WO2007009233A2

WO2007009233A2 - Peptide-based cytokine vaccines in the treatment of autoimmune and inflammatory diseases

Info

Publication number: WO2007009233A2
Application number: PCT/CA2006/001170
Authority: WO
Inventors: Zhikang Peng; Yanbing Ma
Original assignee: University Of Manitoba
Priority date: 2005-07-18
Filing date: 2006-07-18
Publication date: 2007-01-25
Also published as: WO2007009233A3

Abstract

Methods and materials involved in the development of peptide-based cytokine vaccines for treatment of autoimmune and inflammatory diseases are herein described. These cytokines include tumour necrosis factor alpha (TNFα ), interferon gamma (IFNϜ ), interleukin (IL) 1, 2, 6, 10, 12, 15, 18 and their receptors. In the invention, using TNFα as an example, the selected TNFα peptide is fused to a carrier protein, hepatitis B core antigen (HBcAg), forming a fusion protein - chimeric HBcAg - that is presented as virus-like particles. Administration of the vaccine without adjuvant induces high titers of autoantibodies, which neutralize self-TNFα that is increased in a number of diseases.

Description

Peptide-based cytokine vaccines in the treatment of autoimmune and inflammatory diseases

PRIOR APPLICATION INFORMATION This application claims the benefit of US Provisional Patent Application

60/699,871 , filed July 18, 2005. BACKGROUND OF THE INVENTION

In a number of chronic diseases, such as autoimmune diseases, inflammatory diseases, asthma, and some infections and cancers, cytokines and inflammatory factors are found to be significantly increased.¹' ² These increased cytokines are involved with the pathogenesis of the diseases. Strategies of blocking one of these cytokines with specific monoclonal antibodies or their soluble receptors have shown to be successful in treatment. These cytokines include tumour necrosis factor alpha (TNFα), interferon gamma (IFNγ), interleukin (IL)-I , IL-2, IL-6, IL-10, IL-12, IL-15, and IL-18. For example, TNFα, IFNγ and IL-12 are significantly increased in the following diseases, and treatment with monoclonal antibodies against TNFα resulted in significant clinical improvement in each case. These diseases include rheumatoid arthritis,³' ⁴ inflammatory bowel disease,⁵ Crohn's disease,⁶ graft versus host disease,⁷ cancer,⁸' ⁹ cancer-induced cachexia,⁹' ¹⁰ ankylosing spondylitis,¹¹ psoriasis,¹² systemic lupus erythematosus¹³ as well as allergic asthma.¹⁴' ^1δ Treatment with monoclonal antibodies to cytokines other than TNFα, which include interferon gamma (IFNγ), interleukin (IL) 1 , 2, 6, 10, 12, 15, 18 and their receptors, such as IL-2R alpha and IL-2R beta, are also effective in some of the above diseases as well as in HIV infections and some cancers. Below are some examples showing clinical effectiveness of treatment with monoclonal antibodies to the target cytokine or its receptor. Administration of mAbs to TNFV (the mouse/human chimeric mAb infliximab or the human mAb adalimumab) or soluble TNFV receptors (p55 receptors onercept and p75-Fc fusion protein etanercept) has been highly effective in treating active Crohn's disease, maintaining remission, closing fistulas, maintaining fistula closure, and treating ankylosing spondylitis,⁵ which induces a significant improvement of symptoms and quality of life in 65% of patients refractory to 5-ASA drugs, corticosteroid and/or immunomodulators.¹⁶ This also resulted in an approximate 60% reduction from baseline in endoscopic lesions accompanied by a marked reduction of inflammatory infiltrate in mucosal biopsies,¹⁷ and showed successful corticosteroid withdrawal in 73% of patients.¹⁸ In a multicenter, randomized, placebo-controlled, double-blind, phase 2 clinical trial, administration of a human monoclonal antibody against IL-12 (ABT-874/J695) induces clinical responses and remissions in patients with active Crohn's disease.¹⁹ Administration of a humanized monoclonal antibody to IL-6 receptor has shown to be effective in the treatment of Crohn's disease and rheumatoid arthritis,²⁰' ²¹ while administration of a humanized monoclonal antibody to IL-2R alpha (daclizumab) is effective in the treatment of multiple sclerosis and leukemia. Targeting IL-15 and its receptor system are also effective in the treatment of inflammatory autoimmune diseases.²²

Although in the above-mentioned studies administration of either the mAb to cytokine/receptor or soluble receptors provides a novel treatment for the diseases, the agents all act as passively administered neutralizing blockages with a short half-life. For example, the half-life of a humanized monoclonal antibody against TNFV (adamilumab) is 18 - 20 days and that of etanercept (soluable TNFV receptors) is several days. Repeated injections are required to maintain their effects, since improvements seen are reversed upon the discontinuation of treatment. Side effects include induction of anti-chimeric antibodies and acute infusion reactions.⁵ In the infliximab (an mAb against TNFα) therapy, 28% to 68% of treated patients are found to have anti-chimeric antibodies, and the development of such antibodies may be associated with a loss of response to infliximab.⁵ Infusion reactions occur in 17% of infliximab-treated patients.⁵ Another highly relevant concern is the extremely high cost (US$12-15,000 annually per patient) associated with such passively administered therapeutics.

In order to overcome the disadvantages in the currently used mAbs to cytokines or soluble cytokine receptors strategies, vaccines against cytokines have been developed. The immune system can identify and destroy foreign microbes as well as foreign substances while sparing the body's own tissues. For the immune system to recognize self-proteins, the protein's structure must be altered. Studies have demonstrated that if a self-protein is coupled to a foreign protein or modified by inserting a foreign peptide containing T cell epitopes, the self-component in the conjugate or the modified self-protein is recognized as foreign by the immune system of the host, and antibodies against self-epitopes are generated. According to this principle, vaccines against TNFV have been successfully investigated. Neutralizing antibodies to TNFV are raised by immunization with a TNFV vaccine, a modified TNFV inserted with a hen egg lysozyme or an ovalbumin peptide.²³ Vaccination with the TNFV vaccine decreased the severity of collagen-induced arthritis, ameliorated the symptoms of experimental cachexia²³ and airway inflammation¹⁵ accompanying high titer antibody responses to TNFV in mice. Furthermore, in clinical trials the administration of another cytokine vaccine, modified interferon alpha (IFNα), for the treatment of H I V- 1 -infected patients has been successful and has reached phase ll/lll stages.²' ²⁴Compared with currently used mAbs to cytokines, this active immunization strategy using vaccines has the following advantages:

1. It provides long-term protection with a few injections, while infliximab must be administered every 8 weeks; 2. The antibodies induced by the vaccine are natural human antibodies which are more effective and longer lasting than the artificial chimeric antibodies; 3. Costs are low. The vaccine itself is less costly in comparison with infliximab, which is estimated over US$11 ,000 annually per patient at a low dose of 3 mg/kg; 4. No side effects occur, such as infusion reactions and the induction of anti- chimeric antibodies.

