# Patent

## Title
Cytotoxic T cell epitope peptide for SARS coronavirus, and use thereof

## Abstract

The present invention aims to provide a novel CTL epitope peptide of the SARS coronavirus. The present invention provides a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2009/070043 filed Nov. 27, 2009, claiming priority based on Japanese Patent Application No. 2008-304965, filed Nov. 28, 2008, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a cytotoxic T cell epitope peptide for the SARS coronavirus and use thereof.
BACKGROUND ART
Severe Acute Respiratory Syndrome (SARS) is an emerging infectious disease with a high lethality caused by the novel SARS coronavirus (SARS-CoV). Since the outbreak of the disease in 2003 in China, not less than 8000 people have been infected and about 800 people have died. However, no effective prophylactic or therapeutic method for the disease exists so far. After the outbreak of SARS, the SARS virus, which is the causative virus of SARS, was identified (Non-patent Document 1), and its base sequence has been determined (Non-patent Document 2).
The SARS coronavirus is a novel species of coronavirus belonging to Coronaviridae, a group of single-stranded (+) RNA viruses. The genome size of the SARS coronavirus is 29.7 kb, which is very large (Non-patent Document 2), and the genome encodes 23 putative proteins. As major structural proteins, there are Spike (1256 aa), Nucleocapsid (423 aa), Membrane (222 aa) and Small Envelope (77 aa). As nonstructural proteins, there are two polyproteins pp1a (4382 aa, SEQ ID NO:31; GenBank Accession No. AAP13439) and pp1b (2696 aa), and from these polyproteins, individual proteins are cleaved out by proteases in a site-specific manner.
CITATION LIST
Non Patent Literature


    Non-patent Document 1: Ksiazek, T. G. et al., N. Engl. J. Med. 348:1953-1966 (2003)
    Non-patent Document 2: Marra, M. A. et al., Science 300:1399-1404 (2003)
    Non-patent Document 3: Yang, Z. Y. et al., Nature 428:561-564 (2004)
    Non-patent Document 4: Wang. Y. D. et al., J. Virol. 78:5612-5618 (2004)
    Non-patent Document 5: Chen, H. et al., J. Immunol. 175:591-598 (2005)
    Non-patent Document 6: Zhou, M. et al., J. Immunol. 177:2138-2145 (2006)


SUMMARY OF INVENTION
Technical Problem
It has been reported so far that a virus-neutralizing antibody is induced by a DNA vaccine encoding the Spike protein (Non-patent Document 3). Although humoral immunity and cell-mediated immunity are necessary for effective defense reaction, cell-mediated immunity has been less studied compared to humoral immunity in the field of the SARS coronavirus. Cytotoxic T lymphocytes (CTLs) play an important role for protection against viruses in cell-mediated immunity, and therefore a new therapeutic method may be provided by controlling the CTL activity specific to the SARS coronavirus. From this viewpoint, identification of a strong CTL epitope peptide having a high antigenicity among the SARS coronavirus proteins has been demanded. Several types of partial peptides derived from the Spike protein, which is a structural protein, have been identified so far as CTL epitope peptides specific to the SARS coronavirus (Non-patent Documents 4 to 6). However, CTL epitope peptides derived from nonstructural proteins have not been reported yet.
The present invention aims to provide a novel CTL epitope peptide of the SARS coronavirus. More particularly, the present invention aims to provide a peptide comprising a novel CTL epitope derived from the SARS coronavirus pp1a protein, a peptide-bound liposome and an antigen presenting cell. Furthermore, the present invention aims to provide an inducing agent for HLA-A2-restricted CTLs, which comprises the peptide, the peptide-bound liposome or the antigen presenting cell as an active ingredient and is specific to the SARS coronavirus. The present invention also aims to provide a vaccine and the like for therapy or prophylaxis of infection by the SARS coronavirus.
Solution to Problem
The present inventors carried out the following studies in order to identify a CTL epitope derived from the SARS coronavirus, which has not been reported so far. First, by focusing on the pp1a protein, which is a nonstructural protein of the SARS coronavirus, 30 kinds of peptides were selected among peptides predicted as candidates of the epitope. Subsequently, the binding affinity to the HLA-A2 molecule, which is a MHC class I molecule, was confirmed for the peptides, and 9 kinds of peptides among the epitope candidate peptides were found to have significant CTL inducing activities. Furthermore, it was revealed that, by immunization with peptide-bound liposomes containing the peptides, the CTL induction is initiated and the CTL response is activated in vivo. Based on these findings, the present inventors succeeded in providing peptides comprising novel CTL epitopes derived from the SARS coronavirus pp1a protein, thereby completing the present invention.
That is, the present invention provides a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24. Furthermore, the peptide of the present invention preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 15, 17 and 24, and more preferably comprises the amino acid sequence shown in SEQ ID NO:24. The peptide of the present invention is characterized in that it comprises a cytotoxic T cell epitope specific to the SARS coronavirus, and that it comprises an HLA-A2-restricted cytotoxic T cell epitope.
The present invention provides a peptide-bound liposome, wherein a peptide is bound to the surface of a liposome, wherein the liposome comprises a phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond, and a stabilizer, and wherein the peptide is at least one peptide selected from the above-described peptide.
The above-described phospholipid preferably comprises a C14-C24 acyl group containing one unsaturated bond, and more preferably comprising an oleoyl group. Furthermore, the above phospholipid is preferably at least one selected from the group consisting of diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine and maleimide-diacylphosphatidylethanolamine.
If the phospholipid comprised in the liposome has such a constitution, CTLs for killing pathogen-infected cells can be efficiently enhanced, and prophylaxis and therapy of infectious diseases becomes possible.
The above-described stabilizer is preferably a cholesterol. By this constitution, the above liposome can be more stabilized.
The above-described peptide is preferably bound to the phospholipid comprised in the liposome. By this way, the peptide can be presented on the surface of the liposome, thereby CTLs specific to the SARS coronavirus can be induced more effectively.
Furthermore, the peptide-bound liposome of the present invention preferably comprises the following constituents:
(A) 1 to 99.8 mol % of a phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond; and
(B) 0.2 to 75 mol % of a stabilizer.
Furthermore, the peptide-bound liposome of the present invention preferably comprises the following constituents:
(I) 1 to 85 mol % of an acidic phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond;
(II) 0.01 to 80 mol % of a neutral phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond, at a concentration of;
(III) 0.2 to 80 mol % of a phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond, wherein the phospholipid is bound to at least one peptide selected from the above-described peptide; and
(IV) 0.2 to 75 mol % of a stabilizer.
The present invention provides an antigen presenting cell prepared by contacting a cell expressing a cell surface antigen HLA-A2 with at least one peptide selected from the above-described peptide in vitro. The cell is preferably autologous, and more preferably allogenic.
Furthermore, the present invention provides an inducing agent for HLA-A2-restricted CTLs specific to the SARS coronavirus comprising at least one peptide selected from the above-described peptide, the peptide-bound liposome or the antigen presenting cell as an active ingredient.
The present invention provides a vaccine for prophylaxis of infection by the SARS coronavirus comprising at least one peptide selected from the above-described peptide, the peptide-bound liposome or the antigen presenting cell as an active ingredient.
Furthermore, the present invention provides a method for providing immunity to a subject who needs to be given immunity against the SARS coronavirus comprising administering at least one peptide selected from the above-described peptide, the peptide-bound liposome or the antigen presenting cell to the subject.
The present invention provides a method for providing immunity to a subject who needs to be given immunity against the SARS coronavirus comprising: collecting cells from the subject; preparing antigen presenting cells by contacting the cells with at least one peptide selected from the above-described peptide in vitro; and reinjecting the antigen presenting cells to the subject. The cells are preferably lymphoid monocytes, and more preferably dendritic cells.
Advantageous Effects of Invention
The present invention provides a peptide comprising a novel CTL epitope derived from the SARS coronavirus pp1a protein, a peptide-bound liposome and an antigen presenting cell. Since the CTL induction is initiated and the CTL response is activated in vivo by the peptide, the peptide-bound liposome or the antigen presenting cell, any of the peptide, the peptide-bound liposome and the antigen presenting cell of the present invention can be used as a CTL inducing agent and/or a vaccine for elimination of the SARS coronavirus. Furthermore, since nonstructural proteins are synthesized earlier than structural proteins during the process of virus infection, a virus elimination effect at the initial stage of virus infection can be expected by activating the immune reaction using a CTL epitope derived from the pp1a protein, which is a nonstructural protein.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows staining plots for CD8 and IFN-γ by flow cytometry in mice immunized with a peptide-bound liposome. In FIG. 1, the legends “No pep” and “with pep” each indicate that the immunization was conducted using a liposome alone and a peptide-bound liposome, respectively. In FIG. 1, the legends “Lip-pp1a-xxxx” each indicate a peptide-bound liposome wherein the peptide is a SARS coronavirus pp1a epitope having a sequence shown in Table 1.
FIG. 2 shows staining plots for CD8 and IFN-γ by flow cytometry in mice immunized with a spike-1203-peptide-bound liposome. In FIG. 2, the legends “No pep” and “with pep” each indicate that the immunization was conducted using a liposome alone and a peptide-bound liposome, respectively. In FIG. 2, the legend “Lip-SARS Spike 1203” indicates a peptide-bound liposome wherein the peptide is a SARS Spike 1203 epitope of SEQ ID NO: 34 (FIAGLIAIV).
FIG. 3 shows staining plots for CFSE (carboxy fluorescein diacetate succinimidyl ester) by flow cytometry in mice which were booster-immunized with CFSE-labeled antigen presenting cells. In FIG. 3, the legends “No pep” and “with pep” each indicate that the immunization was conducted using a liposome alone and a peptide-bound liposome, respectively. In FIG. 3, the legends “Lip-pp1a-xxxx” each indicate a peptide-bound liposome wherein the peptide is a SARS coronavirus pp1a epitope having a sequence shown in Table 1.



