KR20230054225A - Immunogenic Composition - Google Patents

Abstract

The present invention provides an immunogenic composition. More specifically, the present invention relates to an immunogenic composition comprising: a) self-assembled multimeric protein assemblies including (i) a fusion protein in which RBD protein having SEQ ID NO: 4 or at least 75 % sequence identity thereto and SEQ ID NO: 5 or a protein having at least 75 % sequence identity thereto are linked through a linker, and (ii) a protein having SEQ ID NO: 1 or at least 75 % sequence identity thereto; and b) a preservative. The immunogenic composition of the present invention can be used to prevent coronavirus infection-19 (COVID-19).

Description

Immunogenic Composition

The present invention relates to immunogenic compositions.

The new coronavirus (2019-nCoV) that occurred in Wuhan, China in 2019 was named COVID-19 in the sense that human infection was first confirmed in the second half of 2019, and it is a virus that causes respiratory diseases in humans and animals. It is known that various symptoms appear after an incubation period of up to two weeks at work. The main symptoms include lethargy, high fever of 37.5 degrees or higher, cough, sore throat, phlegm, muscle pain, headache, shortness of breath and pneumonia, and respiratory failure due to lung damage. Similar diseases include MERS and SARS, which have a lower mortality rate than MERS or SARS, but are known to be much more contagious. There is an increasing demand in the medical industry for the production of efficient vaccines for the prevention of COVID-19 and the production of vaccine antigen proteins.

Accordingly, the present inventors developed an immunogenic composition as a result of diligent efforts to develop a coronavirus vaccine and completed the present invention.

US 2021-0338807 A1

It is an object of the present invention to provide immunogenic compositions.

One aspect of the present invention is an immunogenic composition.

In one embodiment, the immunogenic composition comprises a. (i) SEQ ID NO: 4 or an RBD protein having at least 75% sequence identity therewith and SEQ ID NO: 5 or a protein having at least 75% sequence identity therewith connected via a linker, and (ii) SEQ ID NO: 1 or 75% therewith self-assembled multimeric protein assemblies, including proteins with aberrant sequence identity; and b. Contains preservatives.

In the immunogenic composition according to the above embodiment, the multimeric protein assembly is composed of 20 trimers of the fusion protein of (i) and 12 pentamers of the protein of (ii).

The immunogenic composition according to any one of the preceding embodiments, wherein the preservative is 2-Phenoxyethanol or Thimerosal.

In the immunogenic composition according to any one of the preceding embodiments, the RBD protein is included in an amount of 10 to 50 ug/dose.

In the immunogenic composition according to any one of the preceding embodiments, the single administration dose is 0.5 ml unit.

In the immunogenic composition according to any one of the preceding embodiments, the RBD protein is included in an amount of 10 to 50ug/0.5ml.

An immunogenic composition according to any one of the preceding embodiments, wherein a. The multimeric protein assembly of is included in the content of 25 to 150 ug / dose.

An immunogenic composition according to any one of the preceding embodiments, wherein a. The multimeric protein assembly of is included in an amount of 25 to 150 ug/0.5ml.

An immunogenic composition according to any one of the preceding embodiments, wherein a. The multimeric protein assembly of is included in an amount of 50 to 300 ug/ml.

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises an adjuvant.

An immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is a mineral-containing composition, an oil emulsion, a saponin preparation, a virus-like particle, a bacterial or microbial derivative, a bioadhesive, a mucoadhesive, a liposome, a polyoxyethylene ether , polyoxyethylene esters, polyphosphazenes, muramyl peptides, imidazoquinolone compounds, thiosemicarbazone compounds, tryptanthrin compounds, human immunomodulators, lipopeptides, benzonaphthyridine, microparticles and immunostimulatory polynucleotides.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is selected from an oil-in-water emulsion and an aluminum salt.

An immunogenic composition according to any one of the preceding embodiments, wherein the oil-in-water emulsion adjuvant is AS03 or MF59.

An immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is an aluminum salt.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is an aluminum salt, and the immunogenic composition comprises 2-phenoxyethanol in an amount of 2.5 to 7.5 mg/dose.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is an aluminum salt, and the immunogenic composition comprises 2-phenoxyethanol in an amount of 2.5 to 7.5 mg/0.5 ml.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is an aluminum salt, and the immunogenic composition comprises 2-phenoxyethanol at a concentration of 5 to 15 mg/ml.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is an aluminum salt, and the immunogenic composition comprises thimerosal in an amount of 25 to 100 ug/0.5ml.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is an aluminum salt, and the immunogenic composition comprises thimerosal in an amount of 25 to 100 ug/dose.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is an aluminum salt, and the immunogenic composition comprises thimerosal at a concentration of 50 to 200 ug/ml.

An immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03, and the immunogenic composition comprises 2-phenoxyethanol in an amount of 2.5 to 7.5 mg/dose.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03, and the immunogenic composition comprises 2-phenoxyethanol in an amount of 2.5 to 7.5 mg/0.5 ml.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03, and the immunogenic composition comprises 2-phenoxyethanol in an amount of 2.5 to 5 mg/dose.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03, and the immunogenic composition comprises 2-phenoxyethanol in an amount of 2.5 to 5 mg/0.5 ml.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03, and the immunogenic composition comprises 2-phenoxyethanol at a concentration of 5 to 10 mg/ml.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03, and the immunogenic composition comprises thimerosal in an amount of 25 to 50 ug/dose.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03, and the immunogenic composition comprises thimerosal in an amount of 25 to 50 ug/0.5 ml.

The immunogenic composition according to any one of the preceding embodiments, wherein the adjuvant is AS03, and the immunogenic composition comprises thimerosal at a concentration of 50 to 100 ug/ml.

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition is for prevention of COVID-19 (Coronavirus Infectious Disease-19).

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or an RBD protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly consisting of 12 pentamers of a protein having at least 75% sequence identity thereto, b. 2-phenoxyethanol and c. aluminum salt or AS03 adjuvant, and the RBD content of the immunogenic composition is 10 to 50 ug/dose.

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or an RBD protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly consisting of 12 pentamers of a protein having at least 75% sequence identity thereto, b. 2-phenoxyethanol and c. aluminum salt or AS03 adjuvant, and the RBD content of the immunogenic composition is 10 to 50 ug/0.5ml.

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or a protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly consisting of 12 pentamers of a protein having at least 75% sequence identity therewith in an amount of 27 to 135 ug/dose, b. 2-phenoxyethanol and c. aluminum salt or AS03 adjuvant.

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or a protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly composed of 12 pentamers of a protein having at least 75% sequence identity therewith in an amount of 54 to 270 ug/ml, b. 2-phenoxyethanol and c. aluminum salt or AS03 adjuvant.

An immunogenic composition according to any one of the preceding embodiments, wherein the composition comprises 2-phenoxyethanol in an amount of 5.0 to 15.0 mg/ml.

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or a protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly consisting of 12 pentamers of a protein having at least 75% sequence identity therewith, b. Thimerosal and c. aluminum salt or AS03 adjuvant, and the RBD content of the immunogenic composition is 10 to 50 ug/dose. The immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or a protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly consisting of 12 pentamers of a protein having at least 75% sequence identity therewith, b. Thimerosal and c. aluminum salt or AS03 adjuvant, and the RBD content of the immunogenic composition is 10 to 50 ug/0.5ml.

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or a protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly consisting of 12 pentamers of a protein having at least 75% sequence identity therewith in an amount of 27 to 135 ug/dose, b. Thimerosal and c. aluminum salt or AS03 adjuvant.

An immunogenic composition according to any one of the preceding embodiments, wherein the immunogenic composition comprises a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or a protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly composed of 12 pentamers of a protein having at least 75% sequence identity therewith in an amount of 54 to 270 ug/ml, b. Thimerosal and c. aluminum salt or AS03 adjuvant.

An immunogenic composition according to any one of the preceding embodiments, wherein the composition comprises Thimerosal in an amount of 50 to 200 ug/ml.

Another aspect embodying the present invention is a vaccine composition for preventing COVID-19 comprising the immunogenic composition.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the contents of the present invention.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be included in this invention.

As used herein, the term "about" may be preceded by a specific numerical value. As used in this application, the term "about" includes not only the exact number that follows the term, but also a range that is or is close to that number. It can be determined whether the number is close to or nearly the specific number mentioned, given the context in which it is presented. As an example, the term “about” can refer to a range of -10% to +10% of a numerical value. As another example, the term "about" can refer to a range of -5% to +5% of a given numerical value. However, it is not limited thereto.

As used herein, the term “and/or” when used herein is to be understood as specifically disclosing each of the two specified features or elements, either with or without the other. Thus, the term "and/or" as used herein in phrases such as "A and/or B" includes "A and B", "A or B", "A" (alone), and "B" (alone). It is intended to include Likewise, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term "consisting essentially of" means that a non-specified ingredient may be present if the characteristics of the claimed subject matter are not substantially affected by the presence of the non-specified ingredient.

As used herein, the term “consisting of” means that the proportions of the specified component(s) total 100%. Ingredients or features that come after the term “consisting of” may be essential or mandatory. In some embodiments, in addition to the ingredients or features that come under "consisting of", any other ingredients or non-essential ingredients may be excluded.

As used herein, the term "comprising" refers to the presence of any of the features, steps or components described below the term, and does not exclude the presence or addition of one or more features, steps or components. Components or features described herein under "comprising" may be required or mandatory, but in some embodiments may further include other optional or non-essential components or features.

As used herein, the term “comprising” may, in some embodiments, be modified to refer to “consisting essentially of” or “consisting of”.

With reference to amino acid sequences herein, a polypeptide "comprising" the amino acid sequence set forth in a particular SEQ ID NO, a polypeptide "consisting of' the amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide or protein that "has" the amino acid sequence set forth in a particular SEQ ID NO. Even if it is described, as long as it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the corresponding sequence number, proteins having amino acid sequences in which some sequences have been deleted, modified, substituted, conservatively substituted or added can also be used in this application. It is self-evident that it can For example, if the amino acid sequence has an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution to the N-terminus and/or C-terminus of the amino acid sequence. It may, but is not limited thereto.