Figure 1 shows the concept of passive immunizatoin with mAbs and active immunization with vaccines.

In the above-mentioned pioneering anti-cytokine vaccine studies, the vaccines are all modified cytokines, made by inserting a foreign peptide into the whole cytokine molecule,¹⁵' ²³' ²⁴ Thus, the vaccine-induced polyclonal antibodies are directed to all epitopes of the whole target molecule. As will be appreciated by one of skill in the art, these antibodies may cross-react with other cytokines. This is a particular concern when this strategy is used in humans. Only in one study, the vaccine was made by coupling TNFα peptides to a carrier protein, keyhole limpet hemocyanin (KLH).²⁵ However, the immunogenicity of the vaccine was very low in spite of the addition of complete Freund's adjuvant.²⁵ In the present invention, the vaccine consists of small cytokine peptides derived from receptor binding sites integrated into a unique carrier protein, hepatitis B core antigen, which presents itself as a virus-like particle and are highly immunogenic. Compared with the modified cytokine vaccines such as the AutoVac TNFα vaccine that is currently under development¹⁵' ²⁶ or an IFNα vaccine²⁴ and other peptide-based TNFα vaccines tested in animals,²⁵ our invention of peptide-based cytokine vaccines has the following advantages (Figures 2 and 3):

1. No cross-reaction to other cytokines due to the fact that the vaccine is peptide-based. The modified cytokine vaccines such as the AutoVac TNFα vaccine, are constructed using a modified whole TNFα molecule, i.e. a

TNFα molecule inserted with a peptides containing T epitopes.¹⁵' ²⁶ Antibodies induced by this type of vaccine are against all epitopes in the TNFα molecule. Therefore, it is possible that the vaccine-induced polyclonal antibodies cross-react with the epitopes in other cytokines, causing side effects - particularly in humans. In contrast, polyclonal antibodies induced by the peptide-based vaccine are directed only to the small peptide contained in the vaccine. Therefore our peptide-based TNFα vaccines are much safer than the modified TNFα vaccine, the AutoVac TNFα vaccine, which is currently in phase Il human studies. Figure 2 shows the differences between the AutoVac TNFα vaccine and our vaccine.

2. No adjuvant is required to elicit a strong immune response because the vaccine presents itself as capsid-like particles. Our peptide-based vaccine consists of the hepatitis B core antigen (HBcAg) as a carrier protein and a TNFα peptide. The hepatitis B core antigen (HBcAg) virus capsid, consisting of 240 or 180 molecules of the core antigen protein, has demonstrated great potential to break B cell tolerance and produce strong antibody responses to the inserted antigens. The inserted peptide is natively arrayed in a highly repetitive and ordered fashion on the surface of the virus-like particles that are highly immunogenic, inducing strong and specific immune responses to the TNFα peptides. Our experimental data have shown that when immunization with the vaccine without adjuvant, high titers of antibody responses to TNFα were induced (Table 1), which are as strong as the antibody responses induced by vaccine plus an adjuvant such as complete Freund's adjuvant, novasome or CpG oligodeoxynucleotides (Figure 4). The responses last for more then 7 months and significantly increased upon a boost injection (Figure 4). In contrast to our results, in a recent report, a TNFα peptide-based vaccine, made by coupling TNF peptides to the carrier protein KLH,²⁵ which is not presented as capsid-like particles, had a weak immunogenicity. Among the four TNFα peptide-based KLH vaccines tested, only one induced a low titer antibody response under the help of a strong adjuvant - complete Freund's adjuvant, which can only be used in animals, not humans. This makes the invented vaccine more suitable for human use than either the AutoVac TNFα vaccine¹⁵' ²⁶ or the peptide/KLH conjugates ²⁵ since both vaccines require an adjuvant to enhance immunogenicity. Figure 3 shows the difference between KLH-TNFα peptide conjugate and our vaccine.

Autoimmune and inflammatory diseases are chronic immunological inflammatory disorders, requiring long-term treatment. The proposed new strategy has the opportunity to provide a long-lasting impact with fewer side effects and low cost in the treatment of these diseases as compared to currently used passive immunization with monoclonal antibodies.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a reagent comprising: a TNFV-derived peptide; and a carrier protein.

According to a second aspect of the invention, there is provided a method of inducing an immune response in an individual, comprising: administering to an individual in need of such a treatment, an effective amount of a composition comprising: a TNFV-derived peptide; and a carrier protein.

According to a third aspect of the invention, there is provided a method of treating, ameliorating or preventing autoimmune and inflammatory diseases comprising: administering to an individual in need of such a treatment, an effective amount of a composition comprising: a TNFV-derived peptide; and a carrier protein.

According to a fourth aspect of the invention, there is provided an expression system comprising: a nucleic acid molecule deduced from a peptide selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID No. 1, amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ

(SEQ ID No. 2, amino acids 16-26 of TNFα); 6 or more consecutive residues of

EINRPDYL (SEQ ID No. 3, amino acids 135-142 of TNFα); 6 or more consecutive residues of LNRRANALLANG (SEQ ID No. 4, amino acids 28-39 of TNFα); and 6 or more consecutive residues of ELRDNQLW (SEQ ID No. 5, amino acids 41-49 of TNFα).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Concept of passive immunization with humanized mAbs to TNFα

(left) and active immunization with TNFα vaccines (right). Figure 2. A comparison of AutoVac TNFα vaccine and our peptide vaccine.

Figure 3. A comparison of KLH-TNFα peptide conjugate and our peptide vaccine.

