Surface-modified iron oxide nanoparticles and vaccine against SARS-cov-2 containing said surface-modified iron oxide nanoparticles

Abstract

The problem addressed is to provide surface-modified iron oxide nanoparticles for use in a vaccine exhibiting an infection preventive effect on SARS-CoV-2.　The problem can be solved by surface-modified iron oxide nanoparticles comprising SARS-CoV-2 spike protein and iron oxide nanoparticles, where the spike protein of SARS-CoV-2 is bound to the surface of the iron oxide nanoparticles.

Description

Surface-modified iron oxide nanoparticles and vaccine against SARS-CoV-2 containing the surface-modified iron oxide nanoparticles

[0001] The disclosure in this application relates to surface-modified iron oxide nanoparticles and vaccines against SARS-CoV-2 comprising the surface-modified iron oxide nanoparticles.

[0002] Vaccines against COVID-19, which develops as a result of infection with the SARS-CoV-2 virus, are known. COVID-19 vaccines currently approved in Japan include mRNA vaccines (Pfizer and Moderna) that use the full-length spike protein mRNA as a material, adenovirus vector vaccines (AstraZeneca and Johnson & Johnson) that use the full-length spike protein mRNA as a material, and a protein subunit vaccine (Novavax) that uses the full-length spike protein as a material. Non-Patent Document 1 describes that vaccination is more than 90% effective in preventing severe illness.

[0003] HM El Sahly et. al., “Efficacy of the mRNA-1273 SARS-CoV-2 Vaccine at Completion of Blinded Phase”, N Engl J Med 2021; 385:1774-1785

[0004] All currently approved vaccines are intended to prevent the disease from becoming severe. All are administered by intramuscular injection, and prevent the disease from becoming severe after infection by activating T / B cells in secondary lymphoid tissues and inducing the production of spike protein-specific IgG antibodies in the blood.

[0005] In addition to preventing the worsening of symptoms after infection with COVID-19, it is also desirable to develop a vaccine that is effective in preventing infection with COVID-19. However, at present, no vaccine that is effective in preventing infection with COVID-19 is known.

[0006] The present application has been made to solve the above-mentioned problems. As a result of extensive research, the present inventors have newly discovered that (1) surface-modified iron oxide nanoparticles can be prepared by binding the spike protein of SARS-CoV-2 to the surface of iron oxide nanoparticles, (2) when a vaccine containing the surface-modified iron oxide nanoparticles is administered to the nasal mucosa or respiratory tract, it induces the secretion of spike protein-specific IgA antibodies (hereinafter sometimes simply referred to as "IgA antibodies") into the mucosa, and (3) the induced IgA antibodies exert a preventive effect against infection with SARS-CoV-2.

[0007] That is, an object of the disclosure of the present application is to provide surface-modified iron oxide nanoparticles for use in vaccines that are effective in preventing infection with SARS-CoV-2, and vaccines containing the surface-modified iron oxide nanoparticles.

[0008] (1) Surface-modified iron oxide nanoparticles comprising a SARS-CoV-2 spike protein and iron oxide nanoparticles, wherein the SARS-CoV-2 spike protein is bound to the surface of the iron oxide nanoparticles. (2) The surface-modified iron oxide nanoparticles according to (1) above, wherein the SARS-CoV-2 spike protein is bound to the iron oxide nanoparticles via an arginine sequence added to its terminus. (3) The surface-modified iron oxide nanoparticles according to (1) above, wherein the SARS-CoV-2 spike protein is bound to the iron oxide nanoparticles via a histidine sequence not containing arginine added to its terminus. (4) The surface-modified iron oxide nanoparticles according to (3) above, wherein the number of histidines contained in the histidine sequence is 5 to 35. (5) The surface-modified iron oxide nanoparticles according to (1) above, wherein X is the average particle diameter of the iron oxide nanoparticles and Y is the length of the spike protein, and X + 2Y is 90 nm to 110 nm. (6) The surface-modified iron oxide nanoparticles according to (1) above, wherein the number of spike proteins bound to the surface of the iron oxide nanoparticles is 5 to 50. (7) The surface-modified iron oxide nanoparticles according to (1) above, wherein the iron oxide nanoparticles are surface-modified with carboxy groups. (8) A vaccine against SARS-CoV-2, comprising the surface-modified iron oxide nanoparticles according to any one of (1) to (7) above as an active ingredient. (9) The vaccine according to (8) above, which is administered to the nasal mucosa or respiratory tract. (10) The vaccine according to (9) above, which induces the production of secretory IgA antibodies by administering the vaccine to the nasal mucosa or respiratory tract. (11) The vaccine according to (8) above, wherein the iron oxide nanoparticles have an adjuvant function.

