SARS-CoV-2 Vaccine

Abstract

A vaccine that comprises, as an active ingredient, a viral vector which comprises a sequence containing any of the following amino acid sequences (a) to (c) and in which an induced neutralizing antibody presents, on the viral particle envelope, a protein inhibiting the binding of SARS-CoV-2 receptor binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2). (a) An amino acid sequence represented by SEQ ID NO: 1; (b) an amino acid sequence represented by SEQ ID NO: 1 in which one to several amino acids have been deleted, inserted, substituted or added; and (c) an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence represented by SEQ ID NO: 1.

Description

[Technical Field]

[0001] The present invention relates to a vaccine against the novel coronavirus (SARS-CoV-2). [Background technology]

[0002] The novel coronavirus (SARS-CoV-2) is an enveloped virus with a positive-sense single-stranded RNA genome, a 5'-terminal cap structure, and a 3'-terminal poly(A) sequence. Its genome is approximately 30 kb, which is extremely long for a virus. Although the virus has its own RNA polymerase with a repair function, genome mutations are inevitable. The genome consists of 14 open reading frames (ORFs) encoding 27 proteins. These proteins consist of four structural proteins: membrane (M), envelope (E), spike (S), and nucleocapsid protein (N). Spike protein is primarily used as a vaccine antigen. Spike protein consists of an S1 region containing the receptor-binding domain (RBD) and an S2 region required for viral membrane fusion with the host cell membrane.

[0003] Infection of host cells by SARS-CoV-2 begins with binding of the RBD to human angiotensin-converting enzyme 2 (hACE2) on the host cell. The RBD has two states, up and down, and when in the up state, it can bind to hACE2. This binding exposes the S2' domain, which is cleaved by transmembrane serine protease 2 (TMPRSS2), resulting in cell-virus fusion. There is also an alternative infection route via the late endosomal cathepsin pathway, but the former route is believed to allow faster viral entry into cells.

[0004] Among the pathologies that SARS-CoV-2 infection can cause to worsen, pneumonia presenting with acute respiratory distress syndrome (ARDS) deserves special attention. The SARS-CoV-2 receptor, ACE2, is expressed on the surface of airway epithelial cells, including type II alveolar epithelium, and infects airway epithelial cells via the RBD of SARS-CoV-2. Diffuse alveolar damage due to ARDS is a characteristic feature of SARS-CoV-2-infected pneumonia. Generally, the histological appearance of diffuse alveolar damage progresses over time from the exudative stage to the organizing stage and then to the fibrotic stage. However, in SARS-CoV-2 infection, these different stages of lesions often progress simultaneously within the same lung lobe, resulting in severe disease.

[0005] The neutralizing antibody effect of the vaccine is to inhibit the binding of RBD to hACE2. The following Spike protein mutations have been reported to date, and are expected to affect the neutralizing antibodies induced by the vaccine. These mutations are the UK variant (alpha): N501Y / D614G mutation, the South African variant (beta): E484K / N501Y / D614G mutation, and the Japanese variant: E484K mutation.

[0006] In addition, the following variants have been recently recognized by the WHO as Variant of Interest (VOI) or Variant of Concern (VOC): Indian variant (kappa): L452R / E484Q / D614G, Indian variant (delta): L452R / T478K / D614G, and Indian variant (delta plus) K417N / L452R / T478K / D614G.

[0007] For example, the N501Y mutation is an amino acid mutation in the region primarily involved in the binding between RBD and hACE2, and is said to increase the binding affinity by 7-fold compared to the Wuhan type by forming a mutual bond between the tyrosine (Y) side chain of RBD and the amino acids Y41 and K353 of hACE2.

[0008] Antibodies evaluated as having the strongest neutralizing effect against SARS-CoV-2 (IC 50 At a concentration of 2 ng / mL, molecular-level analysis of binding to Spike protein revealed that amino acids in the heavy and light chains of the antibody interact with E484 of the Spike protein. In other words, with the E484K mutation, the interaction with the antibody seen with E484 is reduced with K484, and it is possible that the antibody with the highest neutralizing activity induced by Wuhan SARS-CoV-2 vaccination is rendered dysfunctional as a neutralizing antibody with the E484K mutation.