Figure 4. IgG responses after immunization with a virus-like particle cytokine vaccine (chimeric HBcAg) with or without adjuvant. A mouse cytokine peptide-based vaccine was constructed by inserting a mouse cytokine peptide (14 amino acid residues) into the carrier protein HBcAg, expressed by E. coli, and purified by sucrose gradient centrifugation. The vaccine is presented as virus-like particles. Groups of BALB/c mice were subcutaneously immunized 3 times with the vaccine alone or with the vaccine plus one of the following adjuvants: novasome, alum and complete Freund's adjuvant. Ten days after the 7^th month, all mice were boosted with the cytokine vaccine without adjuvant. Serum IgG responses to the mouse cytokine were measured using ELISA. Figure 5. Human TNFα and mouse TNFα sequences and key peptides selected for vaccine preparation.

Figure 6. Identification of the chimeric HBcAg / TNFα peptide fusion protein under an electron microscope. The fusion protein is presented as capsid-like particles as indicated by the arrow.

Figure 7. Vaccination with a mouse TNFα vaccine prevents subsequent airway inflammation in asthmatic mice. (A) Protocol. Mice were subcutaneously immunized with a mouse TNFα vaccine (HBcAg/TNFα peptide #1 or HBcAg/TNFα peptides #1+#2) and then sensitized with ovalbumin (OVA) plus alum intraperitoneal^. Airway inflammation was induced by nasal administration of ovalbumin. Mice receiving immunization with the carrier protein (native HBcAg) and sensitized and intranasally challenged with ovalbumin served as controls. (B) Anti-TNFα titers. Serum TNFα-specific IgG titers were measured by ELISA. (C) Bronchoalveolar lavage fluid (BALF) eosinophils and cytokines. One week after the intranasal challenge, BALF and sera were collected. Eosinophils were stained and counted. BALF TNFα, IL-13 and IL-12 levels were measured by ELISA.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. As used herein, "effective amount" refers to the administration of an amount of a given compound that achieves the desired effect.

As used herein, "purified" does not require absolute purity but is instead intended as a relative definition. For example, purification of starting material or natural material to at least one order of magnitude, preferably two or three orders of magnitude is expressly contemplated as falling within the definition of "purified".

As used herein, the term "isolated" requires that the material be removed from its original environment.

As used herein, the term "treating" in its various grammatical forms refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent other abnormal condition.

As used herein, "conservative substitution" refers to substitution of an amino acid residue with another amino acid residue which has similar properties such that one of skill in the art would anticipate or predict that the secondary structure and hydropathic nature of the polypeptide would be substantially unchanged.

As used herein, "variant" refers to allotypes known in the art that comprise one or more amino acid changes within a given sequence. As used herein, "autoimmune or inflammatory disease" refers to a disease or disorder wherein cytokine levels are elevated. These diseases include but are by no means limited to rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, graft versus host disease, cancer, cancer-induced cachexia, ankylosing spondylitis, psoriasis, systemic lupus erythematosus as well as allergic asthma. The invention relates to TNFα peptide based vaccines and the methods and materials involved in the development of these vaccines for treatment of autoimmune and inflammatory diseases, for example, many of autoimmune and inflammatory diseases having elevated TNFα levels, which may also be referred to as TNFα-related diseases. In the invention, selected TNFα-derived peptide fragments are fused through molecular engineering methods to a carrier protein or chemically coupled to a carrier protein in a preferred embodiment, hepatitis B core antigen (HBcAg), hepatitis B surface antigen or other virus carrier proteins, forming a fusion protein - chimeric HBcAg that is presented as virus-like particles. Administration of the vaccine induces autoantibodies, which neutralize self-TNFα, which as discussed above, is increased in a number of diseases.

TNFα, a Th1 cytokine, is a 17 kDa pro-inflammatory cytokine produced by monocytes, macrophages and T cells that can affect proliferation, differentiation and function of virtually every cell type. TNFα cytokine level is increased in various diseases such as rheumatoid arthritis, inflammatory bowel disease, graft versus host disease, and some cancers. As will be appreciated by one of skill in the art, immunologically down-regulating the levels of TNFα is a better approach for the treatment of these diseases than the currently available pharmaceutical therapies. This is supported by the successful use of humanized monoclonal antibodies to TNFα or soluble TNFα receptors as passive immunization in the treatment of these diseases. However, a short half-life, side effects such as infusion reactions, and extremely high cost limit the use of this approach, because all these diseases are chronic and require long-term treatment. To overcome these disadvantages, we have developed an active immunization strategy using peptide-based vaccines against TNFα. These vaccines will induce autoantibodies, which will neutralize TNFα, leading to long-term improvement of these diseases.

The vaccine prepared should produce high titers of neutralising antibodies against TNFα. The binding of the antibodies to the TNFα should prevent the interaction of TNFα with receptors and reduce serum and tissue levels due to the elimination of the antibody-antigen immune complexes by the immune system. The key step to induce high titer antibodies in the active immunization strategy is to design an effective vaccine. Modified cytokine vaccines have been developed,¹⁵' ²⁶ as discussed previously, these vaccines have two significant disadvantages; a. it may have cross-reaction to other cytokines due to the fact that the vaccine is whole molecule-based; b. it has a low immunogenicity and therefore strong adjuvants are required to elicit immune responses. To overcome these disadvantages, we have invented peptide-based cytokine vaccines. When designing a peptide-based vaccine against a self-protein, the following factors should be taken into consideration.

(1) The size of the peptide. Small peptide-based vaccines have several advantages for limiting any possible cross-reactivities. Some epitopes in linear peptides ranging between 6 - 20 consecutive amino acid residues are fully antigenic. (2) Location on the native protein and cross-reaction of the peptide. It is preferable to choose peptides derived from receptor binding sites to avoid possible activation of cells when antibodies react with cell-bound TNFα. Cross- reactivity of the chosen peptides to other cytokines, especially with those of the same family, should also be avoided. (3) The carrier protein. The selection of the appropriate carrier protein is important for increasing vaccine antigenicity. The most commonly used carrier proteins are bacterial proteins that humans commonly encounter, such as Tetanus toxoid or Diphtheria toxoid. Virus-like particles can induce potent B cell responses even in the absence of adjuvants.²⁷These large particles are highly antigenic because they improve the presentation of the epitopes to cells of the immune system. Immunization with the self-antigen displayed on the surface of a virus-like particle has been proven to produce high titer antibodies to the self-antigen.²⁷ The hepatitis B core or surface antigens (HBcAg or HBsAg) virus capsid, consisting of 240 or 180 molecules of the core antigen protein, has demonstrated great potential to break B cell tolerance and produce strong antibody responses to the inserted antigens. The inserted peptide or polypeptide is natively arrayed in a highly repetitive and ordered fashion on the surface of the virus-like particles.^27"29

According to the above principles, five key peptides ranging between 9 and

12 amino acid residues were selected from the receptor binding sites of the human

TNFV molecule (Table 1) (Figure 5). The selection of key peptides was based on the crystal or high-resolution solution structures of the TNFα molecule and the structures of their receptor complexes,^30"32 mutational analysis,³³ epitopic regions for antibodies against TNFV,³⁴ and antigen prediction software. Accordingly, 5 chimeric HBcAg fusion protein vaccines were constructed and produced . Four of the 5 fusion proteins presented as virus-like particles (Table 1) (Figure 6).

