Virosomes containing hemagglutinin extracted from influenza virus produced in the cell lines, compositions, containing said virosomes, means of manufacturing and applications

FIELD: medicine.

SUBSTANCE: group of inventions is referred to the area of medicine, namely to the area of virusology, and is related to virosomes containing hemagglutinin extracted from influenza virus produced in the cell lines, compositions, containing said virosomes, means of manufacturing and applications. The essence of the invention including virosomes containing hemagglutinin extracted from influenza virus produced in the bird cell lines, compositions, containing said virosomes, application of virosome as a vessel, set, methods of vaccination, methods of treatment and methods of virosome production.

EFFECT: production of virosomes having improved merging capability and increased immunogenicity.

21 cl, 3 tbl, 5 dwg

 

The technical field

The present invention relates to the fields of immunology and vaccinology. In particular, the invention relates to improved virosomal, compositions comprising virosome, and their application.

Background of invention

One of the primary goals of medicine is the development of modern vaccines for the prevention and effective delivery of therapeutic substances for the treatment of diseases. Currently virosome known as vesicles, which can be used as vehicles for delivery of antigens and/or as carriers of therapeutic agents.

Virosome are complexes consisting of lipids and at least one viral envelope protein, which receive in vitro. Lipids or produce eggs or plants, or get artificially, and some lipids derived from the same virus, derived from a protein shell. In essence, virosome are restored (reconstructed) empty viral shells without nucleocapsid, containing the genetic material of the virus(viruses). Virosome not capable of self-renewal and are mere bubbles, with the ability to merge. The functionality of such virosa is that the activity of their membranes to merge very similar to low Zn the values of pH the ability of intact virus to cause fusion of the membranes, which provides extremely viral fusion protein. Like viruses, virosome quickly internalized into cells by a mechanism mediated receptor endocytosis or by fusion with the cell membrane.

Mainly used virosome are so-called immunostimulating reconstructed virosome influenza (IRIV). IRIV is a single-layer spherical bubbles with a mean diameter of 150 nm, consisting of a lipid bilayer composed of phospholipids, mainly phosphatidylcholine (PC) and phosphatidylethanolamine (RE). Virosome IRIV contain functional viral envelope glycoproteins - hemagglutinin (ON) and neuraminidase (NA) of influenza virus embedded in the membrane, representing a phospholipid bilayer.

Biologically active hemagglutinin not only provides structural stability and homogeneity of the compositions containing virosome, but also makes a significant contribution to the immunological characteristics, because it maintains the ability of the virus to cause fusion. If necessary, IRIV contain molecules of hemagglutinin more than one strain of the virus and thus represent a chimeric IRIV.

IRIV were developed through the introduction of hemagglutinin (HA) of influenza virus strain And in liposomes consisting of phosphatidylcholine. Glycoprotein hemagglutinin membrane Viru is and influenza-specific manner directs virosome to antigen presenting cells (APS) and mediates the fusion of their membranes them Eidolon. This process ensures optimal processing and presentation of antigens to immunocompetent cells. The activation of the production of cytokines by T-lymphocytes, which, in turn, stimulates the production of large quantities of specific antibodies by b-lymphocytes. Moreover, stimulation of b-lymphocytes as a result of direct interaction with the complex antigen-virosome.

Virosome are highly effective systems adjuvant/carrier in the modern vaccination/therapy has excellent characteristics as a means of delivery of antigen and high immunogenic potential, at the same time minimising the risk of side effects. In addition, virosome have the effect of adjuvant (WO 92/19267), the effect of TRANS-adjuvant (application for European patent EP 05027624) and nonspecific immunostimulant (European patent EP 06027120) effect.

More than 50 years influenza vaccine produced in the cells of chick embryos. However, the traditional standard method is extremely time-consuming and laborious. Currently, the production of vaccines in the embryo takes up to 9 months from the date of allotment of the new identified virus strain to obtain the final product. This can complicate the solutions to unexpected problems, such the AK detection of pandemic strains, failures in the production and seasonal variation of influenza virus strains. Moreover, traditional methods based on embryos requires huge amounts of eggs, transfer of virus isolation in eggs and additional cleaning to reduce the amount of impurities embryonic proteins and minimize the risk of Allergy to albumin of the eggs.

The method is based on the use of cell lines is faster and more flexible in relation to the propagation of viruses and allows to obtain strains that cannot effectively be grown in chicken embryos (e.g., Hong Kong bird flu in 1997). In addition, the use of cell lines upon receipt of the viruses has some advantages from the point of view of safety of the resulting vaccine: the vaccine contains no additives antibiotics, there is no need for toxic preservatives such as thiomersal), the level of endotoxin reduced, eliminated the occurrence of allergies to egg proteins, growth of the virus occurs in an environment that does not contain protein and whey (no auxiliary agents/BSE), high degree of purification of the vaccine.

Recently, considerable efforts have been made to develop systems of cell cultures for vaccine production. A large part of these systems is based on the lines of mammalian cells such as Vero cells, MDCK, BHK and PerC6. Published several reports what s on vaccine development in cultures of mammalian cells. However, a significant drawback of antiviral vaccines, obtained in the above-mentioned cell cultures, is the risk of autoimmune reactions to proteins in mammalian cells.

The merger of virosome necessary for the effective delivery of antigens/medicines (Schoen P et al, 1999). Accordingly, there is a need to develop a method of producing virosa with improved performance fusogenicity (the ability to merge) and immunogenicity.

The invention

The present invention solves the above problem by creating new virosome containing the hemagglutinin of influenza virus obtained in cell lines of birds. Such new virosome characterized as an enhanced ability to cause the merger and increased immunogenicity compared to virosome containing the hemagglutinin of influenza virus, obtained with the standard method in chicken embryos.

Thus, the first aspect of the present invention relates to virosomes containing hemagglutinin, and hemagglutinin isolated from influenza virus obtained in cell lines of birds.

"Cell line birds" in the context of the present invention is a cell culture selected on the basis of the homogeneity of the cell population obtained from a generally homogeneous fabric birds (such as authority). This term is not included the t birds ' eggs, such as, for example, chicken. Thus, the hemagglutinin isolated from influenza virus obtained in cell line birds"means that the hemagglutinin isolated from viruses grown in cell culture birds, and not from viruses grown in eggs (chicken embryos). The preferred cell line birds include, but are not limited to the above, a primary cell line, such as chick embryo fibroblasts (CEF); permanent/an immortalized cell line, for example, DF-1 (US 5672485), PBS (US 5989805) and HD11.

In addition, the present invention relates to virosomes containing hemagglutinin, and the ability to merge these virosa at least 50% higher than the ability to merge virosome containing hemagglutinin isolated from influenza virus grown in chicken embryos, and has the same primary structure, or peptide sequence. According to a preferred variant implementation of virosome according to the present invention have a significantly higher immunogenicity than virosome containing hemagglutinin derived from influenza viruses grown in chicken embryos. In the preferred case, the ability to merge virosome according to the present invention, at least 30% above the ability to merge virosome containing hemagglutinin isolated from influenza virus grown in the mammalian cells.

It was unexpectedly found that the ability of virasam to merge depends on the process of growing influenza virus, which receive virosome. According to a preferred variant implementation of the hemagglutinin, which is part of virosome, separated from influenza viruses obtained in cell lines. Preferably isolated from the hemagglutinin of influenza viruses grown in cell line birds.

Patent application WO 2006/108846 (author Vivalis) refers to the use of stem cells of the embryo of a bird, preferably a cell line EVH, to produce viral vectors and viruses. However, in WO 2006/108846 does not contain any prerequisites for use virosome hemagglutinin derived from viruses grown in cell lines.

Virosome can represent the chimeric virosome, in which the hemagglutinin selected from at least two different influenza virus strains. In addition, virosome can be dried. In a preferred embodiment of the invention virosome loaded with antigen. In a more preferred embodiment, virosome according to the present invention is not loaded/empty.

According to another aspect of the present invention relates to compositions comprising virosome according to the present invention. In a preferred embodiment, the specified composition which is a vaccine. In another preferred embodiment, the composition is immunogenic and additionally includes a liposome and at least one molecule of the antigen. In the preferred case, this at least one molecule of the antigen is encapsulated in a liposome. to implement at least one molecule of the antigen in the liposome.

According to further aspect the present invention relates to the use of virosome according to the present invention as a means of delivery of antigen in the pharmaceutical composition to obtain an immune response against antigens of different origin. Virosome according to the present invention can also be used to obtain pharmaceutical compositions for vaccination or immunization. In addition, the present invention relates to immunostimulatory virosomal, not loaded with antigen. Accordingly, the present invention relates to the use of virosome according to the present invention as a non-specific immunostimulating agents to obtain pharmaceutical compositions for stimulating an effective immune response against antigens of different origin. Finally, the invention relates to the use of virosome according to the present invention in obtaining pharmaceutical compositions for the treatment or prevention of diseases or disorders.

According to another aspect of this invention relates to sets, including virosome or composition according to the present invention.

Another aspect includes a method of vaccination or immunization of a patient virosome or compositions according to the present invention, including an introduction of these virosome or compositions to a patient to stimulate an immune response. Also in the present invention includes a method of treating or preventing diseases or disorders (such as infectious disease and/or malignant tumor) in a patient using virosome or compositions according to the present invention, including the introduction of these virosome or compositions indicated patient.

According to another aspect of the present invention relates to a method of producing virosome according to the present invention, comprising the processing steps of the whole virus influenza a detergent or the short-chain phospholipid, and separating the fractions containing the hemagglutinin, and the removal of the detergent, resulting in recovery (formation) virosome. Alternatively, the phase separation can be done by adding phospholipids. The present invention also relates to virosomal obtained in the mentioned way.

A brief description of graphic materials

Figure 1 on the azan Western blot analysis of drugs virosa using hemagglutinin from line infected with a strain of New Caledonia flu And chicken cells (lanes 1 and 4), duck cells (lanes 2 and 5) or virus obtained by multiplication of fertilized eggs (lanes 3 and 6). Blot a was obtained with the use of specific influenza a polyclonal rabbit serum, the blot was obtained using monoclonal antibodies that recognize a specific epitope on the subunit 1 of the hemagglutinin.

