Peptide B-Vaccines Protecting from Allergy
Development of B-Vaccine for the Protection from Allergy Induced by House Dust Mites, Which Will Be Used for the Treatment of Asthma and Atopic Dermatitis
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Institute of Bioorganic Chemistry, Russia, Moscow
- Institute of Genetics and Selection of Industrial Microorganisms, Russia, Moscow\nResearch Center of Toxicology and Hygienic Regulation of Biopreparations, Russia, Moscow reg., Serpukhov
- Medical University of Vienna, Austria, Vienna\nUS Department of Health & Human Services / National Institute of Health / National Cancer Institute / Division of Clinical Sciences, USA, MD, Bethesda\nMedical College of Wisconsin, USA, WI, Milwaukee\nMax Plant Institute of Colloids and Interfaces, Germany, Potsdam
Project summaryAIM. The aim of this project is the development of a new approach for the production of improved, safe, and efficient recombinant peptide vaccines for the treatment of different forms of allergy. The particular model selected for this research is house dust mite allergy as it often progresses to asthma and atopic dermatitis.
BACKGROUND. Allergic diseases represent a major health threat to humans. Severe forms of allergy such as asthma and atopic dermatitis (AD) require long-life treatment with anti-histamine and corticosteroid medications. Because asthma is not a curable condition, the development of strategies for prevention of the disease has a high priority. AD is a common precursor to the development of asthma. Allergy to house dust mites (HDM) often manifests early during childhood and can progress to asthma and AD. It is shown that 45% of patients with AD and more than 60% of asthmatics are sensitive to HDM. Allergen-specific immunotherapy (SIT) is one of the significant approaches towards the treatment of IgE-mediated allergy induced by HDM and its control. The mechanisms involved in SIT-induced responses are complex and still speculative. Immunological events associated with successful SIT include an increase in allergen specific “blocking” IgG, reduction in cytokine production and induction of regulatory or suppressor cells. Adverse reactions resulting from SIT are the main concern when natural extracts are used. The most severe side effects include anaphylaxis induced via IgE mediated degranulation of mast cells leading to release of vasoactive mediators, and IgE facilitated antigen uptake by dendritic cells. Due to the risk of IgE mediated adverse reactions SIT consists of allergen extract injections in low doses and can last for up to three years. Successful SIT is directly associated with the dose of allergen extract used for vaccination. Production of peptide vaccines, which do not bind IgE will permit to increase antigen concentration used for SIT and shorten the time needed for the treatment.
Allergic diseases are the result of chronic immune system activation induced by proteins penetrating through mucosal tissues. These proteins having perse origins and properties in most cases are presented by immune competent cells to T-helper cells, which induce humoral immune response leading to IgE production. Subsequent cycles of immune system activation lead to the recognition of additional new proteins from the complex allergens. Allergy is characterized by B cell switch to IgE driven by IL-4/IL-13 produced by CD4+ T cells. IgE production is found in allergy and not in normal immune response to pathogens. The difference in these responses is the concentration of protein antigens, contacting immune system. Pathogens replicate inside the body by this mean increasing significantly the concentration of antigens. It can be hypothesized that due to the low concentration of proteins in allergens, immune response is not focused to dominant antigens and many proteins are recognized. High IL-4/IL-13 level produced in genetically predisposed persons by all responding T cells facilitates B cell switch to IgE while low concentration of specific proteins recognized by inpidual T and B cells prevents affinity maturation of IgG. As allergens are mostly extracellular antigens humoral immune response should be protective as it is protective against extracellular bacteria. Previously we have demonstrated a protective effect of IgG to a single major Aspergillus fumigatus (Af) allergen Asp f 2 on the allergy induced by this saprophytic fungus. A batch of Af crude extract used for this work contained around 10% of Asp f 2. However, IgG to Asp f 3, concentration of which was below 3%, was only partially protective. Af crude extract as well as other allergens contains more than 20 proteins with different concentration. It can be hypothesized that induction of high affinity IgG to 4-5 major proteins will focus immune response to these proteins and prevent both IgE production and minor antigen recognition. Allergy to Af can progress to very severe forms such as allergic broncho-pulmonary aspergillosis, however it rarely appears as a primary course of allergy. On the contrary, hyperreactivity to HDM or pollen rather often manifests as monospecific allergy. It was shown that treatment of patients, hyperreactive to a limited number of allergens, usually prevents allergy progression and recognition of new allergens. Taking into account this fact, as well as the financial aspects of future commercialization in the case of successful results, it will be more reasonable to estimate the effect of this new approach in the model of allergy induced by HDM.
SCOPE OF ACTIVITY. The aim of this project is the development and study of multivalent peptide vaccine, which contains B-cell epitopes of 4 major HDM allergens expressed in an immunogenic carrier. Saccharomyces cerevisiae virus-like particles (VLP) were selected as immunogenic carrier for HDM peptides. The vaccines against human immunodeficiency virus (HIV) and the malarias, created on the basis of VLP, are in the middle of clinical trials. Four major D.pteronyssinus (Dp) allergens able to bind IgE in 60-90% of sera from asthma patients with HDM sensitivity were preliminary selected for this research. B-cell epitopes of Dp will be selected by a computer analysis.