Table 1. Core sequences selected from human TNFα receptor binding sites and expression of chimeric HBcAg vaccines containing human TNFα peptides

Preparation of vaccine conjugates. As discussed previously, HBcAg has been chosen as the carrier protein. Two methods will be used to prepare the peptide-HBcAg conjugate. One is to use molecular engineering methods to form a chimeric HBcAg containing TNFV peptides, and the other method is to use chemical linking methods in the case that the chimeric HBcAg fails to form virus- like particles.

To increase the antigenicity and ensure better quality control, the genetic linking method was used to insert the key peptide into the carrier HBcAg to form a virus-like fusion protein. The DNA sequences coding the key peptides were synthesized and inserted into the polynucleotide sequence corresponding to the immunodominant region of HBcAg by introduction of a restriction enzyme site, as described below. This allows expressed peptides to latch to the surface of HBcAg particles. The chimeric HBcAg cDNA may be inserted into the pThioHis prokaryotic expression vector. The proper insertion of the key peptide can be confirmed by restriction endonuclease digestion and polymerase-chain reaction with specific primers for HBcAg and different DNA sequences of the target peptides, respectively. The E. CoIi expressed chimeric HBcAg was purified by ammonium sulphate precipitation and sucrose gradient sedimentation. As shown below, the chimeric HBcAg containing TNFα peptides elicited high titers of antibody responses in mice without use of any adjuvant (Table 2).

Table 2. Titers of antibodies to human TNFV in mice immunized with a human TNFV vaccine

Mouse #1 Mouse #2 Mouse #3 Mouse #4

Vaccine HBcAg / 1 : 80,000 1 : 160, 000 1 : 640,000 1 : 160,000 peptide #1

HBcAg / 1 : 160,000 1 : 160, 000 1 : 320,000 1 : 320,000 peptide #2

HBcAg / 1 : 80,000 1 : 640, 000 1 : 80,000 1 : 40,000 peptide #3

In other embodiments, the peptide may comprise 7 or more consecutive residues, 8 or more consecutive residues, 9 or more consecutive residues, or 10 or more consecutive residues of any one of SEQ ID No. 1-5 or SEQ ID No. 1-4 (where appropriate), or variants thereof. As will be appreciated by one of skill in the art, in embodiments wherein the vaccine is a DNA vaccine or wherein the peptides are produced as genetic fusions co-synthesized with a carrier as discussed below, the nucleic acid sequence may be based on or deduced from any one of the above-described peptides, as described below. In other embodiments, the peptide may consist essentially of or may consist of 6 or more consecutive residues, 7 or more consecutive residues, 8 or more consecutive residues, 9 or more consecutive residues, or 10 or more consecutive residues of any one of SEQ ID No. 1-5 or SEQ ID No. 1-4 (where appropriate), or variants thereof. In other embodiments, the peptide may consist essentially of or may consist of any one of SEQ ID No. 1-5 or SEQ ID No. 1-4 inclusive. As will be appreciated by one of skill in the art, in embodiments wherein the vaccine is a DNA vaccine or wherein the peptides are produced as genetic fusions co-synthesized with a carrier as discussed below, the nucleic acid sequence may be based on or deduced from any one of the above-described peptides, as described below. As discussed herein, SEQ ID No. 1-5 or SEQ ID No. 1-4 are human-derived peptide sequences. As will be appreciated by one of skill in the art, the corresponding proteins in other evolutionarily related organisms may have identical or closely related or homologous sequences over the regions corresponding to the peptides designated as SEQ ID No. 1-5 or SEQ ID No. 1-4. These are also considered to be variants within the scope of the invention. As used herein, evolutionarily related organisms includes for example, but by no means limited to human, rat, mouse and dog.

It is of note that It is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. As will be appreciated by one of skill in the art, in the instant invention, the "biological function" is the immunogenicity of the peptide and as such a "biologically equivalent" peptide would cross-react with antibodies raised against a peptide as described above. In one aspect of the invention, the above-described peptides may include peptides that differ by conservative amino acid substitutions. The peptides of the present invention also extend to biologically equivalent peptides that differ by conservative amino acid substitutions. As used herein, the term "conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function, in this case the folding of the epitope. In making such changes, substitutions of similar amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6) are assigned to amino acid residues (as detailed in United States Patent No. 4,554, 101 , incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); GIu (+3.0); Ser (+0.3); Asn (+0.2); GIn (+0.2); GIy (0); Pro (-0.5); Thr (- 0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); VaI (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: lie (+4.5); VaI (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); GIy (-0.4); Thr (-0.7); Set (- 0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); GIu (-3.5); GIn (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, VaI, Len, lie, Phe, Trp, Pro, Met; acidic: Asp, GIu; basic: Lys, Arg, His; neutral: GIy, Ser, Thr, Cys, Asn, GIn₁ Tyr,

Alternatively, the peptides may be cross-linked to a carrier protein, as discussed below. In other embodiments, nucleic acid molecules deduced from the above-described peptides are prepared and inserted into expression vectors such that the peptides are produced fused to or inserted within suitable carrier proteins, as discussed herein. It is also of note that in some embodiments, a suitable adjuvant may also be used in combination with the vaccine, as discussed below. Fusion proteins have several advantages over conjugates including better quality control, increased antigenicity, and the possibility of combining DNA immunization with protein boosters. Two commonly used protein fusion partners of this type are hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg).³⁵' ³⁶ although other suitable fusion partners known in the art may also be used. The recombinant HBsAg containing foreign peptides induces a broad but specific immune response to the foreign peptide, because the inserted peptide or polypeptide is natively displayed on the surface of HBsAg or HBcAg particles which improves presentation of the peptide to cells of the immune system. In a preferred embodiment, the plasmid expressing chimeric HBcAg or HBsAg protein is constructed by inserting a cDNA fragment encoding one of the peptides described herein into the polynucleotide sequence corresponding to the immunodominant region of HBcAg or HBsAg. As will be apparent to one of skill in the art, the expression system will include all appropriate control sequences for transcription, translation and replication for use in a given host cell. The fusion protein can be produced in any suitable expression systems or the expression vector can be used for DNA immunization, as described below. It is of note that the carrier proteins discussed above for chemical fusion may also be used for genetic fusions, as may other suitable carrier proteins known in the art.