One aspect of the present invention is

a. (i) SEQ ID NO: 4 or an RBD protein having at least 75% sequence identity therewith and SEQ ID NO: 5 or a protein having at least 75% sequence identity therewith connected via a linker, and (ii) SEQ ID NO: 1 or 75% therewith self-assembled multimeric protein assemblies, including proteins with aberrant sequence identity; and

b. An immunogenic composition comprising a preservative is provided.

In one embodiment, the multimeric protein assembly of the present invention may be a protein assembly disclosed in WO2019-169120, US 9630994 B2, WO2021-163481, WO2021-163438, PCT/US2021/037341.

The fusion protein of (i), which is one component of the multimeric protein assembly of the present invention, can be used to prepare a multimeric protein assembly displaying an antigen or antigen fragment used in the preparation of an immunogenic composition. In the fusion protein, the protein of SEQ ID NO: 4 is a fragment of SARS-CoV-2 Spike protein, which may also be referred to as "RBD" protein. The RBD protein may exhibit immunogenicity. As used herein, the term "immunogenicity" refers to the ability of a specific protein or a specific region thereof to elicit an immune response against a specific protein or a protein comprising an amino acid sequence having a high degree of identity with the specific protein. do. Thus, the immunogenic protein that may be included in the multimeric protein assembly is SEQ ID NO: 4 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, It may have sequences with greater than 97%, 98% or 99% identity.

The protein of SEQ ID NO: 5 of the present invention is linked to an immunogenic protein to form the structure of a multimeric protein assembly and to display an antigen. The protein of SEQ ID NO: 5 may include any modification that does not disrupt assembly into a multimeric protein assembly, and may include, for example, amino acid addition, deletion, insertion, substitution, or modification. In one embodiment, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO: 5 or thereto. It can have sequences with identity. In any one of the embodiments described above, the protein that forms the structure of the multimeric protein assembly and displays the antigen linked to the immunogenic protein has the amino acid sequence of SEQ ID NO: 5, 17, 18, 19, 20 or 21 It may include, consist essentially of, or consist of the sequence.

In any one of the foregoing embodiments, a range of proteins of SEQ ID NO: 5 of the present invention contain amino acid sequences identical to SEQ ID NO: 5 at least 1, 2, 3, 4 or 5 identified interface positions. include In the protein, the interface residues identified as being present at the interface to be assembled to form the nanostructure may be 25, 29, 33, 54, and 57 from the N-terminus of SEQ ID NO: 5.

The linker may be of any suitable length. For example, the linker may be a peptide linker, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 It may be composed of one or more amino acids. In one embodiment, it may be a Gly-Ser linker containing glycine and/or serine residues. In any one of the foregoing embodiments, the linker is a (GGGGS)n, (GSSGSS)n, (GSSSSSS)n, (SSSSSSS)n, (GSGGSGSGSGGSGSG)n, (GGSGGSGS)n or (GSGGSGSG)n linker. can be In any one of the foregoing embodiments, the linker may comprise a helical linker sequence. Specifically, the helix linker may be represented by an EKAAKAEEAAR sequence. As a specific example of the linker, the sequence of amino acids 237 to 263 from the N-terminus of SEQ ID NO: 6 may be cited, but is not limited thereto.

Connection of the antigenic protein and the nanostructure may be achieved by expressing a fusion protein fused to the N- or C-terminus, such as, for example, the fusion protein of SEQ ID NO: 6, but is not limited thereto, and post-translational covalent attachment. or can be achieved by non-covalent attachment. Also, depending on how the antigenic protein is attached, the antigenic protein can be displayed in any orientation.

In any one of the foregoing embodiments, the fusion protein of (i) is SEQ ID NO: 6 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, It may have sequences with at least 95%, 96%, 97%, 98% or 99% identity. The protein of SEQ ID NO: 6 herein may be referred to as "Component A". A plurality of isolated protein monomers of SEQ ID NO: 6 may self-assemble through interactions to form a trimer. This assembly can be achieved through non-covalent protein-protein interaction reactions.

In the multimeric protein assembly of the present invention, the protein of (ii) may be used to prepare a multimeric protein assembly displaying an antigen or antigen fragment used in the preparation of an immunogenic composition. Specifically, it may be the protein represented by SEQ ID NO: 1 of (ii), but those having the same function as above are included without limitation.

In one embodiment, the protein of (ii) is SEQ ID NO: 1 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% or more identical sequences. In any one of the foregoing embodiments, the protein of (ii) comprises, or consists essentially of, the amino acid sequence of SEQ ID NO: 1, 9, 10, 11, 12, 13, 14, 15 or 16 It may consist essentially of or consist of. In any one of the preceding embodiments, the protein of (ii) has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 identified interfaces ( interface) contains the same amino acid sequence at the position. In the protein, the interface residues identified as being present at the interface to be assembled to form the nanostructure are 24, 28, 36, 124, 125, 127, 128, 129, 131, 132, 133, 135, and 139. The protein of SEQ ID NO: 1 herein may be referred to as "Component B". A plurality of isolated protein monomers of SEQ ID NO: 1 may self-assemble through interactions to form a pentamer. This assembly can be achieved through non-covalent protein-protein interaction reactions.

Such Component B and Component A may self-assemble to form an immunogenic composition, and for the purpose of the present application, may be one component of a vaccine against coronavirus.

The proteins (i) and (ii) of the present invention may contain any modifications that do not perturb assembly into multimeric protein assemblies, for example including amino acid additions, deletions, insertions, substitutions or modifications. can Examples include cases involving conservative sequence modifications.

In the present invention, "conservative sequence modifications" may refer to amino acid modifications that do not significantly affect or change the structural properties of a protein of interest. Such conservative modifications include amino acid substitutions, additions and deletions. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Such substitutions are generally based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Families of amino acid residues with similar side chains have been defined in the art. This family includes basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), non-charged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. For example, tyrosine, phenylalanine, tryptophan, histidine).

The multimeric protein assembly of the present invention may be formed by self-assembly of a trimer of Component A and a pentamer of Component B. The multimeric protein assembly formed by self-assembly of Component A and Component B may also be referred to as "Nanoparticle", "Nanoparticle", or "Nanostructure".

Specifically, the multimeric protein assembly of the present invention may have a structure including (i) 20 trimers of the fusion protein (Component A) and (ii) 12 pentamers of the protein (Component B). there is. Thus, the multimeric protein assembly of the present invention contains 60 copies of Component A and 60 copies of Component B. Meanwhile, the multimeric protein assembly may be formed by non-covalent interactions between (i) and (ii). That is, it may be that no covalent bond is formed between (i) and (ii).

The multimeric protein assembly of the present invention may have an icosahedral symmetrical structure. The multimeric protein assembly of the present invention has an average diameter of about 20 nm to about 80 nm, wherein the lumen may be about 15 nm to about 32 nm horizontally.

The multimeric protein assembly of the present invention may be prepared by mixing the fusion protein of (i) and the protein of (ii) in a molar ratio of 1:10 to 2:1. For example, in the mixing step, the molar ratio of the fusion protein of (i) and the protein of (ii) is about 1:9, 1:8, 1:7, 1:6, 1:5.5, 1:5, 1: 4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2.4, 1:2.3, 1:2.2, 1:2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1.05:1, 1.1:1, 1.15:1, 1.2:1, 1.25:1 or about 1.3 : can be 1. In any one of the embodiments described above, the molar ratio of the fusion protein of (i) to the protein of (ii) may be about 1:0.9 to 1.1, and may be 1:1.0 to 1.1.

The time for performing the mixing reaction is not particularly limited, but may be performed in excess of 1 hour. For example, it may be performed for about 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours or about 4 hours. For example, greater than about 0.5 hours and less than 4 hours, greater than about 0.5 hours and less than 3.5 hours, greater than about 1 hour and less than 3.5 hours, greater than about 1.5 hours and less than 3 hours, greater than 1.5 hours and less than 2.5 hours, greater than 1.8 hours and less than 2.2 hours, or 1.9 hours. It can be performed for more than 2.1 hours or less.

The multimeric protein assemblies of the present invention can be used as components of immunogenic compositions. The immunogenic composition can be used to raise antibodies in mammals.

The immunogenic composition of the present invention may be provided as a single dose or multidose dose. “Multi-dose” or “multi-dose” refers to a unit of preparation comprising more than one (eg, two or more) inoculated doses that can be administered to one subject or to more than one subject. In one embodiment, a single dose of the immunogenic composition of the present invention may be 0.5ml unit.

An immunogenic composition of the present invention may include an immunologically effective amount of a multimeric protein assembly. The term "immunologically effective amount" is an amount effective to elicit an antibody response to an antigen when administered to a subject. The immunologically effective amount depends on the health and physical condition of the individual being treated, the age of the individual, the ability of the individual's immune system to synthesize antibodies, the level of protection required, the formulation of the composition, the treating physician's assessment of the medical situation, and other relevant factors. may vary depending on The immunologically effective amount of the immunological composition of the present invention can be expressed in terms of the content of multimeric protein assemblies or antigen content in the assemblies, and can also be expressed in terms of the mass of protein per dose. The antigen in the assembly refers to SEQ ID NO: 4 or an RBD protein having at least 75% identity therewith.

In one embodiment, the immunogenic composition of the present invention may include RBD protein in an amount of about 10 to 50 μg/dose. In any one of the foregoing embodiments, a single dosage unit of the composition of the present invention may be a 0.5 ml unit. In any one of the foregoing embodiments, the immunogenic composition of the present invention may include an amount of about 10 to 50 μg/0.5 ml of RBD protein.

In any one of the foregoing embodiments, the amount of RBD included in the immunogenic composition is about 1 ug/ml to 200 ug/ml, for example, about 5 ug/ml to 150 ug/ml, 10 ug/ml to 200 ug/ml. ml to 100 ug/ml or 20 ug/ml to 100 ug/ml. However, it is not necessarily limited to this, and as described above, when mixed with other auxiliary ingredients, the content can be appropriately adjusted within the range of maintaining a single dose. As one embodiment of the above-described embodiments, the present invention The immunogenic composition of may include the multimeric protein assembly in an amount of about 25 to 150 ug/dose. In any one of the foregoing embodiments, the immunogenic composition of the present invention may comprise a multimeric protein assembly in an amount of about 25 to 150 ug/0.5ml.