Figure 2 shows the ability to merge virosa flu. Top: Graphic ability to merge the results are shown in Table 2, Experiment 2, Example 4.5. Lower part: the Ratio of the ability to merge virosome influenza isolated from cells and eggs. Bars represent the average ratio of the activity of samples at different dilutions, corresponding to the concentration of hemagglutinin, from 1 to 6 mcg/ in total, equal to 0.8 ml.

Figures 3 and 4 illustrate the results of the study immunogenicity in mice. According to figure 4 is visible to the increased immunogenicity of virosome containing hemagglutinin isolated from influenza virus obtained in cell line birds, and containing a heterologous antigen (UK39). Figa shows that the source of the virus (cell line/cell culture or egg), used to obtain virosome according to the invention, has no substantial effect on the titer of antibodies to hemagglutinin isolated from eggs, after one IMM the organization. Figv demonstrates improved immunogenicity of hemagglutinin: a higher titer of antibodies to hemagglutinin selected from EVH, after the first immunization virosome containing hemagglutinin derived from viruses obtained in cells EVH. Figure 4 shows the individual titers of antibodies specific to the heterologous antigen UK39. The results obtained by calculating the dilution corresponding to the optical density equal to 20% of the maximum optical density value for the control of serum present in each tablet. In the example shown the difference that was observed between virosome containing hemagglutinin isolated from viruses obtained in eggs, and virosome containing hemagglutinin isolated from viruses obtained in cell lines, in regard to the immunogenicity of heterologous antigen UK39, was significant: p=0.002 for the line of chicken cells in comparison with eggs and p=0.009 for cell culture ducks in comparison with eggs, using Wilcoxon criterion.

Figure 5 shows the increased induction of CD8+ T-linfocitos specific to the heterologous antigen (not the hemagglutinin) virosome containing hemagglutinin isolated from viruses obtained in cell lines of birds and loaded heterologous antigen, in comparison with virosome containing hemaggluti the Institute of viruses, isolated from eggs.

Detailed description of the invention

Definition

In the present description, the term "virosome" refers to the bubble (vesicles)resulting from treatments in vitro and consisting of a lipid and at least one envelope protein of the virus. Lipids or isolated from a biological source (e.g., eggs, plants, animals, cell cultures, bacteria, viruses, etc), or obtained synthetically (chemical synthesis). Virosome can be a restored shell of the virus, which can be obtained from various viruses and which does not contain infectious nucleocapsid and genetic material of the virus source, for example, immunostimulirutuyu reconstructed virosome influenza (IRIV). Thus, virosome is a special type of lipid vesicles consisting of lipid membranes and at least one envelope protein of the virus. The term "protein shell of the virus" in the present description refers to any protein encoded by enveloped virus, which fully or partially received virosome according to the present invention and which is present in the lipid membrane of virosome. Protein shell of the virus sometimes function as "fusion proteins of the virus" (fusion proteins), in the case when they are involved in the fusion of viruses or virosome with the membranes of target cells.

In rosoma according to the present invention may contain more than one type envelope protein. The source of these additional proteins found in the membrane of virosome not necessarily come from enveloped viruses, can be any living organism (including microorganisms, such as bacteria, fungi or parasites).

Protein shell can be recombinant proteins, provided that their biochemical properties allow a physical connection to the lipid membrane. Such membrane proteins are responsible for the functionality of virosome.

Unlike viral systems virosome safe, because infectious nucleocapsid virus removed. Currently virosome mainly used as vaccines after the introduction of antigen into the surface or in the cavity of virosome. In contrast to virus-like particles virosome not form spontaneously in the process of recombinant protein expression in a suitable expression system, and are the product of the controlled process in vitro, providing large-scale industrial production of virosome.

In the present description, the term "delivery vehicle antigen" refers to virosome containing one specific disease antigen is placed in the cavity virosome or embedded in its surface.

In the present description, the term "ability to merge (fusion activity) refers to the ability of virosome to merge kletochnoi and/or synthetic membrane. While in vivo virosome merge with the outer membrane of the cell or with membrane of endosome, fusion with liposomes is an established model system to determine the ability to merge virosa in vitro (Smith, J M and others, 2003). It is shown that the fusion of influenza virus and virosome with liposomes has characteristics similar to the fusion of biological membranes targets (Stagmann, T. and others, 1989).

The term "cell membrane" (the cell membrane) in the present description refers to the biological membrane, present in cells in vivo, such as the outer cell membrane or the membrane of endosomes inside the cell. The term "synthetic membrane, in contrast, refers to artificial membranes, such as lipid membranes of liposomes. An example of a synthetic membranes are membranes of liposomes, consisting only of phosphatidylcholine (PC) and DPPG (dipalmitoylphosphatidylcholine) and does not contain proteins normally present in the cell membranes.

The ability to merge viruses and virosome usually measured by fluorescent resonance energy transfer (FRET) (Struck DK and others, 1981). This method is used photophysical process that causes a decrease in fluorescence of a single element (donor) by transferring the excitation energy to another entity (the acceptor) without radiation. You need to range ususkani the donor overlaps the absorption spectrum of the acceptor. The effect of reduction of fluorescence depends on the distance between two molecules: every event that causes a change of the distance between molecules suppresses the effect of damping (reduction of fluorescence), resulting in the release of energy, which can be measured. Thus, this method represents a valuable in vitro test for the study of many biological phenomena, such as the fusion of viral particles and biological cell membranes. Various techniques have been developed to study the merger, based on fluorescence resonance energy transfer (FRET) to demonstrate the ability to fusion of viral membranes (viruses or virosa) with liposomes or shades of red blood cells (empty cells in vitro (Smit JM and others, 2003). Some of these methods include staining of membranes targets (liposomes), other - labeling of the samples, namely viruses or virosome. However, the need for labeling the sample is incompatible with the quality control of pharmaceutical products according to cGMP. A more sensitive test for the determination of the merger, based on fluorescence resonance energy transfer without labeling of the samples, was developed by Pevion Biotech (M. Amacker and others, 2005).

The ability to merge virosome according to the present invention can be measured IU the Odom fluorescence resonance energy transfer (FRET), as described in the examples below. To determine whether increased if the ability to merge virosome according to the present invention compared with other virosome, perform the following steps: (a) measuring the ability to merge virosome containing various amounts of hemagglutinin isolated from viruses obtained in cell lines, and corresponding virosome containing the same amount of hemagglutinin isolated from viruses obtained in eggs; (b) determining the ratio values the ability to merge identified in step (a) (i.e. virosome containing the hemagglutinin from the cell line, and virosome containing the hemagglutinin from the egg); and (C) averaging the obtained relations. Thus, to compare the values the ability to merge you need to make several measurements with different numbers of hemagglutinin for each type of virosome. According to a preferred variant of implementation, the ability to merge measure for virosome containing 3-6 μg in a total volume of 0.8 ml Sample calculations are given in section 4.5 section the Examples below. The ability to merge the considered virosome "50% above, if the average ratio has a value of ≥1.5.

In the present description, the term "immunogenicity" refers to the ability of a particular substance (antigen) to induce an immune response. To determine oblad the em does virosome according to the present invention is increased (i.e. superior), the subject is subjected to immunization with virosomes or composition according to the present invention containing the hemagglutinin, or ha in combination with more specific (heterologous) antigen, and measure the titer of antibodies to the hemagglutinin or the specified antigen in the serum of the specified entity. For comparison, another subject subjected to immunization same virosomes containing hemagglutinin isolated from viruses obtained in the egg. Virosome has "greatly improved" or "significantly increased" immunogenicity, if the application of the Wilcoxon criterion to titers of antibodies to virosomal according to the present invention (with a hemagglutinin selected from the viruses obtained in cell line) and virosome with hemagglutinin isolated from viruses obtained in the egg, gives a p-value below 0.05. Example calculations are given in section 5.1 section below for Examples.

The terms "isolated from cell line", "received cell line", "produced in cell line" are used interchangeably and mean that anything obtained or produced in a cell line or cell culture.

The term "loaded antigen" in the present description refers to virosomes containing additional antigen, non-hemagglutinin (i.e., "heterologous antigen or antigen that is different from the hemagglutinin"). Antigen mo is et to be embedded in virosome (for example, inside the cavity), adsorbed on the/associated with the surface of virosome integrated into the lipid membrane virosome etc. Virosome with embedded antigen can be used as a delivery vehicle antigen.

In the present description, the term "Chimera virosome" refers to virosome containing hemagglutinin derived from at least two different strains of influenza virus.

In the present description, the terms "light" or "empty" in relation to virosomal interchangeably and refer to the fact that characterized thus chromosomes do not contain specific for any disease antigens cavity and do not bear such antigens in the lipid bilayer. Thus, the "unloaded" or "empty" virosome contain only the solution inside the cavity and does not contain protein, except for the hemagglutinin protein of viral envelope and possible traces of neuramidase, in the membrane.

In the present description, the terms "treatment", "therapy", "treatment" and the like refer to actions against already present diseases or disorders or diseases or disorders in respect of which suggest that it is already present, regardless of whether manifested the symptoms. In this case, the "treatment" or "therapy" refer to the removal of disease or impairment, or at ENISA least the decrease in severity of disease symptoms or disorders; however, if symptoms are already present, they soften, and if no symptoms, reduce the severity of emerging symptoms or eliminate them completely. The terms "prevention", "prevent", "preventing", "prevention" in the present description relate to the actions taken in order to prevent the occurrence of the disease in the subject if there is no suspicion that in the past, this subject has already been specified disease, but suggest that the entity is threatened or is in danger of occurrence of the specified diseases. In addition, these terms are relevant to the prevention of a disease, if the patient has already undergone appropriate vaccination/immunization, the effect of which, however, madagasacar.