[0009] By using a vaccine prepared using the surface-modified iron oxide nanoparticles disclosed in this application, a preventive effect against infection with SARS-CoV-2 can be obtained.

[0010] Figure 1 is a photograph (substitute for a drawing) showing the results of Example 1, in which a fixed amount of S-Arg-His was conjugated to different amounts of iron oxide nanoparticles (IONPs), and the proteins not bound to the iron oxide nanoparticles in the supernatant were separated on a polyacrylamide gel and the S protein was stained with Coomassie brilliant blue. Figure 2A is a graph showing the results of immunization of mice following administration of S-Arg-His (a fixed amount of iron oxide nanoparticles) and measurement of the S protein-specific antibody titer in Example 2. Figure 2B is a graph showing the results of immunization of mice following administration of S-Arg-His (varying amounts of iron oxide nanoparticles) and measurement of the S protein-specific antibody titer in Example 2. Figure 3 is a graph showing the results of confirming the change in antibodies induced when the amount of S protein administered was increased while the amount of S protein bound per iron oxide nanoparticle was kept the same in Example 3. Figure 4 is a graph showing the results of immunization of mice following administration of S-His and measurement of the S protein-specific antibody titer in Example 4. Figure 5 is a graph showing the results of calculating the neutralizing antibody titers in the BALF and serum collected in Example 4 in Example 5. Figure 6 is a drawing-substitute photograph, a Western blotting photograph showing the difference in S protein expression levels between the presence and absence of an Arg tag in a reference example. Figure 7 is a graph showing the results of measuring antibody production levels when the vaccine dose and administration interval were changed in Example 6. Figure 8A is a graph showing the results of measuring antibody titers in bronchoalveolar lavage fluid (BALF) and serum one month after the final vaccine administration in Example 7. Figure 8B is a graph showing the results of measuring antibody titers in bronchoalveolar lavage fluid (BALF) and serum three months after the final vaccine administration in Example 7. Figure 9 is a graph showing the results of measuring antibody titers when only S protein and only PBS were administered one month after the final vaccine administration in Example 7. FIG. 10A is a graph showing the results of measuring TNF-α concentrations in the supernatants obtained when a mouse macrophage cell line was cultured with IONPs at various concentrations in Example 8.Figure 10B is a graph showing the results of measuring the expression levels of each protein, CD40, CD80, CD86, and MHC class II (IA / IE), which are macrophage activation markers, when a mouse macrophage cell line and IONPs were cultured in Example 8.

[0011] The surface-modified iron oxide nanoparticles (hereinafter may be referred to as "modified nanoparticles") and the vaccine against SARS-CoV-2 (hereinafter may be referred to as "vaccine") disclosed in the present application are described in detail below.

[0012] In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. Furthermore, in this specification, numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") are to be interpreted as indicating numerical values, numerical ranges, and properties that include errors generally accepted in the technical field.

[0013] (Embodiments of Modified Nanoparticles) The modified nanoparticles according to the embodiments include spike proteins (hereinafter, sometimes referred to as "S proteins") of SARS-CoV-2 and iron oxide (IO) nanoparticles, with the S proteins bound to the surfaces of the iron oxide nanoparticles.