DESCRIPTION OF EMBODIMENTS
The best mode for carrying out the invention will be described in detail below.
The peptide in the present invention comprises a CTL epitope derived from the SARS coronavirus pp1a protein, and particular examples thereof include peptides containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24, and peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24. Furthermore, an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 15, 17 and 24 is preferred, and the amino acid sequence shown in SEQ ID NO:24 is more preferred. These peptides comprise an HLA-A2-restricted CTL epitope, more particularly, an HLA-A*0201-restricted CTL epitope. The peptide of the present invention may be used in various forms (e.g., unmodified, fusion, glycosylated and nonglycosylated), and may contain a C-terminal modification (amidation, esterification, modification with aldehyde or the like), N-terminal modification (acetylation, biotinylation, fluorescent labeling or the like) and chemical modification of a functional group (phosphorylation, sulfation, biotinylation or the like).
The peptide may be synthesized according to a method used in conventional peptide chemistry. Examples of the known method of peptide synthesis include those described in literatures (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, MARUZEN Co., Ltd., 1975; Fundamentals and Experiments of Peptide Synthesis, MARUZEN Co., Ltd., 1985; Development of Pharmaceuticals, Continued Edition, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991) and the like.
The peptide of the present invention as described above may be used solely or in combination of plural types of peptides as an inducing agent for HLA-A2-restricted CTLs specific to the SARS coronavirus and/or as a vaccine for therapy or prophylaxis of infection of the SARS coronavirus. That is, since the peptide of the present invention can bind to a HLA-A2 molecule and is presented by cells that express the HLA-A2 molecule, thereby strongly induce CTLs, the peptide of the present invention can be used as a CTL inducing agent and/or a vaccine for elimination of the SARS coronavirus.
The liposome used for the peptide-bound liposome of the present invention is a phospholipid bilayer membrane having a closed space.
The liposome used for the peptide-bound liposome of the present invention comprises: a phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond; and a stabilizer. A phospholipid comprising a C14-C24 acyl group containing one unsaturated bond is preferably used as the phospholipid.
The carbon number of the acyl group in the phospholipid comprising a C14-C24 acyl group containing one unsaturated bond is preferably 16 to 22, more preferably 18 to 22, and most preferably 18. Particular examples of the acyl group include palmitoleoyl, oleoyl and erucoyl, and most preferably oleoyl. The carbon number of the hydrocarbon group in the phospholipid comprising a C14-C24 hydrocarbon group containing one unsaturated bond is preferably 16 to 22, more preferably 18 to 22, and most preferably 18. Particular examples of the hydrocarbon group include tetradecenyl, hexadecenyl, octadecenyl, C20 monoene, C22 monoene and C24 monoene and the like. The unsaturated acyl groups or unsaturated hydrocarbon groups bound to the 1-position and the 2-position of the glycerin residue comprised in the phospholipid may be either the same or different. In view of industrial productivity, the groups at the 1-position and the 2-position are preferably the same.
In view of enhancement of the CTL activity to a practically sufficient level, the phospholipid preferably comprises a C14-C24 acyl group containing one unsaturated bond. In cases where the carbon number of the acyl group is less than 13, the liposome may be unstable, or the CTL activity enhancement effect may be insufficient. In cases where the carbon number of the acyl group is more than 24, the liposome may be unstable.
Examples of the phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or comprising a C14-C24 hydrocarbon group containing one unsaturated bond include acidic phospholipids, neutral phospholipids, and reactive phospholipids comprising a functional group to which a peptide can be bound. Their types and ratios may be selected depending on various demands.
Examples of the acidic phospholipids which may be used include phosphatidylserine, phosphatidylglycerol, phosphatidic acid and phosphatidylinositol. In view of enhancement of the CTL activity to a practically sufficient level, industrial supply capacity, quality for use as a pharmaceutical, and the like, diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid and diacylphosphatidylinositol comprising a C14-C24 acyl group containing one unsaturated bond are preferably used. Since an acidic phospholipid gives an anionic group on the surface of a liposome, a negative zeta potential is given on the surface of the liposome. Therefore, the liposome gains charge repulsion and can exist as a stable formulation in an aqueous solvent. Thus, an acidic phospholipid is important in view of ensuring the stability of the liposome in an aqueous solvent.
Examples of the neutral phospholipids include phosphatidylcholine. The neutral phospholipids which may be employed in the present invention may be used by selecting their types and amounts appropriately within the ranges in which enhancement of the CTL activity can be achieved. Compared to an acidic phospholipid and a phospholipid to which a peptide is bound, a neutral phospholipid has a higher function to stabilize a liposome and hence can enhance the stability of the membrane. From this viewpoint, the liposome to be used for the peptide-bound liposome of the present invention preferably contains a neutral phospholipid. The amount of the neutral phospholipid to be used can be determined after securing the contents of the acidic phospholipid for achievement of the CTL activity enhancement effect, the reactive phospholipid for the peptide bond and the stabilizer.
The peptide of the present invention is bound to the surface of the liposome by being bound to a phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond, wherein the phospholipid is comprised in the liposome. As the phospholipid for such binding of the peptide, a reactive phospholipid comprising a functional group to which the peptide can be bound is used. The type and the amount of the reactive phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or comprising a C14-C24 hydrocarbon group containing one unsaturated bond are appropriately selected depending on various demands. Similarly to the case of the above-described phospholipid, it is also not preferred that the carbon number of the unsaturated acyl group or the unsaturated hydrocarbon group comprised in the phospholipid is more than 24 or less than 14 in the case of the reactive phospholipid.
Examples of the reactive phospholipid include phosphatidylethanolamine and terminally modified derivatives thereof. Furthermore, phosphatidylglycerol, phosphatidylserine, phosphatidic acid and phosphatidylinositol, and terminally modified derivatives thereof may also be used as the reactive phospholipid. In view of industrial availability, simplicity of the process of binding with a peptide, yield and the like, phosphatidylethanolamine or a terminally modified derivatives thereof is preferably used. Phosphatidylethanolamine has an amino group to which an antibody can be bound at its terminus. Furthermore, in view of enhancement of the CTL activity to a practically sufficient level, stability in a liposome, industrial supply capacity, quality for use as a pharmaceutical, and the like, diacylphosphatidylethanolamine comprising a C14-C24 acyl group containing one unsaturated bond or a terminally modified derivatives thereof is most preferably used.
Diacylphosphatidylethanolamine can be obtained by, for example, using diacylphosphatidylcholine as a crude material and carrying out a base-exchange reaction of choline and ethanolamine using phospholipase D. More particularly, a solution of diacylphosphatidylcholine in chloroform is mixed at an appropriate ratio with water in which phospholipase D and ethanolamine are dissolved, to obtain a crude reaction product. The crude reaction product is purified with a silica gel column using a chloroform/methanol/water solvent, thereby obtaining the diacylphosphatidylethanolamine of interest. Those skilled in the art can carry out the purification by appropriately selecting the conditions for purification by the column, such as the composition ratio of the solvent.
Examples of the terminally modified derivatives include a terminally modified diacylphosphatidylethanolamine produced by binding one of the termini of a divalent reactive compound to the amino group of diacylphosphatidylethanolamine. Examples of the divalent reactive compound which may be used include compounds comprising, at least one terminus, an aldehyde group or a succinimide group which can react with the amino group of diacylphosphatidylethanolamine. Examples of the divalent reactive compound comprising an aldehyde group include glyoxal, glutaraldehyde, succindialdehyde and terephthalaldehyde. Preferred examples thereof include glutaraldehyde. Examples of the divalent reactive compound comprising a succinimide group include dithiobis(succinimidylpropionate), ethylene glycol-bis(succinimidylsuccinate), disuccinimidyl succinate, disuccinimidyl suberate and disuccinimidyl glutarate.
Furthermore, examples of the divalent reactive compound comprising a succinimide group at one terminus and a maleimide group at the other terminus include N-succinimidyl4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate, N-succinimidyl-4-(p-maleimidophenyl)acetate, N-succinimidyl-4-(p-maleimidophenyl)propionate, succinimidyl-4-(N-maleimidoethyl)-cyclohexane-1-carboxylate, sulfosuccinimidyl-4-(N-maleimidoethyl)-cyclohexane-1-carboxylate, N-(γ-maleimidobutyryloxy)succinimide and N-(ε-maleimidocaproyloxy)succinimide. By using such a divalent reactive compound, a terminally modified diacylphosphatidylethanolamine comprising a maleimide group as a functional group is obtained. By binding a functional group at one terminus of such a divalent reactive compound to the amino group of diacylphosphatidylethanolamine, a terminally modified diacylphosphatidylethanolamine can be obtained.
Examples of the method for binding the peptide to the surface of the liposome include a method wherein a liposome comprising the above reactive phospholipid is prepared and the peptide is then added thereto to bind the peptide to the reactive phospholipid in the liposome. Furthermore, by preliminarily binding the peptide to the reactive phospholipid and mixing the obtained peptide-bound reactive phospholipid with a phospholipid other than a reactive phospholipid and a stabilizer, a liposome in which the peptide is bound to its surface can also be obtained. The method for binding a peptide to a reactive phospholipid is well-known in the art.
The liposome to be used for the peptide-bound liposome of the present invention comprises at least 1 type, for example, 2 or more types, preferably 3 or more types of phospholipid(s) comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond. For example, the liposome to be used in the peptide-bound liposome of the present invention comprises at least 1 type, for example, 2 or more types, preferably 3 or more types of phospholipid(s) comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond selected from diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine and maleimide-diacylphosphatidylethanolamine.
Furthermore, the liposome to be used for the peptide-bound liposome of the present invention preferably comprises at least one type of each of:
acidic phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond;
neutral phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond; and
reactive phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond.
In the present invention, sterols and tocopherols may be used as the stabilizer of the liposome. The sterols may be those generally known as sterols, and examples thereof include cholesterol, sitosterol, campesterol, stigmasterol and brassicasterol. In view of availability and the like, cholesterol is especially preferably used. The tocopherols may be those generally known as tocopherols, and preferred examples thereof include commercially available α-tocopherol in view of availability and the like.
Furthermore, as long as the effect of the present invention is not adversely affected, the liposome to be used for the peptide-bound liposome of the present invention may contain known liposome constituting components that can constitute a liposome.
Examples of the composition of the liposome to be used for the peptide-bound liposome of the present invention include the following:
(A) 1 to 99.8 mol % of a phospholipid comprising: a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond; and
(B) 0.2 to 75 mol % of a stabilizer.
The content of each component is represented as mol % with respect to the total constituting components of the peptide-bound liposome.
In view of the stability of the liposome, the content of the component (A) is preferably 10 to 90 mol %, more preferably 30 to 80 mol %, still more preferably 50 to 70 mol %.
In view of the stability of the liposome, the content of the component (B) is preferably 5 to 70 mol %, more preferably 10 to 60 mol %, still more preferably 20 to 50 mol %. In cases where the content of the stabilizer is more than 75 mol %, the stability of the liposome is deteriorated, which is not preferred.
The component (A) comprises the followings:
(a) a phospholipid, to which a peptide is not bound, comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond; and
(b) a phospholipid, to which a peptide is bound, comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond.
The content of the component (a) is usually 0.01 to 85 mol %, preferably 0.1 to 80 mol %, more preferably 0.1 to 60 mol %, still more preferably 0.1 to 50 mol %.
The content of the component (b) is usually 0.2 to 80 mol %, preferably 0.3 to 60 mol %, more preferably 0.4 to 50 mol %, still more preferably 0.5 to 25 mol %. In cases where the content is less than 0.2 mol %, the amount of the peptide becomes low, and therefore it is difficult to activate CTLs to a practically sufficient level. In cases where the content is more than 80 mol %, the stability of the liposome becomes low.
The phospholipid of the component (a) usually includes the above-mentioned acidic phospholipid and neutral phospholipid. The phospholipid of the component (b) includes the above-mentioned reactive phospholipid.
The content of the acidic phospholipid is usually 1 to 85 mol %, preferably 2 to 80 mol %, more preferably 4 to 60 mol %, still more preferably 5 to 40 mol %. In cases where the content is less than 1 mol %, the zeta potential becomes small and the stability of the liposome becomes low, and it is difficult to activate CTLs to a practically sufficient level. On the other hand, in cases where the content is more than 85%, the content of the peptide-bound phospholipid in the liposome becomes low as a result, and therefore it is difficult to activate CTLs to a practically sufficient level.
The content of the neutral phospholipid is usually 0.01 to 80 mol %, preferably 0.1 to 70 mol %, more preferably 0.1 to 60 mol %, still more preferably 0.1 to 50 mol %. In cases where the content is more than 80.0 mol %, the contents of the acidic phospholipid, the peptide-bound phospholipid and the liposome stabilizer comprised in the liposome become low, and it is difficult to activate CTLs to a practically sufficient level.
The peptide-bound phospholipid is obtained by binding a peptide to the reactive phospholipid, and the ratio of binding of the reactive phospholipid to the peptide may be selected within the range in which the effect of the present invention is not adversely affected, by taking the type of the functional group used in the binding, conditions of the binding treatment, and the like into consideration appropriately. For example, in cases where the terminally modified diacylphosphatidylethanolamine obtained by binding one terminus of a divalent reactive compound disuccinimidyl succinate to the terminal amino group of diacylphosphatidylethanolamine is used as the reactive phospholipid, 10 to 99% of the reactive phospholipid can be bound to the peptide depending on selection of various conditions of the binding treatment. In such cases, the reactive phospholipid which is not bound to the peptide is comprised in the liposome as an acidic phospholipid.
Examples of a preferred mode of the peptide-bound liposome of the present invention include the following composition:
(I) 1 to 85 mol % of an acidic phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond;
(II) 0.01 to 80 mol % of a neutral phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond;
(III) 0.2 to 80 mol % of a phospholipid, to which at least one peptide selected from the above-described peptide is bound, comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond; and
(IV) 0.2 to 75 mol % of a stabilizer.
(100 mol % in total)
Examples of a more preferred mode of the liposome to be used for the peptide-bound liposome of the present invention include the following composition:
the component (I) at a concentration of 2 to 80 mol %;
the component (II) at a concentration of 0.1 to 70 mol %;
the component (III) at a concentration of 0.3 to 60 mol %; and
the component (IV) at a concentration of 10 to 70 mol %.
(100 mol % in total)
Examples of a still more preferred mode of the liposome to be used for the peptide-bound liposome of the present invention include the following composition:
the component (I) at a concentration of 4 to 60 mol %;
the component (II) at a concentration of 0.1 to 60 mol %;
the component (III) at a concentration of 0.4 to 50 mol %; and
the component (IV) at a concentration of 20 to 60 mol %.
(100 mol % in total)
Examples of an especially preferred mode of the liposome to be used for the peptide-bound liposome of the present invention include the following composition:
the component (I) at a concentration of 5 to 40 mol %;
the component (II) at a concentration of 0.1 to 50 mol %;
the component (III) at a concentration of 0.5 to 25 mol %; and
the component (IV) at a concentration of 25 to 55 mol %.
(100 mol % in total)
The carbon number of the unsaturated acyl group or the unsaturated hydrocarbon group to be comprised in the phospholipid in the liposome used in the peptide-bound liposome of the present invention is characteristically 14 to 24, but a phospholipid comprising an unsaturated acyl group or unsaturated hydrocarbon group comprising a carbon number of less than 14 or more than 24 may also be comprised to an extent at which the effect of the present invention is not adversely affected. The ratio of the number of the C14-C24 unsaturated acyl group or unsaturated hydrocarbon group with respect to the total number of all the unsaturated acyl groups or unsaturated hydrocarbon groups comprised in the phospholipid in the liposome used in the peptide-bound liposome of the present invention is, for example, 50% or more, preferably 60% or more, more preferably 75% or more, still more preferably 90% or more, most preferably 97% or more (for example, substantially 100%).
The liposome to be used for the peptide-bound liposome of the present invention may also comprise a lipid other than a phospholipid, comprising a C14-C24 acyl group or hydrocarbon group, as long as the effect of the present invention is not adversely affected. The content of the lipid is usually 40 mol % or less, preferably 20 mol % or less, more preferably 10 mol % or less, still more preferably 5 mol % or less (for example, substantially 0 mol %).
The liposome to be used in the present invention can be obtained by, for example, a method wherein a phospholipid as a constituting component, reactive phospholipid, stabilizer, peptide and the like are used and are blended and processed in an appropriate way, followed by adding the resulting product to an appropriate solvent. Examples of the production process include the extrusion method, vortex mixer method, ultrasonic method, surfactant removal method, reverse-phase evaporation method, ethanol injection method, prevesicle method, French press method, W/O/W emulsion method, annealing method and freeze-thaw method. The particle diameter of the liposome is not restricted, however, in view of the stability during storage, the particle diameter is, for example, 20 to 600 nm, preferably 30 to 500 nm, more preferably 40 to 400 nm, still more preferably 50 to 300 nm, most preferably 70 to 230 nm.
In the present invention, in order to enhance the physicochemical stability of the liposome, sugars or polyols may be added to the internal aqueous phase and/or the external aqueous phase during or after preparation of the liposome. In particular, in cases where the liposome needs to be stored for a long time or during formulation, a sugar or a polyol may be added/dissolved as a protective agent for the liposome, followed by removing water by freeze-drying to prepare a lyophilized product of the phospholipid composition.
Examples of the sugars include monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose and xylose; disaccharides such as saccharose, lactose, cellobiose, trehalose and maltose; trisaccharides such as raffinose and melezitose; oligosaccharides such as cyclodextrin; polysaccharides such as dextrin; and sugar alcohols such as xylitol, sorbitol, mannitol and maltitol. Among these sugars, monosaccharides and disaccharides are preferred, and glucose and saccharose are especially preferred in view of availability and the like.
Examples of the polyols include glycerin compounds such as glycerin, diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin, octaglycerin, nonaglycerin, decaglycerin and polyglycerin; sugar alcohol compounds such as sorbitol and mannitol; ethylene glycol; diethylene glycol; triethylene glycol; tetraethylene glycol; pentaethylene glycol; hexaethylene glycol; heptaethylene glycol; octaethylene glycol and nonaethylene glycol. Among these, glycerin, diglycerin, triglycerin, sorbitol, mannitol, and polyethylene glycols having molecular weights of 400 to 10,000 are preferred in view of availability. The concentration of the sugars or polyols to be contained in the internal aqueous phase and/or the external aqueous phase is, for example, 1 to 20% by weight, preferably 2 to 10% by weight.
When the peptide-bound liposome of the present invention is produced, the peptide-bound liposome can be simply obtained by preparing a liposome to which the peptide has not been bound yet and then binding the peptide thereto. For example, a liposome which contains a phospholipid, stabilizer and a reactive phospholipid for binding the peptide on the surface of the membrane is prepared as, for example, a liposome liquid, and sucrose, which is one of the sugars, is added to its external aqueous phase to about 2 to 10% by weight and dissolved. This sugar-added formulation is transferred to a 10 ml glass vial and the vial is placed in a shelf freeze dryer, followed by cooling to, for example, −40° C. to freeze the sample, thereby obtaining a lyophilized product by a conventional method. The obtained lyophilized product can be stored for a long time since water has been removed, and, when necessary, by adding a particular peptide and carrying out the following steps, the peptide-bound liposome of the present invention can be simply and rapidly obtained. In cases where the interaction between the peptide and the liposome is strong and strong instability is caused, it is a very simple way to store the liposome at the stage of a lyophilized product like this and to use after binding the peptide when necessary.
The liposome to be used for the peptide-bound liposome of the present invention may comprise a phospholipid to which a peptide is bound. Examples of the method to obtain the liposome containing a phospholipid to which a peptide is bound include the methods by the following (A) and (B).

