WO2023101007A1

WO2023101007A1 - Antigen-protein expression vector and utilization thereof

Info

Publication number: WO2023101007A1
Application number: PCT/JP2022/044526
Authority: WO
Inventors: 真之助鈴木; 賢太郎鷲澤; 薫瀧澤; 教暢町田; 好司草野; 竜太郎島崎; 豊隆森
Original assignee: 株式会社アイロムグループ
Priority date: 2021-12-03
Filing date: 2022-12-02
Publication date: 2023-06-08

Abstract

The present invention provides: a vector that expresses a fusion protein containing a secretion signal, an antigen-protein fragment, and a trimer forming domain; and usages thereof. Said vector has an excellent ability to secrete and release an antigen-protein fragment to the cell exterior. The present invention is capable of: maximizing the expression by minimizing a vaccine antigen and adding a trimer forming domain thereto; and maximizing secreted and released amounts of expression products by additionally adding a secretion signal. The present technology is assumed to be useful as a vector technology that can be programmed so as to induce both humoral immunity and cell-mediated immunity.

Description

Antigen protein expression vector and its use

The present invention relates to antigen protein expression vectors. Specifically, the present invention relates to, but is not limited to, vectors capable of strong expression, intracellular persistence, extramembrane secretion and release of protein factors that induce vaccine effects, and their use. In addition, the present invention is not limited thereto, but for example, for the purpose of inducing strong humoral and cell-mediated immunity as an infectious disease vaccine, the vaccine antigen protein is expressed intracellularly after inoculation, The present invention relates to a vaccine vector technology, etc., in which a portion of the vaccine is left inside the cell and a portion is secreted and released outside the cell.

For the purpose of manufacturing vaccines that induce humoral and cell-mediated immunity to protect against infectious diseases, target pathogenic viruses are attenuated or inactivated and inoculated without inducing pathogenicity. , attenuated vaccines (live vaccines) and inactivated vaccines are known as techniques for inducing immunogenicity (Crott S. et al., The Journal of Immunology 171: 4969-4973, 2003 doi: 10.4049/jimmunol .171.10.4969; Bandyopadhyay A. S. et al., Clinical Infectious Diseases 67(S1): S35-S41, 2018 doi.org/10.1093/cid/ciy633).

When an attenuated strain vaccine or an inactivated vaccine is produced and inoculated, pathogen-specific antibodies are induced, but since the whole pathogen is used as a vaccine, antibodies against pathogen components that are not involved in the infection mechanism are also induced. As a result, the proportion of neutralizing antibodies in the induced antibodies that are able to inhibit infection is low.

Among the antibodies without neutralizing activity (non-neutralizing antibodies) induced by attenuated strain vaccines and inactivated vaccines, antibodies (bad antibodies) that cause antibody-dependent promotion of infection may occur. This reaction is thought to be induced by the Fc domain of non-neutralizing antibodies bound to pathogenic viruses binding to the Fc receptors of monocytes and macrophages, and the pathogenic viruses proliferating in those cells (dengue virus For example: Katzelnick L. C. et al., Science 358(6365): 929-932,2017; Implications for COVID-19: Iwasaki, A., Yang, Y., Nat. Rev. Immunol. 20: 339 -341, 2020 doi.org/10.1038/s41577-020-0321-6). In this way, it is necessary to avoid exacerbation of post-infection pathological conditions in vaccine recipients compared to vaccine recipients. is not preferred. However, the current situation is that it is sometimes included for manufacturing reasons.

As a practical technology for vaccines that induce humoral and cellular immunity to protect against infectious diseases, by inoculating adenovirus vectors with genes derived from the spike protein of the target pathogen virus or synthetic mRNA, Spike gene-loaded vaccines that induce humoral and cell-mediated immunity are known. Three vaccines approved in Japan against the SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) virus use these techniques (Watanabe, Y. et al., ACS Cent. Sci. 7: 594-602, 2021 doi.org/10.1021/acscentsci.1c00080; Corbett K. S. et al., Nature 586: 567-571, 2020 doi.org/10.1038/s41586-020-2622-0; Vogel A. B. et al., Nature 592: 283-289, 2021 doi.org/10.1038/s41586-021-03275-y).

It has been demonstrated that inoculation of these vaccines induces humoral immunity and cell-mediated immunity, resulting in a high infection prevention effect. However, they use the entire spike protein (1273 amino acid residues) of the SARS-CoV-2 virus as an antigen, and a receptor-binding domain (RBD: 227 amino acid residues) that interacts with the ACE2 receptor on the cell surface that binds early in infection. Including regions other than the group (319-545)). If antibodies that bind to regions other than the RBD are induced as non-neutralizing antibodies, we must be aware that they may act as bad antibodies and cause a reaction that promotes antibody-dependent infection. This is because antibody-dependent promotion of infection by antibodies against spikes of the closely related SARS coronavirus has been reported (Examples of SARS-CoV spikes: Wang, S. F. et al., Biochem. Biophys. Res. Commun. 451(2): 208-214, 2014 doi.org/10.1016/j.bbrc.2014.07.090; Liu L. et al., JCI Insight 4(4): e123158, 2019 doi.org/10.1172 /jci.insight.123158). In fact, an antibody has been found that binds to the amino-terminal N-terminal domain (NTD) of the RBD region in SARS-CoV-2, and when this antibody binds to the spike protein of SARS-CoV-2, the RBD region is released, It has been reported that binding to the ACE2 receptor is enhanced (Liu, Y. et al., Cell 184(13): 3452-3466, 2021 doi.org/10.1016/j.cell.2021.05.032) . In addition, it has been reported that the rise of antibodies against SARS-CoV-2 overlaps with the time of severe disease in COVID-19 patients, and antibody-dependent promotion of infection is suspected as the cause of this severe disease (Zhao, Y 71(16): 2027-2034, 2020 doi.org/10.1093/cid/ciaa344; Seow J. et al., Nat Microbiol. 5(12): 1598-1607, 2020 doi:10.1038/s41564-020-00813-8). Therefore, to eliminate this risk, it is not preferable to include regions that give rise to non-neutralizing antibodies in vaccine antigens.

In addition, these vaccines enter cells at the injection site and express spike protein antigens. A secretory signal is located at the amino terminus of the spike protein antigen, followed by the S1 region containing the RBD, flanked by a cleavage motif followed by the S2 region, and at the carboxy terminus by the transmembrane domain. In both adenoviral vector vaccines and synthetic mRNA vaccines, the spike protein antigens expressed are anchored on the cell membrane of the cells into which they have been introduced and protrude outward (Watanabe, Y. et al., ACS Cent. Sci. 7: 594-602, 2021 doi.org/10.1021 /acscentsci.1c00080; Corbett K. S. et al., Nature 586: 567-571, 2020 doi.org/10.1038/s41586-020-2622- 0; Vogel A. B. et al., Nature 592: 283-289, 2021 doi.org/10.1038/s41586-021-03275-y). Such anchored spike proteins are thought to be found and phagocytosed by professional antigen-presenting cells (B cells, macrophages, and dendritic cells) to present antigens to immune cells such as B cells and T cells. However, since the membrane of a single cell presents many anchor spikes, the spike protein molecules cannot diffuse sufficiently, and phagocytosis by antigen-presenting cells is thought to be insufficient (see Fig. 5-1, left).

Recently, an mRNA vaccine expressing RBD with an amino-terminal secretion signal has been reported (Tai, W. et al., Cell Research 30: 932-935, 2020 doi.org/10.1038/s41422-020- 0387-5). Similar to this report, an mRNA vaccine was also reported in which a trimerization domain, foldon, was added to the carboxy terminus of the RBD with a secretion signal, which has been used to mimic the spike structure of pathogenic viruses (Vogel A. B. et al., Nature 592: 283-289, 2021doi.org/10.1038/s41586-021-03275-y). Each mRNA is introduced into the cell by the lipid bilayer capsule, then the vaccine antigen is expressed, and secreted and released outside the cell. It is thought that the amount of free vaccine antigen is small, it cannot spread sufficiently, phagocytosis by antigen-presenting cells becomes insufficient, and as a result, the lifespan of immunity does not extend. The extracellular diffusion of vaccine antigens, which leads to phagocytosis by antigen-presenting cells sufficient to extend immune longevity, is thought to require the coupling of strong expression of vaccine antigens and subsequent liberation of extracellular secretion (Fig. 5-1 (see right), but there is no technology that can meet that requirement.

As mentioned in the two paragraphs above, it has been found that the amount of antibody decreases in about half a year after vaccination with these vaccines that use the entire spike protein as a vaccine antigen. This could be attributed to two problems: 1) the non-diffusibility of the vaccine antigen proteins mentioned in the two paragraphs above, and 2) the induction of B cells that produce non-neutralizing antibodies as mentioned in the three paragraphs above. is.
In secondary lymph nodes, antigen presentation occurs to CD4+ helper T (Th) cells, which, after germinal center migration, stimulate proliferation of B cells derived from the same antigen. Somatic mutations are induced in antibody gene variable regions in B cells that have undergone proliferation stimulation, and through interactions with follicular dendritic cells derived from the same antigen molecule, B cells that possess high-affinity antibody genes are generated. They are selected to become long-lived plasma cells and memory B cells that produce a large amount of high-affinity antibodies, and the longevity of immunity is established.
Therefore, 1) the non-diffusibility of the vaccine antigen is thought to reduce repeated interactions between antibody-producing B cells and follicular dendritic cells, both derived from the same antigen, occurring in the germinal center, and thus high affinity It is thought that selection of sexual B cells becomes insufficient. In addition, 2) the proliferation of non-neutralizing antibody-producing B cells antagonizes and hinders the proliferation of neutralizing antibody-producing B cells in the germinal center, resulting in B cells that produce antibodies with high neutralizing ability (anti-infection ability). It is thought that the selection of As a result, it is thought that the formation of long-lived plasma cells and memory B cells that produce large amounts of antibodies has not been established.

Viral vector-type vaccines such as adenoviral vectors and Sendai virus vectors induce cell-infected virus vectors during booster vaccinations because antibodies and CTLs specific to the proteins of the viral vectors themselves are induced in the initial inoculation. There is a concern that the booster effect of vaccine antigens by booster vaccination will not be exerted because CTLs act to eliminate infected cells after infection. There is also concern that the immunity induced by similar viruses in the past may act on the proteins of the virus vector itself (cross-immunity), thereby suppressing the immunogenicity of vaccine antigens.

The social issue (1) is that the vaccine cannot be distributed worldwide due to the limited number of vaccine formulations. As of September 2021, more than 50% of people in developed countries have been vaccinated twice, and a third dose is already being considered. WHO has requested that the third vaccination in developed countries be delayed until the end of the year in order to advance the number of people who have been vaccinated once in developing countries in September 2021 at about 10%. If the lifespan of immunity induced by vaccination can be extended, the number of vaccinations can be reduced, and limited vaccine preparations can be spread throughout the world.

The social issue (2) is that the country-by-country herd immunization, which was expected for vaccines, has not yet been achieved. In order to prevent the spread of infection, developed countries are currently trying to achieve collective immunization (non-spread of infection) through vaccination. Using the herd immunity threshold % = (1-1/R ₀ ) x 100 (R ₀ : basic reproduction number) as a formula to predict the minimum proportion of immune carriers that a population is protected from infectious diseases, 1 If 1 infected person infects 3 people, R ₀ is 3. In this case, if 67% of the population can acquire immunity, it is assumed that the infection will not spread. Immunocompromised patients, such as those treated with anticancer drugs, are not expected to induce immunity through vaccines, so they must avoid infection by restricting their behavior, but herd immunization is thought to protect such people.
The following conditions are considered necessary to achieve herd immunization. Immunity acquired by vaccination has a long lifespan, and the infection ends during that time. However, even in developed countries where the vaccination rate has reached the majority, the spread of infection has not subsided.
One possible reason for this is the short life span of acquired immunity, as described above. This is because, in secondary lymph nodes, presentation of vaccine antigens to CD4+ helper T (Th) cells occurs, which, after germinal center migration, stimulate proliferation of B cells derived from the same antigen. Proliferation-stimulated B cells can inhibit infection, but they have a short lifespan. To prolong their lifespan, repeated interactions between antibody-producing B cells and follicular dendritic cells derived from the same antigen are required. is necessary, but the non-diffusibility of vaccine antigens may reduce interactions between immune cells derived from the same antigen.
Until September 2021, intramuscular injection has been the route of choice for inactivated vaccines, spike-loaded adenovirus vaccines, and spike-loaded mRNA vaccines against SARS-CoV-2. Vaccination by injection cannot induce immunity in the mucosal region including the upper respiratory tract, so it acquires only the ability to prevent onset and exacerbation, but may not acquire the ability to prevent infection itself. This is because the virus that invades the respiratory tract of injectable vaccinees infects mucosal epithelial cells, suppresses the induction of interferon, does not cause fever, and spreads secondary virus particles as a seemingly uninfected person. When virus particles reach the lower respiratory tract, IgG antibodies and cytotoxic T cells may act to prevent the onset and severity of the disease. Therefore, there is a concern that it will be difficult to stop the spread of infection by injection vaccination alone, and herd immunization will not be achieved.

The social issue (3) is that excessive expectations are placed on vaccine passports. A vaccine passport issued after intramuscular injection of a vaccine against SARS-CoV-2, a respiratory infection, means that it has the ability to prevent onset and severity as described in the paragraph above. However, it is thought that acquiring the ability to prevent infection does not necessarily mean that it is an issue that should be reconsidered globally.

As described above, in attenuated strain vaccines and inactivated vaccines, the ratio of neutralizing antibodies to specific antibodies induced after vaccination is low. Accordingly, there is a high risk that non-neutralizing antibodies will cause antibody-dependent enhancement of infection. In addition, in adenoviral vaccines and mRNA vaccines that use the entire antigen from the spike protein secretion signal to the transmembrane domain, non-neutralizing antibodies against regions other than the RBD of the S1 and S2 domains cause antibody-dependent enhancement of infection. It is dangerous and also has the problem that antibodies to the NTD region of the S1 domain promote infection. In addition, with adenovirus-type vaccines and mRNA-type vaccines, spike proteins are tethered to the cell membrane after inoculation, which prevents the release of spike protein molecules, resulting in insufficient phagocytosis by antigen-presenting cells.
From these vaccinations, it is considered difficult to achieve the medical problems of high neutralizing antibody ratio, avoidance of antibody-dependent enhancement of infection, and longevity of immunity. In addition, vaccination against respiratory infections by injection cannot be expected to prevent infection, which is a medical problem.
If the medical challenges of extending the lifespan of immunity and acquiring the ability to prevent infection are not achieved, it will be difficult to achieve the social challenges of expanding vaccination to the entire world and mass immunization. use can be dangerous.

An object of the present invention is to provide an antigen protein expression vector and its use. Although the present invention is not limited thereto, in a preferred embodiment, for example, for the purpose of inducing high immunogenicity as an infectious disease vaccine, a vaccine antigen protein is expressed intracellularly after inoculation. , a vaccine vector technology, etc., in which a part of the vaccine remains in the cell and a part of it is secreted and released outside the cell.

In order to develop a more effective antigen protein expression vector, the present inventors constructed an antigen protein expression vector using the RNA virus spike protein as an example. Sendai virus that encodes a fusion protein (S1-foldon) in which the antigen is S1 (without transmembrane domain) containing the spike protein secretion signal to just before the cleavage motif, and foldon, a trimerization sequence, is added to it. A fusion protein (S -RBD-foldon) was constructed. For comparison, a Sendai virus vector was also constructed that encodes a protein (S-RBD) in which a secretion signal is added to a fragment containing RBD in the same manner as the above S-RBD-foldon, but no foldon is added.

When these vectors were introduced into cells and the expression from the vector was examined, the fusion protein (S1-foldon), which was a fusion protein (S1-foldon) with a foldon added to the S1 protein, was compared with the fusion protein (S1-foldon), which was a foldon-added RBD with a secretion signal -RBD-foldon) was confirmed to increase the expression level from several times to about 10 times (Example 2b). In addition, the fusion protein (S1-foldon), in which a foldon was added to the S1 protein, released a secretion signal, whereas the amount of protein released outside the cell was about 1/10 of the amount of protein remaining inside the cell. In the case of a fusion protein (S-RBD-foldon) in which a foldon was added to the RBD, most of the expressed fusion protein was secreted extracellularly, and specifically, the protein that was secreted and released extracellularly remained in the cell. It was found to be several to about 10-fold or more (3.8- to 19-fold) higher than protein (Example 2b). Combined with these characteristics, the fusion protein (S-RBD-foldon) with a foldon added to the RBD with a secretion signal is secreted extracellularly, compared to the fusion protein with a foldon added to the S1 protein (S1-foldon). It was found that the released protein increased at least several tens of times.

Surprisingly, the expression level of the secreted RBD protein with foldon (S-RBD-foldon) was 5-10 times higher than that of the RBD protein without foldon (S-RBD). Example 2b). Moreover, the ratio of secreted and released proteins (secretion release rate) to the expressed proteins is as high as when foldon is added (S-RBD-foldon) and when foldon is not added (S-RBD) ( about 80% or more).

Thus, a fragment of an antigenic protein with a secretion signal and an appended trimerization sequence may be an antigenic protein comprising long polypeptides, such as the entire extracellular region of the antigen, or without the addition of the trimerization sequence. It was found that the expression level was remarkably increased when expressed from a vector, and a large amount of the expression product was secreted and released extracellularly compared to the antigen protein fragment.

A Sendai virus vector that encodes a fusion protein (S1-foldon) in which a foldon is added to the S1 protein (not including the transmembrane domain) containing the secretion signal to just before the cleavage motif, or a foldon is added to the RBD to which the secretion signal is added. Sendai virus vectors encoding a fusion protein (S-RBD-foldon) were intranasally inoculated into rats, and IgG antibodies in the induced serum were examined. 1/5, significantly higher IgG antibodies were induced in individuals inoculated with a vector encoding S-RBD-foldon than in individuals inoculated with a vector encoding S1-foldon. It turned out (Example 3c). In addition, in both single and multiple inoculations, inoculation of vectors encoding S-RBD-foldon resulted in induced neutralizing antibody activity, despite the smaller amount of viral vector inoculated. , was confirmed to exhibit high neutralizing antibody activity (Example 3d). This result indicates that inoculation of a vector encoding a secretory-free form of an antigenic protein fragment with a secretion signal and an added trimerization sequence yields a protein with an added trimerization sequence to an antigenic protein containing S1. It shows that significantly higher humoral immunity can be induced than when inoculating a vector encoding

Sendai virus vector encoding S1-foldon containing S1, or Sendai virus vector encoding a fusion protein (S-RBD-foldon) in which a foldon is added to RBD with a secretion signal to examine the induction effect of cell-mediated immunity. was intranasally inoculated into rats, and spleen cells were used to examine the number of CTL cells that respond to peptide stimulation. As a result, in both single and multiple inoculations, the amount of viral vector to be inoculated was smaller with the S-RBD-foldon-encoding vector than with the S1-foldon-encoding vector. Nevertheless, it was found that a significantly higher CTL stimulatory effect could be induced (Example 3e). This result indicates that inoculation of a vector encoding a secretory-free form of an antigenic protein fragment with a secretion signal and an added trimerization sequence yields a protein with an added trimerization sequence to an antigenic protein containing S1. It shows that a significantly higher cell-mediated immunity can be induced compared to inoculation of a vector encoding .