In the present description, the term "pharmaceutical" refers to the characteristics of substances and/or drugs, which provide the possibility of introducing a living animal, preferably a human.

The term "potentiating", "immunopathology", "stimulating", "immunity", etc. in the present description relate to the substance or reinforcing effect on immune function, which can ensure the destruction or removal of antigenaemia pathogens or PLN is kachestvennyh tumors and/or cause immunity to them.

In the present description, the term "non-specific" or "non-specific" refers to a General immunostimulatory activity virosome according to the present invention means that the stimulation of the immune system to prevent, withstand and/or eliminate any of the many diseases or disorders, and not any one specific. Specific immunostimulirutuyu activity, in contrast, refers to stimulation of the immune system to prevent, withstand and/or eliminate one specific disease. Thus, vaccination against a particular disease is an example of achieving specific immunostimulating activity.

The term "disease" or "violation" in this description refers to an abnormal condition of the body or psyche, calling the inconvenience. Diseases and disorders are divided into infectious and non-infectious, neoplastic, immune or metabolic.

Influenza viruses

Influenza viruses (Orthomyxoviridae) is enveloped RNA viruses with RNA, a negative circuit, and a segmented genome. Influenza viruses are divided into two kinds: the first includes the influenza viruses a and b, the second includes the influenza virus C. This division is based on significant antigenic differences in their nucleoproteids and proteins metrics. Also, these three types of viruses R is slideouts on pathogenicity and organization of the genome. Type a is found in many warm-blooded animals, and types b and C are primarily human pathogens. Influenza viruses And then divide by the antigenic characteristics of membrane glycoproteins hemagglutinin and neuraminidase, protruding from the surface of the virion. Currently, there are 15 subtypes of hemagglutinin and 9 neuraminidase subtypes. Influenza a viruses infect many species of animals, including birds, pigs, horses, humans and other mammals. Waterfowl are the natural reservoir for all known subtypes of influenza a and possibly the source of the genetic material for the emergence of strains that cause influenza pandemic in humans.

Influenza viruses accumulate point mutations during replication because of their complex RNA polymers there is no mechanism to correct errors. Mutations, which change amino acids in the antigenic sites of the envelope glycoproteins, can provide a selective advantage to the virus strain, which consists in avoiding existing immunity. Hemagglutinin is a major antigenic determinant of influenza virus, which recognize and bind neutralizing antibodies. The hemagglutinin molecule initiates infection by binding with the receptor (residues of sialic acid) on certain (respiratory) host cells

The hemagglutinin molecule consists of two different domains: the root structure, protruding on the surface of the virion and consisting of H2 and part of the HA1 polypeptide, and a globular head that is composed entirely of HA1.

Antibodies to the protein TO inhibit the binding of receptor and are very effective in preventing re-infection with the same strain. Hemagglutinin may elude existing immunity due to the drift of antigens, in which mutations of already existing gene hemagglutinin inhibit the binding of antibodies and antigenic shift, in which the virus acquires a new subtype hemagglutinin. Such changes are more characteristic for ha than for neuraminidase. Changes in other proteins of influenza virus occur much less often. The influence of antigenic drift is most pronounced in strains of influenza that infect humans, less than typical strains infecting pigs and horses, and to a lesser extent the inherent strains, affecting birds.

Strains of influenza can be characterized genetically by a comparison of individual gene segments.

While the development of vaccines against influenza strains that cause annual epidemics continues, the world is concerned about the threat of pandemic influenza. Authorities and medical services around the world today are working on the article is of ategy in preparation for the pandemic form of the flu.

Virosome

Virosome according to the present invention can be used to deliver compounds (e.g., immunogenic molecules, medicines and/or gene) in the target cell. In comparison with liposomes of virosome have the advantage of the opportunity to effectively penetrate into the cell, which provides the protein shell of the virus, and with the subsequent release of the contents of virosome in a cage. Furthermore, if the membrane virosome built some active protein shell of the virus, such virosome can release the contents into the cytoplasm immediately after fusion with the cell, thus avoiding the destruction of the drug in the acidic environment of endosome.

Virosome according to the present invention is particularly useful in the field of vaccination, when the task consists in stimulation of the immune response to an antigen associated with a particular disease or disorder. In such cases, the antigen is usually encapsulated in or associated with virosomes, which delivers the antigen to the immune system of the host, which was subjected to vaccination. Thanks to this particular antigen reached a prophylactic and/or therapeutic effect is sure to be specific in relation to diseases or disorders associated with which this antigen.

In addition, virosome can be simultaneously is agrogene several different epitopes of b - and T-lymphocytes (Pδltl-Frank and other (1999)), including the universal epitopes of T-helper cells (Kumar and others (1992)) and other well-known experts in this field. Thus, virosome are highly effective adjuvants in modern vaccination, has excellent characteristics as a means of delivery of antigens and high immunogenic potential, at the same time minimizing the risk of side effects.

The functionality of immunostimulatory reconstructed virosome influenza (IRIV) is manifested in the fact that their ability to membrane fusion to a large extent similar to the well known, dependent on low pH the ability to merge membranes of intact virus provided solely by the protein shell of the virus. Like viruses, virosome flu quickly internalized mechanism receptorpositive endocytosis or opsonization. Unlike viral systems virosome secure because of them removed infectious nucleocapsid virus. Thus, virosome according to the present invention are promising transport systems to deliver a wide range of different compounds, prisoners in the internal environment inside virosa in the aquatic environment inside virosome or embedded in its membrane. Joint implementation of various receptors in the membrane of virosome provides targeting virosa in various cells and tissues, virosome used as vaccines after antigen binding to their surface, or after encapsulation of antigen in the cavity virosome, or use their adjuvant effect in the case of the introduction in combination with liposomes associated with antigen.

IRIV reconstructed from the shell of influenza virus and use receptor-mediated endocytosis, as well as their viral counterparts. The mediator binding of influenza virus to receptors and its fusion with the membrane of endosomes is a basic glycoprotein shell of the virus - hemagglutinin (Bungener and others (2002)). As in the case of viral vectors, slightly acidic pH cavity of endosome triggers the fusion of membranes virosome and endosome and release of the encapsulated material, such as DNA, RNA or proteins in the cytoplasm of a cell. Accordingly, exogenous antigens encapsulated in virosomes reach the path of major histocompatibility complex I (GCGS I) without the necessity of de novo protein synthesis. In the process of fusion proteins present on the surface of virosome remain in the cavity of endosome and so, apparently, become available for the path GCGS I.

It is shown that commercially available vaccines based virosa (INFLEXAL®V, EPAXAL®very effective and safe (Gluck R. and others (2000)). Potential virosa as delivery systems has been demonstrated for vaccines n the basis of nucleic acids and proteins for example, in the case of malaria (Pőltl-Frank and others (1999)). According to the results of recent studies also possible to conclude that vaccines containing synthetic peptides and injected subcutaneously using virosomal able to induce effective cytotoxic immune response (Amacker and others (2005)).

Getting virosa

Getting virosa is a procedure well known to specialists in this field. Applied methods of obtaining virosa described, for example, in EP 538437 and Mischler and Metcalfe (2002).

Virosome according to the present invention can be reconstructed from the viral lipids and membrane proteins after solubilization of influenza virus octadecenyl-monododecyl ether, sedimentation of nucleocapsid (viral glycoproteins and lipids remain at the top of the fraction) and the removal of detergent from the upper fraction of the hydrophobic resin (Bio-Beads SM2). Ways of getting flu virosa described in WO 92/19267, virosome other representatives of the family in WO 04/071492.

Getting virosome containing hemagglutinin isolated from different strains of influenza virus can be made by using equal amounts of protein of these viruses, solubilizing non-ionic detergent and octadecenyl-monododecyl ether. After removal of the detergent resin, Bio-Beads SM2, it becomes possible formation of virosome containing different types of be the Cove shell.

Protocols obtain virosome containing material derived from chicken eggs and cell lines that are identical.

Subtypes of influenza virus, which can be obtained virosome according to the present invention, include H1N1, H1N2 influenza, influenza H2N2, H3N2 influenza, influenza H3N8, H5N1 influenza, influenza H5N2, influenza H5N3, influenza H5N8, flu H5N9, influenza H7N1, influenza H7N2, influenza H7N3, influenza, H7N4, H7N7 influenza, influenza H9N2 and/or flu H10N7. In addition, at least one envelope protein of a virus can be obtained from the following strains: A/Bangkok/1/79, influenza a/Beijing/32/92, influenza A/Brazil/11/78, influenza A/California/7/2004 (H3N2), influenza A/Chile/1/83, influenza A/Kriscenski/4/85, influenza A/English/42/72, influenza A/Fujian/411/2002 (H3N2), influenza A/Guijosa/54/89, influenza A/Hong Kong/1 /68, influenza a/Johannesburg/33/94, influenza A/Leningrad/360/86, influenza A/Mississippi/1/85, influenza A/Moscow/10/99 (H3N2), influenza A/new Caledonian/20/99 (H1N1), influenza A/Panama/2007/99-RESVIR - 17), influenza A/Philippines/2/82, influenza A/port Chalmers/1/73, influenza A/Scottish/840/74, influenza A/Shandong/9/93, influenza A/Shanghai/11/87, influenza A/Cichanski/2/87, influenza A/Singapore/6/86, influenza A/Sydney/5/97, influenza A/Texas/1/77, influenza A/Soviet/90/77, influenza A/Victorian/3/75, influenza A/Wisconsin/67/2005 (H3N2), influenza A/Wuhan/359/95, influenza A/Violinski/3/2003 X - 147), influenza a/Hong Kong/330/2001, flu/Zilinsky/20/2003, flu/Malaises the s/2506/2004, the flu/Shanghai/361/2002, influenza a/Beijing/262/95, flu/Victorian/98926/70, influenza a/Singapore/222/79, flu/Soviet/100/83, flu/Amalatsky/16/88, influenza a/Panama/45/90, influenza a/Hong Kong/5/72, the influenza a/Ann Urbanski/1/86, influenza A/Bavarian/7/95, flu/Shandong/7/97) and/or/Sansosti/10/2003.