    (A) A liposome containing a phospholipid, a reactive phospholipid and a stabilizer is prepared, and a peptide and a divalent reactive compound are added thereto, followed by linking a functional group of the reactive phospholipid to a functional group of the peptide via the divalent reactive compound. As the divalent reactive compound, the one used for preparation of the terminally modified derivatives of the reactive phospholipid may be similarly used. Particular examples of the divalent reactive compound comprising an aldehyde group include glyoxal, glutaraldehyde, succindialdehyde and terephthalaldehyde. Preferred examples thereof include glutaraldehyde. Examples of the divalent reactive compound comprising a succinimide group include dithiobis(succinimidylpropionate), ethylene glycol-bis(succinimidylsuccinate), disuccinimidyl succinate, disuccinimidyl suberate and disuccinimidyl glutarate. Furthermore, examples of the divalent reactive compound comprising a succinimide group at one terminus and a maleimide group at the other terminus include N-succinimidyl-4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate, N-succinimidyl-4-(p-maleimidophenyl)acetate, N-succinimidyl-4-(p-maleimidophenyl)propionate, succinimidyl-4-(N-maleimidoethyl)-cyclohexane-1-carboxylate, sulfosuccinimidyl-4-(N-maleimidoethyl)-cyclohexane-1-carboxylate, N-(γ-maleimidobutyryloxy)succinimide and N-(ε-maleimidocaproyloxy)succinimide. By using such a divalent reactive compound, a terminally modified derivatives of a reactive phospholipid comprising a maleimide group as a functional group (e.g., phosphatidylethanolamine) is obtained.
    (B) A method wherein a liposome containing a phospholipid, a reactive phospholipid and a stabilizer is prepared, and a peptide is added thereto, followed by linking a functional group of the reactive phospholipid contained in the liposome to a functional group of the peptide, and thereby binding the peptide to the liposome.