Thus, a vector having a secretion signal and encoding a secretory-release antigen protein fragment to which a trimerization sequence has been added has a high ability to express the antigen protein fragment and a high ability to release the antigen protein fragment from extracellular secretion. It was found to have an excellent ability to induce immune responses to both sexual immunity and cell-mediated immunity. By using the vector of the present invention, it is expected that both humoral immunity and cell-mediated immunity can be efficiently induced, and excellent protective immunity against infectious diseases and the like can be imparted.

That is, the present invention relates to antigen protein expression vectors and the like useful as vaccine antigens that induce immunogenicity, and more specifically to the inventions described in the claims. Inventions comprising any combination of two or more of the inventions recited in claims that cite the same claim are also inventions contemplated herein. That is, the present invention relates to the following inventions.
[1] An antigen expression vector comprising a nucleic acid encoding an extramembrane-releasable fusion protein comprising a secretory signal, an antigenic protein fragment, and a trimerization domain.
[2] the antigen-expressing vector of [1], wherein the fusion protein does not contain a transmembrane domain;
[3] The antigen-expressing vector of [1] or [2], which is a minus-strand RNA viral vector.
[4] the antigen expression vector of any one of [1] to [3], wherein the trimerization domain is the trimerization domain (foldon) of T4 phage fibritin;
[5] The antigen-expressing vector of any one of [1] to [4], wherein the antigen is derived from an infectious pathogen.
[6] the antigen-expressing vector of any one of [1] to [5], wherein the antigen is derived from a virus;
[7] The antigen-expressing vector of any one of [1] to [6], wherein the antigen protein fragment is an extracellular region of a membrane protein or a fragment thereof.
[8] The antigen expression vector of any one of [1] to [7], wherein the length of the antigen protein fragment is 500 amino acids or less.
[9] The antigen-expressing vector of any one of [1] to [8], wherein the antigen protein is an RNA virus spike protein.
[10] the antigen-expressing vector of [9], wherein the antigenic protein fragment is a fragment of the extracellular region containing the receptor-binding domain of RNA virus spike protein;
[11] The antigen-expressing vector of any one of [1] to [10], wherein the expression product containing the antigen protein fragment is distributed both intracellularly and extracellularly in the vector-introduced cell.
[12] The antigen expression vector of [11], wherein the expression product released outside the cell is larger than the expression product that remains in the cell.
[13] the expression level of the expression product containing the antigen protein fragment is increased compared to a control antigen expression vector containing a nucleic acid encoding a protein that contains a secretory signal and an antigen protein fragment and does not contain a trimerization domain; [1 ] to [12].
[14] A vaccine comprising the antigen-expressing vector of any one of [1] to [13].
[15] the vaccine of [14] for inducing both humoral and cell-mediated immunity;

The present invention also includes the following inventions.
[1] An antigen expression vector comprising a nucleic acid encoding an extramembrane-releasable fusion protein comprising a secretory signal, an antigenic protein fragment, and a trimerization domain.
[2] The antigen-expressing vector of [1], wherein the fusion protein does not contain a transmembrane domain.
[3] The antigen-expressing vector of [1] or [2], which is a minus-strand RNA viral vector.
[4] the antigen-expressing vector of [3], wherein the minus-strand RNA viral vector is a paramyxovirus vector;
[5] The antigen-expressing vector of [4], wherein the Paramyxovirus vector is Sendavirus.
[6] the antigen expression vector of any one of [1] to [5], wherein the trimerization domain is the trimerization domain (foldon) of T4 phage fibritin;
[7] The antigen-expressing vector of any one of [1] to [6], wherein the antigen is derived from an infectious pathogen, infectious microorganism, or infectious virus.
[8] The antigen-expressing vector of any one of [1] to [6], wherein the antigen is derived from a virus.
[9] The antigen-expressing vector of [8], wherein the virus has an envelope derived from a biological membrane.
[10] the antigen-expressing vector of [8] or [9], wherein the virus is an RNA virus;
[11] the antigen-expressing vector of [9] or [10], wherein the RNA virus is a positive-strand RNA virus;
[12] the antigen-expressing vector of [11], wherein the RNA virus is a positive-strand single-stranded RNA virus;
[13] the antigen-expressing vector of any one of [9] to [12], wherein the RNA virus is a coronavirus;
[14] the antigen expression vector of [13], wherein the coronavirus is SARS-CoV-2;
[15] the antigen-expressing vector of any one of [1] to [14], wherein the antigen protein fragment is an extracellular region of a membrane protein or a fragment thereof;
[16] the antigen expression vector of any one of [1] to [15], wherein the length of the antigen protein fragment is 500 amino acids or less;
[17] the antigen expression vector of [16], wherein the length of the antigen protein fragment is 400 amino acids or less;
[18] the antigen expression vector of [17], wherein the length of the antigen protein fragment is 300 amino acids or less;
[19] The antigen-expressing vector of any one of [1] to [18], wherein the antigen protein is a viral spike protein.
[20] the antigen expression vector of [19], wherein the antigen protein fragment is a fragment of the extracellular region containing the host cell-binding domain of the spike protein;
[21] the antigen-expressing vector of [19], wherein the antigenic protein fragment is a fragment of the extracellular region containing the receptor-binding domain of RNA virus spike protein;
[22] the antigen-expressing vector of [21], wherein the receptor is ACE2;
[23] the antigen-expressing vector of [21] or [22], wherein the antigen protein fragment is a fragment of the extracellular region containing the receptor-binding domain of the spike protein of coronavirus RNA virus;
[24] the antigenic protein fragment is the 328th to 531st amino acid sequences of SEQ ID NO: 2 (SEQ ID NO: 6), or within 5, preferably within 4, 3, 2, or 1 amino acid sequence therefrom; The antigen expression vector of any one of [1] to [23], which comprises an amino acid sequence with amino acid substitution, deletion and/or addition, or a fragment of any of these amino acid sequences.
[25] the antigenic protein fragment is the 319th to 545th amino acid sequence of SEQ ID NO: 2 (SEQ ID NO: 4), or within 5, preferably within 4, 3, 2, or 1 amino acid sequence therefrom; The antigen expression vector of any one of [1] to [24], which is an amino acid sequence with amino acid substitution, deletion and/or addition, or a fragment of any of these amino acid sequences.
[26] the antigenic protein fragment is the 328th to 531st amino acid sequences of SEQ ID NO: 2 (SEQ ID NO: 6), or within 5, preferably within 4, 3, 2, or 1 amino acid sequence therefrom; The antigen expression vector of any one of [1] to [24], which is an amino acid sequence with amino acid substitution, deletion and/or addition.
[27] the antigen-expressing vector of any one of [1] to [26], wherein the expression product containing the antigen protein fragment is distributed both intracellularly and extracellularly in the vector-introduced cell;
[28] The antigen-expressing vector of [27], wherein the amount of expression product released outside the cell is greater than the amount of expression product that remains in the cell.
[29] the expression product released outside the cell is 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, or 9-fold or more than the expression product that remains in the cell; The antigen expression vector of [28].
[30] compared to a control antigen expression vector containing a nucleic acid encoding a protein that contains a secretory signal and an antigenic protein fragment and does not contain a trimerization domain, the expression level of the expression product containing the antigenic protein fragment is increased, [1 ] to [29].
[31] the expression level of the expression product containing the antigen protein fragment is at least 1.5 times greater than that of a control antigen expression vector containing a nucleic acid encoding a protein containing a secretion signal and an antigen protein fragment but not containing a trimerization domain; The antigen expression vector of [30], which is at least 3-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold.
[32] the amount of an expression product containing an antigenic protein fragment is increased by the addition of a trimerization domain, and the expression product is distributed both intracellularly and extracellularly in vector-introduced cells [1] to [31] ].
[33] the amount of an expression product containing an antigenic protein fragment is regulated by addition or deletion of a trimerization domain, and the expression product is distributed both intracellularly and extracellularly in vector-introduced cells [1] to [ 32], the antigen expression vector according to any one of the above.
[34] the amount of the expression product containing the antigen protein fragment is regulated by the addition or deletion of the trimerization domain, so that the amount of the expression product distributed both intracellularly and extracellularly in the vector-introduced cell and vector production; The antigen expression vector of any one of [1] to [33], which is sex-regulated.
[35] the amount of the expression product containing the antigen protein fragment is regulated by the size of the antigen protein fragment, and the expression product is distributed both intracellularly and extracellularly in vector-introduced cells, [1] to [34] ].
[36] the amount of the expression product containing the antigen protein fragment is regulated by the size of the antigen protein fragment, so that the amount of the expression product distributed both intracellularly and extracellularly in the vector-introduced cell and the vector productivity; is regulated, the antigen expression vector of any one of [1] to [35].
[37] the antigen expression vector of any one of [31] to [36], wherein the trimerization domain is the trimerization domain (foldon) of T4 phage fibritin;
[38] A fusion protein comprising an antigen protein fragment and a trimerization domain expressed from the antigen expression vector of any one of [1] to [37].
[39] A vaccine comprising the antigen-expressing vector of any one of [1] to [37].
[40] the vaccine of [39] for inducing both humoral and cellular immunity;
[41] the vaccine of [39] or [40] for multiple inoculations;
[42] the vaccine of any one of [39] to [41], which is nasally administered;
[43] A method for producing an antigen protein fragment, which comprises the step of introducing the antigen-expressing vector of any one of [1] to [37] into a cell.
[44] A method for producing an extracellularly released antigen protein fragment, which comprises the step of introducing the antigen-expressing vector of any one of [1] to [37] into a cell.
[45] A method for extracellularly releasing an antigen protein fragment, which comprises the step of introducing the antigen-expressing vector of any one of [1] to [37] into a cell.
[46] A method for inducing immunity, which comprises the step of inoculating the antigen-expressing vector of any one of [1] to [37] or the vaccine of any one of [39] to [42].
[47] Humoral immunity against the antigen, cell-mediated Methods of induction of immunity, or both.
[48] Use of the antigen-expressing vector of any one of [1] to [37] for inducing humoral immunity, cell-mediated immunity, or both against the antigen.
[49] Use of the antigen-expressing vector of any one of [1] to [37] in the production of a vaccine.
[50] the use of [49], wherein the vaccine induces humoral immunity, cellular immunity, or both against the antigen;

Any technical matter described in this specification and any combination thereof are intended in this specification. Inventions excluding any matter described herein or any combination thereof in those inventions are also intended herein. In addition, regarding the present invention, a specific aspect described in the specification not only discloses it, but also inventions excluding that aspect from higher inventions disclosed in this specification including that aspect Disclosure.

According to the present invention, for example, in one preferred embodiment thereof, a vaccine antigen derived from the extramembrane domain of an infectious disease pathogen protein is strongly expressed, and while a part of the expressed antigen remains intracellularly, it is abundantly secreted and released outside the membrane. It has become possible to enhance immunogenicity by allowing The present invention is expected to find particular application in the field of infectious immunology. In one preferred aspect of the present invention, cancer cell-specific antibodies and killer T cells are induced by strong expression, intracellular persistence, and extramembrane secretion release of the extramembrane domain of cancer cell-specific membrane proteins. Use in the field of cancer immunology, use in the field of dysfunction therapy that induces the regeneration of dead cell tissues by strong expression and extra-membrane release of protein-based tissue regeneration factors such as cell growth factors, protein-based differentiation factors Strong expression and extramembrane release of this protein can be used in various applications such as regenerative medicine that enhances differentiation induction of adjacent cells.

Solution of the risk of increased antibody-dependent infection due to non-neutralizing antibodies For example, in vaccines against pathogenic viruses, etc., there has been the problem of the risk of increased antibody-dependent infection due to non-neutralizing antibodies. Neutralization of specific antibodies induced after inoculation in attenuated strain vaccines and inactivated vaccines, as well as in adenovirus-type vaccines and mRNA-type vaccines that use the entire antigen from the spike protein secretion signal to the transmembrane domain There is a problem that the ratio of antibodies is low. Since the neutralizing ability (anti-infection ability) of antibodies is essentially their ability to inhibit binding at the point of contact between viruses and cells, in order to avoid the risk of antibody-dependent enhancement of infection by non-neutralizing antibodies, virus It is useful to select only side binding sites as vaccine antigens. In the SARS-CoV-2 spike protein, the region is the reported RBD domain (319-545) (Yang, J. et al., Nature 586: 572-577, 2020 doi.org/10.1038/s41586 -020-2599-8) or, more preferably, RBD (328-531), which is a further narrowed version of this, can be used, and the use of such short antigen protein fragments enhances antibody-dependent infection. It is possible to minimize the possible induction of non-neutralizing antibodies.

Solving the unachieved problem of immune longevity (1)
As already mentioned, the factors that prevent the extension of immune longevity are 1) the lack of production of antigen proteins after vaccination, the production of antigen proteins only for a short period of time, and the inability to efficiently secrete and release antigen proteins. 2) induction of non-neutralizing antibody-producing B cells.
Regarding 1), in a preferred embodiment, in order to overcome the non-diffusibility of vaccine antigens, it is necessary to leave secretory signals while deleting transmembrane domains to promote secretion and release of vaccine antigens from cells. Furthermore, it is conceivable to add a secretory signal to the further refined vaccine antigen fragment. As a result, the vaccine antigen fragment is released outside the infected cells, and the opportunity for phagocytosis by antigen-presenting cells increases, leading to the induction of B cells and follicular dendritic cells derived from the same antigen, and the interaction between them. It is expected to frequently lead to the selection of high-affinity antibody-producing B cells. As for 2), in a preferred embodiment, for example, in SARS-CoV-2, etc., by expressing only the ACE2 receptor-binding domain essential for binding to host cells as a vaccine antigen, a neutralizing antibody can be produced. Only B cells are stimulated to proliferate, and selection of antibody-producing B cells with high ability to block infection is achieved. By improving the problems 1) and 2), the selected B cells become long-lived plasma cells and memory B cells, and the longevity of immunity is established.

Solving the unachieved problem of immune longevity (2)
As shown in Example (4), the smaller the molecular weight, the larger the secreted release amount. Utilizing this rule, it becomes possible to regulate the ability to induce humoral immunity by regulating the amount of vaccine antigen secreted and released. Two types of vaccine antigen vectors with the same secretory signal and the same foldon sequence were prepared and compared in terms of their ability to induce humoral immunity. Do you get it. The following techniques are provided based on these verification results. That is, in a preferred embodiment, by minimizing the peptide length of the vaccine antigen, the amount of the vaccine antigen is increased in the cells after inoculation. Achieve longer life.

Solving the unachieved challenges of extending immune lifespan (3)
Conventionally, the trimerization domain foldon is used to mimic the trimeric structure of pathogenic virus spikes (WO2011008974A2). However, as shown in Example (2), in the present invention, a vector expressing a fusion protein in which this trimerization factor foldon is added to the carboxy terminus of the vaccine antigen S-RBD is prepared, and the antigen protein is expressed and Unexpectedly, we found that the addition of foldon increased the amount of expressed vaccine antigen. Furthermore, it was confirmed that there was no difference in secretion release rate between the presence and absence of foldon. The following techniques are provided based on these discoveries. That is, in a preferred embodiment, the addition of foldon to the vaccine antigen increases the amount of the vaccine antigen in the cells after inoculation, and in conjunction with this, increases the extracellular diffusivity, thereby increasing the immunity. Achieve longevity.

Solving the problems of suppressing the booster effect by immunization with viral vector proteins and suppressing the ability to induce immunity by cross-immunization A strong booster effect was confirmed by repeated inoculation of a Sendai virus vector loaded with a vaccine antigen. By secreting and liberating the virus, it is possible to avoid the reduction of the booster effect of Sendai virus itself by antibodies and CTLs, and it is also possible to avoid the reduction of effects due to existing cross-immunity with similar viruses.

Solution to the Problem of Vaccine Antigen Vector Production Efficiency As shown in Example (4), it was found that a vector with a high secretory release amount resulted in reduced vector productivity during the production process. Shifting the mounting position of the vaccine antigen gene downstream has been known as a technique for suppressing the expression level, but in the present invention, as described in the above paragraph, the foldon sequence that increases the amount of the vaccine antigen is removed. We hypothesized that vector productivity could be improved if the amount of vaccine antigen in the producing cells could be reduced by this method. When the productivity of vaccine antigen vectors was compared, it was found that removal of the foldon increased the vector productivity. Based on these verification results, the following vector productivity control techniques are provided. That is, by adding or removing the foldon sequence of the vaccine antigen vector, the amount of vaccine antigen is increased or decreased in production cells in the production culture process, and the amount of secretion and release is increased or decreased in conjunction with vector production. vector productivity can be adjusted to the extent that it suppresses or enhances the immunogenicity and maintains high immunogenicity.

Solving social issues As mentioned above, there are various difficulties in achieving social issues such as the global expansion of vaccination and the acquisition of herd immunity. It is believed that there is. However, according to the present invention, in one preferred embodiment thereof, vaccine antigens capable of inducing neutralizing antibodies are narrowed down, and a secretion signal is added to them without adding a membrane permeation domain, for example, a vector having airway affinity (minus chain RNA virus vector, etc.) can induce strong expression in mucosal epithelial cells and release a large amount of neutralizing antibody-inducing antigen in the mucosal region. Then, by increasing the chance of phagocytosis by antigen-presenting cells, the induction of B cells and follicular dendritic cells derived from the same antigen and the interaction of these two occur frequently, resulting in high-affinity antibody-producing B cells. Selection is induced, and they become long-lived plasma cells and memory B cells, and the longevity of immunity is established. Therefore, the present invention is considered to contribute to solving these social problems.

FIG. 2 shows the structure of a vaccine antigen vector that induces both humoral and cell-mediated immunity. FIG. 2 shows an insertion sequence of an S1-foldon-carrying Sendai virus vector. FIG. 2 shows an insertion sequence of an S-RBD-foldon-carrying Sendai virus vector. FIG. 2 shows an insertion sequence of an S-RBD-loaded Sendai virus vector. FIG. 3 shows the expression, intracellular persistence, and extramembrane secretory release of a vaccine antigen vector. FIG. 3 shows the expression, intracellular persistence, and extramembrane secretory release of a vaccine antigen vector. FIG. 2 shows humoral immunity induction by vaccine antigen vector technology. FIG. 2 shows humoral immunity induction by vaccine antigen vector technology. FIG. 2 shows cell-mediated immunity induction by vaccine antigen vector technology. FIG. 2 shows cell-mediated immunity induction by vaccine antigen vector technology. Immunity induction method: It is a figure which shows the problem of a conventional method, and the superiority of this invention. In the figure, dark triangles represent antigen proteins, and light triangles represent viral membrane proteins possessed by viral vectors. Antigen-presenting cells are represented in the form of major sectors of circles. When the antigen protein (dark triangle) is expressed in a membrane-tethered form (left panel), the antigen protein is localized on the surface of vector-infected cells and released virus particles, whereas the antigen protein fragment In the vector of the present invention (right panel) in which a large amount of is secreted and released, the released antigen protein fragments diffuse far from the infected cells and are efficiently phagocytosed by the antigen-presenting cells, resulting in the induction of a high immune response. be.