[0014] There are many mutant strains of SARS-CoV-2, including the alpha, delta, and omicron strains, and these mutant strains have several amino acid changes in the S protein. The S protein used in the modified nanoparticles according to the embodiment may be the S protein of all currently known mutant strains, or may be the S protein of a mutant strain that will emerge in the future. Information on the amino acid sequence and genome base sequence of the S protein is available, but is not limited to, from https: / / viralzone.expasy.org / 9556, etc. The full-length S protein may be used, or a portion may be deleted or mutated as long as it functions as a vaccine. Furthermore, as described below, an amino acid sequence may be added to facilitate binding to iron oxide nanoparticles.

[0015] The S protein may be produced by a known method based on the above sequence. Examples of methods include, but are not limited to, producing the S protein by transforming bacterial, insect, or animal cells with a plasmid incorporating the genomic sequence, or synthesizing the S protein by cell-free protein synthesis. Alternatively, a commercially available S protein may be used.

[0016] Iron oxide nanoparticles function as adjuvants when administered with a vaccine. Examples of iron oxide nanoparticles include maghemite (γ-Fe) nanoparticles having a diameter of 1 nm to 5 μm. ２ O ３ ) and / or magnetite (Fe ３ O ４ ) particles.

[0017] In recent years, iron oxide nanoparticles have attracted increasing interest in the biomedical field due to their unique properties, such as superparamagnetic properties, biocompatibility, and non-toxicity. For example, iron oxide nanoparticles have already been used as contrast agents in magnetic resonance imaging (MRI), and their biosafety and uptake by phagocytes have already been demonstrated. Therefore, iron oxide nanoparticles are a highly safe adjuvant.

[0018] The diameter (average particle size) of the iron oxide nanoparticles is not particularly limited as long as it is within a range that allows antibodies to be produced in vivo when used as a vaccine. If the iron oxide nanoparticles are too small, it becomes difficult for a sufficient amount of S protein to bind to the surface, so the diameter is not particularly limited, but examples include 2 nm or more, 4 nm or more, 6 nm or more, 8 nm or more, and 10 nm or more. On the other hand, the upper limit of the diameter is not particularly limited, but examples include 5 μm or less, 3 μm or less, 1 μm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, and 200 nm or less.

[0019] The diameter of SARS-CoV-2 is said to be approximately 60 nm to 140 nm, with an average of approximately 100 nm. Furthermore, the size (length) of the S protein is said to be approximately 20 nm to 23 nm. Therefore, if iron oxide nanoparticles correspond to the SARS-CoV-2 envelope, the diameter of the iron oxide nanoparticles would be approximately 14 nm to 100 nm. Therefore, the lower limit of the diameter (average particle size) of iron oxide nanoparticles can be 14 nm or more, 18 nm or more, 22 nm or more, 26 nm or more, 30 nm or more, 34 nm or more, 38 nm or more, 42 nm or more, 46 nm or more, 50 nm or more, or 54 nm or more. Meanwhile, the upper limit of the diameter of iron oxide nanoparticles can be 100 nm or less, 96 nm or less, 92 nm or less, 88 nm or less, 84 nm or less, 80 nm or less, 76 nm or less, 72 nm or less, 68 nm or less, 64 nm or less, or 60 nm or less.

[0020] Furthermore, the size of the modified nanoparticles according to the embodiment is preferably close to the size of SARS-CoV-2. For example, when the average particle size (diameter) of the iron oxide nanoparticles is defined as X and the length (size) of the S protein is defined as Y, X+2Y (corresponding to the size of the modified nanoparticles) can be 60 nm to 140 nm, 70 nm to 130 nm, 80 nm to 120 nm, 85 nm to 115 nm, 90 nm to 110 nm, or 95 nm to 105 nm.

[0021] The surface of iron oxide nanoparticles is not particularly limited as long as it can bind to S protein. Iron oxide nanoparticles may be self-prepared or commercially available. Many types of iron oxide nanoparticles are commercially available, and commercially available iron oxide nanoparticles are known to be surface-modified or unmodified. The surface of iron oxide nanoparticles used as raw materials may be modified or unmodified. As used herein, "modified surface" refers to a surface coated with a component other than iron (Fe) and oxygen (O), which are components of iron oxide. "Unmodified surface" refers to nanoparticles whose surface is not coated with a component other than iron (Fe) and oxygen (O), which are components of iron oxide, but is composed of iron and oxygen atoms. Examples of modified surfaces include, but are not limited to, coating with biocompatible materials such as polysaccharides (e.g., dextran) or lipid molecules, and modification with functional groups (e.g., carboxyl groups or amino groups).