[0009] There are various modalities of vaccines against the wild-type novel coronavirus (SARS-CoV-2) that use the Spike protein as an antigen, including mRNA vaccines, recombinant viral vector vaccines, whole particle inactivated vaccines, and protein component vaccines. Among them, the mRNA vaccines from Pfizer and Moderna have been reported to be over 90% effective (see non-patent document 1). [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] The Advisory Committee on Immunization Practices' Interim Recommendation for Use of Pfizer-BioNTech COVID-19 Vaccine - United States, December 2020. Oliver SE, Gargano JW, Marin M, Wallace M, Curran KG, Chamberland M, McClung N, Campos-Outcalt D, Morgan RL, Mbaeyi S, Romero JR, Talbot HK, Lee GM, Bell BP, Dooling K. MMWR Morb Mortal Wkly Rep. 2020 Dec 18;69(50):1922-1924. Summary of the Invention [Problem to be solved by the invention]

[0011] All of these vaccines are administered intramuscularly. However, side effects such as anaphylaxis, blood clots, and cardiomyopathy have been reported due to vaccine administration, and mRNA vaccines are known to cause stronger side effects after the second dose. Many people refuse to be vaccinated due to side effects, making it difficult to achieve herd immunity. Going forward, COVID-19 vaccines will require a variety of modalities, including administration methods other than intramuscular administration, reduced side effects, and the ability to administer multiple doses.

[0012] The present invention was made in consideration of the above circumstances, and aims to provide a SARS-CoV-2 vaccine, which is a new modality. [Means for solving the problem]

[0013] That is, the present invention includes the following aspects. [1] A vaccine comprising as an active ingredient a viral vector that comprises a sequence containing any one of the amino acid sequences (a) to (c) below, and that displays on the viral particle envelope a protein that induces neutralizing antibodies that inhibit the binding of the receptor binding domain (RBD) of SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2). (a) the amino acid sequence represented by SEQ ID NO: 1; (b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1; (c) an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1

[0014] [2] The vaccine described in [1], which has a D614G mutation in the amino acid sequence represented by SEQ ID NO: 1.

[0015] [3] A vaccine described in [1] or [2], which has at least one mutation selected from the group consisting of K417N, L452R or L452Q, T478K, E484K, E484Q, F490S, and N501Y in the amino acid sequence represented by SEQ ID NO: 1.

[0016] [4] The vaccine according to any one of [1] to [3], wherein the transmembrane (TM) sequence and cytoplasmic tail (CT) sequence of the protein are replaced with the TM and CT sequences of human parainfluenza type 2 virus.

[0017] [5] The vaccine according to any one of [1] to [4], wherein the viral vector is human parainfluenza type 2 virus.

[0018] [6] The vaccine according to [5], wherein the human parainfluenza type 2 virus used as the viral vector is a non-replicating type in which the F gene has been deleted from the genome.

[0019] [7] The vaccine according to [5] or [6], wherein the genome of the human parainfluenza type 2 virus used as the viral vector is inactivated.

[0020] [8] The vaccine according to any one of [1] to [7], which is for nasal spray administration. [Effects of the Invention]

[0021] According to the present invention, a SARS-CoV-2 vaccine, which is a new modality, can be provided. [Brief explanation of the drawings]