In any one of the foregoing embodiments, the immunogenic composition of the present invention may comprise a multimeric protein assembly in an amount of about 50 to 300 ug/ml.

In any one of the above embodiments, the content of the multimeric protein assembly may be about 54 to 270 ug/ml.

For example, the content of the multimeric protein assembly contained in one dose of the immunogenic composition of the present invention may be about 27 to 135 ug. The content of the multimeric protein assembly may be about 27 to 135ug/0.5ml.

For example, when the immunogenic composition of the present invention is provided in multiple doses and the dose is a dose that can be inoculated twice, the content of the multimeric protein assembly may be about 50 to 140 ug/ml. For example, when the immunogenic composition of the present invention is provided in multiple doses and the dose is a dose that can be inoculated twice, the content of the multimeric protein assembly may be about 54 ug/ml to 135 ug/ml. The two doses may be in units of 1 ml.

However, it is not limited thereto, and considering the case of mixing with other auxiliary ingredients, the content can be appropriately adjusted according to the volume within the range of maintaining a single dose. For example, when an adjuvant is administered in the same volume ratio as the composition containing the multimeric protein assembly, the content of the multimeric protein assembly in the immunogenic composition in a state independently of the adjuvant is 2 of the above-mentioned content. it can be a boat That is, when the composition containing the multimeric protein assembly of the present invention is provided in a dose that can be inoculated twice, and an adjuvant of the same volume ratio is administered independently of this, the content of the multimeric protein assembly is about 108 to 270 may be ug/ml. However, it is not particularly limited thereto.

The immunogenic composition provided in the present invention may include a preservative.

In the present invention, the term "preservative" means a substance that inhibits the growth of microorganisms in the immunogenic composition and has anti-fungal and/or antibacterial action. Examples of preservatives used in immunogenic compositions are 2-phenoxyethanol, formaldehyde, chlorobutanol, m-cresol, methylparaben, propylparaben, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thiourea There are merosal, phenyl mercury nitrate, etc.

In one embodiment, the preservative included in the immunogenic composition of the present invention may be 2-phenoxyethanol or thimerosal.

"Thimerosal" is an ethyl mercury derivative compound (Ethyl(2-mercaptobenzoato-(2-)-O,S) mercurate(1-) sodium) also referred to as "Thiomersal". Thimerosal has been used for the purpose of preventing the growth of contaminating microorganisms and maintaining sterility during storage or use of vaccine products, and many pentavalent vaccines (D, T, P, Hib , including HBsAg) contains thimerosal as a preservative.

In any one of the above-described embodiments, the content of thimerosal may be about 5 to 150 ug/dose, for example, 25 to 100 ug/dose. In any one of the above embodiments, the content of thimerosal may be about 5 to 150ug/0.5ml, for example, 25 to 100ug/0.5ml.

In any one of the above embodiments, the content of thimerosal may be about 10 ug/ml to 300 ug/ml, for example, 50 ug/ml to 200 ug/ml.

2-Phenoxyethanol (2-PE) is a phenol ether that is a combination of phenol and ethylene glycol, and is widely used as a preservative for cosmetics and transdermal drugs. 2-Phenoxyethanol is also used as a preservative in vaccine injections. For example, inactivated polio vaccine (IPV) has a problem in that potency decreases by more than 50% in a week even when refrigerated when thimerosal is included, so it is common to use 2-PE as a preservative.

In any one of the above-described embodiments, the content of 2-phenoxyethanol may be about 0.5 mg to 10 mg/dose, for example, 2.5 mg to 7.5 mg/dose. In any one of the above-described embodiments, the content of 2-phenoxyethanol may be about 0.5 mg to 10 mg/0.5ml, for example, 2.5mg to 7.5mg/0.5ml.

In any one of the above embodiments, the content of 2-phenoxyethanol may be about 1 mg/ml to 20 mg/ml, for example, 5 mg/ml to 15 mg/ml.

In any one of the above embodiments, the immunogenic composition provided in the present invention may include an adjuvant.

In the present invention, "adjuvant" refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention. The adjuvant is often provided to enhance the immune response.

In any one of the preceding embodiments, the adjuvant is a mineral-containing composition, an oil emulsion, a saponin preparation, a virus-like particle, a bacterial or microbial derivative, a bioadhesive, a mucoadhesive, a liposome, a polyoxyethylene ether, Polyoxyethylene esters, polyphosphazenes, muramyl peptides, imidazoquinolone compounds, thiosemicarbazone compounds, tryptanthrin compounds, human immunomodulators, lipopeptides, benzonaphthyridine, microparticles and immunity It may be selected from stimulatory polynucleotides.

In any one of the embodiments described above, the immunogenic composition provided in the present invention is an aluminum salt, an oil-in-water emulsion, a TLR7 agonist (such as imidazoquinoline or imiquimod) or any of these Combinations may be included.

In any one of the above-described embodiments, the composition of the present invention is provided in a state in which the composition comprising the multimeric protein assembly is mixed with an adjuvant, or the multimeric protein assembly and the adjuvant are each independently, for example For example, it may be provided in a state contained in a separate container.

In any one of the foregoing embodiments, the composition of the present invention may include an aluminum salt as an adjuvant.

Examples of aluminum salts include hydroxides (eg oxyhydroxides), phosphates (eg hydroxyphosphates, orthophosphates) or mixtures thereof. Examples of these include chapters 8 & 9 of Vaccine Design. (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum] is incorporated by reference. The salt may take any suitable form (eg, gel, crystalline, amorphous, etc.), wherein, for example, antigen adsorption to the salt may be achieved. The concentration of Al ⁺⁺⁺ in the composition for administration to a patient can be appropriately adjusted.

For example, the content of the aluminum salt per dose may be about 5 mg/ml or less, 4 mg/ml or less, 3 mg/ml or less, or about 2 mg/ml or less.

For example, the composition of the present invention may be provided in a state in which the aluminum salt and the multimeric protein assembly are mixed, or the multimeric protein assembly and the aluminum salt may be provided independently, for example, in separate containers. Not limited.

For example, the weight ratio of the multimeric protein assembly and the aluminum salt included in the immunogenic composition is about 20:50 to 1:50, for example, about 20:50 to 2:50, 20:50 to 3:50, 20:50 to 4:50, 20:50 to 5:50, 20:50 to 6:50, 20:50 to 7:50, 20:50 to 8:50, 20:50 to 9:50, 19:50 to 2 :50, 19:50 to 3:50, 19:50 to 4:50, 19:50 to 5:50, 19:50 to 6:50, 19:50 to 7:50, 19:50 to 8:50 , 19:50 to 9:50, 18:50 to 2:50, 18:50 to 3:50, 18:50 to 4:50, 18:50 to 5:50, 18:50 to 6:50, 18 :50 to 7:50, 18:50 to 8:50 or about 18:50 to 9:50.

In the present invention, an “oil-in-water (O/W)” emulsion refers to one liquid (oil phase) dispersed in another liquid (water phase). In one embodiment, an oil-in-water emulsion used as an adjuvant may include a biocompatible oil and a surfactant. In one embodiment, the oil-in-water emulsion of the present invention may support squalene. Examples of such squalene-containing oil-in-water emulsion adjuvants include, but are not limited to, AS03, MF59, AF03, AddaVax, GLA-SE, and the like.

In any one of the foregoing embodiments, the composition of the present invention may include AS03 as an adjuvant.

AS03 is a squalene-containing adjuvant, which is a mixture of DL-α-tocopherol, squalene and polysorbate 80. AS03 is known to contain about 10.69 mg of squalene, about 11.86 mg of DL-alpha-tocopherol, and about 4.86 mg of polysorbate 80 per dose.

For example, in the composition of the present invention, the multimeric protein assembly and AS03 may be provided independently, for example, in a state in which they are contained in separate containers. In one example, AS03 can be administered in the same volume as the multimeric protein assembly. However, it is not limited thereto.

In any one of the above embodiments, the immunogenic composition may contain a preservative and an adjuvant in specific amounts.

In any one of the above embodiments, when the immunogenic composition includes an aluminum salt and 2-phenoxyethanol, the 2-phenoxyethanol may be included at a concentration of about 5.0 to 15.0 mg/ml. The immunogenic composition may be provided in a single or multiple administration dose. Specifically, one or two administration doses may be provided. In any one of the foregoing embodiments, when the immunogenic composition provides two doses and comprises an aluminum salt and 2-phenoxyethanol, the 2-phenoxyethanol is about 5 to 15 mg/day. may be included in ml amounts. The two doses may be in units of 1 ml.

In any one of the above embodiments, when the immunogenic composition includes an aluminum salt and 2-phenoxyethanol, the weight ratio of the multimeric protein assembly and 2-phenoxyethanol included in the immunogenic composition is about It may be 0.1:100 to 10:100. For example about 0.2 to 10:100, about 0.3 to 10:100, about 0.4 to 10:100, about 0.5 to 10:100, about 0.2 to 9:100, about 0.3 to 9:100, about 0.4 to 9: 100, about 0.5 to 9:100, about 0.6 to 4:100, about 0.7 to 4:100, about 0.8 to 4:100, about 0.2 to 8:100, about 0.3 to 8:100, about 0.4 to 8:100 , about 0.5 to 8:100, about 0.6 to 8:100, about 0.7 to 8:100, about 0.8 to 8:100, about 0.2 to 7:100, about 0.3 to 7:100, about 0.4 to 7:100, About 0.5 to 7:100, about 0.6 to 7:100, about 0.7 to 7:100, about 0.8 to 7:100, about 0.2 to 6:100, about 0.3 to 6:100, about 0.4 to 6:100, about 0.5 to 6:100, about 0.6 to 6:100, about 0.7 to 6:100, about 0.8 to 6:100, about 0.2 to 5:100, about 0.3 to 5:100, about 0.4 to 5:100, about 0.5 to 5:100, about 0.6 to 5:100, about 0.7 to 5:100, about 0.8 to 5:100, about 0.2 to 4:100, about 0.3 to 4:100, about 0.4 to 4:100, about 0.5 to 4:100, about 0.6 to 4:100, about 0.7 to 4:100, about 0.8 to 4:100, about 0.2 to 3:100, about 0.3 to 3:100, about 0.4 to 3:100, about 0.5 to 3 :100, about 0.6 to 3:100, about 0.7 to 3:100, or about 0.8 to 3:100. For example, it may be about 0.36 to 5.4:100, but is not limited thereto.