[0022] The binding of S protein to the surface of iron oxide nanoparticles is not particularly limited as long as it can bind S protein to the surface of the iron oxide nanoparticles. Although not limited, a sequence consisting of arginine, histidine, or a mixture of arginine and histidine (hereinafter referred to as the "additional sequence") can be added to the C-terminus of S protein. When iron oxide nanoparticles pre-modified with carboxy groups are used, the additional sequence portion can be bound to the carboxy groups. When iron oxide nanoparticles not modified with carboxy groups are used, carboxy groups can be introduced onto the surface of the iron oxide nanoparticles and then the additional sequence portion can be bound to the carboxy groups. The length of the additional sequence is not particularly limited as long as it can bind S protein to the carboxy groups. In the case of arginine only or histidine only, examples of the length of the additional sequence include, but are not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 residues. The number of amino acids contained in the arginine sequence and histidine sequence may be any number selected from the above numbers, and may be described as, for example, 6 to 8. When arginine and histidine are mixed, it is desirable to mix them in blocks. When arginine and histidine are mixed, the length may be the same as that of the above-mentioned arginine alone or histidine alone.

[0023] When producing S protein using the S protein expression plasmid to which the above-mentioned additional sequence has been added, the yield of S protein is improved when only histidine is used as the additional sequence. Therefore, costs can be reduced by producing modified nanoparticles using S protein to which only a histidine sequence has been added as the additional sequence.

[0024] Other than the above-mentioned binding of the additional sequence to the carboxy group, there is also a method of binding the S protein to iron oxide nanoparticles using various linkers.

[0025] There are no particular limitations on the number of S proteins bound to the surface of iron oxide nanoparticles, as long as the number is within the range that allows antibody production. It is said that there are 26±15 S proteins on the surface of SARS-CoV-2 (H. Yao, et al., "Molecular Architecture of the SARS-CoV-2 Virus," 2020, Cell 183, 730-738). Therefore, the number of S proteins bound to the surface of iron oxide nanoparticles may be close to the number of S proteins in actual SARS-CoV-2. Although not limited thereto, for example, the lower limit may be 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more, and the upper limit may be 50 or less, 49 or less, 48 ​​or less, 47 or less, 46 or less, 45 or less, 44 or less, 43 or less, 42 or less, 41 or less, 40 or less, 39 or less, 38 or less, 37 or less, 36 or less, 35 or less, 34 or less, 33 or less, 32 or less, 31 or less, 30 or less, 29 or less, 28 or less, 27 or less, or 26. The number of S proteins may be any number selected from the above-mentioned lower limit and upper limit, for example, 5 to 35. The above number represents the average number of S proteins bound to the surface of the prepared modified nanoparticles.

[0026] (Embodiments of Vaccines) Next, vaccines according to embodiments will be described. The vaccines contain, as an active ingredient, any of the modified nanoparticles described in the embodiments of the modified nanoparticles above. The vaccines may be composed solely of modified nanoparticles, or may contain additives. The vaccines disclosed in the present application are intended to induce the production of secretory IgA antibodies by administration to the nasal mucosa or respiratory tract, rather than by intramuscular injection. There are no particular limitations on the additives as long as they are suitable for administration to the nasal mucosa or respiratory tract. Examples of additives include, but are not limited to, adjuvants such as aluminum salts; stabilizers such as proteins, amino acids, sugars, and gelatin; preservatives such as antibacterial agents, thimerosal, phenoxyethanol, and formalin; buffers; mucosal protectants; and the like.

[0027] The vaccine may be in the form of powder (lyophilized), liquid, precipitate, or the like.

[0028] The dosage of the vaccine can be determined appropriately by a doctor, taking into consideration factors such as age.

[0029] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application.