Hereinafter, embodiments of the present invention will be described in detail.

In the present invention, the term "vaccine" refers to a composition for eliciting an immune response against a target antigen, for example, a composition used for prevention or treatment of infectious diseases, infectious diseases, cancer, and the like. Vaccines contain or are capable of expressing a target antigen or fragment thereof, thereby having the ability to induce an immune response against the target antigen. For example, for the prevention or treatment of pathogenic virus infection, transmission, and epidemics, the vaccine composition of the present invention is formulated as a vaccine comprising a target antigen or fragment thereof, or a nucleic acid expressing the target antigen or fragment thereof. can be This vaccine can be used in any desired form. The vaccine composition of the present invention is particularly useful for the prevention and/or treatment of infection by viruses such as coronaviruses or microorganisms, replication in the body, or diseases caused by them.

An "antigen" is a molecule containing one or more epitopes (parts of an antigen recognized by antibodies or immune cells) that can stimulate the host's immune system to induce an antigen-specific immune response. say. The antigen used in the present invention is an antigen capable of inducing a humoral or cell-mediated immune response. The antigen of the present invention is preferably an antigen capable of inducing at least humoral immune response, more preferably an antigen capable of inducing both humoral and cell-mediated immunity. The antigen of the present invention is not particularly limited as long as it can induce an immune response, but usually one epitope in the protein is about 7 to about 15 amino acids, for example, at least 8, 9, 10, 12, or contains 14 amino acids. In the present invention, the epitope includes not only an epitope formed from the primary structure but also an epitope dependent on the three-dimensional structure of the protein. Antigens capable of eliciting an immune response are also referred to as immunogens.

The present invention provides an antigen expression vector comprising a nucleic acid encoding a fusion protein capable of extramembrane releasability, comprising a secretory signal, an antigenic protein fragment, and a trimerization domain. The fusion protein encoded by this vector is capable of extramembrane releasability and is secreted and released from the cell upon expression in the cell. The secretory signal may be cleaved off during secretion. Thus, the secreted fusion protein may be a fusion protein that lacks a secretory signal and contains an antigenic protein fragment and a trimerization domain.

An antigenic protein fragment refers to a portion of a protein that has antigenicity or immunogenicity, that is, a portion of a naturally occurring antigenic protein that is not the full length. The length of the antigen protein fragment can be selected as appropriate, but is preferably 70% or less, such as 60% or less, 50% or less (half or less), 40% or less, 35% or less of the length of the naturally occurring antigen protein. % or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. Here, when a naturally occurring antigen protein is, for example, once produced as a proprotein (precursor) and exhibits its activity only after being cleaved, the protein after cleavage is regarded as a naturally occurring antigen protein, On the other hand, it is preferable to use fragments with lengths in the above proportions. For example, the spike protein (S) of a virus can be cleaved into S1 and S2. In this case, S1 and S2 are the full-length naturally occurring antigen proteins, and fragments having the above proportions of the length. can be used. Antigenic protein fragments are preferably 500 amino acids or less, such as 450 amino acids or less, 400 amino acids or less, 350 amino acids or less, 300 amino acids or less, 280 amino acids or less, 250 amino acids or less, or 230 amino acids or less. Particularly preferred are antigenic protein fragments of 220 amino acids or less, for example antigenic protein fragments of 215 amino acids or less, 210 amino acids or less, or 205 amino acids or less are particularly preferred.

The total length of the fusion protein, which further comprises the secretory signal and the trimerization domain, is e.g. 550 amino acids or less, e.g. 280 amino acids or less. In particular, it is preferably 270 amino acids or less, and particularly preferably 265 amino acids or less, 260 amino acids or less, or 250 amino acids or less.

The length of the entire fusion protein excluding the secretory signal is, for example, 550 amino acids or less, such as 500 amino acids or less, 450 amino acids or less, 400 amino acids or less, 350 amino acids or less, 330 amino acids or less, 300 amino acids or less, or 280 amino acids or less. In particular, it is preferably 270 amino acids or less, and particularly preferably 265 amino acids or less, 260 amino acids or less, 255 amino acids or less, 250 amino acids or less, 245 amino acids or less, or 240 amino acids or less.

There are no particular restrictions on the origin of the antigen protein fragment, and any desired protein to induce an immune response can be used. For example, proteins of infectious microorganisms (including bacteria, fungi, viruses) and cancer-specific proteins can be used as target proteins. By using a protein of a pathogenic infectious microorganism as an antigen protein, a vaccine useful for prevention or treatment against the pathogenic microorganism can be produced. As the origin of the antigen protein, for example, a membrane protein is useful, and in particular, a membrane protein having an extracellular domain can be used as a suitable origin of the antigen protein fragment. Membrane proteins include membrane proteins possessed by viruses, membrane proteins specifically expressed in cancer cells, and the like. When targeting the extracellular domain of a membrane protein, all or part of the extracellular domain, preferably part, is used as the antigen protein fragment. The length of the antigen protein fragment can be selected as appropriate, but is preferably 70% or less, such as 60% or less, 50% or less (half or less), 40% or less, or 35% or less of the total extracellular domain length. , 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

The fusion protein of the present invention is a protein that is secreted and can be released outside the membrane. It does not contain a domain or a membrane anchoring domain. For example, when the antigen protein to be targeted is a membrane protein, it is preferable to use as the antigen protein fragment a fragment that does not contain a domain that retains the protein in a biological membrane, such as a transmembrane domain or a membrane anchoring domain.

Suitable antigen proteins in the present invention include proteins of enveloped viruses. Here, an enveloped virus refers to a virus having an envelope of a lipid bilayer membrane derived from a biological membrane. Such viruses include RNA viruses, DNA viruses, retroviruses, etc. RNA viruses include coronaviruses (coronaviridae viruses) including SARS virus and MERS (Middle East Respiratory Syndrome) virus, and influenza virus. orthomyxovirus (orthomyxoviridae virus), hepatitis C virus, Japanese encephalitis virus, flavivirus (flaviviridae virus) including Zika virus, togavirus (togaviridae virus) including rubella virus, measles virus and paramyxoviridae viruses, including human respiratory syncytial virus, rhabdoviruses, including rabies virus, bunyaviridae viruses, including Crimean-Congo hemorrhagic fever virus, and Ebola virus. and filoviruses (Filoviridae viruses), including Marburg virus, hepatitis D virus, and the like.

DNA viruses include herpesviruses (herpesviridae viruses), including varicella-zoster virus, poxviruses (poxviridae viruses), including smallpox virus, and hepadnaviruses, including hepatitis B virus (hepadnavirus). viruses of the family Viridae). Retroviruses include retroviruses (Retroviridae viruses), including lentiviruses (viruses of the genus Lentivirus) such as human immunodeficiency virus and adult T leukemia virus.

In addition, virus-derived antigens include, for example, viral proteins of positive-strand RNA viruses and single-stranded RNA viruses, and in particular, viral proteins of single-stranded positive-strand RNA viruses can be preferably used. Specific examples of such viruses include coronavirus, enterovirus, rubella virus, Japanese encephalitis virus, dengue fever virus, hepatitis C virus, norovirus, and the like. Most preferred viral antigens include coronaviruses (coronaviridae viruses), particularly betacoronaviruses, including SARS virus, including SARS-CoV-2, MERS virus. Specific examples include SARS-CoV-2 and coronaviruses derived therefrom. SARS-CoV-2 may be a desired strain, such as, but not limited to, 2019-nCoV/Japan/TY/WK-521/2020 (accession number LC522975).

In the present invention, the antigen protein particularly includes the envelope virus spike protein, and as the antigen protein fragment, a fragment containing a part of the extracellular region of the spike protein can be preferably used. For example, the amino acid sequence of the coronavirus spike (S) protein includes the above SARS-CoV-2 strain S protein amino acid sequence (SEQ ID NO: 2), accession number BCA25674.1, and the coding sequence 21560th to 25378th base sequences of accession number LC522975 (SEQ ID NO: 1). As the S protein and the nucleic acid encoding it, in addition to the nucleotide sequences and amino acid sequences exemplified above, homologous genes and proteins of other strains and species, and variants thereof may be used. Here, homology refers to corresponding amino acid sequences of different viruses. can be easily identified. These nucleic acids and proteins are, for example, one or more (for example, several, within 3, within 5, within 10, within 15, within 20) compared to the base sequences and amino acid sequences exemplified above. Those having sequences with additions, deletions, substitutions, and/or insertions of bases and amino acids, respectively, are included. Such base sequences and amino acid sequences usually exhibit a high degree of identity with the base sequences and amino acid sequences exemplified above. For example, in the present invention, those having high identity with SEQ ID NOs: 1 and 2 can be preferably used.

A high identity is, for example, a sequence with 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, or 96% or more identity. The identity of nucleotide sequences and amino acid sequences can be determined using, for example, the BLASTN and BLASTP programs (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410, 1990). For example, a search can be performed using the default parameters on the BLAST web page of the NCBI (National Center for Biochnology Information) (Altschul S.F. et al., Nature Genet. 3:266-272, 1993; Madden, T.L. et al. al., Meth. Enzymol. 266:131-141, 1996; Altschul S.F. et al., Nucleic Acids Res. 25:3389-3402, 1997; Zhang J. & Madden T.L., Genome Res. 7:649-656, 1997 ). For example, the blast2sequences program (Tatiana A et al., FEMS Microbiol Lett. 174:247-250, 1999), which compares two sequences, can be used to generate a two-sequence alignment and determine sequence identity. A gap is treated in the same manner as a mismatch, and the identity value for the entire nucleotide sequence or amino acid sequence of the antigen gene or protein molecule is calculated. Specifically, for example, the ratio of the number of matching amino acids to the total number of amino acids of a certain protein (mature type in the case of a secretory protein) is calculated. Alternatively, the ratio of the number of matching bases to the total number of bases of the base sequence encoding the protein (in the case of a secretory protein, the mature form) is calculated. A coronavirus antigen comprising an amino acid sequence highly identical to SEQ ID NO: 2, 4, 6, or 8, and a coronavirus antigen gene comprising a nucleotide sequence highly identical to SEQ ID NO: 1, 3, 5, or 7, It can be preferably used in the vaccine of the present invention.

The antigen protein of the present invention is a protein encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a part or all of the nucleotide sequence of the coronavirus antigen gene exemplified above or a nucleic acid comprising a complementary sequence thereof. Examples include proteins having antigenicity. In hybridization, for example, a probe is prepared from either a nucleic acid containing the coding region sequence of an antigen protein gene or its complementary sequence, or a nucleic acid to be hybridized, and whether it hybridizes to the other nucleic acid is examined. It can be identified by detection. Stringent hybridization conditions are, for example, 5x SSC, 7% (W/V) SDS, 100 μg/ml denatured salmon sperm DNA, 5x Denhardt's solution (1x Denhardt's solution contains 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and (containing 0.2% Ficoll) at 50°C, preferably 55°C, more preferably 60°C, more preferably 65°C, followed by hybridization at the same temperature in 2xSSC, preferably in 1xSSC , more preferably in 0.5xSSC, more preferably in 0.1xSSC, with shaking for 2 hours. Specifically, for example, a protein encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising part or all of the nucleic acid having the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7 or a complementary sequence thereof. , antigenic proteins are useful as antigens of the present invention.

In the vector of the present invention, the antigen protein is preferably used as a short fragment containing the target site. As described above, for example, when used as a vaccine that is expected to have a neutralizing effect to prevent infection with viruses, it is necessary to induce antibodies with neutralizing activity and not induce other antibodies (non-neutralizing antibodies) as much as possible. It is desirable to use the shortest possible antigen fragment containing the target site. For example, in viral spike proteins and the like, fragments of extracellular regions containing host cell-binding domains are preferred. Fragments containing (RBD) can be used. For example, in a virus that binds to or interacts with ACE2 of host cells to infect, the ACE2-binding domain of the viral protein can be preferably used.

　In the case of SARS-CoV-2, the RBD is present in the 319th to 545th amino acid sequences of the spike protein (eg, SEQ ID NO: 2). Therefore, a fragment consisting of this amino acid sequence, a spike protein fragment containing this amino acid sequence, a partial sequence of this amino acid sequence, a spike protein fragment containing this partial sequence, or the like can be used. For example, a fragment consisting of the 328th to 531st amino acid sequences produces a highly effective neutralizing antibody. Therefore, a fragment consisting of the 328th to 531st amino acid sequences, a spike protein fragment containing this amino acid sequence, a partial sequence of this amino acid sequence, a spike protein fragment containing this partial sequence, or the like can be preferably used. . Here, the partial sequence is not limited as long as it can produce a neutralizing antibody, for example, 20% or more, 30% or more, 40% of the 319th to 545th amino acid sequence or the 328th to 531st amino acid sequence or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, such as 20 amino acids or more, 30 amino acids or more 40 amino acids or more, 50 amino acids or more, 60 amino acids or more, 70 amino acids or more, 80 amino acids or more, 90 amino acids or more, 100 amino acids or more, 120 amino acids or more, It may be 150 amino acids or more, 180 amino acids or more, or 200 amino acids or more. An example of the above amino acid sequence from 319th to 545th is shown in SEQ ID NO:4, and an example of its coding sequence is shown in SEQ ID NO:3. An example of the above amino acid sequence from 328th to 531st is shown in SEQ ID NO:6, and an example of its coding sequence is shown in SEQ ID NO:5.

The amino acid of the antigen protein fragment or its coding sequence may be appropriately mutated. For example, one or more (e.g. several, preferably within 30, 20 sequences in which no more than 10, no more than 8, no more than 7, no more than 5, no more than 3, no more than 2, or 1 amino acid or base are added, deleted, substituted, and/or inserted can also be used. Such amino acid sequences or base sequences usually have a high degree of identity (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more) to the amino acid sequences or base sequences exemplified above. % or greater than 96% identity). For example, in the present invention, those having high identity with SEQ ID NOS: 1-6 can be preferably used. High identity can be determined as described above. Such antigenic protein fragments are useful for eliciting immunity targeting SARS-CoV-2.

A desired peptide can be used as the secretion signal used in the present invention as long as it can extracellularly secrete the fusion protein of the present invention. If the antigen protein originally has a secretion signal, that secretion signal can be used. For example, in the SARS-CoV-2 spike protein, the first 13 peptides (SEQ ID NO: 8; base sequence: SEQ ID NO: 7) correspond to the secretion signal. For example, by adding it to an antigen protein fragment (eg, the RBD domain or fragment thereof, or a polypeptide comprising the RBD domain or fragment thereof), the antigen protein fragment can be made secretable. However, the present invention is not limited to this, and secretion signals for other desired secretory proteins may be used.

As the trimerization domain used in the present invention, any desired domain can be used as long as it can trimerize the fusion protein of the present invention. Preferably, the trimerization domain (foldon) of the T4 phage fibritin is used. For Foldon, for example, a polypeptide containing the amino acid sequence of SEQ ID NO: 10 or a partial sequence thereof and having trimerization activity can be used. SEQ ID NO: 9 can be exemplified as a coding sequence, but it is not limited to this. Foldon sequences are well known to those skilled in the art, and various variants have been made and used. These variants are also collectively referred to as foldon in the present invention.

Foldon sequences derived from other phages are easily identified by searching nucleotide and protein databases (eg, Enterobacteria phage phiC600P9, Escherichia phage vB_EcoM_FJ1, foldon sequences derived from these phages). In the present invention, these trimerization domains can be used as appropriate. GCN4 Leucine-zipper (Harbury, P. B. et al., A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 262 : 1401-1407, 1993), Lung surfactant protein-derived trimerization motif (Hoppe, H. J.et al., A parallel three stranded alpha-helical bundle at the nucleation site of collagen triplehelix formation. FEBS Lett 344: 191-195 , 1994), trimerization motif derived from the Collagen superfamily (McAlinden, A. et al., Alpha-helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily. J Biol Chem 278: 42200-42207, 2003). ing. In the present invention, trimerization domains other than Foldon, including these, can also be used as appropriate.

The trimerization domain may be added to the antigen protein fragment as appropriate, for example, to the N-terminus or C-terminus of the antigen protein fragment. For example, when the target antigen protein from which the antigenic protein fragment is derived is a membrane protein, a trimerization domain may be added to the original protein on the membrane-proximal side. For example, a trimerization domain can be added to the C-terminus of the antigenic protein fragment. A fusion protein of the invention preferably comprises, in that order, a secretory signal, an antigenic protein fragment, and a trimerization domain.

The fusion protein of the present invention containing an antigenic protein fragment can be expressed from a vector containing a nucleic acid encoding it. Nucleic acid is not limited in its form, and may be DNA or RNA.

A vector in the present invention is a carrier that introduces a nucleic acid into a cell. In the present invention, an expression vector is a carrier for introducing a nucleic acid into a cell, and is a vector capable of expressing a gene incorporated in the nucleic acid in the introduced cell. In the present invention, an expression vector means a vector into which a new nucleic acid is produced in a cell into which it has been introduced. Here, the production of a new nucleic acid means synthesis (biosynthesis) of a new nucleic acid using the nucleic acid contained in the vector as a template in a cell into which the vector has been introduced. Mere modification of the nucleic acid contained in the vector, decomposition of the nucleic acid contained in the vector to generate degradation products, and cleavage to generate cleavage products are not included in the generation of new nucleic acids. do not have. For example, in the present invention, an expression vector means that a nucleic acid contained in the vector is replicated and/or transcribed in a cell into which it has been introduced, or a functional RNA, mRNA, or the like is produced using the nucleic acid contained in the vector as a template. It is something to do. Functional RNA or mRNA itself, or compositions containing them (including lipid mixtures, lipid membrane capsules, etc.) do not produce new nucleic acids in the cells into which they are introduced, and are not expression vectors in the present invention.

There are no particular restrictions on the form of the vector, and any desired vector such as a plasmid, viral vector, or non-viral vector (eg, self-amplifying RNA) can be used. When expressed from a DNA vector, it can be expressed from any desired promoter. Examples of promoters that can be used include, but are not limited to, CMV promoter, CAG promoter, SV40 promoter, RSV promoter, EF1α promoter, and SRα promoter.

A viral vector is preferably used in the present invention. A "viral vector" is a vector that has a genomic nucleic acid derived from the virus, and that can express the gene after the viral vector is introduced into a cell by, for example, integrating a transgene into the genomic nucleic acid. Viral vectors include adenoviral vectors, adeno-associated viral vectors, HSV vectors, retroviral vectors (including lentiviral vectors), and negative-strand RNA viral vectors (including paramyxoviral vectors, particularly Sendai virus vectors). . In the present invention, it is most preferable to use a minus-strand RNA viral vector as the viral vector.