[0022] [Figure 1]This diagram illustrates the four types of modified Wuhan-type SARS-CoV-2 Spike proteins introduced into BC-PIV. Specifically, (1) the S1 region protein, (2) a protein obtained by fusing the S1 region with the TM and CT regions of the hPIV2 F protein, (3) the Wuhan-type Spike protein (mutated at two sites; hereafter, also referred to as 2P substitution), and (4) a protein in which the TM and CT regions of the Wuhan-type Spike protein (3) were replaced with those of the hPIV2 F protein (designated BC-PIV / S2PM). The genes encoding these proteins were inserted into the NotI (upstream region of the gene encoding the NP protein) or MluI (region between the genes encoding the M and HN proteins) restriction enzyme cleavage sites of the F gene-deleted hPIV2 construction plasmid. [Figure 2] This figure shows the fusion of cells transfected with a plasmid encoding the Spike protein of Wuhan SARS-CoV-2 with 2P substitution. [Figure 3] This figure shows the proteins in infected cells and virus particles of BC-PIVs carrying the Spike gene of four types of Wuhan-type SARS-CoV-2 confirmed by Western blotting. [Figure 4] This figure shows that cell fusion is lost in cells infected with BC-PIV carrying the Spike gene / protein of four types of modified Wuhan SARS-CoV-2. [Figure 5A] This figure shows the degree of binding of each of the four types of viruses to hACE-2+Fc tag after adsorption onto a plate. [Figure 5B] This figure shows the degree of binding of each of the three types of inactivated viruses to hACE-2+Fc tag after adsorption onto a plate. [Figure 6] These are images of BC-PIV and BC-PIV / S2PM taken under a conventional electron microscope and an immunoelectron microscope using an anti-S1 antibody. [Figure 7] FIG. 1 shows the hemagglutination activity of BC-PIV / S2PM. [Figure 8]This figure shows the results of ELISA to compare the IgG antibody titers that bind to the RBD protein in the collected sera after BC-PIV / S2PM was administered intranasally or intramuscularly to mice. [Figure 9] This figure shows a comparison by ELISA of IgG antibody titers against S1 or RBD in sera collected from mice intranasally infected with BC-PIV carrying the Spike gene of four types of modified Wuhan-type SARS-CoV-2 with 2P substitutions. [Figure 10] This figure shows the results of ELISA of IgG antibody titers against S1 or RBD in the serum and IgA antibody titers against S1 or RBD in the nasal washes of mice that received a single or two doses of BC-PIV / S2PM intranasally. [Figure 11] This figure shows the evaluation of neutralizing antibody titers in the serum of mice administered twice using a cPass kit by inhibiting the binding of RBD to hACE-2. [Figure 12] This figure shows the induction of neutralizing antibodies confirmed using the cPass kit before examining the SARS-CoV-2 inhibitory effect of the BC-PIV / S2P vaccine in hamsters. [Figure 13] This figure shows the growth of SARS-CoV-2 in hamsters that had been intranasally administered the BC-PIV / S2P vaccine once or twice and then infected with SARS-CoV-2, after which the growth of SARS-CoV-2 was examined by plaque assay using each tissue collected. [Figure 14] This is an explanatory diagram of the Spike protein of the SARS-CoV-2 mutant strains (UK strain, South African strain, Japanese strain, Indian strain) to be introduced into BC-PIV. [Figure 15A] Figure 14 shows the proteins in infected cells and virus particles of BC-PIV carrying the Spike gene of SARS-CoV-2 mutant strains (UK strain, South African strain, Japanese strain, Indian strain) confirmed by Western blotting. [Figure 15B] Figure 14 shows the proteins in infected cells and virus particles of BC-PIV carrying the Spike gene of SARS-CoV-2 mutant strains (UK strain, South African strain, Japanese strain, Indian strain) confirmed by Western blotting. [Figure 16] This is an explanatory diagram of the Spike protein of SARS-CoV-2 mutant strains (Indian strain (delta, delta plus), South American strain (lambda)) to be introduced into BC-PIV. [Figure 17] This is an explanatory diagram of the Spike protein of SARS-CoV-2 mutant strains that are expected to be introduced into BC-PIV. DETAILED DESCRIPTION OF THE INVENTION

[0023] <Vaccines> In one embodiment, the present invention provides a vaccine comprising, as an active ingredient, a viral vector that displays on the viral particle envelope a protein comprising any one of the amino acid sequences (a) to (c) below, and in which the induced neutralizing antibody inhibits the binding of the receptor binding domain (RBD) of SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2): (a) the amino acid sequence represented by SEQ ID NO: 1; (b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1; (c) an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1

[0024] In (a), the amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of the Spike protein of Wuhan SARS-CoV-2, and has the mutations K986P / V987P. In this specification, Wuhan SARS-CoV-2 is also referred to as Wuhan SARS-CoV-2 with 2P substitution.

[0025] Herein, substitution mutations in amino acid sequences may be expressed by the single letter code of the original amino acid, followed by a one- to four-digit number for the position, and then the single letter code of the substituted amino acid. For example, if a mutation occurs at amino acid position 986 in which lysine (K) is substituted with proline (P), it is represented as "K986P," which is synonymous with "substitution of Lys with Pro at amino acid position 986."

[0026] In (b), the number of deleted, inserted, substituted or added amino acids is preferably 1 to 190, more preferably 1 to 120, more preferably 1 to 50, more preferably 1 to 25, even more preferably 1 to 10, and most preferably 1 to 5.