In any one of the above embodiments, when the immunogenic composition includes aluminum salt and 2-phenoxyethanol, the weight ratio of 2-phenoxyethanol and aluminum salt included in the immunogenic composition is about 10:3 to 10:1.

In any one of the above embodiments, when the immunogenic composition includes an aluminum salt and thimerosal, the thimerosal may be included at a concentration of about 50 to 200 ug/ml. The immunogenic composition may be provided in a single or multiple administration dose. Specifically, one or two administration doses may be provided. In any one of the foregoing embodiments, when the immunogenic composition provides two doses and includes an aluminum salt and thimerosal, the thimerosal will be included in an amount of about 50 to 200 ug/ml. can The two doses may be in units of 1 ml.

In any one of the above embodiments, when the immunogenic composition includes an aluminum salt and thimerosal, the weight ratio of the multimeric protein assembly and thimerosal included in the immunogenic composition is about 1:6 to 4 :1 can be For example, it may be about 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, but is not limited thereto.

In any one of the above embodiments, when the immunogenic composition includes AS03 and 2-phenoxyethanol, the 2-phenoxyethanol may be included at a concentration of about 5 to 10 mg/ml. The immunogenic composition may be provided in a single or multiple administration dose. Specifically, one or two administration doses may be provided. The method of any one of the foregoing embodiments, wherein the immunogenic composition provides two doses, comprises AS03 and 2-phenoxyethanol, and comprises a multimeric protein assembly and a preservative, wherein the composition is independent of AS03. When provided as, the 2-phenoxyethanol may be included in an amount of about 5 to 10 mg / ml. The two doses may be in units of 1 ml.

In any one of the above embodiments, when the immunogenic composition includes AS03 and thimerosal, the thimerosal may be included at a concentration of about 50 to 100 ug/ml. The immunogenic composition may be provided in a single or multiple administration dose. Specifically, one or two administration doses may be provided. The method of any one of the foregoing embodiments, wherein the immunogenic composition provides two doses, and comprises AS03 and thimerosal, wherein the composition comprising a multimeric protein assembly and a preservative is provided independently of AS03. If it is, the thimerosal may be included in an amount of about 50 to 100 ug/ml. The two doses may be in units of 1 ml. Routes of administration of the immunogenic composition of the present invention include, for example, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal. It can be administered in any effective and convenient manner, including by route. The immunogenic composition of the present invention may be used as an injectable formulation or as a liquid, but is not limited thereto.

In one embodiment, the immunogenic composition may further include a pharmaceutically acceptable carrier, excipient, binder, carrier, preservative, buffer, tonicity agent, emulsifier or wetting agent.

In any one of the foregoing embodiments, the composition may include a buffer, such as a Tris buffer, a citrate buffer, a phosphate buffer, or a histidine buffer.

An immunogenic composition of the present invention can be administered to an individual to prevent infection in the individual. Thus, an immunogenic composition of the present invention may be a vaccine composition.

The vaccine composition of the present invention can be used to prevent diseases caused by infection with SARS-CoV-2. The term "prevention" of the present invention refers to any action that inhibits or delays the onset of a disease. The term "SARS-CoV-2" of the present invention refers to a virus that causes respiratory disease coronavirus infection-19 (COVID-19). Therefore, the immunogenic composition of the present invention or a vaccine composition comprising the same can be used for the prevention of COVID-19 (coronavirus infection-19).

The immunogenic composition of the present invention can be used for the prevention of COVID-19 (Coronavirus Infectious Disease-19).

1 and 2 are results confirming the induction of an immune response with the prepared multimeric protein assembly and an adjuvant. Aluminum hydroxide was used as an adjuvant.
3 and 4 are results confirming the induction of an immune response with the multimeric protein assembly and the adjuvant. AS03 was used as an adjuvant.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Manufacturing Example 1. Manufacturing of Component A

Component A cDNA (NT-SARS-CoV-2RBD-I53-50A-16GS-he) synthesized by Genscript for the production of Component A (SEQ ID NO: 6), an antigen of SARS-COV-2 recombinant nanoparticle vaccine, was used as a donor plasmid (pJV145) using the SalI/NotI restriction enzyme reaction to construct a recombinant expression vector plasmid (M-2560). As a result of sequencing analysis of Component A cDNA (NT-SARS-CoV-2RBD-I53-50A-16GS-he) inserted into the final expression vector plasmid (M-2560), the nucleotide sequence of the inserted gene is It was confirmed that the base sequence (SEQ ID NO: 7) and 100% match. The vector sequence is shown in SEQ ID NO: 8.

The prepared vector was transfected into the HD-BIOP3 host cell line (HORIZON Discovery) by electroporation. Cell lines with high productivity were selected through fed-batch, and single cell-derived clones were obtained from them to construct a cell bank.

Cells were thawed, seed culture in a flask and main culture were performed in a bioreactor, and the main culture was centrifuged to collect the supernatant, which was then filtered through a 0.5/0.2 μm filter. In order to remove cell line-derived DNA, the filtered main culture medium was treated with benzonase, a DNA cutting enzyme, and reacted.

The recovered culture medium was first subjected to HIC (hydrophobic interaction chromatography) chromatography, treated with 5 M HCl to a pH of 3.5 to inactivate viruses while stirring, and first ultrafiltration was performed using a 100 kDa filter. In the second chromatography, remaining impure proteins were removed using mixed mode resin and Component A was purified. In the third purification process, Component A was purified using a cation exchange column and impurity proteins and nucleic acids were removed. Viruses were removed by performing a virus filtration process using the column flow-through solution of the third purification process. Next, concentration and buffer exchange were performed by performing secondary ultrafiltration. The ultrafiltration-completed sample was filtered to obtain Component A.

Manufacturing Example 2. Manufacturing of Component B

Component B (SEQ ID NO: 1) serving as an antigen carrier for the SARS-CoV-2 recombinant nanoparticle vaccine was prepared as follows.

BL21 competent cells distributed by Thermofisher were transformed with the vector into which the Component B protein expression gene was introduced. As a vector for gene introduction, pET29b+ was used. The prepared plasmid represented by SEQ ID NO: 3 was transformed into E. coli BL21 strain, and an E. coli strain having kanamycin resistance was selected, and the selected E. coli BL21 strain was inoculated into a soybean-based medium containing yeast extract and heated to 200 °C at 37 ° C. Incubation was performed with shaking until the absorbance was 1.8 or more at rpm. When the culture was completed, sterilized glycerol was added to a final concentration of 25% to construct a cell bank for research.

Cells were thawed and cultured in flasks and main cultured in bioreactors. The seed culture was cultured with shaking at 37 ° C. at 200 rpm so that the final absorbance exceeded 1.5, and then the entire amount of the seed culture was inoculated and cultured at 37 ° C. at 200 rpm until the absorbance was 25 (Growth phase). When the absorbance reached 25, IPTG was added to a final concentration of 0.5 mM (induction phase), and after culturing for 10 hours, the main culture was terminated.

Cells were recovered by centrifugation of the main culture medium, suspended in a buffer solution, and disrupted with a high-pressure disruptor. In order to remove cell line-derived DNA, DNA cleavage enzyme, benzonase, was treated and reacted with the cell homogenate to a final concentration of 5 Unit/ml. The cell lysate after treatment with Benzonase was centrifuged, the supernatant was discarded, and inclusion bodies were recovered. The recovered inclusion bodies were washed using a sodium phosphate buffer solution, dissolved using a sodium phosphate buffer solution containing 2 M urea, and filtered through a 0.2 μm filter.

Purification was performed by first chromatography (negative mode) and second chromatography (positive mode) using a column filled with diethylaminoethyl (DEAE) resin. The recovered solution was filtered through 0.2 μm.

The secondary chromatography recovered solution was concentrated (Ultrafiltration) to 5 kg using a tangential flow filtration system equipped with a 100 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m ² , and It was recovered by performing buffer exchange (diafiltration) using 50 mM Tris buffer (500 mM NaCl, including 0.75 w/v% CHAPS). At this time, the transmembrane pressure (TMP) was maintained at 1 bar or less. The recovered liquid was filtered through 0.2 μm.

Manufacturing Example 3. Assembly of Component A and Component B

Component A and Component B obtained from Preparation Examples 1 and 2 were assembled to prepare a multimeric protein assembly Nanoparticle used in a vaccine.

A reaction of mixing Component A stock solution and Component B at room temperature, 1 hour, and a stirring speed of 80 rpm was performed. The mixing ratio of Component A: Component B was 1: 1.1 (molar concentration). Upon completion of the reaction, the reaction solution was filtered through a 0.2 μm filter.

The filtered assemble reaction solution was concentrated (ultrafiltration) to 4 kg using a tangential flow filtration system equipped with a 300 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m ² , and concentrated (ultrafiltration) to 10 times the volume of the concentrated solution. It was recovered by performing buffer exchange (Diafiltration) using 50 mM Tris buffer. At this time, the transmembrane pressure (TMP) was maintained at 1 bar or less. As a result of the UF/DF process, it was confirmed that Component A, which did not assemble, was removed.

Example 1. Establishment of Optimal Content of Vaccine Formulations Containing Nanoparticles

In order to provide a vaccine formulation containing the nanoparticles prepared in Preparation Examples 1 to 3 as an antigen, the antigen response was tested, and the type of adjuvant and the quantity of raw materials included in the formulation were optimized.

Example 1-1. Confirmation of immune response induced by Nanoparticle when administered with aluminum hydroxide adjuvant

Antigen-specific immunoglobulin and neutralizing antibody induction were measured by administering 0.2 μg and 5 μg of antigen (RBD standard) for each inoculation group to mice with the nanoparticle antigens prepared in Preparation Examples 1 to 3 together with aluminum hydroxide adjuvant. . The results are shown in FIG. 1 and the table below.