Examples of the type of the bond in the above (A) and (B) include an ionic bond, hydrophobic bond and covalent bond, and the type of the bond is preferably a covalent bond. Furthermore, particular examples of the covalent bond include a Schiff base bond, amide bond, thioether bond and ester bond. Both of the above two methods allow binding of the peptide to the reactive phospholipid contained in the liposome, thereby forming a phospholipid to which the peptide is bound in the liposome.
In the method (A), particular examples of the method to bind the liposome as a crude material to the peptide via a divalent reactive compound include a method using a Schiff base bond. Examples of the method to bind the liposome to the peptide via a Schiff base bond include a method wherein a liposome comprising an amino group on its surface is prepared and the peptide is added to a suspension of the liposome, followed by adding dialdehyde as the divalent reactive compound to the resulting mixture and binding the amino group on the surface of the liposome to the amino group in the peptide via a Schiff base.
Particular examples of this binding procedure include the following method.

    (A-1) In order to obtain a liposome comprising an amino group on its surface, a reactive phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond (e.g., phosphatidylethanolamine) is mixed with lipids as crude materials for the liposome (e.g., a phospholipid and a stabilizer for a liposome), to prepare a liposome wherein amino groups exist on the surface of the liposome in a certain amount.
    (A-2) A peptide is added to the liposome suspension.
    (A-3) Subsequently, glutaraldehyde is added as the divalent reactive compound, and the reaction is allowed to proceed for a certain length of time, thereby allowing a Schiff base bond to be formed between the liposome and the peptide.
    (A-4) Thereafter, in order to inactivate the reactivity of the excess glutaraldehyde, glycine as an amino group-containing water-soluble compound is added to the liposome suspension and allowed to react therewith.
    (A-5) By a method such as gel filtration, dialysis, ultrafiltration or centrifugation, the peptide unbound to the liposome, the reaction product between glutaraldehyde and glycine, and excess glycine are removed, to obtain a peptide-bound liposome suspension.