[0027] In (c), the identity is preferably 85% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

[0028] The protein used as an antigen in the vaccine of this embodiment is not limited to the Spike protein of Wuhan SARS-CoV-2, but also applies to proteins with identities equal to or greater than those specified in (b) and (c). That is, the proteins used in the vaccine of this embodiment are British type (alpha): N501Y or N501Y / D614G, South African type (beta): E484K / N501Y or E484K / N501Y / D614G, Japanese type: E484K or E484K / D614G, Indian type (kappa): L452R / E484Q or L452R / E484Q / D614G, Indian type (delta): L452R / T478K or L452R / T478K / D614G, Indian type (delta): L452R / T478K or L452R / T478K / D614G, Also applies to: plus): K417N / L452R / T478K or K417N / L452R / T478K / D614G, South American type (lambda): L452Q / F490S or L452Q / F490S / D614G.

[0029] It is preferable that the amino acid sequence represented by SEQ ID NO: 1 has a D614G mutation, and it is more preferable that the amino acid sequence represented by SEQ ID NO: 1 has at least one mutation selected from the group consisting of K417N, L452R or L452Q, T478K, E484K, E484Q, F490S and N501Y in addition to or independently of the D614G mutation. The protein used in the vaccine of this embodiment is also applicable to future predicted mutations as a combination of these mutations. Future predicted mutations include British type + Indian type (alpha + kappa): L452R / E484Q / N501Y (CTC / GAG / AAT → cgg / cag / tat) or L452R / E484Q / N501Y / D614G (CTC / GAG / AAT / GAT → cgg / cag / tat / ggt), British type + Indian type (alpha + delta): L452R / T478K / N501 (CTC / ACA / AAT → cgg / aaa / tat) or L452R / T478K / N501 / D614G (CTC / ACA / AAT / GAT → cgg / aaa / tat / ggt), and British type + Indian type (alpha + delta) plus):K417N / L452R / T478K / N501Y (AAG / CTC / ACA / AAT → aac / cgg / aaa / tat) or K417N / L452R / T478K / N501Y / D614G (AAG / CTC / ACA / AAT / GAT → aac / cgg / aaa / tat / ggt)).

[0030] In the present invention, "the induced neutralizing antibodies inhibit the binding of the receptor binding domain (RBD) of SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2)" means that the neutralizing antibodies induced in the body of a human administered the vaccine inhibit the binding of RBD to hACE2 by binding to RBD. As described below in the Examples, the vaccine of the present invention has neutralizing activity against all mutant SARS-CoV-2.

[0031] The viral vector contained in the vaccine of this embodiment is not limited as long as it presents an antigen protein on an envelope, and examples thereof include Sendai virus and human parainfluenza virus, with human parainfluenza virus being preferred and human parainfluenza type 2 virus being more preferred.

[0032] [BC-PIV vector and VLP. BC-PIV vector] The present inventors have deleted the F gene encoding the membrane fusion protein (F membrane protein or F protein) of human parainfluenza type 2 virus (hereinafter referred to as hPIV2), a negative-stranded RNA virus belonging to the Paramyxovirinae subfamily, Paramyxovirinae genus, Rubulavirus, so that it is no longer functional, established Vero cells into which the F gene is incorporated so that it is constitutively expressed, and constructed a BC-PIV vector designed to prevent the production of secondary infectious particles in the cells.

[0033] This vector is highly safe because it does not produce secondary infectious particles in infected hosts. It can also introduce foreign genes, and by taking advantage of the properties of RNA vectors, high foreign gene expression is observed in infected cells. Furthermore, BC-PIV has the unique ability to deliver antigens, such as foreign antigen genes and large antigen proteins, coexisting with the hPIV2 HN and F membrane proteins on the BC-PIV envelope while maintaining their three-dimensional structure, making it an effective vaccine vector. High-molecular-weight membrane proteins from viruses other than Ebola virus, whose three-dimensional structure is essential for the induction of neutralizing antibodies in vaccines such as Ebola virus, have been successfully introduced into the vector, resulting in the induction of neutralizing antibodies. Furthermore, BC-PIVs with antigens introduced into the envelope can be used as vectors even after the genome is inactivated (VLP. BC-PIV). Because they are non-replicating vectors, they can be inactivated with much lower concentrations of drugs than conventional virus inactivation treatments, while still maintaining the immunogenicity of the introduced antigen.