Groups sample name RBD (ug/dose) Adjuvant IC ₅₀ (PBNA) PRNT ₅₀ One Vehicle 1 0 - 27 ND* 2 Vehicle 2 0 Alum. H 27 NT** 3 Nanoparticle antigen 0.2 - 987 320 4 Nanoparticle antigen 5 - 2,053 1,280 5 Nanoparticle antigen 0.2 Alum. H 2,422 640 6 Nanoparticle antigen 5 Alum. H 7,433 1,280

Through ELISA, it was confirmed that RBD-specific IgG antibodies were induced, and neutralizing antibodies were also induced. It was confirmed that higher antibody levels were formed when the aluminum hydroxide adjuvant was added. In addition, the cellular immunity of Group 6 (RBD 1 dose 5ug, aluminum hydroxide adjuvantide) was evaluated, and the results are shown in FIG. 2 .

Five weeks after administration, spleen cells isolated from the spleen were treated with the stimulatory antigen to identify T cells secreting mouse IFN-gamma and IL-4. As a result, the number of T cells specifically reacting to the RBD antigen increased.

Example 1-2. Confirmation of immune response induced by Nanoparticle when administered with AS03 Adjuvant

Degree of induction of antigen-specific immunoglobulins and neutralizing antibodies shown by administering 0.2 μg, 1 μg, and 5 μg of antigen (RBD standard) for each group inoculated with the nanoparticle antigens prepared in Preparation Examples 1 to 3 together with AS03 Adjuvant was measured, and the results are shown in FIG. 3 and the table below.

Groups sample name RBD
(µg/dose) Adjuvant IC ₅₀ (PBNA) PRNT ₅₀ One Vehicle 1 0 - 27 ND* 2 Nanoparticle antigen 0.2 - 987 1280 3 Nanoparticle antigen One - 673 1280 4 Nanoparticle antigen 5 - 2,053 1280 5 Nanoparticle antigen 0.2 AS03 56,284 >5120 6 Nanoparticle antigen One AS03 36,582 >5120 7 Nanoparticle antigen 5 AS03 42,414 >5120

Through ELISA, it was confirmed that RBD-specific IgG antibodies were induced, and neutralizing antibodies were also induced. It was confirmed that higher antibody levels were formed when the adjuvant AS03 was added. In addition, the cellular immunity of group 7 (RBD 1 dose 5ug, AS03 adjuvant) was evaluated, and the results are shown in FIG. 4 .

At week 5 after administration, splenocytes isolated from the spleen were treated with a stimulatory antigen to identify T cells secreting mouse IFN-gamma and IL-4. As a result, the number of T cells specifically reacting to the RBD antigen increased.

Through this, it was confirmed that the nanoparticles prepared in Preparation Examples 1 to 3 of the present application were suitable for use as the main component of the COVID-19 vaccine.

Example 1-3. Choice of preservative content

The optimal content of 2-phenoxyethanol (2-PE) or thimerosal was confirmed.

Antimicrobial Effectiveness Testing - Alum (Log CFU/ml reduction) Organisms Preservative Con. 6h 24h 7d 14d 28d Staphylococcus aureus Control 0.1 0.3 5.9 5.9 NR 2-PE
(mg/ml) 5 0.2 1.5 5.9 5.9 NR 7.5 0.7 5.9 5.9 5.9 NR 10 3.7 5.9 5.9 5.9 NR 15 5.9 5.9 5.9 5.9 NR Thimerosal
(ug/ml) 50 0.1 2.2 5.9 5.9 NR 100 0.1 2.3 5.9 5.9 NR 150 0.1 2.3 5.9 5.9 NR 200 0.1 2.2 5.9 5.9 NR Saline 0.1 3.0 5.9 5.9 NR Pseudomonas aeruginosa Control 0.3 5.5 5.5 5.5 NR 2-PE
(mg/ml) 5 4.6 5.9 5.9 5.9 NR 7.5 5.9 5.9 5.9 5.9 NR 10 5.9 5.9 5.9 5.9 NR 15 5.9 5.9 5.9 5.9 NR Thimerosal
(ug/ml) 50 4.6 5.9 5.9 5.9 NR 100 5.9 5.9 5.9 5.9 NR 150 5.9 5.9 5.9 5.9 NR 200 5.9 5.9 5.9 5.9 NR Saline 0.7 2.1 5.9 5.9 NR Escherichia coli Control 1.3 5.2 5.2 5.2 NR 2-PE
(mg/ml) 5 1.7 5.2 5.2 5.2 NR 7.5 5.2 5.2 5.2 5.2 NR 10 5.2 5.2 5.2 5.2 NR 15 5.2 5.2 5.2 5.2 NR Thimerosal
(ug/ml) 50 5.2 5.2 5.2 5.2 NR 100 5.2 5.2 5.2 5.2 NR 150 5.2 5.2 5.2 5.2 NR 200 5.2 5.2 5.2 5.2 NR Saline 1.3 0.2 5.2 5.2 NR

Antimicrobial Effectiveness Testing - Alum (Log CFU/ml reduction) Organisms Preservative Con. 6 h 24h 7d 14d 28d Candida
albicans Control 0.0 0.9 1.8 5.6 NI 2-PE
(mg/ml) 5 0.1 0.1 1.0 5.6 NI 7.5 0.3 0.3 2.9 5.6 NI 10 0.2 0.4 5.6 5.6 NI 15 1.9 5.6 5.6 5.6 NI Thimerosal
(ug/ml) 50 0.3 5.6 5.6 5.6 NI 100 0.5 5.6 5.6 5.6 NI 150 0.6 5.6 5.6 5.6 NI 200 0.6 5.6 5.6 5.6 NI Saline 0.0 0.0 0.0 0.6 NI Aspergillus
brasiliensis Control 0.0 0.6 0.6 1.0 NI 2-PE
(mg/ml) 5 0.0 0.6 1.7 3.0 NI 7.5 0.0 0.6 5.7 5.7 NI 10 0.1 0.7 5.7 5.7 NI 15 0.3 1.7 5.7 5.7 NI Thimerosal
(ug/ml) 50 5.7 5.7 5.7 5.7 NI 100 5.7 5.7 5.7 5.7 NI 150 5.7 5.7 5.7 5.7 NI 200 5.7 5.7 5.7 5.7 NI Saline 0.1 0.4 0.2 5.7 NI

AS03 (Log CFU/ml reduction) Organisms Preservative Con. 6h 24h 7d 14d 28d Staphylococcus aureus Control 0.2 0.0 2.9 6.1 NR 2-PE
(mg/ml) 5 0.2 0.2 6.1 6.1 NR 7.5 0.1 0.5 6.1 6.1 NR 10 0.3 1.1 6.1 6.1 NR Thimerosal
(ug/ml) 50 0.1 6.1 6.1 6.1 NR 75 0.1 6.1 6.1 6.1 NR 100 0.2 6.1 6.1 6.1 NR Pseudomonas aeruginosa Control -0.2 -2.5 -1.8 -1.5 NR 2-PE
(mg/ml) 5 5.9 5.9 5.9 5.9 NR 7.5 5.9 5.9 5.9 5.9 NR 10 5.9 5.9 5.9 5.9 NR Thimerosal
(ug/ml) 50 5.9 5.9 5.9 5.9 NR 75 5.9 5.9 5.9 5.9 NR 100 5.9 5.9 5.9 5.9 NR Escherichia coli Control 0.2 -1.6 -1.4 -1.1 NR 2-PE
(mg/ml) 5 1.0 2.3 6.0 6.0 NR 7.5 2.5 6.0 6.0 6.0 NR 10 6.0 6.0 6.0 6.0 NR Thimerosal
(ug/ml) 50 6.0 6.0 6.0 6.0 NR 75 6.0 6.0 6.0 6.0 NR 100 6.0 6.0 6.0 6.0 NR Candida
albicans Control 0.0 -0.3 0.4 -0.8 NI 2-PE
(mg/ml) 5 0.0 0.0 0.1 0.1 NI 7.5 0.1 0.0 0.2 1.5 NI 10 0.1 0.2 0.4 6.0 NI Thimerosal
(ug/ml) 50 0.8 6.0 6.0 6.0 NI 75 0.9 6.0 6.0 6.0 NI 100 1.6 6.0 6.0 6.0 NI Aspergillus
brasiliensis Control 0.1 0.2 0.3 0.8 NI 2-PE
(mg/ml) 5 0.3 0.4 0.7 1.5 NI 7.5 0.2 0.5 1.4 2.8 NI 10 0.3 0.7 2.6 6.1 NI Thimerosal
(ug/ml) 50 6.1 6.1 6.1 6.1 NI 75 6.1 6.1 6.1 6.1 NI 100 6.1 6.1 6.1 6.1 NI

From this, when using 2-PE or thimerosal as a preservative, the content of the finished product was determined as follows:

The preservative content in the finished drug product containing aluminum hydroxide was set to 5 to 15 mg/ml for 2-PE and 50 to 200 ug/ml for thimerosal. The content of the preservative in the drug product without adjuvant mixing was set at 5 to 10 mg/ml for 2-PE and 50 to 100 μg/ml for thimerosal.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