[0030] [Preparation of modified nanoparticles] Example 1 (1) Preparation of plasmids expressing S protein (pαH-S-GSAS / D614G, pαH-S-GSAS / D614G-Arg) Plasmid pαH-S-GSAS / D614G (Gobeil SMC, et al. Cell Reports 2020, 34, 108630) expressing the S protein of SARS-CoV-2 was purchased from Addgene (USA). The S gene of pαH-S-GSAS / D614G has two mutations (GSAS and D614G) in the S protein of the SARS-CoV-2 Wuhan strain, and the transmembrane region and subsequent regions have been deleted. A trimerization sequence and a Strep-Tac II sequence corresponding to eight His residues as tags for protein purification have been added. Meanwhile, pαH-S-GSAS / D614G-Arg, which contains an Arg sequence, was prepared by adding a sequence corresponding to six Arg residues upstream of the eight His residues in the above pαH-S-GSAS / D614G.

[0031] (2) Expression and Purification of S-His and S-Arg-His pαH-S-GSAS / D614G was introduced into HEK293 (human embryonic kidney)-derived FreeStyle 293-F cells (ThermoFisher Scientific, USA), and the transfected cells were cultured. S-His secreted into the culture medium was affinity purified (IBA Lifescience GmbH, Germany) using a Strep-Tac II tag, and purified S-His was obtained by removing low-molecular-weight contaminants by gel filtration or centrifugal concentration. Protein concentration was measured using NanoDrop One. S-Arg-His was also obtained by expressing and purifying pαH-S-GSAS / D614G-Arg using the same procedure as above.

[0032] (3) Binding of S-Arg-His to iron oxide nanoparticles (IONPs) A fixed weight (2 μg) of S-Arg-His was mixed with 0.5 times (1 μg) to 20 times (40 μg) of iron oxide nanoparticles (diameter 50 nm, Micromod Particle Technology GmbH, Germany, surface-carboxylated), left for 30 minutes, and then centrifuged (13,200 rpm, 15 min) to sediment the iron oxide nanoparticles. Proteins not bound to the iron oxide nanoparticles in the supernatant were separated on a polyacrylamide gel, and the S protein was stained with Coomassie brilliant blue.

[0033] The results are shown in Figure 1. As is clear from Figure 1, adding iron oxide nanoparticles in an amount equal to the weight of S protein reduced the amount of unbound S protein, and adding iron oxide nanoparticles in an amount equal to twice the weight of S protein almost eliminated the unbound protein. In other words, it was confirmed that adding iron oxide nanoparticles in an amount equal to approximately twice the weight of S protein caused almost the entire amount of S protein to bind to the iron oxide nanoparticles. Based on this finding, it was confirmed that adjusting the ratio of iron oxide nanoparticles to S protein allows the amount of S protein bound to the iron oxide nanoparticles, in other words, the number of S protein bound to the surface of one iron oxide nanoparticle, to be adjusted.

[0034] [Immunization of Mice by Administration of S-Arg-His and Measurement of S Protein-Specific Antibody Titer] Example 2 Modified nanoparticles were prepared by mixing 6 μg of S-Arg-His with 480 μg of iron oxide nanoparticles and leaving the mixture for 30 minutes. The prepared modified nanoparticles were used as vaccines in Example 2 and in Examples 3 to 5 described below. The prepared modified nanoparticles were administered via the airway to mice (C57BL / 6, male, 7 weeks old, CLEA Japan, n=6). As a control, an experiment was conducted in which the solvent PBS, 480 μg of iron oxide nanoparticles, and 6 μg of S-Arg-His were similarly administered ( FIG. 2A ; in FIG. 2A , iron oxide nanoparticles are referred to as "IONP" and S-Arg-His as "S"). In addition, 6 μg of S-Arg-His was mixed with 2 to 80 times the weight of iron oxide nanoparticles and similarly administered to mice (Figure 2B). Three doses were administered weekly, and bronchoalveolar lavage fluid (BALF) and serum were collected one week after the final dose. S protein-specific anti-IgA and anti-IgG antibodies in BALF and serum were detected using S protein and enzyme-labeled anti-mouse IgA antibody (Southern Biotech, USA) and anti-mouse IgG antibody (Southern Biotech, USA).