In some embodiments, there are provided kits for carrying out the invention. Specifically, the kits may contain one or more of the fusion proteins described above. In other embodiments, the kit comprises expression systems comprising a nucleic acid molecule deduced from the amino acid sequence of one or more the above-described peptides genetically fused to or fused within a suitable carrier (DNA vaccines). As discussed above, these expression systems may be used for direct vaccination or may be used to prepare a peptide vaccine for subsequent injection. The protein vaccine can be used alone or in combination with its DNA form. Different vaccines can be used alone or in combination depending upon their effect in down regulation of inflammatory responses in the patient. It is of note that in some embodiments, the kit may include instructions, either in written or electronic form, describing the preparation and/or administration of the vaccine.

As discussed above, to investigate the in vivo effect of the vaccines, 4 mouse TN Fa peptide-based vaccines were constructed. These mouse peptides are selected from the counterparts of the human key peptides as shown in Table 3 and Figure 4.

Table 3. Mouse TNFα sequences selected from their correspondent regions of human TNFα and expression of chimeric HBcAg fusion proteins.

The 4 mouse vaccines all produced virus-like particles. Mice immunized with any of the 4 mouse TNFα vaccines produced high titers of antibodies to mouse TNFα (titers up to over 128,000). Two mouse TNFα vaccines (equivalent to human peptide #1 and human peptide #1 plus #2) were used in a mouse model of asthma that has been established in our laboratory. Immunization of mice with a mouse TNFα vaccine elicited high titers of antibodies to mouse TNFα (up to 128,000), leading to a decrease of subsequent airway eosinophilia (Figure 7).

Neutralizing antibodies to TNFV can be induced by the vaccines which as discussed above are comprised of small peptides derived from their receptor binding sites.

As discussed above, no adjuvant is required to elicit a strong immune response because the vaccine presents itself as capsid-like particles or virus-like particles. As discussed above, the peptide-based vaccine comprises the hepatitis B core antigen (HBcAg) and a TNFα peptide, selected from SEQ ID No. 1-5 or more preferably from SEQ ID No. 1-4. The inserted TNFα peptides are displayed natively on the surface of HBcAg particles and are highly immunogenic, inducing strong and specific immune responses to the TNFα peptides. As discussed above, the experimental data has shown that immunization with the vaccine alone without adjuvant induced high titers of antibodies which are as strong as the antibody responses induced by vaccine plus an adjuvant such as complete Freund's adjuvant, novasome or CpG oligodeoxynucleotides.

Treatment of the following diseases with mAbs to TNFα along or in a combination of mAb to TNFα with other therapies has been already shown to be effective: rheumatoid arthritis,³' ⁴ inflammatory bowel disease,⁵ Crohn's disease, [Hanauer, 2004 #1503] graft versus host disease,[Jacobsohn, 2004 #1499] cancer, [Szlosarek, 2003 #1646; Anderson, 2004 #1647] cancer-induced cachexia, [Dalum, 1999 #1363;Anderson, 2004 #1498] ankylosing spondylitis, [De Keyser, 2004 #1502] psoriasis, [Scheinfeld, 2004 #1501] systemic lupus erythematosus[Aringer, 2004 #1505] as well as allergic asthma.¹⁴' ¹⁵ As such, thevirus-like particles containing these peptides may be administered to individuals in need of treatment for these diseases.

For example, vaccination of an individual with Crohn's disease with one or more of the above-described peptide-based vaccines will result in one or more of the following outcomes: remission, closure of fistulas, maintaining fistula closures, treatment of ankylosing spondylitis and longer periods symptom-free.

Vaccination of an individual suffering from or suspecting of suffering from rheumatoid arthritis with one or more of the above-described peptide-based vaccines will result in reduction of joint swelling, reduction of pain, and longer periods symptom-free.

Treatment of the above-mentioned diseases will be improved by the active immunization strategy with our peptide-based vaccines compared to either the passive immunization with mAbs or the cytokine vaccines currently under development.

For example, immunization of mice with the vaccine significantly reduced BALF eosinophils in allergic mice as discussed below.

A potential concern with cytokine vaccines is that the injection of TNF vaccines might induce a permanent autoimmune condition, as some anti-bacterial and anti-virus vaccines do, which would eliminate all TNF which is required to maintain normal functions. However, cytokine vaccines downregulate only over- expressed cytokines not those in normal tissues, as they only target the cytokines ectopically accumulated in the extracellular compartment but not those between cells which occur in normal tissues.¹' ² The titers of cytokine vaccine-induced antibodies which last about 4 months are reversible and able to be adjusted by the frequency of immunization as the immunogenicity of cytokine vaccines is less than that of microbe vaccines.¹' ² Therefore, cytokine vaccines should be safe and effective, which have been documented by studies in animal and human trials.²³' ^24τ ²⁶

EXAMPLE 1 - Construction, expression, purification, and identification of 5 recombinant human TNFα vaccines (Table 1)

Construction of recombinant plasmid. The polynucleotides encoding for Hepatitis B virus core antigen were cloned into plasmid pThioHisA (Invitrogen, Inc.) between the restriction enzyme sites for Nde I and Pst I such that the insert was under the control of a strong promoter Ptrc, resulting a new recombinant plasmid which expresses HBV core antigen.