<110> SK bioscience Co., Ltd. <120> Immunogenic composition <130> KPA211656-KR-P6 <150> KR 10-2021-0137835 <151> 2021-10-15 <150> KR 10-2021-0150799 <151> 2021-11-04 <150> KR 10-2021-0160649 <151> 2021-11-19 <160> 21 <170> KoPatentIn 3.0 <210> 1 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> Component B <400 > 1 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 2 <211> 474 < 212> DNA <213> Artificial Sequence <220> <223> Component B <400> 2 atgaaccagc acagtcacaa ggaccacgaa acggtaagaa tagcggtagt gcgtgcgcgt 60 tggcatgcgg aaattgtgga cgcctgtgtg agtgcgtttg aagccgcgat gcgttgatatt 120 ggcgtggtgatgtc gtgccgg gtgcgtacga aattccactg 180 catgcgcgta ccctggcgga aaccggccgt tatggcgcgg tgttaggcac cgcctttgtg 240 gtgaatggtg gcatttatcg tcacgaattt gtggcgagcg cggttattaa cggcatgatg 300 aatgtgcagc tgaacacggg cgtgccagtg ttaagtgccg tgctgacccc acacaactat 360 gataaaagca aagcccatac cctgctgttc ttagcgctgt ttgcggtgaa aggcatggaa 420 gcattgccgcgtg gcggctgcgtgg cgactgcgcgtgc a agattgcggc gtga 474 <210> 3 <211> 5712 <212> DNA <213> Artificial Sequence <220> <223> plasmid <400> 3 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttcccccg tcaagggct120 ctttctcgcc 0 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctattct tagt6tttatct3 cggtct tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg aattaattctcatagagtaac t 600 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aatagagtgt0 gagaccat8gt agagacgtcaaa atccggtg agaatggcaa aagtttatgc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 aattacaaac aggaatcgaa tgcaaccggc gcagcagacatag tgcc2acatag tgcc2acatag ttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaattccgt cagccagttt agtctgacca tctcatcct0 gt1gtacatcattg agtacatcattg gaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380 cccttgtatt actgtttatg taagcagaca gttttattgt 4tcat0 cctacaa aa gttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560 gtggtttgtt tgccggatca agagctacca acttttttc cgaagg16gtaac cgaagg16gtaac atac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800 cagcggtcgg gctgaacggg gggttcgtgc acacagccca ggtcatagagcg aacgaccggaact 18 ggtcatagga acctac tgagaaagcg ccacgcttcc cgaagggaga 1920 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040 cgtcgattcatat cggagggccatt gggagggggctt acgc cagcaacgcg 2100 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagc0 ccttt2cttg gcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460 gacagccctgtctca ggagctgca tgtgtcagag 2520 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcct0gaggtcc0gagtact 27 ggatt tctgttcatg ggggtaatga taccgatgaa 2760 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880 tcaatggtccatta atgcttctag a gcagcatcc 2940 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcagacg 3120 gcagccacgtcct gatc atgcgcaccc gtggggccgc 3180 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300 gctccgagcat gctccgagcat cgccggaccact cctgtcctac 3360 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540 cctgtcgtgc cagctgggcg aatgaatgggcgt aatgaatgggcgt cgtat 3600 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggttgcccc agcaggcgaa 3720 aatcctgtcc taatactggcggt gt 3780 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960 tcggctgaat ttgagattgccga accgagagattt gccg 4020 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140 ggtcagagac atcaagaaat aacgccggaa cattagtgca atcc4 acccagtcaat tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380 gggccagact ggaggtggca acgccaatca gtacggttgcct 440 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560 catactctgc gacatcgtat aacgttactg 62 gtttactcactgt gggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag ccc8ccgaagtgg gc8ccgaagtggc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc 4980 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctactagaa 5040 ataggacttagaa agcacagt cacaaggacc 5100 acgaaacggt aagaatagcg gtagtgcgtg cgcgttggca tgcggaaatt gtggacgcct 5160 gtgtgagtgc gtttgaagcc gcgatgcgtg atattggcgg cgatcgtttt gccgtggatg 5220 tgtttgatgt gccgggtgcg tacgaaattc cactgcatgc gcgtaccctg gcgttaggata80 gcgttaggata80 gtta ggcaccgcct ttgtggtgaa tggtggcatt tatcgtcacg 5340 aatttgtggc gagcgcggtt attaacggca tgatgaatgt gcagctgaac acgggcgtgc 5400 cagtgttaag tgccgtgctg accccacaca actatgataa aagcaaagcc cataccctgc 5460 gctgttcttgtaga gctgttcttgtaga gaagcggc gcgtgcctgc gtggagattt 5520 tagcggcccg tgaaaagatt gcggcgtgac tcgagcacca ccaccaccac cactgagatc 5580 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac 5640 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa 57t2gggaa <107t2gggaa 5640 ct2> 211> 204 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 RBD <400> 4 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 1 5 10 15 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 20 25 30 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 35 40 45 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 50 55 60 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 65 70 75 80 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 85 90 95 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 100 105 110 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 115 120 125 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 130 135 140 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 145 150 155 160 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 165 170 175 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 180 185 190 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr 195 200 <210> 5 <211> 204 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 5 Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu Arg 1 5 10 15 Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe Ala 20 25 30 Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala Asp 35 40 45 Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile Ile 50 55 60 Gly Ala Gly Thr Val Thr Ser Val Glu Gln Ala Arg Lys Ala Val Glu 65 70 75 80 Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile Ser 85 90 95 Gln Phe Ala Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met Thr 100 105 110 Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu Lys 115 120 125 Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met Lys 130 135 140 Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn Leu 145 150 155 160 Asp Asn Val Ala Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly Val 165 170 175 Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys Ala 180 185 190 Lys Ala Phe Val Glu Lys Ile Arg Gly Ala Thr Glu 195 200 <210> 6 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Component A <400> 6 Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val 1 5 10 15 Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly Thr 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Gly Ser Gly 225 230 235 240 Gly Ser Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Glu Lys Ala Ala 245 250 255 Lys Ala Glu Glu Ala Ala Arg Lys Met Glu Glu Leu Phe Lys Lys His 260 265 270 Lys Ile Val Ala Val Leu Arg Ala Asn Ser Val Glu Glu Ala Ile Glu 275 280 285 Lys Ala Val Ala Val Phe Ala Gly Gly Val His Leu Ile Glu Ile Thr 290 295 300 Phe Thr Val Pro Asp Ala Asp Thr Val Ile Lys Ala Leu Ser Val Leu 305 310 315 320 Lys Glu Lys Gly Ala Ile Ile Gly Ala Gly Thr Val Thr Ser Val Glu 325 330 335 Gln Ala Arg Lys Ala Val Glu Ser Gly Ala Glu Phe Ile Val Ser Pro 340 345 350 His Leu Asp Glu Glu Ile Ser Gln Phe Ala Lys Glu Lys Gly Val Phe 355 360 365 Tyr Met Pro Gly Val Met Thr Pro Thr Glu Leu Val Lys Ala Met Lys 370 375 380 Leu Gly His Thr Ile Leu Lys Leu Phe Pro Gly Glu Val Val Gly Pro 385 390 395 400 Gln Phe Val Lys Ala Met Lys Gly Pro Phe Pro Asn Val Lys Phe Val 405 410 415 Pro Thr Gly Val Asn Leu Asp Asn Val Ala Glu Trp Phe Lys Ala 420 425 430 Gly Val Leu Ala Val Gly Val Gly Ser Ala Leu Val Lys Gly Thr Pro 435 440 445 Asp Glu Val Arg Glu Lys Ala Lys Ala Phe Val Glu Lys Ile Arg Gly 450 455 460 Ala Thr Glu 465 <210> 7 <211> 1404 <212> DNA <213> Artificial Sequence <220> <223> Component A <400> 7 atgggaatcc tgccaagccc tggaatgcca gccctgctgt ccctggtgtc tctgctgagc 60 gtgctgctga tgggatgcgt ggcagagacc ggaacaaggt tccctaacat caccaacctg 120 tgcccattcg gcgaggtgtt taacgccaca cgctttgcct ccgtgtatgc ctggaaccgg 180 aagagaatct ctaattgcgt ggccgactat agcaatagtgctgctc ttaagt gctatggcgt gtctcccacc aagctgaacg acctgtgctt cacaaacgtg 300 tacgccgaca gctttgtgat ccggggcgat gaggtgagac agatcgcacc aggacagacc 360 ggcaagatcg cagactacaa ctataagctg cctgacgatt tcacaggctg cgtatagatcg tgcatagatcgcc 420 ggcggcaact acaattatct gtacaggctg 480 ttccgcaaga gcaacctgaa gccatttgag cgggacatca gcaccgagat ctaccaggca 540 ggctccacac catgcaacgg agtggagggc ttcaattgtt attttcccct gcagagctac 600 ggcttccagc ctaccaatgg cgtgggctat cagccataca gagtggtggt gctgtccgtc agagct60 gccagct60 gccagct60 gccagct60 gtgc ggacctaaga agtccacagg cggctctgga 720 ggaagcggat ccggaggatc cggaggatct ggaagcgaga aggcagcaaa ggcagaggag 780 gcagcaagga agatggagga gctgttcaag aagcacaaga tcgtggccgt gctgagagcc 840 aactctgtgg aggaggcagt cggtcgat ggggcc9 aggt 00 atcgagatca cctttacagt gcccgacgcc gataccgtga tcaaggccct gtccgtgctg 960 aaggagaagg gagcaatcat cggagcagga accgtgacat ctgtggagca ggcaaggaag 1020 gcagtggagt ccggagccga gtttatcgtg tctcctcacc tggatgagga gatctctcccatcag 1080 cctggcgtga tgaccccaac agagctggtg 1140 aaggccatga agctgggcca caccatcctg aagctgttcc caggagaggt ggtgggacca 1200 cagtttgtga aggccatgaa gggcccattc cccaatgtga agtttgtgcc tacaggcggc 1260 gtgaacctgg acaatgttcggggcaggc agagtagct ggggagtggga 1320 tctgccctgg tgaagggaac cccagatgag gtgagggaaa aggccaaggc ctttgtggag 1380 aagatcaggg gagcaacaga gtga 1404 <210> 8 <211> 10704 <212> DNA <213> Artificial Sequence <220> <223> Plasmid (M-2560) <400> 8 tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc gccgcat0ggcct2 atcaggcggcct aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcccc acttcgccgaa 420 ccata agagcccc act ca gttccgccca ttctccgccc 480 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 540 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctagctggt 600 tctttccgcc tcagaaggta cctaaccaag ttcctctttc agaggacct6gtcaccacct6gtcacccat6gtcacccat6gtc cag caagttccca cttgaacaaa aacatcaagc aaatgtactt 720 gtgcctgccc cagggtgaga aagtccaagc catgtatatc tgggttgatg gtactggaga 780 aggactgcgc tgcaaaaccc gcaccctgga ctgtgagccc aagtgtgtag aagagttacc 840 tgatgttaggaccctt tgatgttaggacct ct g ggctccaaca gtgacatgta 900 tctcagccct gttgccatgt ttcgggaccc cttccgcaga gatcccaaca agctggtgtt 960 ctgtgaagtt ttcaagtaca accggaagcc tgcagagacc aatttaaggc actcgtgtaa 1020 acggataatg gacatggtga gcaaccagca cccctggttt ggaatggaac aggagtatac 1080 tctgatggtcga acctagatggtcct cctagatggtccga ggctttc ctgggcccca 1140 aggtccgtat tactgtggtg tgggcgcaga caaagcctat ggcagggata tcgtggaggc 1200 tcactaccgc gcctgcttgt atgctggggt caagattaca ggaacaaatg ctgaggtcat 1260 gcctgcccag tgggaattagga atgagacatcatagga 320 ctgggtggcc cgtttcatct tgcatcgagt atgtgaagac tttggggtaa tagcaacctt 1380 tgaccccaag cccattcctg ggaactggaa tggtgcaggc tgccatacca actttagcac 1440 caaggccatg cgggaggaga atggtctgaa gcacatcgag gaggccatcg agaaactaag 1500 caagcggcac cggtacccagta ttggcgacctaca gcccg 1560 tcgtctgact gggttccacg aaacgtccaa catcaacgac ttttctgctg gtgtcgccaa 1620 tcgcagtgcc agcatccgca ttccccggac tgtcggccag gagaagaaag gttactttga 1680 agaccgccgc ccctgtctgcca attaga1ccca gactttgccgacgt 40 atgccttctc aatgagactg gcgacgagcc cttccaatac aaaaactaac gcccgcccca 1800 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcgcaca catcataaga 1860 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1920 gaaatttgtg atgctacataa gctagttagc tttaatta9 gctagttag accatta 0 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa 2040 agcaagtaaa acctctacaa atgtggtaga attctacgta gataaaagtt ttgttacttt 2100 atagaagaaa ttttgagttt ttgttttttt taataaataa ataaattta atta at216atta attaaatta atta tgtaagtgta aatataataa aacttaatat ctattcaaat 2220 taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt acacatgatt 2280 atctttaacg tacgtcacaa tatgattatc tttctagggt taatctagct gcgtgttctg 2340 cagcgtgtcg agcatcttca tctgctccat cacgctgtaa aaccattgagt ct ca4cccct0gagtg tccacgggtt caaaaacgtg aatgaacgag gcgcgctcat atcatgatta 2460 cgccaagcgc gcccgccggg taactcacgg ggtatccatg tccatttctg cggcatccag 2520 ccaggatacc cgtcctcgct gacgtaatat cccagcgccg caccgctgtc attacatacggc gtcattacggac gtc 2520 gtcgccggt attgttcggg ttgctgatgc 2640 gcttcgggct gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg 2700 tggcctgaac gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca 2760 tggtaaacgt gcgttttcgc tcaacgtcaa tgcagcagca 2 c8catagactctcgt gcacat2cctcgt c ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat 2880 agcgccagct tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca 2940 gctagattaa ccctagaaag atagtctgcg taaaattgac gcatgcattc tttactgatact 3 c gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg 3060 cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata 3120 acgaccgcgt gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact 3180 catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata atgtattactat 3280 catacgataa ttatattgtt atttcatgtt 3220 taga tctggggaca gccccccccc aaagccccca 3300 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct 3360 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg 3420 gggaaggtgg cactgggctcttctg ctct ggtgcctctg ctct tt gagcctgcag 3480 acacctgggg ggatacgggg aaaaagcttt aggctgaaag agagatttag aatgacagaa 3540 tcatagaacg gcctgggttg caaaggagca cagtgctcat ccagatccaa ccccctgcta 3600 tgtgcagggt catcaaccag cagcccaggc tgcccagagc cacatccagc ctctggccag60 atgcctacggcccagg t ccttgggcaa cctgttcagt gcgtcaccac 3720 cctctggggg aaaaactgcc tcctcatatc caacccaaac ctcccctgtc tcagtgtaaa 3780 gccattcccc cttgtcctat caagggggag tttgctgtga cattgttggt ctggggtgac 3840 acatgtttggg cacatgtttgggggc gg aagtgcagga 3900 cagcatggac gtgggacatg caggtgttga gggctctggg acactctcca agtcacagcg 3960 ttcagaacag ccttaaggat aagaagatag gatagaagga caaagagcaa gttaaaaccc 4020 agcatggaga ggagcacaaa aaggccacag acactgctgg tccctgtgtc tgagcctgca 4080 tgtttgatg g gtgtaggggaatgg tgtgctgggaatgg cctgg agagatacag 4140 ctgggtcagt aggactggga caggcagctg gagaattgcc atgtagatgt tcatacaatc 4200 gtcaaatcat gaaggctgga aaagccctcc aagatcccca agaccaaccc caacccaccc 4260 accgtgccca ctggccatgt ccctcagtgc cacattcccca 4260 accgtgccca ctggccatgt ccctcagtgc cacattcccca 4cagttctttcca ccccccacct ccgtgggcag ctgtgccact gcagcaccgc tctttggaga 4380 aggtaaatct tgctaaatcc agcccgaccc tcccctggca caacgtaagg ccattatctc 4440 tcatccaact ccaggacgga gtcagtgagg atggggctct agagtcaaca ggaaagttcc 4500 attggagcca agtaccatatgggtacattga tacataa 4560 ggtcaatggg aggtaagcca atgggttttt cccattactg gcacgtatac tgagtcatta 4620 gggactttcc aatgggtttt gcccagtaca taaggtcaat aggggtgaat caacaggaaa 4680 gtcccattgg agccaagtac actgagtcaa tagggacttt catagaggggggttt ttagagggggttt ttagagggggttt g agtcaatggg tttttcccat tattggcacg tacataaggt 4800 caataggggt gagtcattgg gtttttccag ccaatttaat taaaacgcca tgtactttcc 4860 caccattgac gtcaatgggc tattgaaact aatgcaacgt gacctttaaa cggtactttc 4920 ccatagctga ttaatgggaa agtaccgtagca atgacgtag9 ac4920 80 agggcagcca aaacgtaaca ccgccccggt tttcccctgg aaattccata ttggcacgca 5040 ttctattggc tgagctgcgt tctacgtggg tatataagca gagctctccc tatcagtgat 5100 agagatctcc ctatcagtga tagagatcga gctccagcgtc gggact160 ggct160 ggct160 ggct160 gtctgtac gt gccag ctgttggggt gagtggcggg tgtggcttcc gcgggccccg 5220 gagctggagc cctgctctga gcgggccggg ctgatatgcg agtgtcgtcc gcagggttta 5280 gctgtgagca ttcccacttc gagtggcggg cggtgcgggg gtgagagtgc gaggcctagc 5340 ggcaaccccg tagcctcgcc tcgtgtcgcct aggccgtccct aggtgaggcctcc cttgaggcct 5400 gtgccactcc ggccgcacta tgcgtttttt gtccttgctg ccctcgattg ccttccagca 5460 gcatgggcta acaaagggag ggtgtggggc tcactcttaa ggagcccatg aagcttacgt 5520 tggataggaa tggaagggca ggagg0gtc5 ggagg0catcga ctggggcctcg ctggggcctg cgccg gccac ctggatgggg cgaggcctgt ggctttccga agcaatcggg cgtgagttta 5640 gcctacctgg gccatgtggc cctagcactg ggcacggtct ggcctggcgg tgccgcgttc 5700 ccttgcctcc caacaagggt gaggccgtcc cgcccggcac cagttgcttg cgcggaaaga 5760 tggccgctcc cggggccctg ttgcaaggag ctcaacc8ggggc agcaaccc2ggggg agcgggcggg tgagtcaccc acacaaagga agagggcctt gcccctcgcc ggccgctgct 5880 tcctgtgacc ccgtggtcta tcggccgcat agtcacctcg ggcttctctt gagcaccgct 5940 cgtcgcggcg gggggagggg atctaatggc gttggagttt 6 gttggaggttt gacttcaggcattgt gccagcctgg cgctggaagt cattcttgga atttgcccct ttgagtttgg 6060 agcgaggcta attctcaagc ctcttagcgg ttcaaaggta ttttctaaac ccgtttccag 6120 ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg gaaacccgtc 6180 ggcctccgaa cggtactccg ccaccgaggg acctgagcg6 ccatgcatgcga acct4cgagcga acc acctcgt cgacgccgcc accatgggaa tcctgccaag ccctggaatg ccagccctgc 6300 tgtccctggt gtctctgctg agcgtgctgc tgatgggatg cgtggcagag accggaacaa 6360 ggttccctaa catcaccaac ctgtgcccat tcggcgaggt gtctctgctg ccg ct6gt acgccgct20 ggaac cggaagagaa tctctaattg cgtggccgac tatagcgtgc 6480 tgtacaatag cgcctccttc tctaccttta agtgctatgg cgtgtctccc accaagctga 6540 acgacctgtg cttcacaaac gtgtacgccg acagctttgt gatccggggc gatgaggtga 6600 gacagatcgc accaggacag accggcaaga tcgcagacta caactataag ctgcctgacat gcagcctgacg 6660 attgtcaggacatac atct ggattccaaa gtgggcggca 6720 actacaatta tctgtacagg ctgttccgca agagcaacct gaagccattt gagcggggaca 6780 tcagcaccga gatctaccag gcaggctcca caccatgcaa cggagtggag ggcttcaatt 6840 gttattttcc cctgcagagc tacggctaccag gcaggctcca agcctcagcatgtc 6900 acagagtggt ggtgctgtcc tttgagctgc tgcacgcacc agcaaccgtg tgcggaccta 6960 agaagtccac aggcggctct ggaggaagcg gatccggagg atccggagga tctggaagcg 7020 agaaggcagc aaaggcagag gaggcagcaa ggaagatgga ggagctgttc aagaagctg cgttc aagaaggctg cgatcact tggaggaggc catcgagaag gcagtggccg 7140 tgttcgcagg aggagtgcac ctgatcgaga tcacctttac agtgcccgac gccgataccg 7200 tgatcaaggc cctgtccgtg ctgaaggaga agggagcaat catcggagca ggaaccgtga 7260 catctgtgga gcaggcaagg caggacccagg aggagccttagt tc 7320 acctggatga ggagatctcc cagttcgcca aggagaaggg cgtgttttac atgcctggcg 7380 tgatgacccc aacagagctg gtgaaggcca tgaagctggg ccacaccatc ctgaagctgt 7440 tcccaggaga ggtggtggga ccacagtttg tgaaggccat gaagggccca ttccccaatg 7500 tgaagttttgt gggcgtacacacc gtgg ttcaaggcag 7560 gcgtgctggc agtgggagtg ggatctgccc tggtgaaggg aaccccagat gaggtgaggg 7620 aaaaggccaa ggcctttgtg gagaagatca ggggagcaac agagtgagcg gccgccacac 7680 atcataagat acattgatga gtttggacaa gaatgacaa gaatgacaa 7680 atcataagat acattgatga gtttggacaa 0 tttattggg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 7800 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag 7860 gttttttaaa gcaagtaaaa cctctacaaa tgtggtacac aaagtgggga gctctagagg 7920 gacagccccc ccccaaagcc ccccaggatg cccagggatg cccaggggcc8 cccaggggcc8 cccaggggcc8 cccaggggcc8 cgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca 8040 gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct 8100 tctcgctgct ctttgagcct gcagacct ggggggatac gggacca0 ctaag cc8ccag1gcc agcccccagg gatgtaatta cgtccctccc ccgctagggg 8220 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc 8280 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 8340 cgcttctcgc tgctgaacggaac tgctgagggac attga gcctctgggg tcca 8400 tggccggcca tctgcagatc atatgatcgg atgccgggac cgacgagtgc agaggcgtgc 8460 aagcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8520 caattccaca caacatacga gccggaagca taaagtggagga gcctaatggtagga gctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 8640 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 8700 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 8760 tatcagctca ctcaaaggcg gtaatacggt tatccatagcagg8ga gtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 8880 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 8940 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctggactg 90ccctggactgcc gttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 9060 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 9120 gctccaagct gggctgtgg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 9180 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgcag0 gcagcagccag 92gaacgccactg gcagcagattgcca ggtatg taggcggtgc tacagagttc ttgaagtggt 9300 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 9360 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9420 gtggcag cagtgtgatta gctgtcagtag aaaaggatct caagaagatc 9480 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 9540 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 9600 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgctatcaa gaggctat60 tactgt ttcgttcatc catagttgcc tgactccccg 9720 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 9780 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 9840 ccgagcgcag aagtggtctcatatt cccgccgtta gcaactta cc 9900 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9960 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 10020 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa aggtcggttagc tcctgat0ggtc 10020 t aagttggccg cagtgttatc actcatggtt atggcagcac 10140 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 10200 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc cccaggataccaccta 10200 tacggaccacctaccaa aaagt gctcatcatt ggaaaacgtt 10320 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 10380 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 10440 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 10500 tattattactttcaa tattattactttcaa ca gggtattgt ctcatgagcg 10560 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 10620 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 10680 ggcgtatcac gaggcccttt cgtc 9 <2101> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 9 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 10 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 10 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 11 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 11 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala 20 25 30 Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His Glu 115 120 125 Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala 130 135 140 Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 12 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 12 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 13 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 13 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 14 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 14 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 15 <211 > 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 15 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Asp Ser Asp Ala Asp Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 16 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <220> <221> MISC_FEATURE <222 > (9) <223> Xaa is Tyr or His <220> <221> MISC_FEATURE <222> (82) <223> Xaa is Asn or Asp <220> <221> MISC_FEATURE <222> (87) <223> Xaa is Arg or Asp <220> <221> MISC_FEATURE <222> (105) <223> Xaa is Ser or Asp <220> <221> MISC_FEATURE <222> (119) <223> Xaa is Arg or Glu <220> < 221> MISC_FEATURE <222> (121) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (124) <223> Xaa is Ala or Asp <220> <221> MISC_FEATURE <222> (126 ) <223> Xaa is His or Asp <220> <221> MISC_FEATURE <222> (128) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (150) <223> Xaa is Ala or Asn <400> 16 Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Xaa Tyr Xaa Asp Ser Xaa Glu Xaa His Xaa 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Xaa Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 17 <211 > 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 17 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 18 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide < 400> 18 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Asp Ala Leu Val Lys Gly Asp Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Lys Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 19 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 19 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Glu Phe Val Glu Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asp 145 150 155 160 Leu Asp Asp Val Cys Glu Trp Phe Asp Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Asp Ala Leu Val Glu Gly Asp Pro Asp Glu Val Arg Glu Asp 180 185 190 Ala Lys Glu Phe Val Glu Ile Arg Gly Cys Thr Glu 195 200 205 <210> 20 <211> 205 <212> PRT <213> Artificial Sequence <220> <223 > synthetically constructed nanostructure polypeptide <400> 20 Met Lys Met Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Lys Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Lys Ala Leu Val Lys Gly Lys Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Lys Phe Val Lys Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 21 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <220> <221> MISC_FEATURE <222> (126) <223> Xaa is Thr or Asp <220> <221> MISC_FEATURE <222> (139) <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE <222> (142) <223> Xaa is Lys or Glu <220> < 221> MISC_FEATURE <222> (160) <223> Xaa is Asn or Asp <220> <221> MISC_FEATURE <222> (163) <223> Xaa is Asn or Asp <220> <221> MISC_FEATURE <222> (166 ) <223> Xaa is Glu or Lys <220> <221> MISC_FEATURE <222> (169) <223> Xaa is Lys or Asp <220> <221> MISC_FEATURE <222> (179) <223> Xaa is Ser, Lys or Asp <220> <221> MISC_FEATURE <222> (183) <223> Xaa is Lys or Glu <220> <221> MISC_FEATURE <222> (185) <223> Xaa is Thr, Asp or Lys <220> <221> MISC_FEATURE <222> (192) <223> Xaa is Lys or Asp <220> <221> MISC_FEATURE <222> (195) <223> Xaa is Ala, Glu or Lys <220> <221> MISC_FEATURE <222 > (198) <223> Xaa is Glu or Lys <220> <221> MISC_FEATURE <222> (199) <223> Xaa is Lys or Glu <400> 21 Met Lys Met Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Xaa Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Xaa Phe Val Xaa Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Xaa 145 150 155 160 Leu Asp Xaa Val Cys Xaa Trp Phe Xaa Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Xaa Ala Leu Val Xaa Gly Xaa Pro Asp Glu Val Arg Glu Xaa 180 185 190 Ala Lys Xaa Phe Val Xaa Xaa Ile Arg Gly Cys Thr Glu 195 200 205