[0035] As is clear from Figure 2A, airway administration of purified S-Arg-His alone induced almost no antibodies. On the other hand, airway administration of the S-Arg-His-conjugated iron oxide nanoparticles prepared in Example 1 was confirmed to induce IgA and IgG in the bronchoalveolar cells. Furthermore, while almost no IgA was induced in serum, a large amount of IgG was confirmed to be induced. As described above, the S protein-conjugated iron oxide nanoparticle vaccine disclosed in this application induced the secretion of IgA and IgG into the mucosa of the bronchoalveolar cells, confirming that it can be expected to be effective in preventing infection with SARS-CoV-2, and that even if infection does occur, the IgG produced in the serum can also be expected to have a preventive effect on the severity of the disease.

[0036] Next, a fixed amount (6 μg) of S-Arg-His conjugated with iron oxide nanoparticles at 2- to 80-fold weight was administered to mice in the same manner as above, and BALF was collected. The antibody levels in the BALF were measured at 2- to 10-fold and 10- to 80-fold doses. As shown in Figure 2B, even with the same amount of S protein administered, the amount of antibody induced increased with increasing numbers of iron oxide nanoparticles. The increase was dose-dependent from 2- to 10-fold, but the increase was smaller from 10- to 80-fold. This suggests that the number of iron oxide nanoparticles taken up by phagocytes is more important when the same amount of protein is administered.

[0037] Example 3 Next, an experiment was conducted to confirm changes in antibodies induced when the amount of S protein bound per iron oxide nanoparticle was kept the same and the amount of S protein administered was increased. More specifically, the experiment was conducted in the same manner as in Example 2 above, except that the following samples were used: S protein (0 μg) / iron oxide nanoparticles (0 μg) S protein (3 μg) / iron oxide nanoparticles (60 μg) S protein (6 μg) / iron oxide nanoparticles (120 μg) S protein (12 μg) / iron oxide nanoparticles (240 μg) S protein (24 μg) / iron oxide nanoparticles (480 μg)

[0038] The results are shown in Figure 3. As is clear from Figure 3, the IgA antibody titer in BALF increased in a dose-dependent manner with the administered S protein, but plateaued at 12 μg. These results confirmed that when actually administering modified nanoparticles as a vaccine, it is preferable to design the total amount of S protein to be administered and the number of S protein molecules bound to each iron oxide nanoparticle within a predetermined range.

[0039] [Immunization of mice by administration of S-His and measurement of S protein-specific antibody titer] Example 4 An experiment was conducted in the same manner as in Example 2, except that S-His (6 μg) was used instead of S-Arg-His in Example 2, the amount of iron oxide nanoparticles was 20 times higher (120 μg), and PBS, iron oxide nanoparticles alone (120 μg), and S-His alone (6 μg) were used as controls.

[0040] The results are shown in Figure 4. As is clear from Figure 4, when S-His (represented as "S" in Figure 4) was used, IgG and IgA antibodies were induced in the bronchoalveolar cells, as with S-Arg-His. Furthermore, as is clear from Figures 2A and 4, almost no induction of IgG or IgA antibodies was observed in the control group, confirming that it is extremely important to bind S protein to iron oxide nanoparticles.

[0041] Since the molecular weight of the S protein excluding sugar is approximately 432 kDa, the number of molecules per μg is approximately 1.39 × 10 12 On the other hand, since the diameter of the iron oxide nanoparticles used in this example is about 50 nm, the number of molecules per 1 μg is 2.86 × 10 9 Therefore, when 120 μg of iron oxide nanoparticles were added to 6 μg of S-Arg-His, approximately 24 S proteins were calculated to be bound to each iron oxide nanoparticle. The size and number of S proteins bound to the surface of the modified nanoparticles used in Examples 2 and 4 are close to those of actual SARS-CoV-2.

[0042] [Measurement of Neutralizing Antibody Titer] Example 5 To measure the neutralizing activity of antibodies in the BALF and serum collected in Example 4, a pseudovirus of vesicular stomatitis virus (VSV) (St19pv; Tani H, et al. Virol J. 2021, 18, 16) that has S protein on its surface and produces the luminescent protein luciferase after infection was used. BALF or serum was added to Vero cells, which were then infected with St19pv and the amount of luminescence measured. The neutralizing antibody titer was calculated from the attenuation of luminescence due to infection inhibition.

