Laure Delavallée

TNFalpha kinoid vaccination-induced neutralizing antibodies to TNFalpha protect mice from autologous TNFalpha-driven chronic and acute inflammation.

The proinflammatory cytokine TNFα is a potent mediator of septic shock and a therapeutic target for chronic inflammatory pathologies including rheumatoid arthritis and Crohns disease. As an alternative to anti-human TNFα (hTNFα) mAbs and other hTNFα blocker approved drugs, we developed an active anti-hTNFα immunotherapy, based on a vaccine comprised of a keyhole limpet hemocyanin-hTNFα heterocomplex immunogen (hTNFα kinoid) adjuvanted in incomplete Freunds adjuvant. In mice transgenic for hTNFα (TTg mice), hTNFα kinoid vaccination elicited high titers of Abs that neutralized hTNFα bioactivities but did not result in a cellular response to hTNFα. The vaccine was safe and effective in two experimental models. Kinoid-immunized but not control TTg mice resisted hTNFα-driven shock in one model and were prevented from spontaneous arthritis, inflammatory synovitis, and articular destruction in a second model. These data demonstrate an anti-cytokine induction of autoimmune protection against both acute and chronic hTNFα exposure. They show that active vaccination against a human cytokine can be achieved, and that the immune response can be effective and safe.

Interplay between TNF and Regulatory T Cells in a TNF-Driven Murine Model of Arthritis

CD4+CD25+Foxp3+ regulatory T cells (Treg) are involved in several autoimmune diseases, including rheumatoid arthritis. TNF-α blockers induce therapeutic benefits in rheumatoid arthritis via a variety of mechanisms. We aimed to characterize the impact on Treg of TNF-α overexpression in vivo and of TNF-α inhibiting treatments. We used human TNF-α transgenic mice as a model of strictly TNF-α–dependent arthritis. Our study showed that initial Treg frequency was lower in TNF-α transgenic mice than in wild-type mice. However, the course of arthritis was marked by elevation of Treg frequency and a dramatic increase in expression of TNFR2. Antagonizing TNF-α with either the anti-human TNF-α Ab (infliximab) or active immunotherapy (TNF-kinoid) increased the Treg frequency and upregulated CTLA-4, leading to enhancement of suppressor activity. Moreover, both anti–TNF-α strategies promoted the differentiation of a CD62L− Treg population. In conclusion, in an in vivo model of TNF-α–driven arthritis, Treg frequency increased with inflammation but failed to control the inflammatory process. Both passive and active TNF-α–inhibiting strategies restored the suppressor activity of Treg and induced the differentiation of a CD62L− Treg population.

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.

Vaccination with cytokines in autoimmune diseases

Most autoimmune diseases have an unknown etiology, but all involve cytokines cascade in their development. At the present time, several cytokines have been identified as major targets in various autoimmune diseases, involving the development of monoclonal antibodies (MAbs) against those cytokines. Even if MAbs are indeed efficient, the passive immunotherapies also present some disadvantages and are expensive. To counter this, several strategies have been developed, including active immunotherapy, based on the vaccination principle. The aim of such a strategy is to induce a B cell response and to obtain autoantibodies able to neutralize the interaction of the self-cytokine with its receptor. To that purpose, cytokines (entire or peptide) are either coupled with a protein-carrier or virus-like particle, or modified with foreign Th cell epitopes. DNA vaccination can also be used with cytokine sequences. This review focuses on the different vaccination strategies with cytokines (including Tumor Necrosis Factor (TNF)α, Interleukin-1β (IL-1β), IL-17) in different autoimmune diseases in preclinical studies; the benefit/risk ratio of such a strategy and the present development of clinical trials in some autoimmune diseases are also discussed.