Particular examples of the method (B) include a method wherein a reactive phospholipid comprising a functional group that can form an amide bond, thioether bond, Schiff base bond, ester bond or the like is introduced to the phospholipid membrane. Particular examples of such a functional base include succinimide, maleimide, amino, imino, carboxyl, hydroxyl and thiol. Examples of the reactive phospholipid to be introduced to the liposome include the reactive phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond (e.g., phosphatidylethanolamine) whose amino terminus is modified.
A particular example of the binding procedure will now be described referring to a case in which diacylphosphatidylethanolamine is used.

    (B-1) Diacylphosphatidylethanolamine comprising a C14-C24 acyl group containing one unsaturated bond is reacted with disuccinimidyl succinate by a known method at only one terminus, to obtain disuccinimidyl succinate-bound diacylphosphatidylethanolamine comprising a succinimide group as a functional group at the terminus.
    (B-2) The disuccinimidyl succinate-bound diacylphosphatidylethanolamine is mixed with other liposome constituting components (e.g., a phospholipid and a stabilizer) by a known method, to prepare a liposome composition comprising a succinimide group on its surface as a functional group.
    (B-3) To the liposome composition suspension, a peptide is added, and the amino group in the peptide is reacted with the succinimide group on the surface of the phospholipid membrane.
    (B-4) The unreacted peptides, reaction byproducts and the like are removed by a method such as gel filtration, dialysis, ultrafiltration or centrifugation, to obtain a liposome suspension containing a peptide-bound phospholipid.


In cases where a liposome is bound to a peptide, it is practically preferred to use an amino group or a thiol group, which is often comprised as a functional group. In cases where an amino group is used, a Schiff base bond can be formed by reacting it with a succinimide group. In cases where a thiol group is used, a thioether bond can be formed by reacting it with a maleimide group.
The antigen presenting cell in the present invention is a cell prepared by bringing a cell that expresses the cell surface antigen HLA-A2 into contact with one or more types of peptides in vitro. The cell is preferably autologous and/or allogenic. Examples of the cell include cells that express the cell surface antigen HLA-A2 (e.g., HLA-A*0201). The cell is preferably a lymphoid monocyte (T cell, macrophage, B cell, dendric cell or the like), more preferably a dendritic cell.
The antigen presenting cell of the present invention can be used as an inducer of HLA-A2-restricted CTLs specific to the SARS coronavirus, and/or as a vaccine for therapy or prophylaxis of infection of the SARS coronavirus, by administering (injecting) the cell to a subject. That is, the antigen presenting cell of the present invention presents the peptide of the present invention on its surface and can strongly induce CTLs, so that the cell may be used as a CTL inducer and/or as a vaccine for the purpose of elimination of the SARS coronavirus. The antigen presenting cell is preferably prepared using a peptide at a concentration of preferably 1 to 100 μM, more preferably 5 to 50 μM, for example, 10 μM, per 107 cells.
The peptide, the peptide-bound liposome and the antigen presenting cell of the present invention can be used as inducers of HLA-A2-restricted CTLs specific to the SARS coronavirus, and/or as vaccines for therapy or prophylaxis of infection of the SARS coronavirus. The subject may be any animal including human. In a certain mode, the subject is human. The inducer and/or vaccine of the present invention is/are made into a common form as a pharmaceutical composition depending on the substance regarded as the active ingredient, and direct delivery of the composition is generally achieved by parenteral injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or injection to the space between tissues). Examples of other administration methods include mucosal administration (e.g., oral, transnasal or pulmonary), transocular administration, percutaneous administration and administration by suppositories.
That is, in cases where the composition is administered parenterally, it may be administered in a dosage form such as an injection solution, transnasal agent, formulation for topical administration (e.g., percutaneous preparation), or formulation for rectal administration. In cases where the composition is orally administered, it may be administered in a dosage form usually used in the art. Examples of the injection solution include sterile solutions or suspensions, and emulsions, and particular examples thereof include water, water-propylene glycol solutions, buffers and 0.4% physiological saline. Furthermore, in cases where the composition is made into a liquid formulation, it can be stored frozen, or stored after removing water by freeze-drying or the like. When the freeze-dried formulation is to be used, it can be used by adding distilled water for injection or the like thereto and redissolving it. Examples of the formulation for topical administration include creams, ointments, lotions and percutaneous preparations. Examples of the oral preparation or the formulation for rectal administration include capsules, tablets, pills, powders, drops, suppositories and liquids.
The above dosage forms are formulated by methods usually used in the art, together with pharmaceutically acceptable vehicles and additives. Examples of the pharmaceutically acceptable vehicles and additives include carriers, binders, flavoring agents, buffering agents, thickening agents, coloring agents, stabilizers, emulsifiers, dispersants, suspending agents, antiseptics, pH adjusting agents, tonicity adjusting agents and wetting agents. Furthermore, examples of the pharmaceutically acceptable carriers include magnesium carbonate, lactose, pectin, starch and methyl cellulose.
The inducer of the present invention containing as an active ingredient a peptide, a peptide-bound liposome or an antigen presenting cell, and the vaccine of the present invention containing as an active ingredient a peptide, a peptide-bound liposome or an antigen presenting cell may further contain an adjuvant for enhancement of its effect. Examples of the adjuvant include aluminum hydroxide gel, Freund's complete adjuvant, Freund's incomplete adjuvant, pertussis adjuvant, poly(I,C) and CpG-DNA. Among these, CpG-DNA is preferred. CpG-DNA is a DNA containing an unmethylated CpG motif, and it can activate dendritic cells and enhance the CTL induction by the peptide, peptide-bound liposome or antigen presenting cell of the present invention.
The dose of the peptide of the present invention in the formulation, and the number of doses of the formulation vary depending on the symptoms, age, body weight, dosage form and the like, and it is preferred to administer usually 0.01 μg to 1 mg, preferably 0.1 μg to 500 μg, more preferably 1.0 μg to 100 μg of the peptide once in every several days or several months. For example, for the primary immune response (that is, therapeutic or prophylactic administration), 1.0 μg to 500 μg of the peptide is administered to an adult patient, and depending on the response and the conditions of the patient assayed by measurement of the specific CTL activity in blood of the patient, boosting administration of 1.0 μg to 100 μg of the peptide is subsequently carried out according to boosting therapy that continues for several weeks to several months. The number of the antigen presenting cells of the present invention to be administered is preferably 109 to 106, more preferably 108 to 107, and the number of the cells may be appropriately controlled based on the symptoms, age, body weight, dosage form and the like.
EXAMPLES
The present invention will now be described more concretely, but the present invention is not restricted to the Examples below.
Example 1
Prediction of CTL Epitopes
Using two kinds of computer software for prediction of epitopes, BIMAS <http://www-bimas.cit.nih.gov/molbio/hla_bind>) and SYFPEITHI (<http://www.syfpeithi.de>), epitope candidates for pp1a were searched. The search was carried out with the following settings: HLA Molecule, HLA-A*0201; and Peptide Length, 9 to 10 amino acid residues. Thirty kinds of epitope candidate peptides showing high prediction scores in the both analytic methods were selected by the search. The amino acid sequences of these 30 kinds of epitope candidate peptides are shown in Table 1. For each of the 30 kinds of epitope candidate peptides, a synthetic peptide was prepared. Peptides that can actually function as epitopes were searched, and 9 kinds of epitopes were identified. In order to further analyze functions of the 9 kinds of peptides, the Examples 1 to 6 below were carried out.