[0043] The results are shown in Figure 5. The antibodies in the BALF and serum collected in Example 4 exhibited high neutralizing antibody titers. From these results, it was confirmed that viral infection can be prevented by administering the modified nanoparticles produced in Example 4 via the respiratory tract.

[0044] [Regarding differences in S protein expression levels] <Reference Example> pαH-S-GSAS / D614G-Arg (6 Arg - 8 His) and pαH-S-GSAS / D614G (8 His, 0 Arg) prepared in Example 1 were expressed under the same conditions as in Example 1. The culture supernatant was collected and subjected to polyacrylamide electrophoresis, after which the S protein was detected by Western blotting using a tag (Strep-Tactin HRP-conjugate (IBA Lifesciences)) common to the two types of plasmids.

[0045] The results are shown in Figure 6. As is clear from Figure 6, when S protein (S-His) was expressed using pαH-S-GSAS / D614G that did not contain an Arg tag, the expression level was significantly higher than when the Arg tag was included. These results indicate that producing modified nanoparticles using S protein that contains a His tag but does not contain an Arg tag can improve the efficiency of raw material production (leading to reduced vaccine costs).

[0046] [Effects of vaccine dose and administration interval on antibody production amount] Example 6 A vaccine was prepared using S-His (12 μg) and iron oxide nanoparticles (120 μg) in addition to the S-His (6 μg) and iron oxide nanoparticles (120 μg) prepared in Example 4. Next, an experiment was carried out using the two types of vaccine prepared in the same procedure as in Example 4, except that the vaccines were administered three times every other week (6 μg x 3, 12 μg x 3) and twice every other week (6 μg x 2, 12 μg x 2).

[0047] The results are shown in Figure 7. As is clear from Figure 7, no significant difference was observed in the amount of antibody produced even when the dose and administration interval of S-His administered were changed.

[0048] [Measurement of duration of immunity after vaccine administration and confirmation of booster effect by vaccine administration] <Example 7> Next, S-IONP, S(S-His), IONP, and PBS were prepared using the same procedure as in Example 4. Mice were immunized by administering the vaccine via the airway using the same procedure as in Example 4, except that the vaccine was administered twice every other week as in Example 6. Bronchoalveolar lavage fluid (BALF) and serum were collected one month and three months after the final vaccine administration, and antibody titers were measured using the same procedure as in Example 4. Figure 8A shows the results one month later, and Figure 8B shows the results three months later.

[0049] Next, one month after the final vaccination, 6 μg of S protein (S-His) alone or PBS alone as a control was administered, and the antibody titer was measured three days later. The results are shown in Figure 9.

[0050] As is clear from Figures 8A and 8B, serum IgG had relatively high antibody titers even after 1 month and 3 months. Meanwhile, the antibody titers of IgA and IgG in BALF decreased over time. However, as shown in Figure 9, administration of S protein (S-His) alone after 1 month significantly increased the antibody titers of IgA and IgG in BALF. On the other hand, as shown in Figure 2A, administration of S protein alone via the airway did not significantly increase the antibody titers of IgA and IgG in BALF.

[0051] From the above results, it was confirmed that administration of the vaccine disclosed in the present application to a subject once, and administration of the S protein alone functioned as a booster for IgA and IgG production in the bronchoalveolar tract. Since IgA and IgG were produced in the bronchoalveolar tract by administration of the S protein alone, it is believed that the vaccine disclosed in the present application induced memory B cells on the mucosal surface of the bronchoalveolar tract, and secreted IgA and IgG antibodies immediately after infection. Therefore, the vaccine disclosed in the present application is expected to be effective in preventing infection itself, since IgA and IgG antibodies are rapidly secreted upon contact with the S protein even long after vaccine administration.