[0034] In a preferred embodiment, BC-PIV provides a vaccine against SARS-CoV-2, which carries a gene encoding the Spike protein of Wuhan SARS-CoV-2 and its mutant viruses into which a 2P substitution has been introduced, or a gene encoding the Spike protein of Wuhan SARS-CoV-2 and its mutant viruses in which the transmembrane (TM) and cytoplasmic tail (CT) sequences of the intracellular region of the protein (the base sequence is shown in SEQ ID NO: 13) have been replaced with the TM / CT sequence of hPIV2 F (the base sequence is shown in SEQ ID NO: 14) - the PIV2 F TM / CT gene. This includes intranasal and intramuscular administration of the vector. The genes encoding the spike proteins of Wuhan-type SARS-CoV-2 and its variants—PIV2 F TM / CT genes—are incorporated into BC-PIV or VLPs. BC-PIVs are incorporated not only in genetic form but also in protein form. These proteins are recognized by innate and adaptive immune cells at the time of administration, and subsequently interact with the proteins produced by gene transcription and replication, resulting in a stronger vaccine effect.

[0035] In one preferred form, the virus is an enveloped virus that carries the Spike protein of Wuhan SARS-CoV-2 and its mutant viruses, including the extramembrane region, on its viral envelope. In a more preferred form, the virus is a BC-PIV that carries the Spike of Wuhan SARS-CoV-2 and its mutant viruses in both genetic and protein form, or a BC-PIV that carries the Spike-PIV2 F TM / CT of Wuhan SARS-CoV-2 and its mutant viruses in protein form.

[0036] [Carriers and additives] The vaccine of this embodiment may contain a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, those typically used in pharmaceutical preparations can be used without any particular limitation, and examples thereof include solvents such as sterilized water and physiological saline; binders such as gelatin, corn starch, tragacanth gum, and gum arabic; excipients such as crystalline cellulose; and bulking agents such as alginic acid.

[0037] The vaccine of this embodiment may contain additives, such as lubricants such as magnesium stearate, sweeteners such as sucrose, lactose, and saccharin, flavorings such as peppermint and red ginseng oil, stabilizers such as benzyl alcohol and phenol, buffers such as phosphates and sodium acetate, solubilizers such as benzyl benzoate and benzyl alcohol, antioxidants, preservatives, surfactants, and emulsifiers.

[0038] The vaccine of this embodiment is preferably administered intramuscularly or intranasally, more preferably by spraying into the nasal cavity. Examples of solvents used for administration include isotonic solutions containing adjuvants such as physiological saline, glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. Solvents for injections may contain alcohols such as ethanol, polyalcohols such as propylene glycol and polyethylene glycol, nonionic surfactants such as Polysorbate 80 (trademark) and HCO-50, etc. Vaccine formulations for nasal administration may also be in the form of a spray.

[0039] The vaccine of this embodiment is typically administered at a single dose of about 10 ng to 1000 μg of active ingredient per day to an adult (body weight 60 kg), and if necessary, multiple doses may be administered at intervals of 2 to 12 weeks.

[0040] <Treatment method> In one embodiment, the present invention provides a method for treating severe acute respiratory syndrome, comprising administering to a patient in need of treatment the vaccine described above. [Example]