TABLE 1







SEQ ID




Epitope
Sequence
No:
BIMAS
SYFPEITHI



















 1) pp1a-15
QLSLPVLQV
1
160.0
26


 


 2) pp1a-103
TLGVLVPHV
2
160.0
26


 


 3) pp1a-445
TLNEDLLEI
3
98.4
28


 


 4) pp1a-634
KLSAGVEFL
4
463.5
27


 


 5) pp1a-651
FLITGVFDI
5
640.2
27


 


 6) pp1a-1121
ILLAPLLSA
6
71.9
26


 


 7) pp1a-1139
SLQVCVQTV
7
160.0
28


 


 8) pp1a-1288
MLSRALKKV
8
272.0
25


 


 9) pp1a-1652
YLSSVLLAL
9
226.0
28


 


10) pp1a-2187
CLDAGINYV
10
351.9
27


 


11) pp1a-2207
AMWLLLLSI
11
143.8
27


 


12) pp1a-2340
WLMWFIISI
12
1551.9
26


 


13) pp1a-2546
ILLLDQVLV
13
437.5
26


 


14) pp1a-2754
TLLCVLAAL
14
181.8
29


 


15) pp1a-2755
LLCVLAALV
15
118.2
25


 


16) pp1a-2758
VLAALVCYI
16
224.4
26


 


17) pp1a-2990
ALSGVFCGV
17
132.1
25


 


18) pp1a-3444
VLAWLYAAV
18
177.4
27


 


19) pp1a-3459
FLNRFTTTL
19
373.4
27


 


20) pp1a-3560
MLLTFLTSL
20
1174.4
29


 


21) pp1a-3564
FLTSLLILV
21
735.9
25


 


22) pp1a-3616
FLLPSLATV
22
2722.7
33


 


23) pp1a-3687
TLMNVITLV
23
591.9
25


 


24) pp1a-3709
SMWALVISV
24
958.9
28


 


25) pp1a-3730
FLARAIVFV
25
4047.2
29


 


26) pp1a-3745
LLFITGNTL
26
134.4
26


 


27) pp1a-3816
KLNIKLLGI
27
84.0
27


 


28) pp1a-3848
VLLSVLQQL
28
309.1
27


 


29) pp1a-4071
ALWEIQQVV
29
970.0
25


 


30) pp1a-4219
VLGSLAATV
30
118.2
26









Example 2
Measurement of Binding Affinity of Peptide to HLA-A*0201 Molecule
For the 30 kinds of epitope candidate peptides listed in Table 1, the binding affinity to HLA-A*0201, which is a major histocompatibility complex (MHC) class I molecule, was measured. The measurement was carried out using T2 cells, which are human lymphoid cells. T2 cells lack the TAP gene and hence cannot transport self-peptides derived from autoantigens. Therefore, these cells express HLA-A*0201, to which peptides are not bound, on the cell surfaces. When a peptide added to the outside of the cell is bound to HLA-A*0201, an HLA-A*0201 complex is formed and becomes stable. Using this principle, the binding affinity was calculated based on the relationship between the amount of the HLA-A*0201 complex formed and the concentration of the peptide added. As the antibody for detection of the HLA-A*0201 complex, an anti-HLA-A2 monoclonal antibody BB7.2 (ATCC) was used. Furthermore, an epitope of hepatitis C virus (NS3-1585) was used as a control.
More particularly, T2 cells were incubated at 37° C. overnight together with various concentrations of peptides. Thereafter, the cells were allowed to react with the anti-HLA-A2 antibody BB7.2, and then with a FITC-labeled secondary antibody, followed by analyzing the cells by flow cytometry. Using the mean fluorescence intensity (MFI) of the T2 cells pulsed with NS3-1585 as a standard (100%), the peptide concentration at which a mean fluorescence intensity of 50% was achieved was represented as BL50 (half-maximal binding level) for each peptide and shown in Table 2. BL50 values of less than 100 μM, 100 to 200 μM, and more than 200 μM were grouped into 3 categories “High”, “Medium” and “Low”, respectively, and 24 kinds of peptides showed high binding affinities.












TABLE 2








SEQ ID
BL50




Epitope
No:
(μM)
Affinity





















 1) pp1a-15
1
75.7
High



 2) pp1a-103
2
3.1
High



 3) pp1a-445
3
19.2
High



 4) pp1a-634
4
59.8
High



 5) pp1a-651
5
8.4
High



 6) pp1a-1121
6
40.3
High



 7) pp1a-1139
7
4.9
High



 8) pp1a-1288
8
65.1
High



 9) pp1a-1652
9
6.7
High



10) pp1a-2187
10
3.0
High



11) pp1a-2207
11
323.0
Low



12) pp1a-2340
12
2432.8
Low



13) pp1a-2546
13
7.6
High



14) pp1a-2754
14
96.7
High



15) pp1a-2755
15
187.2
Medium



16) pp1a-2758
16
97.3
High



17) pp1a-2990
17
6.2
High



18) pp1a-3444
18
53.0
High



19) pp1a-3459
19
39.6
High



20) pp1a-3560
20
47.1
High



21) pp1a-3564
21
100.4
Medium



22) pp1a-3616
22
31.8
High



23) pp1a-3687
23
22.8
High



24) pp1a-3709
24
6.4
High



25) pp1a-3730
25
25.7
High



26) pp1a-3745
26
153.7
Medium



27) pp1a-3816
27
68.1
High



28) pp1a-3848
28
102.5
Medium



29) pp1a-4071
29
8.3
High



30) pp1a-4219
30
53.3
High










Example 3
Induction of Peptide-Specific CTLs in Mice Immunized Using Antigen Presenting Cells Pulsed with Peptides
In order to investigate whether or not CTLs are induced specifically to the 30 kinds of epitope candidate peptides listed in Table 1, mice were immunized with mouse spleen cells pulsed with the peptides in vitro, and the spleen cells were stimulated with the peptides followed by measuring the CTL induction activities. As the mouse, the HLA-A2 transgenic mouse (HDD II mouse, Institut Pasteur, France; provided by Dr. F. Lemonnier) prepared by knocking out mouse MHC class I and β2-microglobulin (β2-m) in a mouse and introducing HLA-A*0201, which is a type of human MHC class I, and the human β2-m gene was used. Using as an index the ratio of cells in which production of interferon-γ (IFN-γ) was promoted among CD8-positive cells, the CTL induction activity was measured.
Spleen cells prepared from a naive HLA-A2 transgenic mouse were incubated in vitro with each peptide at a concentration of 10 μM at 37° C. for hour. Another individual of a naive HLA-A2 transgenic mouse was immunized with the spleen cells pulsed with the peptide, by intravenous injection.
One week after the immunization, spleen cells were prepared from the immunized mouse, and the cells were suspended in a medium supplemented with 10% fetal calf serum (FCS), followed by plating the cells in a 96-well plate at the cell number of 2×106 cells/well. To each well, each peptide (10 μM, final concentration) and 5 μL of 25-fold diluted GOLGIPLUSTM solution (Japan BD) were added, and the resulting mixture was incubated at 37° C. for 5 hours. Here, GOLGIPLUSTM was used to inhibit secretion of produced IFN-γ by stopping intracellular transport. After washing the cells, 1 μg/106 cells of FcBlock antibody (Japan BD) suspended in 100 μL of FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) was added to the cells and the resulting mixture was incubated at 4° C. for 10 minutes in order to suppress nonspecific reaction by blocking the Fc receptors on the cell surfaces.
Subsequently, for detecting CD8-positive cells, cells were stained by adding 0.5 μg of a fluorescein isothiocyanate (FITC)-labeled CD8 antibody to the cell suspension, and incubating the resulting mixture at 4° C. for 30 minutes, followed by washing the cells twice. Furthermore, using intracellular cytokine staining (ICS), IFN-γ in the cells was stained by the following procedure. First, 100 μL/well of CYTOFIX/CYTOPERMTM solution (Japan BD) was added to the cells, and the resulting mixture was left to stand at 4° C. for 20 minutes to fix the cells and permeabilize the cell membrane. The cells were washed twice, and then collected. Subsequently, 0.5 μg of a phycoerythrin (PE)-labeled anti-IFN-γ antibody was added to the cells, and the cells were incubated at 4° C. for 30 minutes. The cells were washed, and suspended in 100 μL of FACS fix buffer (PBS containing 2% FCS, 0.1% sodium azide and 1% formaldehyde), after which the cells were subjected to flow cytometry analysis.
For each epitope candidate peptide, the ratio (%) of IFN-γ-positive cells among CD8-positive cells is shown in Table 3.