Immunostimulatory reconstructed virosome influenza (IRIV) contain a double lipid membrane consisting essentially of phospholipids, mainly phosphatidylcholine (NS) and phosphatidylethanolamine (RE). Unlike liposomes IRIV contain additional functional viral membrane glycoproteins hemagglutinin and neuraminidase, are embedded in the phospholipid bilayer membrane. Biologically active hemagglutinin makes a significant contribution to immunological properties, because it maintains the ability of viruses to merge.

IRIV are highly effective means to enhance nonspecific immunity. In addition, they have excellent safety features (Glück and others (2000)) and, thus, are suitable for use in medicinal products for nonspecific immunostimulation designed for people.

Virosome according to the present invention may also provide a chimeric virosome. This means that it contains proteins of the shell as a minimum the two influenza virus strains, for example, strains of the X-31 and Singh or any other listed above. In addition, to create a chimeric virosome capable of sequential or separate merge can be used membrane proteins of other viruses, such as the G protein of vesicular stomatitis virus, the E1 protein of the virus Semliki, F protein of Sendai virus, protein G or F of respiratory syncytial virus or protein of E. hepatitis C virus, and many others.

As has been demonstrated previously (Tsurudome et al. 1992), fusion proteins from different strains of viruses vary considerably according to the temperature characteristics of mergers and inactivation. For example, the hemagglutinin X-31 effectively initiates the merge at pH 5.0 and low temperatures, while the strains PR8/34 or A/Singapore at the same acidity require high temperature (>25°C). Thus, membranes of chimeric virosome may contain proteins, initiating fusion at two different temperatures. Different sensitivity to temperature is a particularly useful feature of the fusion proteins, making it easy and convenient to control fusion reactions. For example, virosome containing hemagglutinin virion X-31 and PR8/34, can catalyze two different fusion reactions at pH values 5: at low temperatures (4-10°C) and at elevated (>25°C). However, it is also Sveshnikova proteins merger, having different fusing characteristics, such as sensitivity to temperature, ion concentration, acidity, specificity to the type of cells and tissues.

Proteins merge with different characteristics merge can be isolated from various strains of influenza virus, such as MRC-11, X-97, NIB24, NIB26, X-47, a/Johannesburg/33 and a/Singapore.

In the preferred case, virosome according to the present invention mainly contains a lipid selected from the group comprising cationic lipids, synthetic lipids, glycolipids, phospholipids, cholesterol and derivatives thereof. The mainly phospholipids include phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol with variable residues of fatty acids. Cationic lipids are predominantly selected from the group comprising DOTMA (N-[(1-(2,3-dialerace)propyl]-N,N,N-trimethylammonium chloride), DOTAP (N-[1-(2,3-dialerace)propyl]-N,N,N-trimethylammonium chloride), DODAC (N,N-dioleoyl-N,N-dimethylammonio chloride), DDAB (didodecyldimethylammonium bromide), TC-Chol (cholesteryl N-(trimethylaminoethyl)carbamate chloride), DC-Choi (cholesteryl N-(dimethylaminoethyl)carbamate chloride) or other cationic derivatives of cholesterol, stearylamine or other aliphatic amines, DPPE (dipalmitoylphosphatidylethanolamine), DOGS(dioleoyl-glycero-succinate), DOSPA (2,3-tileorasi-N-[2(sprintersexual)ethyl]-N,N-dimethyl-1-propanenitrile), DOSPER (1,3-dialerace-2-(6-carboxypentyl)propylamide), THDOB (N,N,N',N'-tetramethyl-N,N'-bis (2-hydroxyethyl)-2,3-tileorasi-1,4-butanediamine iodide), DOPA (dioleoyl-sn-glycerol), DOTP (dioctyl terephthalate), DOSC (dealer-succinyl-glycerine), DOTB (dealer-s-(4'-trimethylammonio)-butanoyl-sn-glycerol), DOPC (dioleoyl-sn-glycero-phosphocholine) etc. are the Most preferred options cationic lipid is cationic derivatives of cholesterol, as TC-Chol (cholesteryl N-(trimethylaminoethyl) carbamate) or DC-Chol (cholesteryl N-(dimethylaminoethyl) carbamate). They can be summarized as small monolayer liposomes, mixed with phosphatidylcholine. Virosome according to the present invention may preferably contain phosphatidylcholine derived from egg or, even more preferably 1 oleyl-3-Palmitoyl-Gus-glycero-phosphatidylethanolamine.

Membrane virosome according to the present invention, preferably, contains 1.9-37 mol% DC-Chol or TC-Chol of the total number of lipids in the membrane. According to a particularly preferred variant implementation of the content of DC-Chol or TC-Chol in the membrane is 1.9-16 mol % of total lipids in the membrane. The remaining membrane lipids, in the preferred case, are phospholipids, in particular, phosphatidyl the nom and phosphatidylethanolamine in the ratio of 4:1.

Mamulleri agent can also be used to increase the rigidity and ability virosome to the circuit. Examples coemulsifier agents can serve as esters of cholesterol, charged or neutral, such as cholesterol sulfate and derivative-based sterols, such as derivatives of vegetable origin, for example, sitosterol, stigmasterol and mixtures thereof.

Virosome according to the present invention may, for example, be obtained in a manner analogous to any of the methods of the synthesis of DOTAP-containing virosa described in examples 1-3 and 6 in WO 97/41834, except that DOTAP replace DOSPER and that the concentration of DOSPER in the membrane end virosome selected accordingly, as described in WO 97/41834, and, in particular, it should not be more than 30% of the total mass of all lipid membrane virosome. In essence, the method of producing virosome according to the present invention may consist of the following stages:

a) preparation of buffer solution containing non-ionic detergent, DOSPER and other limes of IDA and at least one envelope protein of a virus;

b) bringing the concentrations of lipids to the values (based on the total weight of the lipids in the membrane) 5-30% DOSPER and, accordingly, 95-70% other mentioned lipids including phosphatidylcholine and its derivatives or, in the alternative, phosphatidate is alamin and/or cationic lipids, excluding DOSPER; and

(C) removing the detergent by dialysis or by treating the solution granules-microparticle, resulting in getting these virosome.

The use of virosome according to the present invention

Virosome according to the present invention can be used in preparation of medicines for treating and/or preventing at least one disease or disorders. The specified at least one disease or disorder can be infectious, non-infectious, neoplastic, immune or metabolic disease or disorder. According to one variant of implementation, the application includes the introduction of virosome according to the present invention healthy subjects who temporarily are at increased risk to acquire one or more infectious diseases or disorders, or (still) healthy subjects immediately before occurrence of the suspected risk of acquiring one or more diseases or disorders, before the onset of symptoms or confirmed diagnosis. Classification of impact on the individual as a therapeutic or prophylactic described above.

The application may also relate to the treatment of one or more existing diseases or disorders, possibly as a subsidiary of therapeutic measures to combat such diseases and or disorders.

According to another variant of implementation of at least one infectious disease or disorder may be a viral disease or disorder, a bacterial disease or disorder, fungal disease or disorder, a parasitic disease or disorder or a prion disease or disorder.

According to the next result of the implementation of the animal is a mammal. The mammal is preferably a: human, chimpanzee, cynomolgus macaque, Gibbon, not a humanoid monkey, makaku rhesus, mouse, rat, cat, dog, horse, rabbit, camel, Lama, gum or a pig. In the preferred case, the gum is a cow, bull, goat, sheep, Buffalo, deer or oxen.

According to the next result of the implementation of the drug is suitable for intramuscular, intradermal, intravenous (e.g., by injection), subcutaneous, vnutriplevralnogo, parenteral administration, topical administration, injection into the ear, joints, eyes, local administration, administration by rinsing, introduction via patch (for example, a skin patch), in the form of a spray (e.g., naso-fringillinae spray), sublingual, oral (e.g., tablets, capsules, pills, suppositories (e.g. rectal or vaginal suppositories) and VI is e drops (for example, eye drops). The drug can be administered once or, if necessary, multiple doses at appropriate intervals determined by the treating physician.

Perhaps neodnokratno application of virosome according to the present invention. Combining virosome according to the present invention with other agents, such as adjuvants or Immunostimulants, can provide synergistic enhancing the overall effect. The overall effect depends on the number and type of input virosome, plot stimulation and co-stimulating agents (infections, exposure to allergens and the like). The effect is temporary, lasting from several hours to several weeks. The duration of the effect depends on the dose, frequency of administration, place of introduction and composition of the administered drug.

Obtained according to the present invention, the drug is administered in pharmaceutically acceptable forms. These forms can, as usual, contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, additional immunostimulatory agents such as adjuvants and cytokines, and, if necessary, other medicinal substances. The preferred number of input virosa dependent diseases or disorders, which should be prevented or cured. Typically, f is considered objective number in the range from 1 ng/kg to 100 μg/kg, where in kilograms specified the weight of the animal. It is believed that the preferred range is from 10 ng/kg to 10 µg/kg Absolute number depends on many factors, including the composition, selected for administration, the amount of administered doses (one or more doses) and individual patient characteristics, such as age, physical condition, body shape, weight, and disease stage.

The path and mode of administration will vary depending on the stage and severity of the disease, which is treatable and should be determined by the skilled practitioner. The drug is prepared in accordance with the present invention suitable for parenteral administration. The drug contains virosome, dissolved or suspended in a suitable carrier, preferably water. Can be used lots of water carriers, for example, water, buffered aqueous solutions, and 0.4% saline, 0.3% solution of glycine, hyaluronic acid and the like, Such compositions can be sterilized by conventional, well known methods, or may be subjected to sterile filtration. The resulting aqueous solutions may be packaged for use as is, or lyophilized, before the introduction of the dried product is mixed with a sterile solution.