[0052] [Confirmation of the adjuvant effect of iron oxide nanoparticles (IONP)] <Example 8> Next, an experiment was conducted to examine the function of iron oxide nanoparticles (IONP), which are a component of the vaccine. The procedure is described below. (1) 1 × 10 ５ RAW264.7 cells (obtained from the RIKEN Cell Bank) were cultured for 24 hours with various concentrations of IONPs and 10 ng / ml of LPS (lipopolysaccharide, derived from Escherichia coli O111, Sigma-Aldrich, L2630) as a positive control, at a concentration of 1 / ml. The TNF-α concentration in the supernatant was measured using an ELISA kit (BioLegend). The results are shown in Figure 10A.

[0053] (2) 1 x 10 ５ RAW264.7 cells, a mouse macrophage cell line, were cultured at 1000 μg / ml of IONPs and 10 ng / ml of LPS (as a positive control) for 24 hours. Cells were then washed three times with 0.1% sodium azide / 1% FBS / PBS and blocked with anti-Fcγ RII / III antibody (clone 2.4G2, purified from a purchased hybridoma (ATCC HB-197)). Then, cells were stained with PE-anti-CD40 antibody (BioLegend), FITC-anti-CD80 antibody (BioLegend), APC / cy7-anti-CD86 antibody (BioLegend), and APC-anti-IA / IE antibody (BioLegend). After washing twice, the cells were analyzed using a BD FACS Verse® flow cytometer (BD Japan). The results are shown in Figure 10B. The three bar graphs in Figure 9B showing the expression levels of CD40, CD80, CD86, and MHC class II (IA / IE) proteins show the results for 10% FBS / RPMI 1640 (Medium) without IONP or LPS on the left, the results for IONP in the center, and the results for LPS on the right.

[0054] As is clear from Figure 10A, the expression level of TNF-α, a cytokine secreted by mouse macrophages, increased with the amount of iron oxide nanoparticles (IONPs). Also, as is clear from Figure 10B, administration of IONPs increased the expression levels of macrophage activation markers CD40, CD80, CD86, and MHC class II (IA / IE) proteins compared to Medium.

[0055] From the above results, it was confirmed that in the vaccine disclosed in this application, iron oxide nanoparticles (IONPs) simultaneously perform two different functions: carrier function for S protein and adjuvant function (immunostimulatory activity).

[0056] The modified nanoparticles disclosed in the present application can be used to prepare vaccines that can prevent infection with SARS-CoV-2, and are therefore useful for the medical industry.

Claims

1. A surface-modified iron oxide nanoparticle comprising a spike protein of SARS-CoV-2 and an iron oxide nanoparticle, the spike protein of SARS-CoV-2 being bound to the surface of the iron oxide nanoparticle.

2. The surface-modified iron oxide nanoparticles according to claim 1, wherein the spike protein of SARS-CoV-2 is bound to the iron oxide nanoparticles via an arginine sequence added to its terminus.

3. The surface-modified iron oxide nanoparticles according to claim 1, wherein the spike protein of SARS-CoV-2 is bound to the iron oxide nanoparticles via a histidine sequence not containing arginine added to its terminus.

4. The surface-modified iron oxide nanoparticles according to claim 3, wherein the number of histidines contained in the histidine sequence is 5 to 35.

5. The surface-modified iron oxide nanoparticles according to claim 1, wherein X+2Y is 90 nm to 110 nm, where X is the average particle size of the iron oxide nanoparticles and Y is the length of the spike protein.

6. The surface-modified iron oxide nanoparticles according to claim 1, wherein the number of spike proteins bound to the surface of the iron oxide nanoparticles is 5 to 50.

7. The surface-modified iron oxide nanoparticles according to claim 1, wherein the iron oxide nanoparticles are surface-modified with a carboxy group.

8. A vaccine against SARS-CoV-2, comprising the surface-modified iron oxide nanoparticles according to any one of claims 1 to 7 as an active ingredient.

9. The vaccine according to claim 8, which is administered to the nasal mucosa or respiratory tract.

10. The vaccine according to claim 9, wherein the production of secretory IgA antibodies is induced by administration of the vaccine to the nasal mucosa or airways.

11. The vaccine according to claim 8, wherein the iron oxide nanoparticles have an adjuvant function.

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