The chimeric protein expression vector was constructed by introducing a new Kpn I site by PCR mutagenesis at nucleotide position between 235 and 237 of the above HBV core antigen, which allows the insertion of exogenous epitopes. The oligonucleotide encoding for the human TNFα antigenic peptide (Table 1) was synthesized. Positive and negative chain oligonucleotides were mixed and denatured at 95°C for 5 min, and then annealed by gradually reducing the temperature to room temperature. The segment and the vector digested with Kpn I were ligated at 16°C overnight and transformed into DH5α competent cells. The recombinants with the correct insertion orientation were identified by restriction endonuclease digestion and PCR. Expression of chimeric HBcAq containing TNF peptide fusion protein, A single colony was picked and added to 2 ml of fresh LB medium and cultured at 37°C overnight. The culture was transferred into fresh LB as 3-5% inoculation volume, and incubated at 37°C with vigorous shaking. When the OD₆₀O reached 0.6-0.8, IPTG was added to a final concentration of 1 mmol/L to induce the expression of recombinant proteins. After 4-6 hours, the bacteria were harvested by centrifuging at 2,000 g and the pellets were re-suspended with 20 mmol/L phosphate saline buffer (pH 7.4), which contains 150 mmol/L NaCI and 10 mmol/L EDTA. The bacteria were sonicated, and after centrifuging, the supernatants were subjected to sucrose gradient centrifuging.

SDS-PAGE and immunoblotting. SDS-PAGE and immunoblot analysis was performed to identify the TNFα antigenicity of the HBcAg/TNFα fusion protein. The primary antibody was the rabbit anti-human TNFα antibody (Peprotech Canada, Inc.), while the secondary antibody was alkaline phosphatase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.). The rabbit anti- human TNFα recognizes 4 of the fusion proteins. The ID numbers of the 4 fusion proteins are #1 , #2, #3, and #5.

Identification of its capsid-like particle properties. Sedimentation analysis using sucrose gradient centrifuging, size exclusion chromatography, and electron microscopy were used to determine if the fusion protein was present as capsid-like particles. The sucrose step gradient was prepared by sequentially loading 1.9 ml of 60%, 50%, 40%, 30%, 20% and 0.5 ml of 10% sucrose solution into a 12 ml ultracentrifuge tube. Two millilitres of the supernatant sample of bacteria lysates were loaded onto the top of the gradient. The recombinant plasmid expressing HBV core antigens, which are known to form particles, served as a control. Centrifuging was conducted for 3 hr at 36,000 rpm at 20⁰C. Twelve 1 ml-fractions were collected from the top to the bottom of the gradient. The 12 fractions were analysed by SDS-PAGE and the fraction containing capsid-like particles (fractions 6) was loaded onto a Sepharose CL-4B column, and also examined by electron microscopy. As shown in Figure 6, the fusion protein is presented as capsid-like particles with a diameter of 20-30 nm, which are highly immunogenic.

Production of mouse anti-human TNFα serum. Groups of four BALB/c mice were vaccinated with the chimeric HBcAg-TNFa fusion protein for a total of three injections (s.c.) with a 3-week interval between 2 injections. Four mice receiving the native HBcAg served as controls. Two weeks after the last injection, blood samples were collected and sera were obtained.

Measurement of serum IqG titers to human TNFα. ELISAs were performed to determine the titre to human TNFα. In brief, microplates were coated with human TNFα (100 ng/well). After washing and blocking with PBS containing 2%

BSA and 0.5% Tween 20, the plates were incubated with test sera and a pooled control serum (2-fold diluted, starting with 1 :500), followed by incubation with an enzyme-conjugated goat anti-mouse IgG. The titer of the test sample was determined at the dilution whose optical density at 410 nm (OD₄IQ) was at least 2.1 folds of the OD₄io of the control serum at the same dilution when its value was around 0.1. The titres to human TNFα were between 1 :40,000 and 640,000 (Table

2)

EXAMPLE 2 - Construction, expression, purification, and identification of recombinant mouse TNFα vaccines

Four mouse TNFα peptide-based vaccines (Table 3) were constructed, expressed, and identified using the methods described in example 2. The sequences of the 4 peptides are listed in Table 3. All 4 mouse TNFV vaccines induced high titers of IgG responses to mouse TNFα (from 64,000 to over 128,000) as measured using ELISAs.

EXAMPLE 3 - Kinetic IgG responses to the cytokine after vaccination with a peptide-based cytokine vaccine with or without adjuvant To determine if addition of an adjuvant will enhance the immunogenicity of the peptide-based vaccines and how long the antibody titers will last, BALB/c mice were immunized with a peptide-based mouse cytokine vaccine that presents as virus-like particles. The vaccine was constructed by inserting a mouse cytokine peptide (14 amino acid residues) into the carrier protein HBcAg, expressed by E. coli, and purified by sucrose gradient centrifugation. Groups of 4 BALB/c mice were subcutaneously immunized 3 times with the vaccine (50 μg) alone or with the vaccine plus one of the following adjuvants: novasome, alum and complete Freund's adjuvant. Ten days after the 7^th month, all mice were boosted with the cytokine vaccine alone without adjuvant. Serum Kinetic IgG responses to the cytokine after vaccination were measured using ELISA. The results showed that immunization with the virus-like chimeric HBcAg particles alone induced high titers of IgG responses to the cytokine and that the IgG responses lasted for more then 7 months and significantly increased upon a boost injection (Figure 4). There were no significant differences in the mean IgG titers between the vaccine group and the groups using vaccine plus an adjuvant, suggesting that this type of cytokine vaccine is highly immunogenic and suitable in human use.

EXAMPLE 4 - Vaccination with a mouse TNFα vaccine downregulates airway inflammation in asthmatic mice

Groups of 4 BALB/c mice were subcutaneously immunized with 50 μg each of a mouse TNFα vaccine (HBcAg/TNFα peptide #1 or combined HBcAg/TNFα peptides #1 +#2) three times at weeks 0, 2 and 4. Mice were sensitized with 2 μg of ovalbumin (OVA) absorbed to 2 mg of alum intraparitoneally at week 3 and 6. Airway inflammation was induced by nasal administration of 50 μg OVA at week 7. Mice receiving immunization with the carrier protein (native HBcAg) and sensitized and intranasally challenged with ovalbumin served as controls. Six days after the intranasal challenge, bronchoalveolar lavage fluid (BALF) and sera were collected. Serum TNFα-specific IgG titers were measured by ELISA. Dfferential cell counts in BALF cells were performed by counting at least 400 cells on cytocentrifuged preparations (Cytospin, Shandon, Runcornm, U.K.). Cells were stained with HEMA 3 (Fisher Diagnostics, Pittsburgh, PA) and differentiated by standard haematological procedures. BALF TNFα, IL-13 and IL-12 levels were measured by ELISAs using paired capture and biotinylated detection antibodies purchased from PharMingen..