Claims (18)

a. (i) SEQ ID NO: 4 or an RBD protein having at least 75% sequence identity therewith and SEQ ID NO: 5 or a protein having at least 75% sequence identity therewith connected via a linker, and (ii) SEQ ID NO: 1 or 75% therewith self-assembled multimeric protein assemblies, including proteins with aberrant sequence identity; and
b. An immunogenic composition comprising a preservative.
The immunogenic composition according to claim 1, wherein the multimeric protein assembly is composed of 20 trimers of the fusion protein of (i) and 12 pentamers of the protein of (ii).
The immunogenic composition of claim 1, wherein the preservative is 2-Phenoxyethanol or Thimerosal.
The immunogenic composition of claim 1 , wherein the RBD protein is included in an amount of 10 to 50 ug/dose.
5. The immunogenic composition according to any one of claims 1 to 4,
c. An immunogenic composition comprising an adjuvant.
6. The method of claim 5, wherein the adjuvant is a mineral-containing composition, an oil emulsion, a saponin formulation, a virus-like particle, a bacterial or microbial derivative, a bioadhesive, a mucoadhesive, a liposome, a polyoxyethylene ether, a polyoxyethylene ester, a polyoxyethylene ester, Phosphazenes, muramyl peptides, imidazoquinolone compounds, thiosemicarbazone compounds, tryptanthrin compounds, human immunomodulators, lipopeptides, benzonaphthyridines, microparticles and immunostimulatory polynucleotides selected from among wherein the immunogenic composition.
6. The immunogenic composition according to claim 5, wherein the adjuvant is selected from an oil-in-water emulsion and an aluminum salt.
8. The immunogenic composition of claim 7, wherein the oil-in-water emulsion adjuvant is AS03 or MF59.
6. The immunogenic composition of claim 5, wherein the adjuvant is an aluminum salt.
10. The method of claim 9, when the preservative is 2-phenoxyethanol, the 2-phenoxyethanol is included in a concentration of 2.5 to 7.5 mg / dose,
Where the preservative is thimerosal, the thimerosal is included in an amount of 25 to 100 ug/dose.
6. The immunogenic composition of claim 5, wherein the adjuvant is AS03.
The method of claim 11, when the preservative is 2-phenoxyethanol, the 2-phenoxyethanol is included in an amount of 2.5 to 5 mg / dose,
Where the preservative is thimerosal, the thimerosal is included in an amount of 25 to 50 ug/dose.
The immunogenic composition according to claim 1, wherein the immunogenic composition is for preventing COVID-19 (Coronavirus Infectious Disease-19).
A vaccine composition for preventing COVID-19 comprising the immunogenic composition of claim 13.
The method of claim 1, wherein the immunogenic composition is
a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or an RBD protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly consisting of 12 pentamers of proteins having at least 75% sequence identity therewith;
b. 2-phenoxyethanol and
c. an aluminum salt or an AS03 adjuvant;
The RBD content of the immunogenic composition is 10 to 50 ug / dose, the immunogenic composition.
16. The immunogenic composition according to claim 15, wherein the composition comprises 2-phenoxyethanol in an amount of 2.5 to 7.5 mg/dose.
The method of claim 1, wherein the immunogenic composition is
a. (i) 20 trimers of fusion proteins in which SEQ ID NO: 4 or an RBD protein having 75% or more sequence identity therewith and SEQ ID NO: 5 or a protein having 75% or more sequence identity therewith are linked via a linker, and (ii) SEQ ID NO: 1 or a self-assembled multimeric protein assembly consisting of 12 pentamers of a protein having at least 75% sequence identity therewith;
b. Thimerosal and
c. an aluminum salt or an AS03 adjuvant;
The RBD content of the immunogenic composition is 10 to 50 ug / dose, Immunogenic composition.
18. The immunogenic composition according to claim 17, wherein the composition comprises thimerosal in an amount of 25 to 100 ug/dose.

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