TABLE 3








SEQ ID
ICS



Epitope
No:
(% in CD8+ cells)




















 1) pp1a-15
1
0.05



 2) pp1a-103
2
0.07



 3) pp1a-445
3
0.08



 4) pp1a-634
4
0.08



 5) pp1a-651
5
0.02



 6) pp1a-1121
6
0.05



 7) pp1a-1139
7
0.07



 8) pp1a-1288
8
0.05



 9) pp1a-1652
9
0.05



10) pp1a-2187
10
*0.19



11) pp1a-2207
11
*0.48



12) pp1a-2340
12
*0.21



13) pp1a-2546
13
*0.17



14) pp1a-2754
14
0.04



15) pp1a-2755
15
*0.18



16) pp1a-2758
16
0.03



17) pp1a-2990
17
*0.16



18) pp1a-3444
18
*0.12



19) pp1a-3459
19
0.03



20) pp1a-3560
20
0.04



21) pp1a-3564
21
0.06



22) pp1a-3616
22
0.07



23) pp1a-3687
23
*0.20



24) pp1a-3709
24
*0.50



25) pp1a-3730
25
0.06



26) pp1a-3745
26
0.03



27) pp1a-3816
27
0.07



28) pp1a-3848
28
0.07



29) pp1a-4071
29
0.06



30) pp1a-4219
30
0.01







*ICS of 0.1% or more






As a result, among the 30 kinds of candidate peptides, 9 kinds of peptides (pp1a-2187, pp1a-2207, pp1a-2340, pp1a-2546, pp1a-2755, pp1a-2990, pp1a-3444, pp1a-3687 and pp1a-3709; SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24) significantly induced IFN-γ-positive cells among CD8-positive cells, so that these 9 kinds of peptides were determined as the epitopes. These included peptides having low binding affinities to HLA-A*0201 (pp1a-2207 and pp1a-2340) and a peptide having a medium binding affinity thereto (pp1a-2755), as shown in Example 2.
Example 4
Preparation of Liposomes and Peptide-Bound Liposomes
For the 9 kinds of CTL epitope peptides that showed especially high CTL induction activities in Example 3 (pp1a-2187, pp1a-2207, pp1a-2340, pp1a-2546, pp1a-2755, pp1a-2990, pp1a-3444, pp1a-3687 and pp1a-3709; SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24), peptide-bound liposomes were prepared by the following method. Furthermore, in the same manner, liposomes to which a helper peptide (amino acid sequence: TPPAYRPPNAPIL; SEQ ID NO:32) was bound were prepared. As the helper peptide, one synthesized based on the HBV core 128 helper peptide (Operon Biotechnologies) used by Dr. A. Sette et al. (e.g., Glenn Y et al., J. Immunol. 162:3915-3925 (1999)) was used.
(4-1) Synthesis of Reactive Phospholipid Composed of Terminally Modified Phosphatidylethanolamine (Succinimidyl Group-Dioleoylphosphatidylethanolamine)
In 50 ml of chloroform, 2 g of dioleoylphosphatidylethanolamine and 180 μl of triethylamine were dissolved/added, and the resulting solution was placed in a 300 ml four-necked flask. While stirring the solution in the flask with a magnetic stirrer at room temperature, a solution separately prepared by dissolving 3 g of disuccinimidyl suberate, which is a divalent reactive compound, in 80 ml of chloroform was added dropwise to the solution according to a conventional method for 4 hours, thereby allowing one terminus of disuccinimidyl suberate to react with the amino group of dioleoylphosphatidylethanolamine. This crude reaction solution was transferred to an eggplant type flask, and the solvent was evaporated with an evaporator. Subsequently, a small amount of chloroform which is enough for dissolving the crude reaction product was added to the flask to obtain a high concentration crude reaction product solution, and this solution was subjected to column chromatography according to a conventional method using silica gel equilibrated with chloroform/methanol/water (65/25/1, volume ratio). Only the fraction of interest that contained dioleoylphosphatidylethanolamine whose amino group is bound to one terminus of disuccinimidyl suberate was recovered, and the solvent was evaporated, to obtain the succinimidyl group-dioleoylphosphatidylethanolamine of interest.
(4-2) Preparation of Lipid Mixture Powder
In an eggplant type flask, 1.3354 g (1.6987 mmol) of dioleoylphosphatidylcholine, 0.2886 g (0.2831 mmol) of succinimidyl group-dioleoylphosphatidylethanolamine prepared in Example 4-1, 0.7663 g (1.9818 mmol) of cholesterol and 0.4513 g (0.5662 mmol) of dioleoylphosphatidylglycerol sodium salt were placed, and 50 ml of a chloroform/methanol/water (65/25/4, volume ratio)-mixed solvent was added thereto, followed by dissolving the reagents at 40° C. Subsequently, the solvent was evaporated under reduced pressure using a rotary evaporator, to prepare a thin lipid membrane. Furthermore, 30 ml of distilled water for injection was added thereto, and the resulting mixture was stirred, to obtain a uniform slurry. This slurry was frozen, and dried in a freeze dryer for 24 hours, to obtain a lipid mixture powder.
(4-3) Preparation of Liposomes
Subsequently, 60 ml of separately prepared buffer A (1.0 mM Na2HPO4/KH2PO4, 0.25 M saccharose, pH7.4) was placed in the eggplant type flask containing the lipid mixture powder, and the lipids were hydrated while stirring the resulting mixture at 40° C., thereby liposomes were obtained. Thereafter, the particle diameters of the liposomes were adjusted using an extruder. The liposomes were first allowed to pass through an 8-μm polycarbonate filter, and then through 5 μm, 3 μm, 1 μm, 0.65 μm, 0.4 μm and 0.2 μm filters in this order. As a result, liposome particles having an average particle diameter of 206 nm (measured by dynamic light scattering) were obtained.
(4-4) Preparation of Peptide-Bound Liposomes
In a 1.5 ml test tube, the liposomes obtained in Example 4-3 were collected, and 3 ml of each peptide solution (1.25 mM)/buffer A separately prepared was added thereto, followed by stirring the resulting mixture at 5° C. for 48 hours to allow the reaction to proceed. This reaction liquid was subjected to gel filtration according to a conventional method using Sepharose CL-4B equilibrated with buffer A. Since the liposome fraction is turbid, the fraction of interest can be easily recognized, but may also be confirmed using a UV detector or the like. The phosphorus concentration of the thus obtained liposome suspension was measured (Phospholipid Test, Wako), and the suspension was diluted with buffer A such that the concentration of phosphorus derived from the phospholipid is 2 mM, thereby obtaining a suspension of each peptide-bound liposome.
Example 5
Induction of Peptide-Specific CTLs in Mice Immunized Using Peptide-Bound Liposomes
For the 9 kinds of CTL epitope peptides that showed especially high CTL induction activities in Example 3 (pp1a-2187, pp1a-2207, pp1a-2340, pp1a-2546, pp1a-2755, pp1a-2990, pp1a-3444, pp1a-3687 and pp1a-3709; SEQ ID NOs: 10, 11, 12, 13, 15, 17, 18, 23 and 24), the CTL induction activity in a mouse immunized using the peptide-bound liposome was measured.
A naive HLA-A2 transgenic mouse was immunized at its foot pad with an immunization solution obtained by mixing the peptide-bound liposomes (25 μl) and the helper peptide-bound liposomes prepared in Example 4 (25 μl) and an oligonucleic acid comprising a CpG motif (5 μg, base sequence: 5′-tccatgacgt tctgatgtt-3′; SEQ ID NO:33) together. After the immunization, the mouse was kept for 1 week, and the CTL induction activity was then measured by the same method as in Example 3. As a control, liposomes to which the peptide is not bound were used instead of the peptide-bound liposomes. As the oligonucleic acid comprising a CpG motif, one prepared by gene synthesis (Hokkaido System Science Co., Ltd.) based on Nagata. T. et al., Vaccine 25:4914-4921 (2007) was used.
Staining plots for CD8 and IFN-γ by flow cytometry in mice immunized using peptide-bound liposomes are shown in FIG. 1. Each dot inside the boxed area in the upper right of each graph corresponds to an IFN-γ-positive cell among CD8-positive cells, and the value shown in the boxed area indicates the ratio (%) of the IFN-γ-positive cells among the CD8-positive cells. As a result, it was revealed that immunization with the contained peptide-bound liposomes causes CTL induction.
Comparative Example
As a comparative control experiment for Example 5, the following experiment was carried out. Measurement of the CTL induction activity was carried out by the same method as in Example 4 except that, instead of a CTL epitope peptide of pp1a, a CTL epitope peptide of the Spike protein of SARS-CoV (spike-1203, amino acid sequence: FIAGLIAIV; SEQ ID NO:34) was used and that a naive HLA-A2 transgenic mouse was immunized by intramuscular injection at thigh muscle.
Staining plots for CD8 and IFN-γ by flow cytometry in mice immunized using the spike-1203-peptide-bound liposomes are shown in FIG. 2. As a result of the analysis, the ratio (%) of IFN-γ-positive cells among CD8-positive cells was 0.29 when the known CTL epitope peptide of the Spike protein (spike-1203) was used. When compared to the results in Example 4, it was revealed that 8 kinds of CTL epitope peptides derived from pp1a (pp1a-2187, pp1a-2207, pp1a-2340, pp1a-2546, pp1a-2755, pp1a-2990, pp1a-3687 and pp1a-3709; SEQ ID NOs: 10, 11, 12, 13, 15, 17, 23 and 24) show CTL induction activities higher than that of the CTL epitope peptide of the Spike protein.
Example 6
In Vivo CTL assay in Mice Immunized Using Peptide-Bound Liposomes
For 5 kinds of CTL epitope peptides that showed high CTL induction activities in Example 4 (pp1a-2187, pp1a-2340, pp1a-2755, pp1a-2990 and pp1a-3709; SEQ ID NOs: 10, 12, 15, 17 and 24), the CTL response activity in a mouse immunized using the peptide-bound liposome was measured in vivo.
The principle of measurement of the CTL response activity in vivo was as follows. Target cells which were not pulsed with the peptide (negative control) were labeled with carboxy fluorescein diacetate succinimidyl ester (CFSE) at a low concentration, and, on the other hand, target cells pulsed with the peptide were labeled with CFSE at a high concentration (10-fold). The same number of cells of the 2 kinds of cell populations were mixed together, and transferred to a mouse preliminarily immunized using the peptide-bound liposomes. Thereafter, spleen cells were collected from the mouse to which the mixed cells were transferred, and a spleen cell suspension was prepared, followed by measuring the ratio of the transferred cells labeled with CFSE by flow cytometry analysis. In a mouse having no CTL response activity to the antigen peptide, equal amounts of the CFSE-labeled transferred cells are collected. On the other hand, since, in a mouse having the CTL response activity to the antigen peptide, the target cells covered with the antigen peptide are lysed, the degree of the lysis in vivo can be measured by quantifying decrease in the cells highly labeled with CFSE by flow cytometry.
More particularly, the measurement was carried out by the following method.
A mouse for the transfer was preliminarily prepared by immunizing a mouse with the peptide-bound liposomes prepared using each peptide and inducing the peptide-specific CTLs. As a control, a mouse immunized with liposomes to which the peptide was not bound, instead of the peptide-bound liposomes, was used. One week after the immunization, spleen cells were prepared from another individual of the naive mouse, and the cells were suspended in RPMI-1640 (Sigma) at the cell number of the medium is 2×108 cells/2 mL. A 1 mL aliquot of the resulting suspension was taken into each of two tubes. To only one of the tubes, the peptide was added to a final concentration of 10 μM, and the two tubes were incubated at 37° C. for 1 to 2 hours. After washing the cells once, the cells were suspended in 20 mL of PBS/0.1% BSA and then briefly vortexed. Thereafter, to the tube containing the cells pulsed with the peptide, 10 μL of 5 mM CFSE was added, immediately followed by adding 1 μL of 5 mM CFSE to the tube containing the cells which were not incubated with the peptide. The both tubes were vortexed to suspend the cells, and incubated in a water bath at 37° C. for 10 minutes. The both cell populations were centrifuged and washed once, followed by counting the live cell numbers and suspending each cell population in HBSS to a concentration of 5×107 cells/mL. Equal amounts of the cell suspensions were mixed together to prepare an immunization liquid, and 200 μL (1×107 cells/individual) of the resulting cell suspension was intravenously injected to a mouse preliminarily immunized, thereby carrying out the cell transfer. Twelve hours after the transfer, spleen cells were collected from the mouse, and suspended in 1 mL/spleen of PBS/1% FBS/5 mM EDTA. The cells were centrifuged and suspended in 1 mL of FACS fix buffer, and 100 μL of this spleen cell suspension was diluted with 2 mL of FACS buffer, followed by subjecting the resulting dilution to flow cytometry analysis.
Staining plots for CFSE by flow cytometry in mice to which the target cells labeled with CFSE were transferred are shown in FIG. 3. The rightmost peak in each graph indicates the cell population pulsed with the peptide, and the peak in its left indicates the cells which were not pulsed with the peptide. It was revealed that, in each CTL epitope peptide, the mouse immunized with the peptide-bound liposomes (right graph) shows a decreased number of the cells pulsed with the peptide compared to the control immunized with the liposomes (left graph). The peptide-specific lysis rates (%) of the target cells by CTLs were: pp1a-2187, 79.2%; pp1a-2340, 68.5%; pp1a-2755, 70.5%; pp1a-2990, 73.4%; and pp1a-3709, 96.3%; showing that all of these peptides have high CTL response activities. Among the peptides, pp1a-3709 was revealed to have an especially high CTL response activity. Therefore, it was revealed that these CTL epitope peptides (pp1a-2187, pp1a-2340, pp1a-2755, pp1a-2990 and pp1a-3709; SEQ ID NOs: 10, 12, 15, 17 and 24) are dominant peptides having high CTL response activities.