The viral vector may be a replication-incompetent vector, but preferably a replication-incompetent (replication-deficient) viral vector is used. In the virus of the present invention, replication-incompetent (or “replication-deficient” or “replication-deficient”) refers to the inability to replicate infectious viral particles in cells infected with the viral vector, and the viral genome in the infected cells. Even if the virus replicates, it is judged to be replication-incompetent (replication-deficient) unless it can replicate infectious virus particles. For example, genes essential for the formation of infectious virus particles, specifically proteins present on the surface of virus particles (for example, envelope proteins such as F and HN in the case of paramyxoviruses) are deleted or deleted from the virus genome. Thus, a replication-deficient virus can be obtained.

When using a minus-strand RNA viral vector in the present invention, the minus-strand RNA viral vector to be used is not particularly limited, but for example, a paramyxovirus vector can be preferably used. Paramyxovirus refers to viruses belonging to the family Paramyxoviridae or derivatives thereof. The family Paramyxoviridae is a member of the Mononegavirus group with non-segmented negative-strand RNA in the genome, and belongs to the subfamily Paramyxovirinae (genus Respirovirus (also called genus Paramyxovirus), genus Rubulavirus). , and the genera Mobilivirus) and the subfamily Pneumovirinae (including the genera Pneumovirus and Metapneumovirus). Viruses included in Paramyxoviridae viruses include, specifically, Sendai virus, Newcastle disease virus, Mumps virus, Measles virus, Respiratory syncytial virus. virus), rinderpest virus, distemper virus, simian parainfluenza virus (SV5), human

parainfluenza virus types

1, 2, and 3. More specifically, for example Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV), canine distemper virus (CDV), dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MeV), rinderpest virus (RPV), Hendra virus (Hendra), Nipah virus (Nipah), human parainfluenza virus-2 (HPIV-2 ), simian parainfluenza virus 5 (SV5), human parainfluenza virus-4a (HPIV-4a), human parainfluenza virus-4b (HPIV-4b), mumps virus (Mumps), and Newcastle disease virus (NDV). Rhabdoviruses include Vesicular stomatitis virus, Rabies virus, etc. of the Rhabdoviridae family.

In general, the genomic RNA of minus-strand RNA viruses, including paramyxoviruses, is the minus strand (negative strand), and proteins, etc. are encoded as antisense sequences on the genomic RNA. In the present invention, such cases are also referred to as "encoding" the protein. In addition, when a protein is encoded as an antisense sequence in the minus-strand RNA genome, it is also said that the gene for the protein is incorporated in the genome. "Upstream" of the genome (minus strand) of a negative-strand RNA virus refers to the 3' side of the genome, and "downstream" refers to the 5' side. Using the minus-strand RNA genome as a template, plus-strand (positive-strand) RNA genome (also called antigenome) is replicated, and transcription occurs using the minus-strand RNA genome as a template to generate sense-strand RNA. In the present invention, the minus-strand RNA genome and the plus-strand RNA genome are sometimes collectively referred to as "genomes".

A paramyxovirus vector is a chromosomal non-integrating virus vector, and since the vector is expressed in the cytoplasm, there is no risk of the transgene being integrated into the host's chromosome (nuclear-derived chromosome). Therefore, the safety is high, and the vector can be removed from infected cells. In the present invention, paramyxovirus vectors include not only infectious viral particles, but also viral cores, complexes of viral genomes and viral proteins, complexes composed of non-infectious viral particles, etc., which are introduced into cells. Included are complexes that are capable of expressing genes carried by the . For example, in paramyxoviruses, the ribonucleoprotein (virus core portion), which consists of the paramyxovirus genome and the paramyxovirus proteins (NP, P, and L proteins) that bind to it, is introduced into the cell to produce a transgene within the cell. (WO00/70055). Introduction into cells may be performed using an appropriate transfection reagent or the like. Such ribonucleoproteins (RNPs) are also included in the paramyxovirus vectors of the present invention. In the present invention, the paramyxovirus vector is preferably a particle in which the RNP described above is derived from the cell membrane and wrapped in a biomembrane.

When a paramyxovirus vector is used as the expression vector of the present invention, particularly preferred paramyxoviruses are viruses belonging to the subfamily Paramyxovirinae (including the genera Respirovirus, Rubulavirus, and Mobilivirus). , more preferably a virus belonging to the genus Respirovirus (also referred to as the genus Paramyxovirus). Respirovirus viruses to which the present invention can be applied include, for example, human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 3 (HPIV-3), and bovine parainfluenza virus type 3 (BPIV-3). , Sendai virus (also called murine parainfluenza virus type 1), measles virus, simian parainfluenza virus (SV5), and simian parainfluenza virus type 10 (SPIV-10). In the present invention, the paramyxovirus is most preferably Sendai virus.

Paramyxoviruses generally contain a complex consisting of RNA and protein (ribonucleoprotein; RNP) inside the envelope. The RNA contained in RNP is a single-stranded RNA of (-) strand (negative strand), which is the genome of Paramyxovirus, and this single-stranded RNA binds to NP protein, P protein, and L protein to forming The RNA contained in this RNP serves as a template for transcription and replication of the viral genome (Lamb, R.A., and D. Kolakofsky, 1996, Paramyxoviridae: The viruses and their replication. pp.1177-1204. In Fields Virology, 3rd Fields, B. N., D. M. Knipe, and P. M. Howley et al. (ed.), Raven Press, New York, N. Y.).

The "NP, P, M, F, HN, and L genes" of Paramyxovirus refer to the genes encoding the nucleocapsid, phospho, matrix, fusion, hemagglutinin-neuraminidase, and large proteins, respectively. Nucleocapsid (NP) protein is a protein that binds to genomic RNA and is essential for genomic RNA to have template activity. In general, the NP gene is sometimes written as "N gene". Phospho (P) proteins are phosphorylated proteins that are the small subunits of RNA polymerase. Matrix (M) proteins function to maintain the virus particle structure from the inside. The fusion (F) protein is a membrane fusion protein involved in host cell entry, and the hemagglutinin-neuraminidase (HN) protein is involved in host cell binding. The large (L) protein is the large subunit of RNA polymerase. Each gene described above has an individual transcription control unit, and a single mRNA is transcribed from each gene, and a protein is transcribed. From the P gene, in addition to the P protein, a nonstructural protein (C) that is translated using a different ORF and a protein (V) that is produced by RNA editing during reading of the P protein mRNA are translated. For example, each gene in each virus belonging to the subfamily Paramyxovirinae is generally represented in the order of encoding from the beginning (3') of the genome as follows.
Respirovirus N P/C/V M F HN - L
Rubulavirus N P/V M F HN (SH) L
Mobilivirus genus N P/C/V M F H - L

For example, the accession number of the base sequence database for each gene of Sendai virus is M29343, M30202, M30203, M30204, M51331, M55565, M69046, X17218 for the N gene, and M30202, M30203, M30204, M55565 for the P gene. M69046 , X00583, X17007, X17008, for M gene D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, X53056, for F gene D00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152 , X02131, D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, X56131 for HN gene, D00053, M30202, M30203, M30204 for L gene , M69040, X00587, X58886. As examples of viral genes encoded by other viruses, for the N gene, CDV, AF014953; DMV, X75961; HPIV-1, D01070; HPIV-2, M55320; HPIV-3, D10025; NDV, AF064091; PDPR, X74443; PDV, X75717; RPV, X68311; SeV, X00087; SV5, M81442; 1869; DMV, Z47758; HPIV -l, M74081; HPIV-3, X04721; HPIV-4a, M55975; HPIV-4b, M55976; Mumps, D86173; 30202; SV5, AF052755 and Tupaia, AF079780, for the C gene CDV, AF014953; DMV, Z47758; HPIV-1, M74081; HPIV-3, D00047; MeV, ABO16162; 079780, about the M gene is CDV, M12669; DMV Z30087; HPIV-1, S38067; HPIV-2, M62734; HPIV-3, D00130; HPIV-4a, D10241; HPIV-4b, D10242; ; NDV, AF089819; PDPR, Z47977; PDV, X75717; RPV, M34018; SeV, U31956; and SV5, M32248, for the F gene CDV, M21849; -3, X05303 , HPIV-4a, D49821; HPIV-4b, D49822; Mumps, D86169; MeV, AB003178; NDV, AF048763; PDPR, Z37017; PDV, AJ224706; and SV5, AB021962, HN (H or G) Genes CDV, AF112189; DMV, AJ224705; HPIV-1, U709498; HPIV-2. D000865; HPIV-3, AB012132; MeV, K01711 NDV, AF204872; PDPR, X74443; PDV, Z36979; RPV, AF132934; SeV, U06433; 18; HPIV-2, Mumps, AB040874; MeV, K01711; NDV, AY049766; PDPR, AJ849636; PDV, Y09630; However, a plurality of strains are known for each virus, and genes comprising sequences other than those exemplified above exist depending on strain differences. A Sendai virus vector having a viral gene derived from any of these genes is particularly useful as a viral vector for producing the vaccine of the present invention. For example, the Sendai virus vector of the present invention has 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the coding sequence of any of the above viral genes. may contain a base sequence with In addition, the Sendai virus vector of the present invention, for example, the amino acid sequence encoded by the coding sequence of any of the above viral genes, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, Alternatively, it may contain a nucleotide sequence encoding an amino acid sequence with 99% or more identity. In the present invention, the Sendai virus vector has, for example, an amino acid sequence encoded by the coding sequence of any of the above-mentioned viral genes, having 10 amino acids or less, preferably 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, It may include a nucleotide sequence encoding an amino acid sequence in which no more than 5, no more than 4, no more than 3, no more than 2, or one amino acid is substituted, inserted, deleted, and/or added.

The sequences referred to by database accession numbers such as base sequences and amino acid sequences described in this specification refer, for example, to sequences as of the filing date and priority date of the present application. can be specified as the sequence at any point in time, preferably as the sequence as of the filing date of the present application. The sequence at each point in time can be identified by referencing the revision history of the database.

The minus-strand RNA virus used in the present invention may be a derivative, and the derivative includes a virus whose viral gene has been modified so as not to impair the ability of the virus to introduce genes, a virus that has been chemically modified, and the like. .

In addition, viruses used as viral vectors may be derived from natural strains, wild strains, mutant strains, laboratory passage strains, artificially constructed strains, and the like. For example, Sendai virus includes, but is not limited to, Z strain (Medical Journal of Osaka University Vol.6, No.1, March 1955 p1-15). That is, the virus may be a virus having a structure similar to that of a virus isolated from nature or a virus artificially modified by genetic recombination, as long as the virus particles of interest can be produced. For example, wild-type viruses may have mutations or deletions in any of their genes. For example, a virus having a deletion in at least one gene encoding a viral envelope protein or coat protein or a mutation such as a stop codon mutation that suppresses the expression thereof can be preferably used. A virus that does not express such an envelope protein is, for example, a virus that can replicate its genome in infected cells but cannot form infectious virus particles. Such transmissibility-deficient viruses are suitable as highly safe expression vectors. For example, in paramyxovirus vectors, viruses that do not encode either the F or HN envelope protein (spike protein) gene or the F and HN genes in their genomes can be used (WO00/70055 and WO00/70070; Li, H.-O. et al., J. Virol. 74(14) 6564-6569 (2000)). A virus can amplify its genome in an infected cell if its genomic RNA encodes at least the proteins required for genome replication (eg, the N, P, and L proteins). In order to produce envelope protein-deficient and infectious virus particles, for example, the defective gene product or a protein capable of complementing it is exogenously supplied in virus-producing cells (WO00/70055 and WO00/ 70070; Li, H.-O. et al., J. Virol. 74(14) 6564-6569 (2000)). On the other hand, non-infectious virus particles can be recovered by not complementing the defective viral proteins at all (WO00/70070).

In addition, in the production of the virus of the present invention, a virus carrying a mutant viral protein gene may be used. For example, many mutations, including attenuating mutations and temperature-sensitive mutations, are known in viral structural proteins (NP, M) and RNA synthetase (P, L). Minus-strand RNA viruses having these mutant protein genes can be suitably used in the present invention, depending on the purpose. In the present invention, viruses with reduced cytotoxicity may be used. Cytotoxicity can be measured, for example, by quantifying the release of lactate dehydrogenase (LDH) from cells. The degree of attenuation of cytotoxicity can be evaluated, for example, by infecting human-derived HeLa cells (ATCC CCL-2) or monkey-derived CV-1 cells (ATCC CCL 70) at an MOI (infectious titer) of 3 and culturing for 3 days. Viruses in which the amount of LDH released in the fluid is significantly reduced compared to the wild type, for example, viruses in which the amount is reduced by 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 50% or more can be used. . Mutations that reduce cytotoxicity also include temperature sensitive mutations. A temperature-sensitive mutation is one that has significantly reduced activity at the normal temperature of the virus host (e.g. 37°C to 38°C) compared to lower temperatures (30°C to 36°C, e.g. 30°C to 32°C). . Such proteins with temperature-sensitive mutations are convenient because viruses can be produced under the permissive temperature (low temperature). For example, when using a virus with a temperature-sensitive mutation, the growth rate or gene expression level is at least 1/2 or less, preferably at least 1/2 when infected at 32°C in cultured cells, when infected at 32°C. One-third or less, more preferably 1/5 or less, more preferably 1/10 or less, more preferably 1/20 or less can be used.

For example, many mutations, including attenuating mutations and temperature-sensitive mutations, are known in viral structural proteins (NP, M) and RNA synthetase (P, L). Viral vectors and the like having these mutated protein genes can be suitably used in the present invention depending on the purpose.

Specifically, for example, in Sendai virus vectors, the M gene mutation is arbitrarily selected from the group consisting of positions 69 (G69), 116 (T116), and 183 (A183) in the M protein. Site amino acid substitutions can be mentioned (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). A genome encoding a mutated M protein in which any one of the above three sites, preferably a combination of any two sites, more preferably all three sites are substituted with other amino acids in the Sendai virus M protein. Viruses possessing the above are preferably used in the present invention depending on the purpose.

Amino acid mutations are preferably substitutions of other amino acids with different side chain chemistries, such as the BLOSUM62 matrix (Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) is 3 or less, preferably 2 or less, more preferably 1 or less, more preferably 0. Specifically, G69, T116, and A183 of the Sendai virus M protein can be replaced with Glu (E), Ala (A), and Ser (S), respectively. It is also possible to use mutations that are homologous to the mutations in the M protein of the measles virus temperature-sensitive strain P253-505 (Morikawa, Y. et al., Kitasato Arch. Exp. Med. 1991: 64; 15-30). be. Mutations may be introduced according to known methods for introducing mutations, for example, using oligonucleotides and the like.

Further, mutations in the HN gene include, for example, amino acid substitutions at positions arbitrarily selected from the group consisting of positions 262 (A262), 264 (G264), and 461 (K461) of the Sendai virus HN protein. (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). A virus having a genome encoding a mutant HN protein in which any one of the three sites, preferably a combination of any two sites, and more preferably all three sites have amino acids substituted with other amino acids is used in the present invention. It is preferably used depending on the purpose. As with the above, substitution of amino acids with other amino acids having different side chain chemical properties is preferred. To give a preferred example, A262, G264, and K461 of the Sendai virus HN protein are replaced with Thr (T), Arg (R), and Gly (G), respectively. In addition, for example, referring to the temperature-sensitive vaccine strain Urabe AM9 of mumps virus, the 464th and 468th amino acids of the HN protein can be mutated (Wright, K. E. et al., Virus Res. 2000: 67 49-57).

The Sendai virus may also have mutations in the P gene and/or the L gene. Specific examples of such mutations include mutation of Glu (E86) at position 86 of SeV P protein and substitution of Leu (L511) at position 511 of SeV P protein with another amino acid. As with the above, substitution of amino acids with other amino acids having different side chain chemical properties is preferred. Specifically, substitution of the 86th amino acid with Lys, substitution of the 511th amino acid with Phe, and the like can be exemplified. In the L protein, substitution of Asn (N1197) at position 1197 and/or Lys (K1795) at position 1795 of the SeV L protein with other amino acids can be mentioned. is preferably replaced with another amino acid of different chemical nature. Specifically, substitution of the 1197th amino acid with Ser, substitution of the 1795th amino acid with Glu, and the like can be exemplified. Mutations in the P gene and L gene can significantly enhance the effects of persistent infectivity, suppression of secondary particle release, or suppression of cytotoxicity. Furthermore, these effects can be dramatically increased by combining mutations and/or deletions of envelope protein genes. In addition, the L gene includes substitution of Tyr (Y1214) at position 1214 and/or Met (M1602) at position 1602 of the SeV L protein with other amino acids. Substitutions with other amino acids with different chemical properties are included. Specifically, substitution of the 1214th amino acid with Phe, substitution of the 1602nd amino acid with Leu, and the like can be exemplified. The mutations exemplified above can be combined arbitrarily.

For example, at least G at position 69, T at position 116, and A at position 183 of the SeV M protein; at least A at position 262, G at position 264, and K at position 461 of the SeV HN protein; L at position 1197 and K at position 1795 of the SeV L protein are substituted with other amino acids, respectively, and the F gene is deleted or deleted, and the cytotoxicity of these Sendai virus vectors and/or F gene-deficient or deleted Sendai virus vectors with similar or greater suppression of NTVLP formation at 37°C can be used in the present invention depending on the purpose.

More specifically, the F gene is deleted, the G69E, T116A, and A183S mutations in the M protein, the A262T, G264R, and K461G mutations in the HN protein, the L511F mutation in the P protein, and the N1197S and N1197S in the L protein. A Sendai virus vector containing the K1795E mutation in its genome can be used. In the present invention, the combination of F gene deletion and these mutations is referred to as "TSΔF".

The viral vector used in the present invention may encode, in its genome, foreign genes and regulatory factors that control viral properties, in addition to viral protein genes. For example, it may encode a degron sequence or miRNA target sequence to regulate the expression of viral proteins.

In the minus-strand RNA virus preferably used in the present invention, at least one envelope gene is deleted or mutated. Such viruses include at least one envelope gene deleted, at least one envelope gene mutated, at least one envelope gene mutated and at least one envelope gene deleted. includes those that are At least one mutated or deleted envelope gene is preferably a gene encoding an envelope-constituting protein, such as the F gene and/or the HN gene in a paramyxovirus vector. For example, one that lacks the F gene or that encodes a loss-of-function mutant F protein can be preferably used. Alternatively, the HN gene may be deleted, or the HN gene may encode a loss-of-function mutant HN protein. In addition, for example, minus-strand RNA viruses lacking the F gene and further lacking the HN gene or having mutations in the HN gene are preferably used in the present invention. Minus-strand RNA viruses lacking the F gene and further lacking the HN gene, for example, are also preferably used in the present invention. Such mutant viruses can be produced according to known methods.