The drug is made with the according to the present invention, may also contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as regulating the pH and buffering agents, agents affecting toychest, moisturizing agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, monolaurate sorbitan, triethanolamine oleate, and many others. In fact, the methods of preparation of formulations suitable for parenteral administration, known and obvious to specialists in this field and are described, for example, in Remington: The Sciene'an Practice of Pharmacy ("Remington's Pharmaceutical Sciene") publishing house Gennaro AR, 20th edition, 2000: Williams & Wilkins PA, USA, included in the present description by reference.

Medicinal product prepared according to the present invention can also be administered orally, for example in the form of tablets, capsules (including compounds with 5 release within the specified time and delayed release), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, as well as in the form of injections. For example, in oral introduction in the form of tablets or capsules, the active drug component can be introduced in combination with suitable for oral administration of pharmaceutically inactive inert carrier, such as 10 ethanol, glycerol, water, etc.

Similarly, medicinal product prepared according to the present invention may be administered intravenously by bolus injection or infusion), administered intraperitoneally, subcutaneously, tapicerki (with or without occlusion), or intramuscularly. In a preferred embodiment, the prepared medicinal product prepared according to the present invention, intramuscularly, subcutaneously, intradermally, through mucosal or percutaneous route. All these forms of well known professionals in the pharmaceutical industry.

The dosage regimen under which the injected medicinal product prepared according to the present invention, are selected in accordance with many factors, including species, age, weight, sex and health status of the patient, the stage and severity of the disease or disorders, as well as the specific type of virosome. The ordinary skilled physician can readily determine and prescribe the effective amount of the drug required to prevent, combat or stop the progression of infectious diseases or disorders. Optimal precision in the determination of the concentration of the drug in compliance with efficiency and without toxicity or with an acceptable toxicity level implies a regime based on the kinetics of delivery of virosome (availability) in the target at ASDI. This process, which takes into account the distribution, equilibrium, and elimination of virosome, is within the competence of the practitioner and may be identified through routine experimentation.

According to one variant of implementation of the medicinal product prepared according to the present invention, can be administered daily in a single dose, or the total daily dose can be defined and divided into several parts, for example, when taking 2, 3 or 4 times a day. According to another variant of implementation of the stipulated weekly or monthly introduction.

Daily dose of the medicinal product prepared according to the present invention is in the range from 10 ng/kg to 10 mg/kg of virosome per adult patient per day. In the case of oral administration of a medicinal product prepared according to the present invention, it is preferably made in the form of tablets containing 0.001-1000 mg, preferably 0.01-100 mg, even more preferably 0.05-50 mg, and most preferably 0.1-20 mg virosa for symptomatic control the dose depending on the signs and symptoms of the patient in the treatment process. So, the tablets can contain 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 10, 20, 50 or 100 mg of virosome. An effective amount of virosome in medicinal product prepared according to this the mu of the invention, usually achieved at a dose of 0.0001 mg/kg to 50 mg/kg of body weight per day. More specifically, the level of dose is in the range from about 0.0001 mg/kg to 7 mg/kg of body weight per day. When introducing children dose can be reduced accordingly.

In addition, the drug is prepared in accordance with the present invention, may be introduced intranasally or by percutaneous known to specialists in this field. When percutaneous introduction in the form of a system for percutaneous delivery of the injection mode, of course, be continuous rather than periodic.

Medicinal product prepared according to the present invention, can be combined with biodegradable polymers used for controlled release of drugs in the body, for example, polylactic acid, polypeptide caprolactone, polyhydroxyalkanoic acid, polyantiserum, polyacetylene, policyidreference, polycyanoacrylate and sewn or amphipatic block copolymers of hydrogels.

A suitable form of the medicinal product prepared according to the present invention, for external injection can be, for example, in the form of a solution, cream, ointment, gel, lotion, shampoo or spray, suitable for application to the skin. These compositions for topical application is, containing medicinal product according to the present invention, typically include about 0.005-5 wt.% the active substance, i.e. virosome, in a mixture with a pharmaceutically acceptable carrier.

Regardless of the method and route of administration of the medicinal product prepared according to the present invention, it is administered in an effective amount. An effective amount is an amount of the pharmaceutical agents, which after a single injection, or together with further doses, stimulates the desired non-specific immune response.

Moreover, the drug is prepared in accordance with the present invention, if necessary or desired, may be added acceptable agents, lubricants, dezintegriruetsja agents or dyes. Suitable binding agents include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural or synthetic resins, such as gum Arabic, tragacanth gum or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and other Lubricants used in the forms of medications, include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Dezintegriruetsja agents include, without limitation,starch, methylcellulose, agar, bentonite, xanthan resin, etc.

Liquid forms of the medicinal product prepared according to the present invention can be appropriately supplemented suspendresume or dispersing agents, such as synthetic and natural resins, for example, tragacantha kamadgiri, methylcellulose and the like, Other dispersing agents suitable for use, are glycerin, etc. For parenteral administration is desirable to use sterile suspensions and solutions. For intravenous use isotonic formulations, usually containing appropriate preservatives. The composition for external use containing the active drug component can be mixed with different materials, media, widely known in this field, such as, for example, alcohols, aloe Vera gel, allantoin, glycerine, oil, vitamins a and E, mineral oil, PPG2 myristyl propionate and the like, to obtain, for example, alcohol solutions, external cleansers, cleansing creams, gels, skin, skin lotions, and shampoos in the form of cream or gel.

In one embodiment, the drug is prepared in accordance with the present invention, may further comprise at least one adjuvant, enhancing or OPOS EBUSY immune response, for example, innate immune response, answer Th1or Th2. Suitable adjuvants can enhance the immune response by activating macrophages and/or stimulation of certain groups of lymphocytes. Acceptable adjuvant may be any ligand capable of activating the receptor recognition of a pathogen. Compounds that activate the immune response, are classified as adjuvants or cytokines. Adjuvants can enhance the immune response by providing a reservoir of antigen (extracellular or inside macrophages), activating macrophages and stimulation of certain groups of lymphocytes.

In this area there are many types of adjuvants; specific examples include beta-blockers (complete and incomplete), mycobacteria such as BCG, M. vaccae or Corynebacterium parvum, toxins cholera or tetanus, heat-sensitive toxin of Escherichia coli, a mixture of Gila saponin as QS-21 (SmithKline Beecham), MF59 (Chiron), and various water-in-oil emulsions (for example, IDEC - AF). Other adjuvants suitable for use include, but are not limited listed): mineral salts or mineral gels such as aluminum hydroxide, aluminum phosphate and calcium phosphate; surfactants, such as lysolecithin, polyols - pluronic, polyanion, peptides, hemocyanin mollusk fissurella, dinitrophenol, immunostimulating agents, such as saponin derivatives is, nuramilovich of dipeptides and tripeptides, short-chain molecules of nucleic acids, such as dinucleotides CPG, oligonucleotides CPG, monophosphoryl-lipid a and polyphosphazene, adjuvant particles and microparticles, such as emulsions, liposomes, virosome, chelates or complex immunostimulatory adjuvants.

Cytokines can also be used because of their ability to stimulate lymphocytes. Professionals in this field are familiar with many of the cytokines that can be used for these purposes, such as interleukin-2 (IL-2), IL-12, colony-stimulating factor granulocyte-macrophage (GM-CSF), and many others. In addition, the ligands of the family of chemokines, such as RANTES (cytokine A5), lipoprotein gram-positive bacteria, a component of the cell wall of the yeast double-stranded RNA, LPS of gram-negative bacteria, flagellin, rich uracil single-stranded viral RNA suppressor small interfering RNA cytokine signaling (SOCS siRNA), epitope Pan DR (PADRE) and their mixtures can also be used.

Medicinal product prepared according to the present invention, can be introduced in combination with a pharmaceutically acceptable carrier suitable for topical application. Also medicinal product prepared according to the present invention can be used for treatment and significant others the malignant tumors, tumors and/or metastases or viral infections in combination with other agents known that they are suitable for the treatment of these diseases. In the combined treatment of more than one active agent, the active substance can be administered simultaneously or separately at different times.

Another aspect of the present invention relates to non-specific stimulation of the immune system of an animal by introducing virosome according to the present invention. It is desirable to enhance the overall resistance to diseases, particularly infectious diseases and neoplasms, by nonspecific stimulation (Wake-up) the body's immune system. Such nonspecific stimulus can be achieved by the introduction of virosome. This introduction, a one-time or periodic, can be carried out before, during and after exposure to the infective agent or diagnosis of disease, as a preventive, metafictional, therapeutic or auxiliary equipment.

Specific embodiments of the invention and the following examples demonstrate the effectiveness of the present invention, but should not be interpreted as limiting the scope of the invention. In cases where specific materials, such indication is given isklucitelno illustrative purposes and is not intended to limit the scope of the invention. Unless otherwise specified, biochemical and related to molecular biology of the procedures performed as described in the following sources: Voet, Biochemistry, Wiley, 1990; Stryer, Biochemistry, W.H. Freeman, 1995; Bodanszky, Peptide chemistry. A Practical Textbook, 2nd ed., Springer-Verlag, Berlin, 1993; Sambrook et al., Molecular cloning, Cold Spring Harbor Laboratory, 2001; Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, 2000. Specialists in this field can develop equivalent tools and reagents requiring additional inventive activity and not going outside the scope of the present invention.

It is obvious many of the options described in this text options compositions and procedures that do not go beyond the scope of the present invention. According to the authors of inventions such variants are included within the scope of the invention.

Examples

Content

1. Viruses

2. The multiplication of viruses

3. Getting virosa

3.1 Reagents

3.2 Obtaining standard virosa (immunostimulatory reconstructed virosa flu, IRIV)

3.3 Obtaining standard virosa with integrated heterologous antigen (IRIV)

3.4 Obtaining standard virosome containing TC-chol-cholesterol-N-(trimethylaminoethyl)-carbamate-chloride (TIRIV)

3.5 Obtain TIRIV containing heterologous antigen

3.6 Heterologous antigens used for picking virosa

4. Analytical is etody

4.1 Electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE)

4.2 determination of the particle size (average diameter:/polydispersity (table 1).