Active immunization to tumor necrosis factor-α is effective in treating chronic established inflammatory disease: a long-term study in a transgenic model of arthritis

IntroductionPassive blockade of tumor necrosis factor-alpha (TNF-α) has demonstrated high therapeutic efficiency in chronic inflammatory diseases, such as rheumatoid arthritis, although some concerns remain such as occurrence of resistance and high cost. These limitations prompted investigations of an alternative strategy to target TNF-α. This study sought to demonstrate a long-lasting therapeutic effect on established arthritis of an active immunotherapy to human (h) TNF-α and to evaluate the long-term consequences of an endogenous anti-TNF-α response.MethodshTNF-α transgenic mice, which spontaneously develop arthritides from 8 weeks of age, were immunized with a heterocomplex (TNF kinoid, or TNF-K) composed of hTNF-α and keyhole limpet hemocyanin after disease onset. We evaluated arthritides by clinical and histological assessment, and titers of neutralizing anti-hTNF-α antibody by enzyme-linked immunosorbent assay and L929 assay.ResultsArthritides were dramatically improved compared to control mice at week 27. TNF-K-treated mice exhibited high levels of neutralizing anti-hTNF-α antibodies. Between weeks 27 and 45, all immunized mice exhibited symptoms of clinical deterioration and a parallel decrease in anti-hTNF-α neutralizing antibodies. A maintenance dose of TNF-K reversed the clinical deterioration and increased the anti-hTNF-α antibody titer. At 45 weeks, TNF-K long-term efficacy was confirmed by low clinical and mild histological scores for the TNF-K-treated mice. Injections of unmodified hTNF-α did not induce a recall response to hTNF-α in TNF-K immunized mice.ConclusionsAnti-TNF-α immunotherapy with TNF-K has a sustained but reversible therapeutic efficacy in an established disease model, supporting the potential suitability of this approach in treating human disease.

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.

Kinoid of human tumor necrosis factor-alpha for rheumatoid arthritis

Introduction: Anti-TNF-α drugs have dramatically changed treatment of rheumatoid arthritis (RA) in terms of both clinical control and articular damage prevention. Despite this, they hold some important drawbacks, such as frequent therapeutic failures and high costs. Anti-TNF-α active immunization, with a therapeutic vaccine against TNF-α, is a promising alternative anti-TNF-α targeting strategy, potentially devoid of treatment limitations of some of current anti-TNF blocking agents. Areas covered: This review covers the preclinical proof-of-concept of anti-TNF-α vaccination with the kinoid of human TNF-α (TNFK) and analyzes the body of evidence forming the rationale for the application of this strategy in RA and other TNF-α-dependent diseases. We describe the theoretical bases of anti-TNF-α active immunization and of experimental data supporting the applicability of TNFK to human disease in terms of both safety and efficacy. Expert opinion: Based on preclinical efficacy and safety data supporting its feasibility in a Phase I – II trial in Crohns disease, anti-TNF-α vaccination with TNFK has entered the phase of clinical development and promises to be a valuable anti-TNF-α targeting strategy in human disease. The focus is made in the first clinical trial in RA (Phase II) on the efficacy in active RA patients having developed antibodies against anti-TNF mAbs.

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.

In Vivo Expansion of Activated Foxp3+ Regulatory T Cells and Establishment of a Type 2 Immune Response upon IL-33 Treatment Protect against Experimental Arthritis

IL-33 is strongly involved in several inflammatory and autoimmune disorders with both pro- and anti-inflammatory properties. However, its contribution to chronic autoimmune inflammation, such as rheumatoid arthritis, is ill defined and probably requires tight regulation. In this study, we aimed at deciphering the complex role of IL-33 in a model of rheumatoid arthritis, namely, collagen-induced arthritis (CIA). We report that repeated injections of IL-33 during induction (early) and during development (late) of CIA strongly suppressed clinical and histological signs of arthritis. In contrast, a late IL-33 injection had no effect. The cellular mechanism involved in protection was related to an enhanced type 2 immune response, including the expansion of eosinophils, Th2 cells, and type 2 innate lymphoid cells, associated with an increase in type 2 cytokine levels in the serum of IL-33–treated mice. Moreover, our work strongly highlights the interplay between IL-33 and regulatory T cells (Tregs), demonstrated by the dramatic in vivo increase in Treg frequencies after IL-33 treatment of CIA. More importantly, Tregs from IL-33–treated mice displayed enhanced capacities to suppress IFN-γ production by effector T cells, suggesting that IL-33 not only favors Treg proliferation but also enhances their immunosuppressive properties. In concordance with these observations, we found that IL-33 induced the emergence of a CD39high Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.

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.