## Claims

1. A peptide-bound liposome, wherein the peptide is covalently bound to the surface of the liposome; the liposome comprises: a phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond, and a stabilizer; and the peptide comprises the amino acid sequence of SEQ ID NO: 24, wherein the peptide-bound liposome induces an immune response that produces human leukocyte antigen type A2 (HLA-A2)-restricted cytotoxic T lymphocytes (CTLs) specific to severe acute respiratory syndrome (SARS) coronavirus.

2. The peptide-bound liposome according to claim 1, wherein the phospholipid comprises a C14-C24 acyl group containing one unsaturated bond.

3. The peptide-bound liposome according to claim 1, wherein the phospholipid comprises an oleoyl group.

4. The peptide-bound liposome according to claim 1, wherein the phospholipid is at least one selected from the group consisting of diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine and maleimide-diacylphosphatidylethanolamine.

5. The peptide-bound liposome according to claim 1, wherein the stabilizer is cholesterol.

6. The peptide-bound liposome according to claim 1, wherein the peptide is covalently bound to the phospholipid on the surface of the liposome.

7. The peptide-bound liposome according to claim 1, wherein the liposome comprises:
(A) 1 to 99.8 mol % of a phospholipid comprising:
a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond; and
(B) 0.2 to 75 mol % of a stabilizer.

8. A peptide-bound liposome according to claim 1 comprising:
(I) 1 to 85 mol % of an acidic phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond;
(II) 0.01 to 80 mol % of a neutral phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond;
(III) 0.2 to 80 mol % of a phospholipid comprising a C14-C24 acyl group containing one unsaturated bond, or a C14-C24 hydrocarbon group containing one unsaturated bond, wherein the phospholipid is bound to the at least one peptide; and
(IV) 0.2 to 75 mol % of a stabilizer.

9. An inducing agent for human leukocyte antigen type A2 (HLA-A2)-restricted cytotoxic T lymphocytes (CTLs) specific to severe acute respiratory syndrome (SARS) coronavirus comprising: the peptide-bound liposome according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier, wherein the agent induces an immune response that produces HLA-A2-restricted CTLs specific to SARS coronavirus.

10. A vaccine comprising: the peptide-bound liposome according to claim 1, and a pharmaceutically acceptable carrier, wherein the vaccine induces an immune response that produces HLA-A2-restricted CTLs specific to SARS coronavirus.

11. The inducing agent for HLA-A2-restricted CTLs specific to the SARS coronavirus according to claim 9, further comprising CpG-DNA.

12. The vaccine according to claim 10, further comprising CpG-DNA.

13. A method of producing an immune response to severe acute respiratory syndrome (SARS) coronavirus in a subject, comprising administering to the subject an effective amount of the vaccine composition of claim 10, thereby inducing the immune response to SARS coronavirus in the subject.

14. The method of claim 13, wherein the subject is human.