The results are shown in Figure 7. Vaccination with a mouse TNFα vaccine elicited high titers of IgG antibodies to mouse TNFα ranging between 40,000 and 128,000, leading to a decrease of TNFα levels in BALF. The levels of BALF IL-13 and IL-12 were also decreased. Airway inflammation was inhibited as shown by the significantly reduced BALF eosinophil count in the vaccinated groups when compared with the control group.

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

References

1. Zagury D, GaIIo RC. Anti-cytokine Ab immune therapy: present status and perspectives. Drug discovery today 2004; 9:72-81. 2. Zagury D, Le Buanec H, Bizzini B, Burny A, Lewis G, GaIIo RC. Active versus passive anti-cytokine antibody therapy against cytokine-associated chronic diseases. Cytokine Growth Factor Rev 2003; 14:123-37.

3. Finckh A, Gabay C, Guerne PA. [Review of the protective effects of tumor necrosis factor inhibitors in rheumatoid arthritis]. Rev Med Suisse Romande 2004; 124:547-50.

4. Hyrich KL, Silman AJ, Watson KD, Symmons DP. Anti-tumour necrosis factor alpha therapy in rheumatoid arthritis: an update on safety. Ann Rheum Dis 2004; 63:1538-43.

5. Sandbom W, William J. Strategies for targeting tumour necrosis factor in IBD. Bailliere's best practice & research. Clinical gastroenterology 2003;

17:105-17.

6. Hanauer SB. Efficacy and safety of tumor necrosis factor antagonists in Crohn's disease: overview of randomized clinical studies. Rev Gastroenterol Disord 2004; 4 Suppl 3:S18-24. 7. Jacobsohn DA, Vogelsang GB. Anti-cytokine therapy for the treatment of graft-versus-host disease. Curr Pharm Des 2004; 10:1195-205.

8. Szlosarek PW, Balkwill FR. Tumour necrosis factor alpha: a potential target for the therapy of solid tumours. Lancet Oncol 2003; 4:565-73.

9. Anderson GM, Nakada MT, DeWitte M. Tumor necrosis factor-alpha in the pathogenesis and treatment of cancer. Curr Opin Pharmacol 2004; 4:314-

20.

10. Datum I, Butler DM, Jensen MR, Hindersson P, Steinaa L, Waterston AM, et al. Therapeutic antibodies elicited by immunization against TNF-alpha. Nat Biotechnol 1999; 17:666-9. 11. De Keyser F, Baeten D, Van den Bosch F, Kruithof E, Verbruggen G, Mielants H, et al. Structure-modifying capacity of anti-tumour necrosis factor-alpha therapy in ankylosing spondylitis. Drugs 2004; 64:2793-811. 12. Scheinfeld N. Adalimumab (Humira): a brief review for dermatologists. J Dermatolog Treat 2004; 15:348-52. 13. Aringer M, Graninger WB, Steiner G, Smolen JS. Safety and efficacy of tumor necrosis factor alpha blockade in systemic lupus erythematosus: an open-label study. Arthritis Rheum 2004; 50:3161-9.

14. Babu K, Arshad SH, Howarth PH, Chauhan AJ, Bell EJ, Puddicombe S, et al. SoIuIe Tumor Necrosis Factor Alpha (TNF - alpha) Receptor 838 (Enbrel) as an Effective Therapeutic Strategy in Chronic Severe Asthma. J

Allergy Clin Immunol 2003; 111 :S277.

15. Zuany-Amorim C₁ Manlius C, Dalum I, Jensen MR, Gautam A, Pay G, et al. Induction of TNF-alpha autoantibody production by AutoVac TNF106: a novel therapeutic approach for the treatment of allergic diseases, lnt Arch Allergy Immunol 2004; 133:154-63.

16. Targan SR, Hanauer SB, van Deventer SJ, Mayer L, Present DH, Braakman T, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. N Engl J Med 1997; 337:1029-35.

17. D'Haens G, Van Deventer S, Van Hogezand R, Chalmers D, Kothe C, Baert F, et al. Endoscopic and histological healing with infliximab anti-tumor necrosis factor antibodies in Crohn's disease: A European multicenter trial.

Gastroenterology 1999; 116:1029-34.

18. Ricart E, Panaccione R, Loftus EV, Tremaine WJ, Sandborn WJ. Infliximab for Crohn's disease in clinical practice at the Mayo Clinic: the first 100 patients. Am J Gastroenterol 2001 ; 96:722-9. 19. Mannon PJ, Fuss IJ, Mayer L, Elson CO, Sandborn WJ₁ Present D, et al.

Anti-interleukin-12 antibody for active Crohn's disease. N Engl J Med 2004;

351 :2069-79. 20. Nishimoto N, Kishimoto T. Inhibition of IL-6 for the treatment of inflammatory diseases. Curr Opin Pharmacol 2004; 4:386-91. 21. Yokota S, Miyamae T, Imagawa T, Iwata N₁ Katakura S, Mori M, et al.

Therapeutic efficacy of humanized recombinant anti-interleukin-6 receptor antibody in children with systemic-onset juvenile idiopathic arthritis. Arthritis

Rheum 2005; 52:818-25.

22. Waldmann TA. Targeting the interleukin-15/interleukin-15 receptor system in inflammatory autoimmune diseases. Arthritis Res Ther 2004; 6:174-7.

24. Gringeri A, Musicco M, Hermans P, Bentwich Z₁ Cusini M, Bergamasco A, et al. Active anti-interferon-alpha immunization: a European-Israeli, randomized, double-blind, placebo-controlled clinical trial in 242 HIV-1- infected patients (the EURIS study). J Acquir Immune Defic Syndr Hum Retrovirol 1999; 20:358-70.

25. Capini CJ, Bertin-Maghit SM, Bessis N, Haumont PM, Bernier EM, Muel EG, et al. Active immunization against murine TNFalpha peptides in mice: generation of endogenous antibodies cross-reacting with the native cytokine and in vivo protection. Vaccine 2004; 22:3144-53. 26. Nielsen FS, Sauer J, Backlund J, Voldborg B, Gregorius K, Mouritsen S, et al. Insertion of foreign T cell epitopes in human tumor necrosis factor alpha with minimal effect on protein structure and biological activity. J Biol Chem 2004; 279:33593-600.

27. Jegerlehner A, Tissot A, Lechner F, Sebbel P, Erdmann I, Kundig T, et al. A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses. Vaccine 2002; 20:3104-12.