For example, in the case of a paramyxovirus vector, in a preferred embodiment, at least one of its own envelope protein genes (eg, F gene) is deleted from the genome, and its own N, P, and L genes are carried. Also, the viral vector preferably carries its own M gene on its genome.

The viral vector of the present invention expressably carries a nucleic acid encoding the fusion protein of the present invention, that is, a fusion protein that can be released outside the membrane, including a secretory signal, an antigenic protein fragment, and a trimerization domain. A nucleic acid encoding the protein of interest can be inserted at the desired location in the genome of the viral vector. For example, in the case of minus-strand RNA viruses, the closer to the 3' end of the genome (minus strand), the higher the expression level can be expected. A nucleotide sequence encoding the fusion protein can be inserted between the gene for the protein (usually N protein). Alternatively, between the genes for the first negative-strand RNA viral protein (usually the N protein) and the second negative-strand RNA viral protein (usually the P protein), the second and third (usually between the P and M), etc. good too.

The vector of the present invention may encode additional genes as long as it encodes the fusion protein of the present invention. For example, the vector may encode other antigen proteins or physiologically active proteins (cytokines, etc.). .

When a nucleic acid encoding these desired proteins is loaded onto a vector, the position thereof may be determined as appropriate. When loaded, the nucleic acid encoding the fusion protein may be inserted upstream of the genome of the paramyxovirus vector (3' side of the viral genome) relative to nucleic acids encoding other antigens. Nucleic acids encoding these proteins can be inserted with their ends flanked by the S (start) sequence and E (end) sequence of the paramyxovirus as appropriate. In paramyxovirus vectors, the S sequence is a signal sequence that initiates transcription, and the E sequence terminates transcription. A region flanked by the S and E sequences constitutes one transcription unit. Between the E sequence of one gene and the S sequence of the next gene, an appropriate spacer sequence (intervening sequence; I) can be inserted (ie EIS sequence).

For example, in the case of Sendai virus vectors, the S sequence is preferably 3'-UCCCWVUUWC-5' (W = A or U; V = A, C, or G) (SEQ ID NO: 11). can. Particularly preferred are 3'-UCCCAGUUUC-5' (SEQ ID NO: 12), 3'-UCCCACUUAC-5' (SEQ ID NO: 13), and 3'-UCCCACUUUC-5' (SEQ ID NO: 14). These sequences are 5'-AGGGTCAAAG-3' (SEQ ID NO: 15), 5'-AGGGTGAATG-3' (SEQ ID NO: 16), and 5'-AGGGTGAAAG-, respectively, when represented by the DNA sequence encoding the plus strand. 3' (SEQ ID NO: 17). The E sequence of the Sendai virus vector is preferably, for example, 3'-AUUCUUUUU-5' (5'-TAAGAAAA-3' in DNA encoding the plus strand). The I sequence may be, for example, any three bases, and specifically 3'-GAA-5' (5'-CTT-3' in plus strand DNA) may be used, but is limited to this. isn't it.

For example, the fusion protein of the present invention targeting the spike protein of SRAS-CoV-2 includes a protein consisting of the amino acid sequence (S-RBD-foldon) of SEQ ID NO: 29 produced in the Examples, or a protein containing the sequence , one or more in the sequence (e.g. several, preferably no more than 30, no more than 20, no more than 10, no more than 8, no more than 7, no more than 5, no more than 3, no more than 2, or 1) A protein comprising a sequence in which amino acids of SEQ ID NO:29 are added, deleted, substituted, and/or inserted, with a high identity to the amino acid of SEQ ID NO:29 (e.g., 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% 93% or more, 95% or more, or 96% or more identity) can be preferably used, and a vector containing a nucleic acid encoding the protein is suitable in the present invention. Specific examples include, but are not limited to, the 15th to 758th base sequences of SEQ ID NO: 28 or the RNA sequences corresponding thereto, or their complementary sequences (for vectors having a minus strand in the genome, etc.). isn't it. When the nucleotide sequence is inserted into a Sendai virus vector, examples of the nucleotide sequence to be inserted include SEQ ID NO: 28 (as a positive strand DNA sequence). can be preferably used in the present invention. However, the present invention is not limited thereto, and those skilled in the art can construct various antigen expression vectors that exhibit similarly excellent effects based on the description of this specification.

By introducing the antigen expression vector of the present invention into cells, a vector-encoded fusion protein containing an antigen protein fragment can be expressed in cells. Although the expressed fusion protein is secreted and released outside the cell, some expression products may remain inside the cell. That is, the vector of the present invention may be an antigen-expressing vector in which an expression product containing an antigen protein fragment is distributed both intracellularly and extracellularly in a vector-introduced cell. In preferred embodiments, more expression product is released outside the cell than is retained in the cell. Here, the expression product refers to a translation product in the case of expression of a gene encoding a polypeptide, and in the case of the present invention specifically refers to a polypeptide containing an antigenic protein fragment. As shown in the Examples, when the fusion protein having the structure of the present invention is expressed, most of the expression products (polypeptides containing antigenic protein fragments) are extracellularly secreted. That is, the amount of expression products (polypeptides containing antigen protein fragments) that are secreted and released outside the cells is three times or more, for example, about 3 to 20 times the amount of expression products that remain in cells. The intracellular retention rate of the entire expression product including those released outside reached 25% or less, for example, 5-25%, and the extracellular secretory release rate reached 79-95%. For example, in the vector of the present invention, the expression product released outside the cell is, for example, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times the expression product that remains in the cell. It may be greater than, 10 times greater, or 15 times greater. In addition, in the vector of the present invention, the percentage of polypeptides containing antigen protein fragments produced from the vector that are released outside the cells is, for example, 60% or more, 65% or more, 70% or more, 75% or more, or 80%. It may be greater than or equal to 85% or greater than or equal to 90%. A secreted protein may be truncated for a secretory signal.

Further, in the present invention, when the fusion protein of the present invention having a secretion signal, an antigenic protein fragment, and a trimerization domain is expressed from a vector, it has the same configuration (same secretion signal and The fusion protein of the present invention, which has a trimerization domain, is larger in molecular size than a control protein having an antigenic protein fragment (polypeptide having an antigenic protein fragment) expressed from the same vector. Although it is expected to be disadvantageous for That is, a vector of the invention encoding a protein comprising a secretory signal, an antigenic protein fragment, and a trimerization domain, a control antigen encoding a protein comprising a secretory signal and an antigenic protein fragment, but no trimerization domain. The expression level of the expression product containing the antigen protein fragment (polypeptide containing the antigen protein fragment) is, for example, 1.5-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more compared to the expression vector , 7x or more, 8x or more, 9x or more, or 10x or more. Expression levels can be compared by, for example, constructing vectors with the same configuration except for the presence or absence of a trimerization domain, and using the same type of cells to determine the expression level (total sum of intracellular and extracellular secretion). expression level) can be measured. Cells to be used can be appropriately selected, for example, LLCMK2 cells (ATCC-CCL-7) can be used, but are not limited thereto.

The present invention provides a method for producing the fusion protein of the present invention comprising a secretory signal, an antigenic protein fragment and a trimerization domain, comprising the step of introducing the antigen expression vector of the present invention into a cell. The present invention also provides a method for producing an antigen protein fragment, comprising the step of introducing the antigen expression vector of the present invention into a cell. The present invention also provides a method for producing extracellularly released antigen protein fragments, comprising the step of introducing the antigen-expressing vector of the present invention into cells. That is, the present invention provides a method for producing a fusion protein comprising an antigenic protein fragment and a trimerization domain, comprising the step of introducing an antigen expression vector of the present invention into a cell. The present invention also provides a method for extracellular secretion and release of an antigen protein fragment, comprising the step of introducing the antigen expression vector of the present invention into a cell. Each method may further comprise the steps of culturing the vector-introduced cells and recovering the produced or secreted expression products, including antigenic protein fragments.

In addition, the vectors of the present invention increase the amount of expression products containing antigen protein fragments by adding trimerization domains, and the expression products are distributed both intracellularly and extracellularly in vector-introduced cells. Therefore, the vectors of the present invention are useful for increasing the expression level of antigen protein fragments, and for distributing expression products both intracellularly and extracellularly in vector-introduced cells. The present invention provides an antigen expression vector in which the amount of the expression product containing the antigen protein fragment is increased by the addition of a trimerization domain, and the expression product is distributed both intracellularly and extracellularly in vector-introduced cells. do. The present invention also provides a method for constructing the antigen protein expression vector of the present invention and an antigen protein expression vector of the present invention for increasing the amount of an expression product containing an antigen protein fragment by adding a trimerization domain. Provide usage and usage instructions. The present invention also provides a method for producing the antigen protein expression vector of the present invention and the use of the antigen protein expression vector of the present invention for distributing the expression product from the vector both intracellularly and extracellularly in a vector-introduced cell. and provide usage instructions. The present invention also relates to a method for increasing the expression level and/or the extracellular secretion release amount when the antigen protein or antigen protein fragment is expressed by adding a trimerization domain to the antigen protein or antigen protein fragment. .

The present invention also provides an antigen expression vector in which the amount of an expression product containing an antigen protein fragment is regulated by addition or deletion of a trimerization domain, and the expression product is distributed both intracellularly and extracellularly in a vector-introduced cell. I will provide a. The invention also provides methods for modulating the amount of expression products, including antigenic protein fragments, by the presence or absence of a trimerization domain. If it is desired to increase the amount of expression product, a protein with a trimerization domain can be expressed, and if it is desired to decrease it, a protein without a trimerization domain can be expressed. The method is also a method for distributing the expression product both intracellularly and extracellularly in vector-introduced cells.

The present invention also found that the amount of an expression product containing an antigenic protein fragment can be regulated by the size of the antigenic protein fragment. The present invention also found that the intracellular and extracellular distribution of expression products in vector-introduced cells can be controlled by the size of the antigen protein fragment. That is, by shortening the antigen protein fragment, it is possible to increase the amount of expression from the vector and to increase the rate of extracellular secretion and release of the expression product. Conversely, by lengthening the antigen protein fragment, it is possible to suppress the expression level from the vector and to reduce the rate of secretion and release of the expression product to the outside of the cell (that is, increase the rate of retention in the cell). . The length of the antigen protein fragment can be adjusted as appropriate to achieve the desired expression level and/or intracellular/extracellular distribution of the expression product.

For example, when the purpose is to increase the expression level and/or to increase extracellular secretion and release, the length of the antigen protein fragment is, for example, 500 amino acids or less, 350 amino acids or less, 300 amino acids or less, 280 amino acids or less, 250 amino acids or less, or 230 amino acids or less. Particularly preferred are antigenic protein fragments of 220 amino acids or less, for example antigenic protein fragments of 215 amino acids or less, 210 amino acids or less, or 205 amino acids or less are particularly preferred.

The total length of the fusion protein, including the secretory signal and the trimerization domain, is e.g. 550 amino acids or less, e.g. Amino acids or less. In particular, it is preferably 270 amino acids or less, and particularly preferably 265 amino acids or less, 260 amino acids or less, or 250 amino acids or less.

Conversely, when the purpose is to suppress the expression level and/or to suppress extracellular secretion and release, the length of the antigen protein fragment may be increased, for example, longer than the above amino acids. can be done.

The present invention also provides a vector that expresses an antigen protein having a trimerization domain by removing the coding region of the trimerization domain from a vector that expresses an antigen protein that does not have a trimerization domain. It was found that the productivity of can be improved. Based on this knowledge, it becomes possible to regulate the productivity of vectors encoding antigen proteins. That is, the present invention provides a method for suppressing or improving vector productivity by adding or removing a trimerization domain-encoding sequence in the antigen protein coding sequence, respectively. By adding or removing the sequence encoding the trimerization domain in the coding sequence of the antigen protein, the expression level of the antigen protein is increased or decreased, respectively, in production cells during the culture process for manufacturing the vector. It is possible to increase or decrease the amount of secretion and release, respectively, or to suppress or improve vector productivity, respectively, and to adjust vector productivity to the extent that high immunogenicity is maintained. The present invention also provides methods for increasing or decreasing the expression level of an antigen protein, for increasing or decreasing the secretory release amount of an expression product, or for suppressing or improving vector productivity. , relates to the use of vectors expressing antigen proteins with or without trimerization domains, and these vectors used in such applications.

For example, the present invention provides an expression product containing an antigen protein fragment that is distributed both intracellularly and extracellularly in a vector-introduced cell by regulating the amount of the expression product by adding or removing a trimerization domain. It relates to vectors expressing antigenic proteins with or without trimerization domains, wherein vector productivity is modulated. In addition, the present invention provides an expression product containing an antigen protein fragment that is distributed both intracellularly and extracellularly in a vector-introduced cell and a vector by regulating the amount of the expression product containing the antigen protein fragment according to the size of the antigenic protein fragment. It relates to vectors expressing antigenic proteins with trimerization domains or antigenic proteins without trimerization domains with modulated productivity.

In the above description, the trimerization domain can be appropriately selected, but it is preferable to use, for example, the trimerization domain (foldon) of T4 phage fibritin.

The vector, vector-introduced cell, or vector expression product of the present invention can be appropriately made into a composition, for example, by combining it with a pharmaceutically acceptable carrier or medium. The composition is, for example, a composition comprising a vector of the present invention, a vector-introduced cell, or an expression product thereof and a desired carrier or medium. A pharmacologically acceptable carrier or medium is appropriately selected, and examples include water (e.g., sterile water), physiological saline (e.g., phosphate-buffered saline), buffer solution, culture medium, glycol, ethanol, glycerol, Lactose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, gelatin, dextran, agar, pectin, polyvinylpyrrolidone, cellulose, methylcellulose, methylmethylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, olive oil, Oils such as peanut oil, sesame oil, mineral oil, etc., and emulsifiers, suspending agents, surfactants, buffers, flavoring agents, diluents, preservatives, stabilizers, excipients, vehicles, preservatives, slowdown agents. Release agents and the like are also included (Remington: The Science and Practice of Pharmacy, 19th edition, Williams & Wilkins, 1995). Any combination of these and other pharmaceutically acceptable carriers or media can be used to form the compositions of the present invention.

The vector of the present invention encoding a fusion protein with a secretory signal, an antigenic protein fragment, and a trimerization domain, in a preferred embodiment, upon inoculation into a subject showed excellent IgG antibody induction (Example 3c), For example, when a viral antigen is targeted, both single and repeated inoculations induce high levels of neutralizing antibodies having neutralizing activity against viral infection of the target virus (Example 3d). Thus, the vectors, vector-introduced cells, or vector expression products of the present invention are useful for inducing an immunological response, particularly a humoral immune response, to a target antigen. In particular, the vectors of the present invention can secrete and release antigen protein fragments extracellularly at high levels, so that the secreted and released antigen proteins do not remain in the cells into which the vectors have been introduced, but spread over a wide area around the cells. expected to do so. This leads to sufficient phagocytosis by antigen-presenting cells, induces antibody-producing B cells and cytotoxic T cells, promotes the formation of long-lived plasma cells and memory B cells that contribute to antibody production, and promotes immunity. It is expected that longer life will be achieved.

In addition, part of the expression product produced from the vector of the present invention remains in cells, and the antigen-specific CTL induced by vector inoculation is higher than that of the vector (S1-foldon) used for comparison ( Example 3f). Thus, the vectors of the present invention are also useful for inducing cell-mediated immune responses against target antigens. That is, the vector of the present invention has excellent properties for inducing both humoral and cell-mediated immunity.

That is, the vectors of the present invention and the compositions of the present invention are particularly useful as vaccines. Since the vaccine of the present invention can efficiently induce humoral and cellular immunity responses to antigen proteins, it is a vaccine for prophylaxis and treatment against, for example, pathogenic microorganisms and infectious microorganisms (including viruses). is particularly useful as Moreover, the vaccine of the present invention is expected to be suitably used for immunotherapy against cancer and the like. The vaccine formulation of the present invention is appropriately produced by combining pharmaceutically acceptable carriers or media such as water, physiological saline, buffers, buffers, salts, excipients, anticoagulants, or combinations thereof. can do. Moreover, the vaccine formulation of the present invention may further contain an adjuvant or an immunostimulant as appropriate. Adjuvants include, for example, alum, aluminum phosphate, Freund's complete and incomplete adjuvants, virosomes, liposomes, lipopolysaccharides, oily or aqueous emulsion type adjuvants, adjuvants selected from these, or other adjuvants, or Any combination thereof may be included as appropriate.

When the antigen-expressing vector, composition, or vaccine of the present invention is administered to a subject, the administration route can be appropriately selected, and can be administered orally or parenterally. Examples of parenteral administration include nasal administration, suction, intranasal administration, intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, intradermal administration, sublingual administration, intravenous administration, enteral administration, and transmucosal administration. administration and the like, but are not limited to them. For formulation as an injection, it can be formulated using a carrier such as distilled water for injection. Aqueous solutions for injection include aqueous solutions containing other ingredients such as saline, glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride and the like. It may also contain alcohol, propylene glycol, polyethylene glycol, nonionic surfactants, and the like.

The present invention provides an immunity induction method comprising the step of administering the antigen-expressing vector, composition, or vaccine of the present invention to a subject. The present invention also provides a method of inducing humoral immunity, cell-mediated immunity, or both against an antigen, comprising the step of administering the antigen-expressing vector, composition, or vaccine of the present invention to a subject. The present invention also provides use of the antigen-expressing vector of the present invention for inducing humoral immunity, cell-mediated immunity, or both against the antigen. The present invention also provides use of the antigen-expressing vector of the present invention in the manufacture of a drug for inducing humoral immunity, cell-mediated immunity, or both against the antigen. The present invention also provides use of the antigen-expressing vector of the present invention in the production of vaccines. The present invention also provides prophylactic or therapeutic methods against infectious diseases or cancer using the antigen-expressing vectors, compositions, or vaccines of the present invention. The present invention also provides antigen-expressing vectors, compositions, or vaccines of the invention for prophylactic or therapeutic use against infectious diseases or cancer. The present invention also provides use of the antigen-expressing vector of the present invention in the production of a drug for use in preventing or treating infectious diseases or cancer. For example, an antigen derived from an infectious organism that is the cause of an infectious disease (here, the infectious organism may be an infectious virus) is used, and for cancer, the cancer of cancer antigens are used.

The antigen-expressing vector, composition, or vaccine of the present invention can be administered, for example, by nasal administration, intranasal administration, or inhalation. As shown in the Examples, the vector of the present invention induces excellent immune responses against viral infections and the like by intranasal inoculation. Specifically, intranasal inoculation of the vaccine of the present invention enables highly efficient induction of humoral immunity against the target antigen. Moreover, not only humoral immunity but also cell-mediated immunity can be induced at a high level. That is, the present invention provides a method for preventing or treating infectious diseases, which comprises the step of nasally inoculating a subject with the vaccine of the present invention. In addition, the present invention induces an immune response (humoral and/or cellular immune response) against an antigenic protein of an infectious organism (including a virus), which comprises the step of nasally inoculating a subject with the vaccine of the present invention. provide a way. For intranasal inoculation, a suitable form of vaccine may be selected as appropriate. For example, the vaccine of the present invention containing a minus-strand RNA viral vector is suitable because it can efficiently elicit an immune response by expressing the target antigen at the site of inoculation. The present invention provides a method for preventing or treating infectious diseases, which comprises the step of intranasally inoculating a subject with the vaccine of the present invention containing a minus-strand RNA viral vector. The present invention also provides immune response (humoral immunity and/or cellular provide a method of inducing an immune response). Infectious organisms can be any desired pathogenic microorganism, but include, for example, viruses, particularly viruses and bacteria that can be transmitted through the respiratory tract, rhinoviruses, coronaviruses, respiratory syncytial viruses, parainfluenza viruses, adenoviruses. Viruses, influenza viruses, enteroviruses, etc., and in particular coronaviruses (including SRAS-CoV-2).