[0041] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0042] [Example 1] Four types of modified Spike proteins introduced into BC-PIV Using the SARS-CoV-2 Spike gene (GenBank. MN908947) codon-optimized from SinoBiological Inc. Cat. VG40591-UT and VG40590-UT / GeneBank. QHD43416.1, SEQ ID NO: 2), genes encoding four modified Wuhan-type SARS-CoV-2 Spike proteins to be introduced into BC-PIV were constructed (see Figure 1). Specifically, genes encoding (1) the S1 region protein, (2) a protein fused with the TM and CT regions of the hPIV2 F protein, (3) the Wuhan-type Spike protein (2P substitution), and (4) a protein in which the TM and CT regions of the Wuhan-type Spike protein (3) were replaced with those of the hPIV2 F protein (designated BC-PIV / S2PM) were constructed. In (3) and (4), the full-length Spike protein was engineered to have two P mutations (K986P / V987P (AAGGTG→cccccc)) in the S2 region to stabilize the protein structure and eliminate its cell fusion ability. The four modified genes were constructed by adding the corresponding restriction enzyme cleavage sequences and tag sequences for BC-PIV introduction to the NotI cleavage site of BC-PIV. The total base length of the BC-PIV gene, including the transgene, was adjusted to a multiple of six (rule of six). BC-PIV can carry heterologous viral multimeric antigens while maintaining their three-dimensional structure on its envelope. If the antigen is not a membrane protein, such as a foreign virus, the transmembrane domain (TM) sequence and the cytoplasmic tail (CT) sequence of the hPIV2 F membrane protein can be added to the antigen, allowing it to be incorporated into the BC-PIV envelope. On the other hand, when the antigen introduced into BC-PIV is a viral membrane protein or membrane-associated protein, the membrane anchoring signal of the introduced antigen may be incorporated into the BC-PIV envelope even if it is left as is, or the membrane anchoring signal may need to be replaced with that of hPIV2 for it to be incorporated into the BC-PIV envelope. This was examined in the construction of (3) and (4).

[0043] [Example 2] Confirmation of loss of fusion ability of modified proteins with 2P mutations We investigated whether introducing a mutation in the Spike protein reported in MERS-CoV, which inhibits cell fusion, into the Spike protein of SARS-CoV-2 would inhibit cell fusion. Using a codon-optimized SARS-CoV-2 Spike gene (GenBank MN908947) as a template, we introduced a conformationally stable Spike gene (SARS-CoV-2 S2P) with a 2P mutation (K986P / V987P (AAGGTG→cccccc)) in the S2 region of the SARS-CoV-2 Spike protein, and the Wuhan Spike gene (SARS-CoV-2 S) into the pcDNA plasmid. Each plasmid was transfected into Vero cells, and after 3 days, the cells were fixed with 4% paraformaldehyde (PFA), permeabilized, and stained with an anti-SARS-CoV S1 antibody (SinoBiological Inc. Cat. 40150-T62-COV2), which also cross-reacts with the SARS-CoV-2 S1 protein, and DAPI (Nacalai tesque Cat. 19174-31) to confirm cell fusion (see Figure 2). The Wuhan-type Spike protein induced cell fusion, as evidenced by the formation of multinucleated giant cells characteristic of cell fusion. The mutant Spike protein, while capable of protein expression, did not result in multinucleated giant cells, and cell fusion did not occur.

[0044] [Example 3] Confirmation of modified Spike proteins by Western blotting of BC-PIVs carrying four types of modified Spike genes BC-PIV viruses were recovered by reverse genetics, a method commonly used to generate negative-stranded viruses, using BC-PIV construction plasmids carrying the four types of modified Spike genes shown in Figure 1. All viruses were successfully recovered, and protein expression and BC-PIV vector uptake were investigated. Hereafter, unless otherwise specified, the four types described in Example 1 will be referred to as (1) "BC-PIV / S1," (2) "BC-PIV / S1M," (3) "BC-PIV / S2P," and (4) "BC-PIV / S2PM." After several passages, each BC-PIV-infected cell and virus were subjected to Western blotting. The virus was prepared by centrifuging the culture supernatant to remove cellular debris, and then passing the supernatant through a 0.8 μm filter. The supernatant was then ultracentrifuged (141,000 g, 4°C, 30 min) and concentrated. Anti-SARS-CoV S1 antibody (SinoBiological Inc. Cat. 40150-T62-COV2), anti-GAPDH-HRP-conjugated antibody, and anti-PIV2 NP (20A) or anti-PIV2 P (211A) were used. The viruses were collected at 10 μl / mL. 6 Western blot analysis was performed on the particles. Expression of the S1 protein was confirmed in all BC-PIV vectors in infected cells. Expression of the S0 protein before cleavage was also confirmed in BC-PIV / S2P and BC-PIV / S2PM (see Figure 3, left). Furthermore, incorporation into viral particles, as shown in Figure 3, right, S1 protein was detected in BC-PIV / S1M, BC-PIV / S2P, and BC-PIV / S2PM, confirming its presence on the particles. Furthermore, in BC-PIV / S2P and BC-PIV / S2PM, the S0 protein before cleavage was also present on the particles. However, in BC-PIV / S2P, less S0 protein was incorporated into vector particles than in BC-PIV / S2PM (see Figure 3, right). Since S1 protein was not detected on the particles in BC-PIV / S1, it is believed that S1 protein is expressed and secreted extracellularly in infected cells.