28. Pumpens P, Razanskas R, Pushko P, Renhof R₁ Gusars I, Skrastina D, et al. Evaluation of HBs, HBc₁ and frCP virus-like particles for expression of human papillomavirus 16 E7 oncoprotein epitopes. Intervirology 2002; 45:24-32.

29. Netter HJ, Macnaughton TB, Woo WP, Tindle R, Gowans EJ. Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes. J Virol 2001 ; 75:2130-41. 30. Eck MJ, Sprang SR. The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding. J Biol Chem 1989; 264:17595- 605.

31. Jones E, Stuart D, Walker N. Stucture, fuction, and mechanism of action. In: Aggarwal B, Vilcek J, editors. Tumor Necrosis Facor. New York: Marcel

Dekker, Inc.; 1992. p. 93-127.

32. Cha SS, Kim JS, Cho HS, Shin NK, Jeong W, Shin HC, et al. High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity. J Biol Chem 1998; 273:2153-60. 33. Van Ostade X, Tavemier J, Prange T, Fiers W. Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis. Embo J 1991 ; 10:827-36.

34. Yone K, Bajard S, Tsunekawa N, Suzuki J. Epitopic regions for antibodies against tumor necrosis factor alpha. Analysis by synthetic peptide mapping. J Biol Chem 1995; 270: 19509-15.

35. Pumpens P, Grens E. HBV core particles as a carrier for B cell/T cell epitopes. Intervirology 2001; 44:98-114.

36. Milich DR, Hughes J, Jones J, Sallberg M, Phillips TR. Conversion of poorly immunogenic malaria repeat sequences into a highly immunogenic vaccine candidate. Vaccine 2001 ; 20:771-88.

Claims

1. A reagent comprising: a TNFV-derived peptide; and a carrier protein. 2. The reagent according to claim 1 wherein the TNFV-derived peptide is selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID NO. 1 , amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ (SEQ ID No.

2, amino acids 16-26 of TNFα); 6 or more consecutive residues of EINRPDYL (SEQ ID No. 3, amino acids 135-142 of TNFα); 6 or more consecutive residues of LNRRANALLANG (SEQ ID No. 4, amino acids 28-39 of TNFα); and 6 or more consecutive residues of ELRDNQLW (SEQ ID No. 5, amino acids 41-49 of TNFα).

3. The reagent according to claim 1 wherein the TNFV-derived peptide is selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID No. 1 , amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ (SEQ ID No. 2, amino acids 16-26 of TNFα); 6 or more consecutive residues of EINRPDYL (SEQ ID No. 3, amino acids 135-142 of TNFα); and 6 or more consecutive residues of LNRRANALLANG (SEQ ID No. 4, amino acids 28-39 of TNFα).

4. The reagent according to claim 1 wherein the carrier protein is selected from the group consisting of hepatitis B core antigen and hepatitis B surface antigen.

5. The reagent according to claim 1 wherein the peptide is fused into an immunodominant region of the carrier protein.

6. A method of inducing an immune response in an individual, comprising: administering to an individual in need of such a treatment, an effective amount of a composition comprising: a TNFV-derived peptide; and a carrier protein.

7. The method according to claim 6 wherein the TNFV-derived peptide is selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID NO. 1 , amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ (SEQ ID No. 2, amino acids 16-26 of TNFα); 6 or more consecutive residues of EINRPDYL (SEQ ID No. 3, amino acids 135-142 of TNFα); 6 or more consecutive residues of LNRRANALLANG (SEQ ID No. 4, amino acids 28-39 of TNFα); and 6 or more consecutive residues of ELRDNQLW (SEQ ID No. 5, amino acids 41-49 of TNFα).

8. The method according to claim 6 wherein the TNFV-derived peptide is selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID No. 1, amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ (SEQ ID No. 2, amino acids 16-26 of TNFα); 6 or more consecutive residues of EINRPDYL (SEQ ID No. 3, amino acids 135-142 of TNFα); and 6 or more consecutive residues of LNRRANALLANG (SEQ ID No. 4, amino acids 28-39 of TNFα).

9. The method according to claim 6 wherein the carrier protein is selected from the group consisting of hepatitis B core antigen and hepatitis B surface antigen.

10. The method according to claim 6 wherein the peptide is fused into an immunodominant region of the carrier protein.

11. A method of treating, ameliorating or preventing an autoimmune or inflammatory diseases comprising: administering to an individual in need of such a treatment, an effective amount of a composition comprising: a TNFV-derived peptide; and a carrier protein.

12. The method according to claim 11 wherein the TNFV-derived peptide is selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID No. 1 , amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ (SEQ ID No. 2, amino acids 16-26 of TNFα); 6 or more consecutive residues of EINRPDYL (SEQ ID No. 3, amino acids 135-142 of TNFα); 6 or more consecutive residues of LNRRANALLANG (SEQ ID No. 4, amino acids 28-39 of TNFα); and 6 or more consecutive residues of ELRDNQLW (SEQ ID NO. 5, amino acids 41-49 of TNFα).

13. The method according to claim 11 wherein the TNFV-derived peptide is selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID NO. 1 , amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ (SEQ ID No. 2, amino acids 16-26 of TNFα); 6 or more consecutive residues of EINRPDYL (SEQ ID No. 3, amino acids 135-142 of TNFα); and 6 or more consecutive residues of LNRRANALLANG (SEQ ID No. 4, amino acids 28-39 of TNFα).

14. The method according to claim 11 wherein the carrier protein is selected from the group consisting of hepatitis B core antigen, hepatitis B surface antigen or other virus antigen.

15. The method according to claim 1 1 wherein the peptide is fused into an immunodominant region of the carrier protein.

16. An expression system comprising: a nucleic acid molecule deduced from a peptide selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID No. 1, amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ

(SEQ ID No. 2, amino acids 16-26 of TNFα); 6 or more consecutive residues of

17. The expression system to claim 16 wherein the peptide is selected from the group consisting of: 6 or more consecutive residues of SRTPSDKPVAH (SEQ ID No. 1 , amino acids 4-14 of TNFα); 6 or more consecutive residues of VANPQAEGQLQ (SEQ ID No. 2, amino acids 16-26 of TNFα); 6 or more consecutive residues of EINRPDYL (SEQ ID No. 3, amino acids 135-142 of TNFα); and 6 or more consecutive residues of LNRRANALLANG (SEQ ID No. 4, amino acids 28-39 of TNFα).