The form of administration of the antigen-expressing vector, composition, or vaccine of the present invention is not particularly limited, and can be used, for example, for single or multiple inoculations. In multiple vaccinations, for example, the vaccine of the present invention may be administered multiple times, or may be administered in combination with other vaccines. For example, in primary inoculation, inoculation may be performed using a non-viral vector vaccine, and in booster inoculation, a vaccine formulation containing a viral vector (preferably a negative-strand RNA viral vector) may be inoculated. Negative-strand RNA viral vectors have no risk of integration into host chromosomes, so they are highly safe and can be administered multiple times (e.g., 2 or more, 3 or more, 4 or more, or 5 or more) It is also suitable for inoculation of

In general, viral vector-type vaccines induce antibodies and CTLs specific to the protein of the viral vector itself, which limits the booster effect of vaccine antigens by booster vaccination and has been induced by similar viruses in the past. There is a concern that the immunogenicity of the vaccine antigen may be suppressed by acting on the proteins of the viral vector itself (cross-immunity). However, as shown in the Examples, when repeated inoculations were performed using a viral vector expressing a fusion protein of a secretory signal, an antigenic protein fragment, and a trimerization domain, repeated inoculations exhibited a strong booster effect. was confirmed. This suggests that the strong immunity-inducing effect of the vaccine of the present invention using a viral vector is not hindered even if vaccination with a similar viral vector has been performed in the past or there is a history of infection with a similar virus. This means that the vaccine of the present invention using a viral vector exhibits excellent effects in multiple inoculations such as booster inoculations. That is, the vaccine of the present invention is useful for exerting a booster effect by multiple or repeated inoculations.

The vaccine composition of the present invention may also be a multivalent vaccine that contains multiple antigens or expresses multiple antigens.

When administering multiple doses of an antigen-expressing vector, composition, or vaccine, the vaccination interval may be determined as appropriate. For example, intervals of 1 week or more, 10 days or more, 2 weeks or more, 20 days or more, 3 weeks or more, 4 weeks or more, or 5 weeks or more with intervals of 4 months or less, 3 months or less, 2 months or less, 9 weeks or less, 8 weeks or less, 7 weeks or less, 6 weeks or less can do. Specifically, it can be 1 to 6 weeks, 10 days to 5 weeks, 2 weeks to 5 weeks, 3 to 5 weeks, or 4 to 5 weeks, but is not limited thereto.

For example, the vaccine of the present invention containing a negative-strand RNA viral vector can induce an immune response against the target antigen even with one inoculation, and induces a marked immune response against the target antigen with two inoculations. When administering multiple doses of a vaccine containing a negative-strand RNA viral vector, the intervals should be, for example, 2-6 weeks, 3-6 weeks, 4-6 weeks, 3-5 weeks, or 4-5 weeks. can be done. In addition, when the vaccine of the present invention containing a DNA vector is administered multiple times, the intervals are, for example, 1 week to 6 weeks, 1 week to 5 weeks, 1 week to 4 weeks, 1 week to 3 weeks, 1 week to It can be 2 weeks.

When a vaccine containing a DNA vector and a vaccine containing a negative-strand RNA viral vector are administered in combination, they can be administered in any desired order. may be inoculated, or a vaccine containing a DNA vector may be inoculated after inoculation with a vaccine containing a negative-strand RNA viral vector.

When administering multiple doses of a vaccine, the antigenic protein fragment contained or expressed in the vaccine administered each time may be the same or different each time. Combining vaccines containing or expressing different antigens can broaden the targeting of immune responses induced in vaccinated individuals. For example, when booster vaccination is performed using the vaccine formulation of the present invention, using a vaccine formulation containing (or expressed) an antigen different from the antigen contained (or expressed) in the vaccine formulation used in primary vaccination good too. For example, even if the antigen is the same pathogen, a protein different from the antigen used in the primary inoculation may be used as the target antigen. ) can be used as an antigen, or a protein (homologous protein, etc.) of a pathogen different from the pathogen to which the antigen used in the primary inoculation belongs can be used as an antigen. In particular, even if the pathogen is of the same kind, a strain different from the pathogen from which the vaccine formulation used in the primary inoculation is derived can be used for the booster inoculation.

When the antigen-expressing vector, composition, or vaccine of the present invention is administered to animals (including humans), the dose varies depending on the disease, patient body weight, age, sex, symptoms, purpose of administration, form of administration composition, It can be determined as appropriate according to the administration method and the like. In addition, the dosage may be appropriately adjusted according to the target animal, administration site, administration frequency, and the like. For example, depending on body weight, 1 ng/kg to 1000 mg/kg, 5 ng/kg to 800 mg/kg, 10 ng/kg to 500 mg/kg, 0.1 mg/kg to 400 mg/kg, 0.2 mg/kg to 300 mg/kg , 0.5 mg/kg to 200 mg/kg, or 1 mg/kg to 100 mg/kg. In addition, when administering the antigen-expressing vector, composition, or vaccine of the present invention containing a negative-strand RNA viral vector, for example, 1x10 ⁴ to 1x10 ¹⁵ CIU/kg, 1x10 ⁵ to 1x10 ¹⁴ CIU/kg, 1x10 ⁶ to 1x10 ¹³ CIU/kg, 1x10 ⁷ to 1x10 ¹² CIU/kg, 1x10 ⁸ to 5x10 ¹¹ CIU/kg, 1x10 ⁹ to 5x10 ¹¹ CIU/kg, or 1x10 ¹⁰ to 1x10 ¹¹ CIU/kg, and 1x10 ⁶ ~ 1x10 ¹⁷ particles/kg, 1x10 ⁷ ~ 1x10 ¹⁶ particles/kg, 1x10 ⁸ ~ 1x10 ¹⁵ particles/kg, 1x10 ⁹ ~ 1x10 ¹⁴ particles/kg, 1x10 ¹⁰ ~ 1x10 ¹³ particles/kg, 1x10 ¹¹ ~ 5x10 ¹² particles/kg kg, or 5x10 ¹¹ to 5x10 ¹² particles/kg. A person skilled in the art can appropriately determine an appropriate dose and administration method in consideration of the patient's body weight, age, symptoms, and the like.

The subject of administration of the antigen-expressing vector, composition, or vaccine of the present invention is preferably mammals (including humans and non-human mammals). Specifically, non-human primates such as humans and monkeys, rodents such as mice, rats and guinea pigs, non-rodent animals such as rabbits, goats, sheep, pigs, cows, dogs and cats mammals), primates of interest, eg, non-human primates such as monkeys, particularly macaques such as cynomolgus and rhesus monkeys, and humans.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Also, all publications and other references cited herein are incorporated as part of this specification.

[Example 1a] Construction of vaccine antigen S1-foldon-loaded Sendai virus vector In order to introduce and express a Sendai virus vector carrying a gene encoding S1-foldon, which is a fusion protein with the merization domain foldon, into cells, a Sendai virus vector carrying S1-foldon was constructed as follows ( Figure 1).
Using the S1-foldon gene having a secretory signal at the N-terminus as a template, 5'-ATATGCGGCCGCGCCACCATGTTCGTCTTTTTGGTGTTG-3' (Not1_Signal_N (SEQ ID NO: 18)) and 5'-CCAGCTCTTGTTGTTCTTATGATAGTAGAC-3' (S1-foldon_A441G_C (SEQ ID NO: 19)) using KOD One ^TM PCR Master Mix-DNA polymerase (TOYOBO Co., Ltd. Code No. KMM-101) for PCR reaction (98°C-2 minutes → 98°C-10 seconds, 55°C-5 seconds, 68°C-10 seconds 40 cycles → 68 ° C.-30 seconds), and using the PCR product (1) of about 330 bases and the S1-foldon gene as a template, 5'-GTCTACTATCATAAGAACAACAAGAGCTGG-3' (S1-foldon_A441G_N (SEQ ID NO: 20) ) and 5'-ATATGCGGCCGCGTGGATGAACTTTCACCCTAAGTTTTTCTTACTACGGCTAACCCAGGAAGGTGGAGAGCAGC-3' (foldon_EIS_Not1_C (SEQ ID NO: 21)), KOD One ^TM PCR Master Mix-DNA polymerase (TOYOBO Co., Ltd. code number KMM-101) for PCR reaction (98 ° C - 2 minutes → 40 cycles of 98°C-10 seconds, 55°C-5 seconds, and 68°C-10 seconds → 68°C-30 seconds) were performed to obtain a PCR product (2) of about 1700 bases. Using ^these two PCR products as templates, PCR reaction ( 98°C-2 minutes → 30 cycles of 98°C-10 seconds, 55°C-5 seconds, 68°C-10 seconds → 68°C-30 seconds) to obtain a PCR product (4) of about 2000 bases. After confirming the size of the PCR product by electrophoresis, it was purified using NucleoSpin ^™ Gel and PCR Clean-up (MACGEREY-NAGEL Catalog No. 740609.250/U0609C).
By the first and second PCRs, PCR products (1) and (2) in which the S1-foldon sequence is divided into two are obtained. ) as a template, a PCR product (3) spanning the entire length of the S1-foldon sequence was obtained and loaded into a Sendai virus vector. The reason for dividing the S1-foldon sequence into two in the first and second PCRs is that there is one A rich sequence (5xA_N_2xA) in the S1-foldon sequence, but on the A rich sequence, This is to avoid errors caused by the RNA-dependent RNA polymerase of Sendai virus, which tend to occur during the production process of Sendai virus vectors. PCR primers were set on the A rich sequence site, and each primer sequence was substituted from A/T to G/C under the restriction of synonymous codons.

[Table 1-1]

NotI-digested and gel-extracted full-length S1-foldon fragment was digested with NotI and BAP-treated plasmid pSeV18+/ΔF(Δ5aa) DNA (F gene deleted and NotI site between leader sequence and N gene After ligation with Sendai virus vector (WO2003/025570, WO2010/008054, DNA encoding positive strand genome of Z strain) (Fig. 1A), confirming the base sequence of cloned S1-foldon, it is suitable for SeV. A plasmid pSeV18+S1-foldon/ΔF(Δ5aa) carrying the modified full-length S1-foldon was obtained. Using this plasmid DNA as a template, Sendai virus reconstruction was performed to obtain an S1-foldon-loaded Sendai virus vector SeV18+S1-foldon/ΔF(Δ5aa).
The inserted sequence (SEQ ID NO:22) is shown in FIG. The inserted sequence consists of the NotI site (underlined), the Kozak sequence (double underlined), the sequence from the start codon to the stop codon of S1-foldon (secretory signal sequence S (uppercase dashed underlined); excluding the secretory signal (S) sequence S1 sequence (no uppercase underline); foldon sequence (uppercase single-dotted underline)), 34 bases (wavy underline) including EIS sequence (bold wavy underline), and NotI site (underlined). The number of bases (NotI site + Kozak sequence + sequence from start codon to stop codon of S1-foldon + 34 bases including EIS sequence) is preferably a multiple of 6 (6n rule), so conform to the 6n rule. Therefore, the regulatory sequence cac (bold) was inserted immediately after the 34 bases containing the EIS sequence.The coding sequence (CDS) of the insert sequence of S1-foldon (SEQ ID NO: 22) is 15 of SEQ ID NO: 22. -2144.The encoded amino acid sequence is shown in SEQ ID NO: 23. Of these, the 1-13th is the signal peptide (derived from the SARS-CoV-2 spike protein), the 14th-679th is the S1 sequence, and the 680- The 710th is the foldon array.

[Example 1b] Construction of Sendai virus vector carrying vaccine antigen S-RBD-foldon Secretion signal (S), receptor binding domain (RBD) of spike protein (S1) of SARS-CoV-2, and trimerization domain In order to introduce and express a Sendai virus vector carrying the gene encoding the foldon fusion protein S-RBD-foldon into cells, we constructed a Sendai virus vector carrying S-RBD-foldon as follows (Fig. 1).
KOD One ^™ PCR Master Mix-DNA polymerase ( TOYOBO Co., Ltd. Code No. KMM-101) PCR reaction (98°C - 2 minutes → 98°C - 10 seconds, 55°C - 5 seconds, 68°C - 1 second for 30 cycles → 68°C - 30 seconds). 5'-CCCTCTGGTATCCTCCAGATTCCCCAACAT-3' (signal_RBD_N (SEQ ID NO: 25)) and 5'-TAACCGGAGCCGGGGGTGCTCTTTCTTGGGG-3' (RBD_foldon_C (SEQ ID NO: 26)) using the 350-base PCR product (1) and the RBD synthetic gene as templates. using KOD One ^TM PCR Master Mix-DNA polymerase (TOYOBO Co., Ltd. Code No. KMM-101) for PCR reaction (98°C-2 minutes → 98°C-10 seconds, 55°C-5 seconds, 68°C-1 second 30 cycles → 68°C-30 seconds), and using the PCR product (2) of about 620 bases and the S1-foldon gene with a foldon tag at the C-terminus as a template, 5'-CCCCAAGAAGAGCACCCCCGGCTCCGGTTA-3' (RBD_foldon_N ( SEQ ID NO: 27)) and foldon_EIS_Not1_C (SEQ ID NO: 21), KOD One ^™ PCR Master Mix-DNA polymerase (TOYOBO Code No. KMM-101) for PCR reaction (98°C-2 minutes → 98°C-10 seconds) , 55°C-5 seconds, 68°C-1 second for 30 cycles → 68°C-30 seconds) to obtain a PCR product (3) of approximately 100 bases. Using ^these three PCR products as templates, PCR reaction ( 98°C-2 minutes → 30 cycles of 98°C-10 seconds, 55°C-5 seconds, 68°C-1 seconds → 68°C-30 seconds) to obtain a PCR product (4) of about 830 bases. After confirming the size of the PCR product by electrophoresis, it was purified using NucleoSpin ^™ Gel and PCR Clean-up (MACGEREY-NAGEL Catalog No. 740609.250/U0609C).

[Table 1-2]

The NotI-digested and gel-extracted full-length S-RBD-foldon fragment was transformed into NotI-digested and BAP-treated plasmid pSeV18+/ΔF(Δ5aa) DNA (F gene deleted, NotI site between leader sequence and N gene). DNA encoding the positive strand genome of Sendai virus vector (WO2003/025570, WO2010/008054, Z strain) (Fig. 1A) and confirming the nucleotide sequence of the cloned S-RBD-foldon. , resulting in the plasmid pSeV18+S-RBD-foldon/ΔF(Δ5aa) carrying the SeV-optimized full-length S-RBD-foldon. Using this plasmid DNA as a template, Sendai virus reconstruction was performed to obtain an S-RBD-foldon-loaded Sendai virus vector SeV18+S-RBD-foldon/ΔF(Δ5aa).
The inserted sequence (SEQ ID NO:28) is shown in FIG. The inserted sequence consists of the NotI site (underlined), the Kozak sequence (double underlined), the sequence from the start codon to the stop codon of S-RBD-foldon (secretory signal sequence S (uppercase dashed underline); RBD sequence (uppercase underlined) ); foldon sequence (one-dot dashed underline)), 34 bases (wavy underline) including EIS sequence (bold wavy underline), NotI site (underline) Total number of bases inserted into Sendai virus vector (NotI site + Kozak sequence + sequence from start codon to stop codon of S-RBD-foldon + 34 bases including EIS sequence) is preferably a multiple of 6 (6n rule). The regulatory sequence cac (bold) was inserted immediately after the 34 bases containing the sequence.The coding sequence (CDS) of the inserted nucleotide sequence of S-RBD-foldon (SEQ ID NO:28) is 15-758 of SEQ ID NO:28. The encoded amino acid sequence is shown in SEQ ID NO: 29. Of these, the 1-13th is the signal peptide (derived from the SARS-CoV-2 spike protein), the 14th-217th is the RBD, and the 218th-248th is the RBD. An array of foldons.

[Example 1c] Construction of Sendai virus vector carrying vaccine antigen S-RBD Equipped with S-RBD gene encoding secretory signal (S) and receptor binding domain (RBD) of spike protein (S1) of SARS-CoV-2 In order to introduce the resulting Sendai virus vector into cells and express it, an S-RBD-loaded Sendai virus vector was constructed as follows (Fig. 1).
Using pSeV18+S-RBD-foldon/dF(Δ5aa) carrying the S-RBD-foldon gene with a secretion signal at the N-terminus as a template, 5′-ACAAGAGAAAAAACATGTATGG-3′ (SeV_F6 (SEQ ID NO: 30)) and 5'-GGAAGTAGCAGTTGAAGCCCTC-3' (RBD_R (SEQ ID NO: 31)) using KOD One ^™ PCR Master Mix-DNA polymerase (TOYOBO Co., Ltd. code number KMM-101) for PCR reaction (98°C-2 minutes → 98 ℃-10 seconds, 55℃-5 seconds, 68℃-10 seconds for 40 cycles → 68℃-30 seconds), PCR product (1) of about 550 bases and RBD without foldon tag at C-terminus 5'-GCAACAACCTGGACAGCAAG-3' (RBD_F (SEQ ID NO: 32)) and 5'-GATAACAGCACCTCCTCCCGACT-3' (SeV_R199 (SEQ ID NO: 33)) were prepared using SeV18 + RBD/dF (Δ5aa) loaded with the gene as a template. Using KOD One ^TM PCR Master Mix-DNA polymerase (TOYOBO Co., Ltd. code number KMM-101) PCR reaction (98°C-2 minutes → 98°C-10 seconds, 55°C-5 seconds, 68°C-10 seconds) 40 cycles→68°C-30 seconds) to obtain a PCR product (2) of about 400 bases. Using ^these two PCR products as templates, a PCR reaction ( 98°C-2 minutes → 40 cycles of 98°C-10 seconds, 55°C-5 seconds, 68°C-10 seconds → 68°C-30 seconds) to obtain a PCR product (3) of approximately 900 bases. After confirming the size of the PCR product by electrophoresis, it was purified using NucleoSpin ^™ Gel and PCR Clean-up (MACGEREY-NAGEL Catalog No. 740609.250/U0609C).