4.3 Simple radial diffusion (SRD, concentration hemagglutinin)

4.4 Western Blot

4.5 analysis of the increased ability to merge by fluorescent resonance energy transfer (FRET)

5. Analysis of immunogenicity

5.1 Improved antigenic characteristics

5.2 Definition of high immunogenicity on painting, staining of interferon-gamma (IFN-y) and increased immunogenicity

5.3 Comparison of hemagglutinin derived from viruses obtained in eggs, and hemagglutinin derived from viruses obtained in cell culture

1. Viruses

Used the influenza virus strains A/new Caledonian/20/99 (H1 N1) and a/ Singapore/6/86 (H1N1).

2. The multiplication of viruses

Viruses were propagated either in allantoine cavity fertilized eggs (Gerhard, 1996), or in the line of embryonic stem cells of birds (WO 2006/108846). The viruses were propagated in allantoine cavity fertilized eggs obtained from Berna Biotech AG (Bern, Switzerland); the viruses were propagated in the line of embryonic stem cells of birds obtained from Vivalis (Roussay, France).

Obtained from eggs, the virus was purified and concentrated using ultracentrifugation in sucrose gradient and ina who was rivervale beta propiolactone (BPL). Derived from cell lines of bird virus (EVH, chicken (EV) or duck) was present in the supernatant of cell culture Ebx infected with influenza A. Prior to analysis the supernatant was concentrated and precipitated by ultracentrifugation. The number of viral proteins was determined by the method of simple radial diffusion (SRD) (Wood and others, 1977).

The ratio of the hemagglutinin/phospholipids was determined by the method according to Böttcher (Böttcher and others, 1961), by determining the content of hemaglutinin by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) and extraction of Kumassi, as described by Ball, 1986.

3. Getting virosa 3.1. Reagents

Octadecenyl-mono(n-dodecyl)ether (OEG, Ci2E8), dimethylsulfoxide (DMSO), hydroxylamine hydrochloride, 1,2-dipalmitoyl-sn-glycero-3-phospho-Gus-(1-glycerol) (PG), acetonitrile, a solution of phosphate of triethylamine (TEAR), sucrose, streptomycin, HEPES, penicillin and RPMI medium were purchased in Fluka Chemie GmbH and Signia (Buchs, Switzerland), respectively. Sucrose (Eur. Phar.) was acquired by Merck (Dietikon, Switzerland). Fluorescence correlation spectroscopy (FCS) was purchased from Gibco BRL (Basel, Switzerland). Egg phosphatidyl choline was acquired in Lipoid (Cham, Switzerland). 1 oleyl-3-Palmitoyl-Gus-glycero-2-phosphoethanolamine was acquired in Bachem (Bubendorf, Switzerland). Bio-particles of Bio-Beads SM2 were purchased from Bio-Rad Laboratores (best Western, Switzerland). 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (N-MCC-PE) were purchased from Avanti Polar Lipids (Alabaster, USA). N-(4,4-Diptera-5,7-diphenyl-Bora-3A,4A-diaza-8-indocin-8-propionyl 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (Bodipy 530/550-DHPE), lissamine-rhodamine 1.2-dihexadecyl-5s-glycero-3-phosphoethanolamine trimethylammonium salt (N-Rh-DHPE) and Biotin-DHPE (N-(biotinyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine, triethylamine salt) were obtained from Molecular Probes Europe (Leiden, Netherlands). The Sephadex (Sephadex G-50 coarse, was acquired Amersham Biosciences (Otelfingen, Switzerland). Interleukin-2. (IL-2) was purchased in EuroCetus B.V. (Amsterdam, Netherlands). N - Succinimidyl-S-acetylthiourea (SATA) is was obtained in Pierce Biotechnology (Rockford, USA). Cholesteryl-N-(trimethylaminoethyl)carbamate chloride (TC-chol) was obtained from Merck Eprova (Schaffhausen, Switzerland).

3.2 Obtaining standard virosa (immunostimulatory reconstructed virosa flu. IRIV)

In total volume of 4 ml was dissolved 32 mg phosphatidylcholine from egg, 8 mg of phosphoethanolamine was dissolved in 3 ml of phosphate-saline buffer containing 100 mm oligoarthritis (PBS/OEG). An inactivated influenza virus containing 2 mg of hemagglutinin, was centrifuged at 100,000 × g for 1 h at 4°C, after which the precipitate was dissolved in 1 ml of PBS/OEG. Phospholipids and viruses, solubilization in detergent, CME is ivali in a total volume of 4 ml and dispersively under the action of ultrasound for 1 min The resulting mixture was centrifuged at 100,000 × g for 1 h at 18°C. Then virosome by removal of the detergent, using 1.5 g of wet granules SM2 Bio-Beads provided education virosa (BioRad, best Western, Switzerland) twice for 1 hour at room temperature, with shaking. Then virosome was subjected to sterile filtration (0.22 μm) and stored at 4°C.

3.3 Obtaining standard virosa with integrated heterologous antigen (I I RIV).

In total volume of 4 ml 32 mg phosphatidylcholine from egg, 8 mg of phosphoethanolamine and the desired amount of coupling of the heterologous antigen and phosphoethanolamine was dissolved in 3 ml of phosphate-saline buffer containing 100 mm oligoarthritis (PBS/OEG). Vaccine strain A/Singapore/6/86 influenza virus containing 2 mg of hemagglutinin, was centrifuged at 100,000 × g for 1 h at 4°C, after which the precipitate was dissolved in 1 ml of PBS/OEG. Phospholipids and viruses, solubilization in detergent, mixed and dispersively under the action of ultrasound for 1 min, This mixture was centrifuged at 100,000 × g for 1 h at 18°C. Then, by removal of the detergent, using 1.5 g of wet granules SM2 Bio-Beads (BioRad, best Western, Switzerland) twice for 1 hour at room temperature with shaking provided education virosa. Then virosome was subjected to sterile filtration (0.22 μm)and stored at 4°C.

3.4 Obtaining standard virosome containing TC-chol - cholesterol-N-(trimethylaminoethyl)-carbamate-chloride (TIRIV)

TIRIV received method by detergent. In total volume of 4 ml 32 mg phosphatidylcholine from egg, 8 mg of phosphoethanolamine, 5 mg cholesterol-T-(trimethylaminoethyl)-carbamate-chloride and 200 mg of sucrose was dissolved in 3 ml of phosphate-saline buffer containing 100 mm oligoarthritis (PBS/OEG). 1-2 mg of hemagglutinin inactivated influenza virus were centrifuged at 100,000 × g for 1 h at 4°C, after which the precipitate was dissolved in 1 ml of PBS/OEG. Phospholipids and viruses, solubilization in detergent, mixed and dispersively under the action of ultrasound for 1 min the mixture was centrifuged at 100,000 × g for 1 h at 18°C. Then, by removal of the detergent, using 1.5 g of wet granules SM2 Bio-Beads (BioRad, best Western, Switzerland) twice for 1 hour at room temperature with shaking produces virosa. Then virosome was subjected to sterile filtration (0.22 μm) and poured into sterile glass vials. The closed tubes were frozen at -70°C, then freeze-dried at -40°C for 20 h and at 10°C for 2 hours covered tubes were stored frozen until use.

3.5 Getting TIRIV containing heterologous antigen

To obtain TIRIV containing the selected heterologic the second antigen, the specified antigen dissolved in water at the desired concentration. Frozen, lyophilized TIRIV removed from the freezer and balanced at room temperature for 2-5 min, and then in the lyophilisate was added an equal amount of dissolved heterologous antigen (4°C). Then the tube briefly (about 10 seconds) were placed in a vortex mixer at medium speed and kept at 4°C until use.

Alternatively, the peptides associated with phosphatidylethanolamine, you can add to TIRIV during the retrieval process described in example 5. The peptide to the desired concentration was added before dispersion under the action of ultrasound and sterile filtration of the solution. The remaining stages of the preparation have not changed. Recovery liofilizirovanny TIRIV carried out with an equal amount of water.

3.6 Heterologous antigens used to obtain virosa

Used heterologous antigens were malaria UK 39 (WO 2004/106366)derived from Plasmodium falciparum (UK 39); and envelope antigen of the hepatitis C virus (HCV core 132).

4. Methods of analysis

4.1 Electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE)

The analyzed samples were mixed with the appropriate buffer for the analysis of production Invitrogen (Basel, Switzerland) with a regenerating agent or without (Invitrogen) and incu is Aravali at 85°C 2 minutes 5-10 μl of the sample was applied to a matrix of polyacrylamide gel (Invitrogen, Basel, Switzerland) and analyzed according to manufacturer's instructions. Then the gels were analyzed by the method of Western-Blot and/or abrasively silver set SilverQuest Kit (Invitrogen, Basle, Switzerland), following the Protocol "speed painting" ("fast staining"), provided by the manufacturer.

4.2 determination of the particle size (average diameter:/polydispersity

The table below shows the concentration of hemagglutinin in mg/ml and an average particle diameter of virosome. The polydispersity, are shown in the last row, is a measure of the homogeneity of particle size in solution. The solution particles with a value of polydispersity below 0.3 is acceptable for virosa used as vaccines, the value below 0.1 indicates significant homogeneity (homogeneity was determined by dynamic light scattering instrument Zetasizer 1000HS).

The size was determined by dynamic light scattering instrument Zetasizer 1000HS (Malvern Instruments)equipped with a standard 10 mW helium-neon laser (λ=633 nm) and avalanche photodiode (APD). 5-20 μl of sample was added to the filtered phosphate-saline buffer in a cuvette with a final volume of 1 ml. Measurements were carried out at a temperature of 25°C and a fixed angle of dispersion of 90°. The size distribution was assessed by appropriate selection of the apt is aximili.