[0045] [Example 4] Confirmation of loss of fusion ability of recovered virus in non-F-expressing cells To examine the kinetics of cell infection and cell fusion by the Spike protein introduced into each BC-PIV, Vero cells were infected with each BC-PIV and, 3 days later, fixed and permeabilized with 4% PFA. The cells were then stained with an anti-SARS-CoV S1 antibody (SinoBiological Inc. Cat. 40150-T62-COV2), DAPI (Nacalai tesque Cat. 19174-3), and an anti-PIV2 NP antibody (20A). SARS-CoV-2 S1 was stained with Alexa-568 (red), PIV2 NP with Alexa-488 (green), and nuclei were detected with DAPI (blue). BC-PIV / S2PM-infected cells did not induce cell fusion or form multinucleated giant cells, confirming that the Spike protein derived from BC-PIV / S2PM, unlike the Wuhan-type Spike protein, lacks cell fusion activity. Even if the modified Spike gene is introduced into PIV2 F gene-deficient BC-PIV, secondary infectious particles will not be produced in cells that do not express PIV2 F protein because the modified Spike protein does not have the ability to replicate autonomously.

[0046] [Example 5] Confirmation of binding of recovered virus to hACE2 We confirmed whether the protein present on each BC-PIV particle could bind to the hACE2 protein, which is the receptor for the Spike protein. 6 TCID 50 Each BC-PIV was immobilized on a 96-well ELISA plate at 1 / well. Human ACE2 (hACE2)-Fc tag (Acro Bosystems, Cat. AC2-H5257) was added at 5 ng, 10 ng, 25 ng, 50 ng, 75 ng, 100 ng, 150 ng, and 200 ng, and incubated with HRP-Protein A (Biolegend, Cat. 689202). After incubation with TMB reagent, the plate was incubated with TMB reagent and the absorbance at 450 nm was measured. Each experiment was performed in triplicate. The absorbance of BC-PIV / S2PM, BC-PIV / S2P, and BC-PIV / S1M increased in a concentration-dependent manner, but no binding was observed for BC-PIV / S1 and BC-PIV (see Figure 5A), confirming that the S1 protein was not present on the BC-PIV / S1 particles. Although BC-PIV / S2P and BC-PIV / S1M had similar binding avidity, BC-PIV / S2PM exhibited a higher binding valency, suggesting that more Spike protein was incorporated into BC-PIV / S2PM than into BC-PIV / S1M. This is presumably because the replacement of the transmembrane cytoplasmic tail with PIV2F TM / CT allows for greater Spike protein incorporation into BC-PIV particles. This result is consistent with the particle Western blot results (BC-PIV / S2P and BC-PIV / S2PM) shown in Figure 3.

[0047] Figure 5B shows the binding of VLP.BC-PIV inactivated with 0.05% β-propiolactone to hACE2 protein. The binding of VLP.BC-PIV / S1M, VLP.BC-PIV / S2P, and VLP.BC-PIV / S2PM to hACE2 protein was highest, in that order, similar to BC-PIV, suggesting that inactivated VLP.BC-PIV may also be used as a SARS-CoV-2 vaccine.

[0048] [Example 6] Virus morphology and hemagglutination activity The morphology of BC-PIV / S2PM was confirmed by electron microscopy. For conventional electron microscopy, BC-PIV / S2PM or BC-PIV-infected cells were fixed in 4% PFA + 2.5% glutaraldehyde (GA), then further fixed in 1% OsO₄, dehydrated in alcohol, and embedded in epoxy resin. Ultrathin sections were prepared and observed under an electron microscope. For immunoelectron microscopy, infected BC-PIV / S2PM or BC-PIV-infected cells were fixed in a mixture of 2% PFA + 0.1% GA, then further fixed in 4% PFA. The sections were then stained with anti-SARS-CoV-2 S1 antibody (Sino Biological Inc. Cat. 40591-T62) and anti-rabbit biotinylated antibody (Vector Laboratories), then reacted with DAB and post-fixed in 1% OsO4. They were dehydrated in alcohol and embedded in epoxy resin. Ultrathin sections were prepared and observed under an electron microscope. In conventional electron microscopy, large protrusions presumably representing Spike protein (see arrow in the upper right of Figure 6) were observed on the outside of the envelope of BC-PIV / S2PM, but no such protrusions were observed in BC-PIV (see arrow in the upper left of Figure 6). Immunoelectron microscopy using anti-SARS-CoV-2 S1 antibodies revealed a positive image of the anti-S1 antibody in BC-PIV / S2PM (see arrow in the lower right of Figure 6), visually confirming that the envelope was covered with Spike protein.