[Table 1-3]

NotI-digested and gel-extracted full-length S-RBD fragment was digested with NotI and BAP-treated plasmid pSeV18+/ΔF(Δ5aa) DNA (F gene deleted and NotI site between leader sequence and N gene After ligation with Sendai virus vector (WO2003/025570, WO2010/008054, DNA encoding positive strand genome of Z strain) (Fig. 1A), confirming the base sequence of cloned S-RBD, it is suitable for SeV. A plasmid pSeV18+S-RBD/ΔF(Δ5aa) carrying the modified full-length S-RBD was obtained. Using this plasmid DNA as a template, Sendai virus reconstruction was performed to obtain an S-RBD-loaded Sendai virus vector SeV18+S-RBD/ΔF(Δ5aa).
The inserted sequence (SEQ ID NO:34) is shown in FIG. The inserted sequence consists of the NotI site (underlined), the Kozak sequence (double underlined), the sequence from the start codon to the stop codon of S-RBD ((secretory signal sequence S (uppercase dashed underline); RBD sequence (uppercase no underline)). , 34 bases (wavy underline) including EIS sequence (bold wavy underline), NotI site (underline) Total number of bases to be inserted into Sendai virus vector (NotI site + Kozak sequence + stop from start codon of S-RBD) The sequence up to the codon + 34 bases including the EIS sequence) is preferably a multiple of 6 (6n rule), so no adjustment sequence insertion is required to comply with the 6n rule. The coding sequence (CDS) of the inserted base sequence (SEQ ID NO: 34) is 15 to 665 of SEQ ID NO: 34. The encoded amino acid sequence is shown in SEQ ID NO: 35. Of these, 1 to 13 are the signal peptide ( SARS-CoV-2 spike protein), 14th-217th is RBD.

[Example 2a] Manipulation of expression of vaccine antigen protein and measurement of intracellular and extracellular secretory levels The cells were seeded at a density of 2.3×10 ⁵ cells/well and adherently cultured for 3 days in a 37° C., 5% CO ₂ incubator.
2) The number of cells in the pre-inoculated wells was measured, and based on this, the cells were infected with a vaccine antigen gene-loaded Sendai virus vector at a multiplicity of infection of 3. Following 1), PBS was replaced, the PBS was completely removed, the vaccine solution was added, the mixture was allowed to stand in a 37°C, 5% CO ₂ incubator, and the mixture was shaken every 15 minutes for adsorption. , was incubated for 1 hour for initiation of infection.
3) After the infection operation of 2), the medium was replaced with PBS, PBS was completely removed, 1 mL/well of medium was added, and the cells were cultured at 37°C in a 5% CO ₂ incubator for 3 days.
4-1) The culture supernatant was recovered, sterilized with a filter, quickly frozen with Dryice/EtOH, and stored at -80°C.
4-2a) On the other hand, in order to detach the adherent cells, add DPBS, remove it, add 250 μL of RIPA buffer (nacalai tesque, Cat# 08714-04), detach the cells with a cell scraper, and place them in a cell suspension. A turbid liquid was collected.
4-2b) This cell suspension was quickly frozen with Dryice/EtOH, thawed at room temperature and mixed. This operation was repeated 3 times.
4-2c) This freeze-thaw solution is centrifuged at 15,000 rpm for 5 minutes at 4°C, the supernatant is collected as a cell extract, and the cell extract and cell pellet are rapidly frozen with Dryice/EtOH and stored at -80°C. stored in
5) Using the culture supernatant of 4-1) and the cell extract of 4-2c) as extracellular secretory free protein and intracellular protein, the amount of vaccine antigen protein was measured by ELISA. In the case of expression from S-RBD-foldon-loaded Sendai virus vectors and S-RBD-loaded Sendai virus vectors, SARS-CoV-2 Spike S1 RBD ELISA Kit (Elabscience, Cat# E-EL-E605) was used, and S1 For expression from the -foldon-loaded Sendai virus vector, the 2019-nCoV S1 Protein ELISA Kit (SignalChem, Cat# C19SD-876) was used.

[Example 2b] Cellular amount and extracellular secretory release amount after expression of vaccine antigen protein

[Table 2-1]
Cellular Amount and Extracellular Secretory Release Amount after Expression of Vaccine Antigen-Loaded Sendai Virus Vector in Cultured Cells

The volume of the vaccine solution added at the time of infection was 100 μL for S1-foldon (S1F7S), 651 μL for S1-foldon (S1F8S), 100 μL for S-RBD-foldon (SRBDF007P), 655 μL for S-RBD-foldon (SRBDF0012S), and 655 μL for S-foldon (SRBDF0012S). -RBD-foldon (SRBDF013S) was 752 µL, S-RBD (SRBD4S) was 100 µL, and S-RBD (SRBD9S) was 100 µL.
[Table 2-2]
Cellular Amount and Extracellular Secretory Release Amount after Expression of Vaccine Antigen-Loaded Sendai Virus Vector in Cultured Cells

The amount of the vaccine solution added at the time of infection was 100 μL for S1-foldon (S1F7002P), 100 μL for S-RBD-foldon (SRBDF015P), and 100 μL for S-RBD (SRBD4002PS).

We quantified the extracellular secretion and intracellular residual amounts of the vaccine antigen protein, and determined the secretory release rate and intracellular residual rate (Fig. 5-1). In the S1-foldon vaccine antigen, the expression amount (extracellular secretion release amount + intracellular residual amount) was around 2 to 4 μg, and the secretory release amount was several to 10 times lower than the intracellular residual amount. In contrast, with the S-RBD-foldon vaccine antigen, the expressed amount was around 25-50 μg, and the secreted free amount was several to 10-fold or more higher than the residual amount.
These results confirm that the S-RBD-foldon, which has a smaller molecular weight, has a significantly higher expression level in the Sendai virus vector. It was found that the secretory release rate was significantly higher than that of In addition, it was found that the expression level of the S-RBD vaccine antigen from which foldon, a trimerization factor, was removed was significantly lower than that with foldon added (for example, 40 to 50 μg; 5 to 7 μg in Table 2-1). bottom. This cannot be expected from the known functionality of foldon.
To confirm the reproducibility of these two findings, we produced new lots of vaccine antigen-loaded Sendai virus vectors for S1-foldon, S-RBD-foldon, and S-RBD, and measured the total expression level (/10 ⁶ cells) were 5.6 μg, 32 μg, and 2.8 μg, respectively (Table 2-2). (3.8 times) and several times higher (7.6 times) (Table 2-2, Figure 5-2), showing the same tendency as Table 2-1 and Figure 5-1.
Based on these two discoveries, the following techniques are provided. In other words, technology that maximizes expression by minimizing the vaccine antigen and adding a foldon to it, and by adding a secretory signal, maximizes the amount of released secretion and secures the residual amount in cells. is.
After expression, about 80% or more of the S-RBD-foldon protein was secreted and released outside the cells, and about 10% or less remained inside the cells. In this way, it is speculated that most of the expressed S-RBD-foldon vaccine antigen protein is secreted and released extracellularly, and part of it remains intracellularly. Released vaccine antigen proteins are phagocytosed by antigen-presenting cells, leading to the induction of antigen-specific antibody-producing B cells and cytotoxic T cells, while vaccine antigens that remain in infected cells are phagocytosed by neighboring cells. It is expected to present antigen to impaired T cells. Thus, the present invention is considered useful in that it can be designed as a vector technology that can be programmed to induce both humoral and cellular immunity. As shown in Example 3, the S-RBD-foldon vaccine antigen is humoral immunity (Tables 3-8, 3-9, 3-12, 3-14) and cell-mediated immunity (Tables 3-16, 3-19 , 3-20) are strongly induced.

[Example 3a] Inoculation dose, schedule and blood collection method in animal immunogenicity test

[Table 3-1]
Inoculation schedule in rat immunogenicity test using Sendai virus vector carrying S1-foldon

[Table 3-2]
Inoculation schedule for mouse immunogenicity test using Sendai virus vector carrying S1-foldon

For rats, 200 μL or 500 μL of blood was collected from the jugular vein of each rat on the 1st, 8th, 15th, 22nd, 29th and 36th days of inoculation (before inoculation) according to the schedule shown in Table 3-1. On day 43, exsanguination and splenectomy were performed. The collected rat blood was centrifuged at 1,700 x g at room temperature for 5 minutes, and the obtained serum was stored frozen.

[Table 3-3]
Inoculation schedule for mouse immunogenicity test using S-RBD-foldon-loaded Sendai virus vector

[Table 3-4]
Inoculation schedule for mouse immunogenicity test using S-RBD-foldon-loaded Sendai virus vector

[Table 3-5]
Inoculation schedule for mouse immunogenicity test using Sendai virus vector carrying S-RBD

For mice, 100 μL of blood was collected from the orbit once each on Day 1, 8, 15, 22, 29, 36, and 43 of the day of inoculation (before inoculation) according to the schedule in Table 3-3 above. Then, on day 57, exsanguination and splenectomy were performed. Collected mouse blood was centrifuged at 6,146 x g at room temperature for 5 minutes, and the resulting serum was stored in a refrigerator.

[Example 3b] Measurement of maximum diluted IgG antibody titer using serum in animal immunogenicity test 1) For measurement of mouse serum, the antigen protein SARS-CoV-2 Spike S1-His Recombinant Protein (Sino Biological Inc. Cat# 40591-V08B1) was diluted with 0.2 M Carbonate-Bicarbonate buffer (pH 9.6) and adjusted to 100 ng/100 μL.
2) The diluted antigen protein was added to a 96-well plate (NUNC, IMMUNO PLATE, MAXISORP, Cat# 439454) at 100 µL/well, sealed to prevent evaporation, and allowed to stand at 4°C for 16 hours.
3) After washing three times with PBS-T, 250 μL/well of 2% BSA/PBS-T was added for blocking at 37° C. for 1 hour.
4) Washed 3 times with PBS-T.
5-1) Mouse serum was serially diluted with 2% BSA/PBS-T, added in duplicate at 100 µL/well, and incubated at 37°C for 2 hours.
5-2) To the negative control, 100 μL/well of 100-fold diluted PBS-inoculated mouse serum was added in duplicate.
6) Washed 5 times with PBS-T.
7) Secondary antibody is Goat Anti-Mouse IgG H&L (HRP) (abcam, Cat# ab205719) diluted to 67 ng/mL with 2% BSA/PBS-T, added 100 μL/well, and incubated at 37°C. Incubated for 1 hour.
8) Washed 5 times with PBS-T.
9) TMB Substrate Kit (ThermoFisher, Cat# 34021) was returned to room temperature, equal amounts of TMB Solution and Peroxidase Solution were mixed, and 100 μL of TMB substrate solution was added to each well.
10) The plate was incubated at 37°C for 15 minutes, and 100 µL of 2M sulfuric acid was added to stop the reaction.
11) Absorbance was measured at 450 nm using a plate reader.
12) Serum dilution in the repeated administration group was 100-fold to 4000-fold for 2w, 100-fold to 7000-fold for 4w, and 100-fold to 100000-fold for 6w and 8w. Regarding the evaluation of the antibody titer sample, the maximum dilution rate at which the cut point (two times the average absorbance value of the negative control sample) or higher was taken as the antibody titer.
In the measurement of rat serum, almost the same method was performed except that the incubation temperature was 25 ° C from 1) to 12), but the secondary antibody in 7) was Mouse monoclonal Anti-Rat IgG H & L (HRP ) (Abcam, Cat# ab106718) was used.

[Example 3c] Comparison of IgG antibody titer maximum dilution using serum: S1-foldon, S-RBD-foldon, S-RBD

[Table 3-6]
Maximum detectable dilution of IgG antibodies in sera from intranasal inoculation study with 4-week booster of S1-foldon vaccine in rats

ND: not detected
[Table 3-7]
Maximum detectable dilution of IgG antibodies in sera from intranasal inoculation study with 4-week booster of S1-foldon vaccine in mice

[Table 3-8]
Maximum detectable dilution of IgG antibodies in sera from intranasal inoculation study with 4-week booster of S-RBD-foldon vaccine in mice

[Table 3-9]
Maximum detectable dilution of IgG antibodies in sera from intranasal inoculation study with 4-week booster of S-RBD-foldon vaccine in mice

[Table 3-10]
Maximum detectable dilution of IgG antibodies in sera from intranasal inoculation study with 4-week booster of S-RBD vaccine in mice

Maximum dilution of S1 protein-specific IgG antibody titers was determined using sera at 2, 4, and 6 weeks post-nasal inoculation from intranasal inoculation studies with a 4-week booster for each vaccine. , S-RBD-foldon (1x10 ⁷ CIU/shot) vs. S1-foldon (2x10 ⁷ CIU/shot; 5x10 ⁷ CIU/shot), S-RBD-foldon showed higher , showed a higher maximum dilution (Fig. 6-1). Considering this result together with the difference in secreted and released amount in Example 2b, it is considered that S-RBD-foldon with a larger secreted and released amount induces vaccine antigen-specific antibodies more strongly.
For the purpose of comparing the immunogenicity of each vaccine in the same animal species (mice), 2 weeks, 4 weeks, Using sera at 6 weeks, maximum dilution of S1 protein-specific IgG antibody titer was calculated as S-RBD-foldon (1x10 ⁷ CIU/shot) (Table 3-9) vs. S1-foldon (1x10 ⁷ CIU/shot). shot) (Table 3-7), S-RBD-foldon showed a higher maximum dilution than S1-foldon with the same inoculum dose (Fig. 6-2). Considering this result together with the difference in secreted and released amount in Example 2b, it is considered that S-RBD-foldon with a larger secreted and released amount induces vaccine antigen-specific antibodies more strongly.
In addition, with the aim of comparing the immunogenicity of each vaccine in the same animal species (mice), 2 weeks after intranasal inoculation and 4 Using sera at weeks and 6 weeks, the maximum dilution of S1 protein-specific IgG antibody titers was calculated as S-RBD-foldon (1x10 ⁷ CIU/shot) (Table 3-9) vs. S-RBD (1x10 ⁷ CIU/shot) (Table 3-10), S-RBD-foldon showed a higher maximum dilution than S-RBD at the same inoculum dose (Fig. 6-2). Considering this result together with the difference in secreted and released amount in Example 2b, it is considered that S-RBD-foldon with a larger secreted and released amount induces vaccine antigen-specific antibodies more strongly.

[Example 3d] Measurement of neutralizing antibody activity using serum in animal immunogenicity test 1) Comparison of neutralizing antibody activity using serum: S1-foldon vs. S-RBD-foldon

[Table 3-11]
Maximum detectable dilution of neutralizing antibody activity in sera from a single intranasal inoculation study of S1-foldon vaccine in rats.

[Table 3-12]
Maximum detectable dilution of neutralizing antibody activity in sera from a single intranasal inoculation study of S-RBD-foldon vaccine in mice.

ND: not detected

SARS-CoV-2 WA1/2020 virus into VeroE6 cells (1000 TCID ₅₀ /mL) using sera at 2, 4 and 6 weeks post-nasal inoculation from a single intranasal inoculation study with each vaccine ) S-RBD-foldon (1x10 ⁷ CIU/shot) vs. S1-foldon (2x10 ⁷ CIU/shot; 5x10 ⁷ CIU/shot). , S-RBD-foldon showed a higher neutralizing antibody titer even at a lower inoculation dose.

[Table 3-13]
Neutralizing Antibody Activity Maximum Detectable Dilution of Serum from Nasal Inoculation Study with Week 4 Booster of S1-foldon Vaccine in Rats

[Table 3-14]
Neutralizing Antibody Activity Maximum Detectable Dilution of Serum from Nasal Inoculation Study with S-RBD-foldon Vaccine Week 4 Booster in Mice

SARS-CoV-2 WA1/2020 virus into VeroE6 cells using sera at 2, 4, and 6 weeks post-nasal inoculation from intranasal inoculation studies with 4-week booster with each vaccine (1000 TCID ₅₀ /mL) The maximum dilution (neutralizing antibody titer) showing neutralizing antibody activity against infection was calculated as S-RBD-foldon (1x10 ⁷ CIU/shot) vs. S1-foldon (2x10 ⁷ CIU/shot; 5x10 ⁷ CIU/shot), S-RBD-foldon showed a higher neutralizing antibody titer even at a lower inoculation dose. Considering this result together with the difference in secreted release amount in Example 2b, S-RBD-foldon with a larger secreted release amount is considered to induce stronger anti-SARS-CoV-2 neutralizing antibody activity. .

[Example 3e-1] Isolation and hemolysis of mouse splenocytes in animal immunogenicity test and measurement of CTL cells by ELISpot assay 1) Spleens excised from mice were immersed in RPMI1640 medium and transported on ice. .
2) Cell strainer 40 µm Nylon (FALCON, Cat# 352340) was placed on a Φ60-mm dish containing 5 mL of 1xPBS(-), and the spleen of 1) was placed therein.
3) The splenocytes were separated by pressing the flat part of a 1-mL syringe plunger against the spleen.
4) The cell strainer was taken out, the splenocytes remaining in the dish were collected in a tube, centrifuged at a low speed, and the supernatant was removed.
5) RBC Lysis Buffer (Bio Legend, Cat# 420301) was diluted 10-fold, added onto the splenocyte pellet of 4), and hemolyzed on ice for 10 minutes with occasional shaking.
6) It was centrifuged at low speed, the supernatant was removed, CTL Test PLUS Medium (CTL, # CTLTP-005) was added, and a cell count was performed.
7) Mouse IFN-γ/IL-2 FluoroSpot Kit (MABTECH, Cat# FS-4142-2) was used on mouse splenocytes for ELISpot assay.
7-1) The plate was washed 5 times with 1xPBS(-) and 200 µL/well, 200 µL/well of 10% FBS-RPMI1640 was added, and the plate was incubated at room temperature for 1 hour for blocking.
7-2) Anti-CD28 mouseAb (0.2 µg/mL) was added to CTL Test PLUS Medium at a double concentration to enhance antigen-specific stimulation.
7-3) 0.5 μL/well (double concentration) of PepTivator SARS-CoV-2 Prot S (Milteny Biotech, #130-126-700, Lot.5200904575) was added to half of them.
7-4) The medium of the plate was removed, and anti-CD28 mouseAb-added medium was added at 100 μL/well, and anti-CD28 mouseAb+PepTivator-added medium was added at 100 μL/well.
7-5) The splenocyte suspension obtained in 6) was seeded in 2 wells at 2.5×10 ⁵ cells/100 μL/well.
7-6) The plate was placed in a humidified incubator at 37°C and cultured for 12-48 hours.
Plates were emptied and washed 5 times with 200 μL/well of PBS to remove cells.
7-7) Dilute R4-6A2-BAM to 1:200 and 5H4-Biotin to 2 μg/mL with 0.1% BSA/PBS and filter antibody solution using 0.2-μm low protein binding filter. was filtered and added at 100 μL/well and incubated for 2 hours at room temperature.
7-8) Washed 5 times with 1xPBS(-).
7-9) Dilute anti-BAM-490 1:200 and SA-550 1:200 with 0.1% BSA/PBS and filter the solution using a 0.2-μm low protein binding filter. , was added at 100 μL/well and incubated for 1 hour at room temperature.
7-10) The plate was covered with aluminum foil to limit exposure. Washed 5 times with 1xPBS(-).
7-11) The plate was emptied, 50 μL/well of fluorescence enhancer-II was added, and the plate was incubated at room temperature for 15 minutes.
7-12) Empty the plate and tap the plate firmly against a clean paper towel to remove residual fluorescence enhancer.
7-13) Remove the plastic underneath the plate and place the plate in the dark to dry.
7-14) Spots were observed with a microscope equipped with filters for FITC (excitation 490 nm/emission 510 nm). Spots identified with the FITC filter represent IFN-γ-producing cells.

