -8, 7 and 37). Sera were sampled from each mouse three times during the experiment (days -44, -2, 48). Results Vpep1-K group showed lower arthritic scores compared to KLH and PBS groups (p<0.05). At histological analysis, inflammation and destruction scores of the paws were lower in Vpep1-K group versus KLH and PBS group (p<0.005). Vpep1-K and Vpep2-K groups showed anti-VEGF Ab production as assessed by ELISA at day -2 and sacrifice. Conclusions Active immunotherapy with the kinoid of a single VEGF-derived peptide (Vpep1-K) induces anti-VEGF antibodies and ameliorates collagen-induced arthritis. Active anti-angiogenic immunotherapy based on peptidic vaccines might be a potential strategy to control chronic inflammation.

Combination of active and passive anti-TNFα treatments in human-TNFα transgenic mice

Background and objectives Anti-tumour necrosis factor α (TNFα) active immunisation with the heterocomplex TNFα-keyhole limpet hemocyanine (TNFK) proved therapeutic efficacy in human TNFα transgenic (TTg) mice spontaneous arthritis, opening the way for the application of this anti-TNFα targeting strategy in human disease (a phase II clinical trial is ongoing at present in rheumatoid arthritis). In this animal study the authors compared the clinical and histological efficacy of different anti-TNFα strategies in TTg mice: active anti-TNFα immunisation with TNFK, passive anti-TNFα immunotherapy with infliximab (IFX) and the association of both. Methods After spontaneous arthritis onset (w0) 48 TTg mice were allocated to receive one of the following treatments: TNFK immunisation at w0, w1, w4 (TNFK) (10 mice), weekly IFX all along the study duration (IFX w0-w15) (10 mice), TNFK immunisation+weekly IFX from w0 to w4 (TNFK+IFX) (10), weekly IFX from w0 to w4 (IFXw0-w4) (8 mice), phosphate-buffered saline (PBS) at w0, w1, w4 (PBS) (10 mice, control group). Animals were killed at w15. Clinical and histological scores were compared for all treatments. Anti-TNFα antibodies levels were assessed by ELISA, the anti-TNF neutralising capacity of sera by L929 bioassay. Histological scores of articular inflammation and destruction (H&E) and of cartilage degradation (safranine O) were quantified after sacrifice. Results All TNFK immunised mice (TNFK and TNFK+IFX groups) produced neutralising anti-TNFα antibodies. All the treatments had comparable overall clinical efficacy versus PBS (p<0.05). All IFX-treated groups had a more rapid clinical improvement versus PBS and TNFK (p<0.05). At the end of the study TNFK, IFXw0-w15 and IFX+TNFK groups had lower clinical scores of arthritis than both IFXw0-w4 and PBS groups (p<0.05). Reduced histological scores of inflammation and destruction and of cartilage degradation were evident for TNFK, IFXw0-w15 and TNFK+IFX versus PBS group. TNFK group had less histological inflammation and destruction even compared to IFX w0-w4 group. Conclusion Active immunisation with TNFK is as effective as weekly IFX on clinical activity and structural damage in TTg arthritis model. The association of a short course of IFX to TNFK results in a more rapid clinical efficacy with the same long-term clinical and histological efficacy than solely TNFK immunisation.

IL-23 specific targeting by active immunisation improves inflammation and destruction in a murine model of rheumatoid arthritis

Interleukin 23 (IL-23) is a pro-inflammatory cytokine known to be essential for the differentiation of the Th17 lymphocytes, a subtype of T lymphocyte implied in auto-immunity. Its subunit, IL-23 p19, is specific of this cytokine. We had previously demonstrated for IL-1β and tumour necrosis factor α, that active immunisation against these cytokines could be protective in animal models of arthritis. The …