The next main tasks will be addressed in this project.
1. Selection of major 4 allergens from HDM, whose epitopes will be used for the peptide vaccine.
2. Selection of inpidual peptides representing potential B-cell epitopes of major HDM allergens.
3. Construction of hybrid genes coding p1 protein with C-terminus localized D. pteronyssinus (Dp) peptides.
4. Production of 4 inpidual VLPs containing two peptides from each Dp protein.
5. Analysis of the ability of IgG and IgA induced in mice immunized with inpidual VLPs-Dp to recognize HDM crude extract and full-length proteins.
6. Estimation of human IgE binding to recombinant VLPs containing Dp peptides.
7. Study of the protective effects of inpidual VLPs-Dp against allergy induced by HDM extract in a mouse model.
8. Design and production of multivalent peptide VLP-based vaccine, containing four recombinant VLPs-Dp, expressing B-cell epitopes of Dp major allergens (B-vaccine).
9. Preclinical study of the B-vaccine.
METHODS. Major allergens were preliminary selected basing on IgE binding data and their sequence availability and included major proteins of D.pteronyssinus Der p 1, 2, 3, and 4. Two possible B-cell epitopes of each protein selected using compute analysis of hydrophilic, acrophilic and surface access protein profiles will be expressed separately in the yeast virus-like particles (VLP). DNA coding peptides will be obtained by the annealing of synthetic oligonucleotide sequences. Mice immunized with inpidual VLPs-Dp or B-vaccine will be subjected to allergy induction protocol. Allergy will be induced by s.c. and i.n. immunization of mice with HDM extract produced in the Mechnikov Institute of Vaccines and Sera (Moscow) supplemented by additional injections with recombinant IL-4. Earlier we have shown that addition of IL-4 to immunizing protocol leads to Af specific IgE induction and lung inflammation characteristic to asthma. HDM specific IgG, IgE and IgA and cytokine IL-4, 5, 10 and TGF-b production will be estimated. Protective effect of VLP-Dp immunization will be also estimated by lung histology. Preclinical study will be conducted according to the established protocols.
EXPECTED RESULTS AND ITS APPLICATION. Previously we have shown that immunization of mice with VLP containing intrinsic Asp f 2 B-cell epitopes induces high level of IgG recognizing Asp f 2. Moreover, our unpublished results demonstrate that immunization with artificial B-cell epitopes selected by computer prediction does induce IgG able to bind Asp f 2. Thus, first expected result is the demonstration that recombinant peptides representing computer selected B-cell epitopes will induce IgG, recognizing corresponding proteins Der p 1, 2, 3, or 4. Secondly, it can be expected that the induction of IgG to these proteins will prevent B-cell switch to IgE due to the down regulation of B cell activation in the presence of high serum concentration of specific IgG. The third result is the demonstration that immunization with B-cell epitopes of major antigens will focus immune response induced by HDM extract to the selected proteins and prevent other protein recognition. Finally, the analysis of cytokine production and lung histology will demonstrate the protective effect of VLP-based vaccine. Preclinical studies on the vaccine safety will be conducted. As a result of this research a new peptide multivalent B-vaccine for the treatment of allergy induced by HDM will be ready for a phase I clinical trial.
COMPETENCE OF THE PROJECT TEAMS. Basic approach to the immune focusing to major allergens and its effectiveness were developed and shown by IBCH team in cooperation with our foreign collaborator Dr.Kurup (USA) in the model of allergy induced by saprophytic fungus Aspergillus fumigatus. In a murine model of asthma we have demonstrated a protective effect of IgG induction to a single Af major allergen Asp f 2 on the IgE production and lung inflammation induced by Af crude extract. However, even recombinant pure full-length proteins can bind IgE in patients and induce adverse reactions. Thus, the usage of peptides, which can model immune response to the full-length proteins but are not able to bind IgE, will permit to prevent side effects of SIT. The development of peptide vaccine containing dominant T and B cell epitopes of Asp f 2 was conducted by combined efforts of IBCH and GOSNIIGENETICA teams. Fifteen recombinant peptides expressed as yeast virus-like particles were obtained and their immunogenic activity studied. Immunization of mice with VLP-Af peptides decreased significantly the lung inflammation in treated mice in comparison with untreated control. These results were shown using an acute model of allergy in mice. Unfortunately, chronic allergy can not be modeled in mice. Prolonged sensitization of mice with allergens induces protective response as it is found in healthy subjects. Thus, the effect of peptide vaccine on chronic forms of allergy observed in humans can be studied only in clinics. To introduce peptide vaccines into clinical practice a preclinical study of vaccine safety is needed. We plan to conduct preclinical investigation in RCTHRB, which has a license for preclinical testing of different preparations and drugs.
ADDRESSING ISTC OBJECTIVES. Allergic diseases represent a major health threat to humans all over the world. Development and study of vaccines protecting from allergy is an important task for many researchers as well as for patients suffering from asthma and atopic dermatitis. Preclinical studies will be conducted by experienced persons previously involved in weapon research.
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