[Example 3e-2] Isolation and hemolysis of rat splenocytes in animal immunogenicity test and measurement of CTL cells by ELISpot assay 1) The spleens excised from rats were immersed in RPMI1640 medium and transported on ice. .
2) Cell strainer 40 µm Nylon (FALCON, Cat# 352340) was placed on a Φ60-mm dish containing 5 mL of 1xPBS(-), and the spleen of 1) was placed therein.
3) The splenocytes were separated by pressing the flat part of a 1-mL syringe plunger against the spleen.
4) The cell strainer was taken out, the splenocytes remaining in the dish were collected in a tube, centrifuged at a low speed, and the supernatant was removed.
5) RBC Lysis Buffer (Bio Legend, Cat# 420301) was diluted 10-fold, added onto the splenocyte pellet of 4), and hemolyzed on ice for 10 minutes with occasional shaking.
6) It was centrifuged at low speed, the supernatant was removed, CTL Test PLUS Medium (CTL, # CTLTP-005) was added, and a cell count was performed.
7) For the ELISpot assay, the Rat IFN-γ ELISpot Plus Kit (MABTECH, Cat# 3220-3APW-2) was used on rat splenocytes.
7-1) The plate was washed three times with 1xPBS(-) and 200 µL/well, 200 µL/well of 10% FBS-RPMI1640 was added, and the plate was incubated at room temperature for 30 minutes for blocking.
7-2) PepTivator SARS-CoV-2 Prot S (Milteny Biotech, #130-126-700, Lot.5200904575) was added at 0.5 μL/well (double concentration) to half.
7-3) The medium was removed from the plate, and 100 μL/well of the medium and 100 μL/well of PepTivator-added medium were added.
7-4) The splenocyte suspension obtained in 6) was seeded in 2 wells at 2.5×10 ⁵ cells/100 μL/well.
7-5) The plate was placed in a humidified incubator at 37°C and cultured for 12 to 48 hours.
Plates were emptied and washed 5 times with 200 μL/well of PBS to remove cells.
7-6) Dilute 1 mg/mL rIFNγ-II-biotin to 1 μg/mL with 0.5% FBS/PBS and filter the antibody solution using a 0.2-μm low protein binding filter at 100 μL/well. added and incubated for 2 hours at room temperature.
7-7) Washed 5 times with 1xPBS(-).
7-8) Streptavidin-ALP was diluted 1000-fold with 0.5% FBS/PBS, the solution was filtered using a 0.2-μm low protein binding filter, added at 100 μL/well and incubated at room temperature for 1 hour. .
7-9) The plate was covered with aluminum foil to limit exposure. Washed 5 times with 1xPBS(-).
7-10) The plate was emptied, BCIP/NBT-plus substrate was added, and the plate was incubated at room temperature for 15 minutes.
7-11) The plate was emptied and the wells washed well with tap water.
7-12) Removed the plastic underneath the plate and placed the plate in the dark to dry.
7-13) Spots were observed with an optical microscope. The observed ALP spots represent IFN-γ-producing cells.

[Example 3f] Comparison of CTL cell numbers by ELISpot assay using splenocytes: S1-foldon vs. S-RBD-foldon

[Table 3-15]
Peptide stimulation effect on the number of CTL cells by ELISpot assay in spleen cells derived from a single intranasal inoculation test of S1-foldon vaccine in rats

[Table 3-16]
Peptide stimulation effect on the number of CTL cells by ELISpot assay in spleen cells derived from a single intranasal inoculation test of S-RBD-foldon vaccine in mice

Number of S1-peptide-stimulated IFN-γ-releasing T cells at 6 weeks (rats) and 8 weeks (mice) after nasal inoculation using spleen cells derived from a single intranasal inoculation test with each vaccine was compared with S-RBD-foldon (1x10 ⁷ CIU/shot) vs. S1-foldon (2x10 ⁷ CIU/shot; 5x10 ⁷ CIU/shot) as a peptide stimulation effect. showed a higher IFN-γ release CTL stimulation rate.

[Table 3-17]
Peptide stimulation effect on the number of CTL cells by ELISpot assay in spleen cells derived from intranasal inoculation test with 4-week booster inoculation of S1-foldon vaccine in rats

[Table 3-18]
Peptide stimulation effect on the number of CTL cells by ELISpot assay in spleen cells derived from intranasal inoculation test with 4-week booster inoculation of S1-foldon vaccine in mice

[Table 3-19]
Peptide stimulation effect on the number of CTL cells by ELISpot assay in spleen cells derived from intranasal inoculation test with 4-week booster inoculation of S-RBD-foldon vaccine in mice

[Table 3-20]
Peptide stimulation effect on the number of CTL cells by ELISpot assay in spleen cells derived from intranasal inoculation test with 4-week booster inoculation of S-RBD-foldon vaccine in mice

[Table 3-21]
Peptide stimulation effect on the number of CTL cells by ELISpot assay in spleen cells derived from intranasal inoculation test with 4-week booster inoculation of S-RBD vaccine in mice

S1-peptide-stimulated IFN-stimulated 6 weeks (rats) and 8 weeks (mice) after nasal inoculation using spleen cells derived from intranasal inoculation tests with 4-week booster inoculation with each vaccine. When comparing the number of γ-released T cells as the peptide stimulation effect between S-RBD-foldon (1x10 ⁷ CIU/shot) and S1-foldon (2x10 ⁷ CIU/shot; 5x10 ⁷ CIU/shot), each vaccine showed , showed an IFN-γ release CTL stimulation rate significantly greater than 1 (Fig. 7-1). Considering this result together with the intracellular residual amount in Example 2b, it is considered that the vaccine antigen-specific CTL are induced by the residual vaccine antigen expressed in the infected cells in the inoculated area.
For the purpose of comparing the immunogenicity of each vaccine in the same animal species (mice), spleen cells derived from an intranasal inoculation study with a 4-week booster with each vaccine were used after intranasal inoculation. The number of S1-peptide-stimulated IFN-γ-released T cells at week 8 (mouse) was calculated as peptide stimulation effect, S-RBD-foldon (1×10 ⁷ CIU/shot) (Table 3-20) vs. S1-foldon (1×10). ⁷ CIU/shot) (Table 3-18), each vaccine showed an IFN-γ release CTL stimulation rate significantly exceeding 1 (Fig. 7-2). Considering this result together with the intracellular residual amount in Example 2b, it is considered that the vaccine antigen-specific CTL are induced by the residual vaccine antigen expressed in the infected cells in the inoculated area.
For the purpose of comparing the immunogenicity of each vaccine in the same animal species (mice), spleen cells derived from an intranasal inoculation study with a 4-week booster with each vaccine were used after intranasal inoculation. The number of S1-peptide-stimulated IFN-γ-released T cells at week 8 (mice) was calculated as peptide stimulation effect, S-RBD-foldon (1×10 ⁷ CIU/shot) (Table 3-20) vs. S-RBD (1×10). ⁷ CIU/shot) (Table 3-21), each vaccine showed an IFN-γ release CTL stimulation rate significantly exceeding 1 (Fig. 7-2). Considering this result together with the intracellular residual amount in Example 2b, it is considered that the vaccine antigen-specific CTL are induced by the residual vaccine antigen expressed in the infected cells in the inoculated area.

In the initial inoculation of the Sendai virus vector vaccine, specific antibodies and CTL are induced against proteins derived from the Sendai virus vector. There was concern that the booster effect of vaccine antigens by booster vaccination would not be exerted due to the action of , but the results in Tables 3-13 and 3-14, etc., confirm that booster vaccination has a strong booster effect on humoral immunity. , and Tables 3-17 to 3-20, etc., it was confirmed that the booster inoculation had a strong booster effect on cell-mediated immunity. Based on these results, the booster effect is that the vaccine antigen is secreted and released from the cells and spreads before the vaccine-introduced cells after the booster vaccination are eliminated by the action of antibodies and CTLs against Sendai virus induced by the first vaccination. It is considered to have been achieved because the

[Example 4a] Measurement of productivity of vaccine antigen-loaded Sendai virus vector 1) For preparation of preMVB seeds, MEM medium containing 10% FBS was used to prepare LLCMK2/LLCMK2 cells, which are LLCMK2 cells expressing Sendai virus F protein. 7×10 ⁶ cells/30 mL of F cell (Li, H.-O. et al., J. Virology 74. 6564-6569 (2000), WO00/70070) culture medium was added to each of two T225 flasks. After seeding, the cells were allowed to stand in a 37°C, 5% CO ₂ incubator and cultured for 3 days.
2) A CVS diluted solution was prepared by adding 2 mL of Cloned virus seed (CVS) of Sendai virus vector loaded with vaccine antigen to 6 mL of MEM medium.
3) Infection was initiated by removing the LLC-MK2/F cell culture medium from the two plates of 1) and adding 4 mL of the vector dilution from 2) to each of the two plates.
4) Cultured for 1 hour at 37°C in a 5% CO ₂ incubator. At that time, T225 was soaked every 15 minutes to prevent drying.
5) 25 mL of MEM medium containing 5.33 mrPU/mL TrypLE Select (ThermoFisher, Cat# 12563011) was added to T225 of 4), and statically cultured overnight at 32°C in a 5% CO ₂ incubator.
6) The medium was replaced with 30 mL of MEM medium containing 5.33 mrPU/mL TrypLE Select, and cultured at 32°C in a 5% CO ₂ incubator for 3 to 4 days.
7) The culture medium was collected every day, the hemagglutination activity of the culture supernatant was measured, and a portion of the culture supernatant in which HA activity was confirmed was used to analyze the base sequence of the loaded gene.
8) The rest of the culture supernatant was stored at -80°C after being rapidly frozen with dry ice/ethanol.
9) As a result of nucleotide sequence analysis, the culture supernatant in which it was confirmed that the nucleotide sequence of the loaded gene was as designed was designated as preMVB.
10) Infectious titers were measured.
11) To prepare the cells for the productivity test (T75 flask), 2.3×10 ⁶ cells/10 mL of LLC-MK2/F cell culture solution was added to the number of test samples + 1 using MEM medium containing 10% FBS. was seeded in each of the T75 flasks, left at rest in a 37°C, 5% CO ₂ incubator, and cultured for 3 days.
12) Cells were recovered from one flask, counted, and the number of cells per flask was calculated.
13) Using MEM medium, a vector solution of 1 mL/T75 was prepared so that the vector amount was equivalent to a multiplicity of infection of 0.5.
14) Except for the LLC-MK2/F cell culture medium of 11), the cells were infected with 1 mL/T75 sample of the vector solution of 13) and cultured at 37°C in a 5% CO ₂ incubator for 1 hour. At that time, the flask was soaked every 15 minutes to prevent drying.
15) MEM medium containing 5.33 mrPU/mL TrypLE Select was added at 10 mL/T75 and cultured at 32°C in a 5% CO ₂ incubator for 24 hours.
16) Replace the medium with 10 mL/T75 of MEM medium containing 5.33 mrPU/mL TrypLE Select. After culturing for 48 hours, collect a part of the culture medium, measure the HA of the supernatant, and infect the rest of the supernatant. Stored at -80°C for titer determination.
17) After culturing for 72, 96, 120, 144, and 168 hours, hemagglutination activity was measured in the same manner as in 16), and the remainder of the supernatant was stored at -80°C for infectious titer measurement.
18) The infection titer was measured.
19) From the measurement results of the hemagglutination activity, the total number of SeV particles/mL was calculated, and the change over time was graphed along with the measurement results of the infection titer, and vector productivity was determined as the maximum infection titer.

[Example 4b] Negative correlation between vector productivity (manufacturing efficiency) of vaccine antigen-carrying Sendai virus vector and extracellular secretion release amount of vaccine antigen

[Table 4-1]
Vaccine antigen expression level and vector productivity of vaccine antigen-loaded Sendai virus vectors in cultured cells

*: The intracellular amount of S1-foldon and S-RBD (μg/10 ⁶ cells) (Table 2-1) relative to the intracellular amount of S-RBD-foldon (μg/10 ⁶ cells) (Table 2-1) 1) Relative value **: The ^{extracellular} secretion release amount of S1-foldon and S-RBD (µg/ 10 ⁶ cells) (Table 2-1) ***: Maximum S1-foldon and S-RBD relative to S-RBD-foldon maximum infectious titer (CIU (infectious particle number)/mL) Relative value of infectious titer (CIU (number of infectious particles)/mL)

As shown in Example 2b, in the S1-foldon and S-RBD-foldon expressed from the Sendai virus vector, the molecular weight is S1-foldon > S-RBD-foldon, whereas the secreted release amount is , S1-foldon < S-RBD-foldon, but when the vector productivity, which indicates the production efficiency of each vector, was measured, S-RBD-foldon decreased to 1/6 that of S1-foldon. (S1-foldon > S-RBD-foldon). Therefore, it is considered that the increase in the amount of secretion and release is the cause of the low productivity of the vector. -foldon > S-RBD, we hypothesized that vector productivity would be S-RBD-foldon < S-RBD, and the productivity of vectors expressing S-RBD-foldon or S-RBD As expected, removing the foldon from the S-RBD-foldon resulted in a 6.5-fold increase in vector productivity (S-RBD-foldon < S-RBD). The following techniques are provided based on these verification results. That is, by adding or removing the foldon sequence of the vaccine antigen vector, the expression level of the vaccine antigen is increased or decreased in production cells in the production culture process, and the amount of secretion and release is increased or decreased in conjunction with the increase or decrease of the vector. Vector productivity can be adjusted to the extent that productivity is suppressed or enhanced and high immunogenicity is maintained.

From the difference in immunity induction in the two vaccines (S-RBD-foldon > S1-foldon), by minimizing the vaccine antigen and adding foldon to it, expression is maximized, and by adding a secretory signal , the released amount of vaccine antigen protein is maximized and the intracellular residual amount is secured. while vaccine antigens retained in infected cells are expected to be presented by those cells to neighboring cytotoxic T cells. Thus, the present invention is considered useful as a vector technology that can be programmed to induce both humoral and cellular immunity.

In this way, from the difference in the ability to induce immunity between the two vaccines (S-RBD-foldon > S1-foldon), by minimizing the vaccine antigen and adding foldon to it, the expression is maximized and the secretion signal The addition maximizes the amount of secretion and release and secures the intracellular residual amount. It is expected that the induction of B cells and follicular dendritic cells derived from the same antigen and frequent interactions between them will lead to the selection of high-affinity antibody-producing B cells (Fig. 8). In addition, by expressing only the ACE2 receptor-binding domain as a vaccine antigen, only neutralizing antibody-producing B cells are stimulated to proliferate, and antibody-producing B cells with high infection-blocking ability can be selected.
Therefore, by minimizing the vaccine antigen and adding a foldon to it, the expression is maximized, and by adding a secretory signal, the amount of released secretion is maximized and the residual amount in the cell is secured. It is believed that the technology of the invention will enable selected B cells to become long-lived plasma cells and memory B cells, thereby achieving long-lived immunity.

Antibodies and CTLs against viral vector-derived proteins are induced in the initial inoculation of a viral vector vaccine. There was concern that the booster effect of vaccine antigens by inoculation would not be exhibited. However, a strong booster effect of humoral immunity and cell-mediated immunity was confirmed by booster inoculation of vaccine antigen-loaded Sendai virus vector with added secretion signal and foldon (Tables 3-13, 3-14, 3-17-3). -20 etc.). These results suggest that secretion and release of vaccine antigens from cells after booster vaccination can ward off the effects of antibodies and CTL against Sendai virus induced by primary vaccination. In addition, it is thought that the effect of cross-immunity due to similar viruses can also be avoided. This technique can be used as a technique for enhancing the booster effect of viral vector vaccines and as a technique for avoiding cross-immunity.

From the difference in the amount of secretion and release (S-RBD-foldon > S-RBD) and the difference in vector productivity (S-RBD-foldon < S-RBD) in the two vaccines with and without Foldon, the foldon of the vaccine antigen vector By adding or removing sequences, the amount of vaccine antigen expressed is increased or decreased in production cells in the manufacturing culture process, which in turn increases or decreases the amount of secreted release, and suppresses or promotes vector productivity. , vector productivity can be adjusted to the extent that high immunogenicity is maintained.

Although preferred embodiments of the invention have been described in detail, it will be apparent to those skilled in the art that these embodiments can be modified. Accordingly, it is contemplated that the present invention may be practiced in ways and aspects other than those specifically described herein. That is, the present invention includes all modifications that fall within the spirit or essential parts of the appended claims.

According to the present invention, for example, vaccine antigens derived from the extramembrane domain of the target antigen protein can be strongly expressed and remain in the cell, while abundant extramembrane secretion and release can enhance immunogenicity. INDUSTRIAL APPLICABILITY The present invention is particularly expected to be used in the field of infection immunology and its clinical application.

Claims

An antigen expression vector comprising a nucleic acid encoding a secretable fusion protein comprising a secretory signal, an antigenic protein fragment, and a trimerization domain.
The antigen expression vector according to claim 1, wherein the fusion protein does not contain a transmembrane domain.
The antigen expression vector according to claim 1, wherein the vector is a negative-strand RNA viral vector.
The antigen expression vector according to claim 1, wherein the trimerization domain is the trimerization domain (foldon) of T4 phage fibritin.
The antigen expression vector according to claim 1, wherein the antigen is derived from an infectious pathogen.
The antigen expression vector according to claim 1, wherein the antigen is derived from a virus.
The antigen expression vector according to claim 1, wherein the antigen protein fragment is the extracellular domain of a membrane protein or a fragment thereof.
The antigen expression vector according to claim 1, wherein the antigen protein fragment has a length of 500 amino acids or less.
The antigen expression vector according to claim 1, wherein the antigen protein is an RNA virus spike protein.
The antigen expression vector according to claim 9, wherein the antigen protein fragment is a fragment of the extracellular region containing the receptor-binding domain of the RNA virus spike protein.
The antigen expression vector according to claim 1, wherein the expression product containing the antigen protein fragment is distributed both intracellularly and extracellularly in the vector-introduced cell.
The antigen expression vector according to claim 11, wherein the expression product secreted extracellularly is greater than the expression product that remains in the cell.
2. The amount of expression product comprising the antigenic protein fragment is increased compared to a control antigen expression vector comprising a protein-encoding nucleic acid comprising a secretory signal and an antigenic protein fragment and not comprising a trimerization domain. antigen expression vector.
A vaccine comprising the antigen expression vector according to any one of claims 1 to 13.
The vaccine according to claim 14, for inducing both humoral and cellular immunity.