4.3 Simple radial diffusion (SRD, concentration hemagglutinin)

Analysis by the method of simple radial immunodiffusion to identify the hemagglutinin in the above-described samples of influenza virus derived from eggs and cell lines was performed according to the procedure described by wood and others (Wood et al, 1977). Virions were destroyed by incubation in 1% Zwittergent (Calbiochem) for 30 min at room temperature and subjected immunodiffusion for 72 h at room temperature agarose gel containing antibodies. Measured diameters of the zones of deposition of complexes of antigen-antibody and determined the content of the antigen in the virus preparations using the calibration curve control sample of whole virus (NIBSC, London) with a known content of hemagglutinin provided by the manufacturer NIBSC. As control samples of whole virus used to determine the amount of hemagglutinin in samples of influenza virus obtained in cell lines used are derived from eggs standard counterparts obtained at NIBSC and used antisera.

Table 1
The definition of medium size and polydispersity IRIV different compositions with heterologous antigen for malaria antigen UK 3. The size and polydispersity were determined by dynamic light scattering using the instrument Zetasizer I OOOHS (Malvem Instruments)
Description sampleThe concentration of hemagglutinin (mg/ml)Environments. aifm. Z
Average (nm)
The polydispersity
1.UK39 moriza0.261160.16
2.UK39 IRIV_0.251170.14
3.UK39 IRIV_0.18116-

4.4 Analysis Western Blot

Comparative analysis of influenza virus obtained in embryonated eggs and cell lines of birds, included SDS-PAGE and analysis by the method of Western Blot preparations of viruses, which allowed to obtain information about the synthesis and processing of the hemagglutinin of the virus in both types of cells: analysis SDS-PAGE was possible to determine the purity and protein content in suspensions of viruses, and to identify proteins/size proteins hemagglutin is on and neuraminidase.

Carried out analysis of the samples on the gel SDS-PAGE as described above. The gels were placed in the appropriate buffer for transfer provided by the manufacturer (Invitrogen, Basel, Switzerland). In parallel polyvinylidenedifluoride (PVDF) membrane (Invitrogen, Basel, Switzerland) were incubated in methanol and placed in a buffer for transfer. 4-5 filters for blotting and 2 sheets of Whatman paper (Biorad, Reinach, Switzerland) for each gel was soaked in the buffer to transfer and receive the immunoblot. The transfer was carried out by the application 25V, 125 mA, 17 watts to each gel for 1 h 30 min, Membranes were rapidly washed in phosphate-buffered saline containing 0.2% Tween 20, non-specific binding of the antibody or serum blocked by incubation in 5% milk in phosphate-buffered saline for 2 hours After re-washing of the membranes in phosphate-buffered saline/0.2% Tween 20, immunoblot incubated with a mixture of primary antibodies and serum dissolved in 0.5% milk in phosphate-buffered saline/0.2% Tween 20, in a ratio of from 1:100 to 1:1000, depending on the antibody at room temperature for 1-2 hours in the Membrane washed three times in phosphate-buffered saline/0.2% Tween 20 for 5 minutes and incubated in the appropriate solution of secondary antibodies labeled with horseradish peroxidase (HRP), diluted in a ratio of from 1:1000 to 1:20000 in 0.5% milk in phosphate-buffered saline/0.2% Tween 20. After 5 times washing the Oia membranes in phosphate-buffered saline/0.2% Tween 20 was carried out by visualization using a set of SuperSignal West Dura (Pierce, Lausanne, Switzerland) according to the manufacturer's instructions.

4.5 analysis of the increased ability to merge by fluorescent resonance energy transfer (FRET)

To measure the ability to merge in vitro by the method of fluorescence resonance energy transfer (FRET) was developed by the following procedure: 0.75 mol.% N-(4,4-Diptera-5,7-diphenyl-Bora-3A,4A-diaza-s-indocin-S-propionyl 1,2-dihexadecyl-sn-glycero-S-phosphoethanolamine (Bodipy 530/550-DHPE) and 0.25 mol.% 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine trimethylammonium salt (N-Rh-DHPE) was implemented in liposomes containing phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (PC/PG) (70:30). Fluorescence analysis was carried out in 5 mm sodium phosphate buffer pH 7.5, 100 mm NaCl, in a final volume of 0.8 ml in 2.5 ml microcuvette polymethyl methacrylate (VWR, Dietikon, Switzerland) with constant stirring. Usually 1 ál of labeled liposomes (0.3 nmol phospholipid) were mixed with 5-20 μl of virosa and stimulated fusion using a 3.75-7 ál of 1 M HCl and the resulting pH 4.5. The increased fluorescence was recorded every 5 seconds when the wavelengths of excitation and emission, respectively 538 nm and 558 nm, with excitation cross section 2.5 nm and emission cross-section 15.0 nm. The measurements were carried out on luminescence spectrometer LS 55 (Perkin Elmer Instruments, Schwerzenbach, Switzerland)equipped with a cuvette holder and magnetic stirrer. EIT is giving the temperature of the instrument was set at 42°C, thus the final temperature of the sample was 35 to 37°C. the Maximum fluorescence at infinite dilution of the sample was obtained by adding buffer Triton X-100 (final concentration 0.5% vol./vol.). For scale calibration fluorescence initial residual fluorescence of the liposomes was equated to zero, and the fluorescence of the sample with infinite dilution was taken as 100% (maximum fluorescence).

The total content of the ha samples for analysis FRET should be 0.5-10 μg. For analysis of the composition of virosome optimum was found as the contents 2-6 µg of hemagglutinin. The concentration of ha in the sample was pre-determined by the method of simple radial diffusion (SRD). Depending on the concentration of ha in each separate sample required for FRET sample volume of virosome is in the range from 3 to 40 ál (respectively, 2-6 µg hemagglutinin). If the volume of sample virosa was below 40 μl, the difference was compensated by the addition of a phosphate-saline buffer.

It is important that the ratio of hemagglutinin and lipid virosa remained unchanged when using different amounts of virosome in the test FRET, for example, in measurements, described in Table 2.

The interpretation of the results FRET

Because percentage values obtained by the method of FRET range, the absolute value of the dia is Altanbulag/threshold has been difficult to establish. In contrast, the ratio of the different samples was relatively stable. The number of the ha used in the analysis should be selected in the range of 3-6 μg in a total volume of 0.8 ml Multiple measurements with different amounts within a specified interval (for example, 3, 4, 5, 6 μg) was allowed to construct the curve, depending on the dose, provide additional information (e.g., saturation of the system).

Table 2
The ability to merge immunostimulatory reconstructed virosome influenza (IRIV) in various compositions with heterologous antigen malaria UK 39. For each composition used 4 of the sample with different contents of hemagglutinin.
The number of hemagglutinin (µg)The ability to merge (%)
Experiment 1Experiment 2
1.UK39 IRIV_5.24639
3.4 2823
1.62926
0.82918
2.UK39 IRIV_5.35454
3.54233
1.82238
1.52813
3.UK39 IRIV_5.21914
3.6158
1.81813
0.965

For each value of the concentration of hemagglutinin studied using FRET, we compared the results (expressed as % ability to merge), obtained for IRIVa containing g is agglutinin, isolated from viruses obtained in eggs, and from viruses obtained in cell lines, the comparison was made by calculating the ratio values for the two corresponding samples for each concentration value. Then calculate the average value of the relationship for different concentrations of hemagglutinin. Raw readings (% ability to merge) significantly vary for different concentrations of hemagglutinin (the dose)and also in different repetitions of the test (test variability) for the same sample. Average ratio, in contrast, showed significantly lower variability and, thus, represents a reliable measure reproducibly to compare the samples.

5 µg: 39%:14%=2,78

3 mcg: 23:8=2,87

2 mcg: 26:13=2,0

1 mcg: 18:5=3.6

The result was the average of 2.81 (figure 2, bottom)

5. Immunogenic methods. The study of the immunogenicity in mice

The reaction of antibodies: Unless otherwise specified, to study the immune response of a group of at least 10 mice of BALB/c mice were immunized intramuscularly with 0.1 ml empty IRIV or IRIV loaded heterologous antigen (MIRIV). In this experiment, as heterologous antigen used malarial antigen UK39. Was carried out by two vaccinations with an interval of three weeks, serum samples were taken across the two weeks after the second vaccination.

The response of CD8+ T cells: Unless otherwise indicated, to study the immune response of transgenic mice HLA-A2 6 were immunized intramuscularly with 0.1 ml empty IRIV or IRIV loaded heterologous antigen (MIRIV). Was carried out by two vaccinations with an interval of three weeks, samples of spleen cells were taken two weeks after the second vaccination.

5.1 Immunological analysis by ELISA (enzyme-linked immunosorbent assay, the reaction of b-cells)

Mice groups of at least 10 individuals of BALB/c were immunized intramuscularly twice with an interval of three weeks, then took samples of serum. Schedule, dosage, and number of immunization procedures may vary, provided that for the comparison groups used the same procedure. Sera were tested by ELISA to determine antibody responses to hemagglutinin of influenza virus (figure 3) and heterologous malaria antigen UK39 derived from Plasmodium falciparum (figure 4). For each serum sample was determined the titer of antibodies to a specific antigen. With this purpose we calculated the dilution corresponding to the value of optical density (OD) 20% of the maximum OD of the control serum samples present in each tablet.

For each group recorded all individual titles . Groups were compared using Wilcoxon criterion to the data (ti is ture serum). If the resulting p value less than 0.05, this means that with 95% probability the two groups were different. In this case, the Wilcoxon test was used to show that improved virosome are significantly more immunogenic, i.e. according to the Wilcoxon criterion for titles serum obtained a p-value below 0.05 when comparing virosa obtained using eggs, and virosome obtained with the use of cell lines.

For the detection of antibodies to heterologous antigens or hemagglutinin was carried out by enzyme-linked immunosorbent assay (ELISA). Briefly, microtiter tablets with 96 cells (Nunc, Fisher Scientific, Wohlen, Switzerland) overnight at 4°C covered the investigated antigen in a suitable buffer system, in the amount of 100 μl/well. For example, complexes of malarial antigen UK39 with phosphatidylethanolamine were applied at a concentration of 10 μg/ml solution in phosphate-buffered saline (pH 7.4) on microtiter tablets Polysorb, and proteins hemagglutinin proteins (inactivated whole virus or samples of virasam) was applied at a concentration of 1 μg/ml solution in M carbonate buffer (pH 9.4) on microtiter tablets Maxisorb.

After coating, the plates were blocked with 5% milk powder in phosphate-buffered saline for at least 2 h at room temperature, then washed three times with phosphate-saline buffer/0.2% Twen 20. Then the plates were incubated with serial dilutions (from 1:50), mouse serum in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% milk powder for 2 h at 37°C. each tablet was present in serum as a positive control. After a cycle of additional washing, the plates were incubated with linked to horseradish peroxidase antibodies goat to mouse Ig (BD Bioscience, Basel, Switzerland) for 1 h at 37°C. After the last rinse cycle is added to the substrate OPD (tablets O-phenylenediamine Fluka, Buchs, Switzerland, 1 tablet per 50 ml of nitrate buffer +20 μl of H2O2), and tablets incubated in the dark at room temperature to obtain a sufficient color in the colorimetric reaction was stopped by adding 100 μl of 1 M H2SO4, and then measured the optical density at 492 nm using a Spectra Max Plus (Molecular Devices, Bucher Biotech, Basel, Switzerland).

Figure 4 shows significant differences in immunogenicity between IIRIV (immunostimulating reconstructed virosome flu, loaded UK39)containing the hemagglutinin of the virus obtained in eggs, and IIRIV containing the hemagglutinin of the virus obtained in cell lines: p=0.002 for culture in cages of chickens in comparison with the culture in eggs, and p=0.009 for cell culture ducks when compared with cultures in eggs. The show is on improved immunogenicity UK39, associated with virosome containing the hemagglutinin of the virus obtained in Ebx, compared with UK39 associated with virosome containing the hemagglutinin of the virus obtained in eggs. The gray dashed line shows the value of OD, allowing it to calculate the titer of antibodies to the UK39, defined as 20% of the maximum value OD492 of control present in each tablet.

5.2 Immunological analysis by staining of intracellular interferon-gamma IFN-v (T-cells)

Staining of intracellular IFNγ: spleen Cells (12×106) were incubated with 10 μg/ml of specific peptide or peptide unrelated to the present invention (negative control)in complete RPMI medium containing 2 mm L-glutamine, 100 u/ml penicillin, 100 μg/ml streptomycin, 5 mm HEPES, 5% serum fetal bovine serum (FCS) and 5×10'5M 2-mercaptoethanol at 37°C and 5% CO2in the presence of 5 μg/ml of Brefeldin And within 4 hours the Cells were stained with antibodies to CD8, associated with isothiocyanato fluorescein (FITC), was permeabilities, stained-linked phosphatidylethanolamine antibodies to IFNy, using a set of Cytofix/Cytoperm, following the manufacturer's instructions (BD Pharmingen, San Diego, USA). The results were read on a flow cytometer BD™ LSR II and analyzed using FlowJo software. The number synthesizing cells, with desiroush IFNy, was calculated as the proportion of IFNy-positive and CD8-positive cells from all CD8-positive cells. The percentage of peptide-specific cells was obtained by subtracting the percentage in samples stimulated with the peptide used as the negative control from the percentage in samples stimulated specific peptide.

Transgenic mouse lines HLA-A2 were immunized twice subcutaneously at an interval of three weeks-boosting reconstructed virosome influenza containing cholesteryl-N-(trimethylaminoethyl)-carbamate-chloride (TC-Chol-IRIV, TIRIV)derived from influenza virus, cultured in cell culture (TIRIV EVH ducks) and TIRIV obtained from obtained in eggs viruses (TIRIV eggs), both variants are equipped with heterologous antigen shell hepatitis C (HCV core 132). The number of hemagglutinin in both cases was the same. Control mice were immunized TIRIV without heterologous antigen. Two weeks after the last immunization was determined by the number

CD8+T lymphocytes specific to the heterologous antigen, a method of dyeing intracellular interferon-gamma. Shown are average values ± standard deviation.

5.3 Comparison of hemagglutinin isolated from viruses obtained in eggs, and hemagglutinin isolated from virus produced in cell culture.

As shown in figure 1, polyclonal and monoclonal antibodies react with the material obtained from eggs obtained from cell lines of birds in different ways. While the polyclonal serum reacts with a hemagglutinin selected from the obtained in cell lines of bird virus, and hemagglutinin selected from the obtained in eggs of the virus, monoclonal antibodies react only with a hemagglutinin selected from the obtained in eggs of the virus.

Deglycosylation hemagglutinin

Different strains of influenza a (A/Singapore (H1N1), A/new Caledonian (H1N1), A/Panama (H3/N2)) amplified in embryonated eggs; hemagglutinin derived from their drugs is recognized MAT, indicating a lack of specificity MAT to strains. For deglycosylation used set Enzymatic protein deglycosylation Kit from Sigma-Aldrich (Buchs, Switzerland). All procedures were performed according to the manufacturer's instructions. Deglycosylation drugs viruses of the three strains in all cases led to a complete loss of signal, while a polyclonal serum specific for hemagglutinin, recognized bands of smaller size, representing deglycosylated protein (table 3).

The passage of influenza virus in a mammalian cells and analysis by the method of Western Blot.

Prepared on the and tablet with 6 holes and cultures of the cell line MDCK or line Vera: in each well were sown 5×10 5cells in the environment Episerf. The next day cells were infected with the virus obtained in embryonated eggs or in cell EVH, with the addition of trypsin or not, that ensured the production of infectious virions (resulting from cleavage of the hemagglutinin, in which the protein becomes active) or limit replication to one passage in the maintenance of hemagglutinin in an inactive conformation IN0in the absence of trypsin), respectively. Scrapings of the cells were taken after 1 day (MDCK) or 3 days (Vera), as soon as viral infection caused lysis of cells or a pronounced cytopathic effect, respectively. After that, the infected cells were studied using Western Blot using polyclonal rabbit serum specific for hemagglutinin, or using specific hemagglutinin MAT.

However, staining with monoclonal antibodies showed a positive result only in the case of virus isolated from infected eggs (control), but was negative in the case of samples hemagglutinin derived from viruses, past the passage in mammalian cells (MDCK cells or Vera: experiment without trypsin, which are formed only viruses that are unable to re-infect cells). Thus, one passage was sufficient is about to eliminate signals from the MAT.

One passage eliminates the possibility of replacing amino acids, and therefore modification of the epitope at the amino acid level as a cause of loss of binding capacity can be excluded. In the absence of trypsin does not occur cleavage of the polypeptide HA0on H1 and H2. The destruction of the epitope in the hemagglutinin cleavage by trypsin as the reason for the impossibility of binding antibodies also can be deleted.

Table 3
Comparison of the ha isolated from influenza viruses obtained in eggs or in cell lines
Influenza virus multiplied inPolyclonal ATMonoclonal-ing ATAfter deglycosylated of: Polyclonal ATMonocle national AT
Cells chickens+-n/an/a
Cells ducks+-n/an/a
Eggs+ ++bands of smaller size-
MDCK+-n/an/a
Veron/a (limited growth)n/an/an/a
1. Eggs+-n/an/a
2. Mammals

1. Virosome containing hemagglutinin isolated from influenza virus obtained in cell line birds.

2. Virosome according to claim 1, characterized in that the ability to merge the specified virosome at least 50% above average ability to merge virosome containing hemagglutinin isolated from influenza virus obtained in eggs of chickens.

3. Virosome according to any one of claims 1 or 2, characterized in that the immunogenicity of the specified virosome significantly higher immunogenicity of virosome containing hemagglutinin isolated from influenza virus obtained in eggs of chickens.

4. Virosome any and what claims 1 or 2, characterized in that the hemagglutinin derived from at least two different strains of influenza virus.

5. Virosome according to any one of claims 1 or 2, characterized in that virosome dried.

6. Virosome according to any one of claims 1 or 2, characterized in that virosome loaded with antigen.

7. Virosome according to any one of claims 1 or 2, characterized in that virosome empty.

8. The composition containing virosome according to any one of claims 1 or 2.

9. The composition according to item 8, which is a vaccine.

10. The composition according to claim 8, characterized in that it is immunogenic and further comprises a liposome and at least one molecule of the antigen.

11. The composition according to claim 10, in which the specified liposome embedded at least one molecule of the antigen.

12. The use of virosome according to any one of claims 1 or 2 as a means of delivery of antigen in the pharmaceutical composition to provide an immune response to an antigen.

13. The use of virosome according to claim 7 as a non-specific immunostimulating agent to obtain pharmaceutical compositions for providing immune responses to antigens of different origin.

14. The use of virosome according to any one of claims 1 or 2 to obtain pharmaceutical compositions for vaccination or immunization.

15. The use of virosome according to any one of claims 1 or 2 to obtain pharmaceutical compositions for l the treatment or prevention of diseases or disorders.

16. Set includes virosome according to any one of claims 1 or 2 or a composition according to any one of p-11.

17. Method of vaccination or immunization of a subject with virosome according to any one of claims 1 to 7 or a composition according to any one of p-11, including the introduction of the specified virosome or specified composition to a subject.

18. A method of treating or preventing diseases or disorders in need thereof of a subject using virosome according to any one of claims 1 or 2 or a composition according to any one of p-11, including the introduction of the specified virosome or composition to the subject.

19. The method according to p, in which the disease or disorder is an infectious disease or cancer.

20. The method of obtaining virosome according to any one of claims 1 or 2, comprising the following steps:
a) processing whole virus influenza a detergent or the short-chain phospholipid,
b) separating the fractions containing the hemagglutinin, with the possible addition of phospholipids,
c) removing the detergent, which leads to the formation of virosome.

21. Virosome obtained by the method according to claim 20.



 

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