[0049] Next, we examined the hemagglutination activity of BC-PIV to confirm whether the Spike protein altered its properties. BC-PIV or BC-PIV / S2PM virus solutions containing the same number of particles were serially diluted 2-fold with cold PBS and mixed with an equal volume of 2% guinea pig red blood cells in a 96-well U-bottom plate. The hemagglutination activity was examined. Experiments were performed in duplicate. BC-PIV demonstrated a hemagglutination value of at least 64 HAU, whereas BC-PIV / S2PM did not. While BC-PIV possesses hemagglutination activity due to the HN membrane protein, which is typically difficult to eliminate, the loss of hemagglutination activity in the BC-PIV / S2PM virus was surprising. This loss of activity was presumably due to the HN protein on the BC-PIV particles being covered by the bulky Spike protein, which prevents red blood cells from agglutinating on the HN protein. This supports the results of Figure 5, which show that BC-PIV / S2PM, which retains a bulkier Spike protein than BC-PIV / S1M, binds more to hACE2. The HN protein of BC-PIV / S2PM is thought to coexist with the bulky Spike protein, but the 2P mutation has been introduced into the Spike protein, and Spike protein-dependent cell fusion was not observed, as shown in Figures 2 and 4. This suggests that cell infection depends on the HN and F proteins derived from BC-PIV, and the functions of both proteins are maintained.

[0050] [Example 8] Confirmation of administration method (intranasal or intramuscular) in mice To investigate the difference in antibody titers induced by the vaccine administration route, the vaccine was administered intranasally or intramuscularly to mice, and the antibody titers of the induced antibodies were evaluated. Five- to seven-week-old BALB / c mice were administered 2 × 10 7 TCID 50BC-PIV / S2PM or BC-PIV was administered intranasally or intramuscularly once. Experiments were performed in triplicate. Thirty-three days after administration, blood was collected from the mice, centrifuged, and serum was collected and heat-inactivated. Antibody titers were determined by ELISA. RBD protein (SinoBiological Inc. Cat. 40592-V02H) was immobilized overnight at 4°C on a 96-well ELISA plate. After blocking, the serum was reacted with diluted serum, treated with HRP-conjugated anti-mouse IgG antibody (Biolegend Cat. 405306), and reacted with TMB reagent. The absorbance at 450 nm was measured. In both nasal and intramuscular administration, BC-PIV / S2PM induced higher IgG antibody titers against the RBD protein than BC-PIV. Furthermore, the absorbance values ​​for nasal and intramuscular administration were nearly equivalent, indicating no difference in antibody induction titers depending on the administration route (see Figure 8). These results demonstrate that nasal administration of a vaccine using BC-PIV is comparable to intramuscular administration in inducing antibodies with sufficient antibody titers.

[0051] [Example 9] Induction of antibodies against S1 and RBD After intranasal administration of four types of BC-PIV vaccines (1) to (4) to mice, serum IgG antibody titers against S1 and RBD were evaluated. Five- to seven-week-old BALB / c mice were intranasally administered 2 × 10 7 TCID 50Mice were intranasally administered either (1): BC-PIV / S1, (2): BC-PIV / S1M, (3): BC-PIV / S2P, (4): BC-PIV / S2PM, BC-PIV, or PBS. Thirty-three days after the initial administration, blood was collected from the mice and centrifuged. Serum was then heat-inactivated and tested for antibody titers by ELISA. S1 protein (SinoBiological Inc. Cat. 40591-V02H) or RBD protein (SinoBiological Inc. Cat. 40592-V02H) was immobilized on a 96-well ELISA plate. After blocking, the plate was reacted with diluted serum, treated with HRP-conjugated anti-mouse IgG antibody (Biolegend Cat. 405306), and reacted with TMB reagent. The absorbance at 450 nm was measured. This experiment was performed in six groups. High antibody induction against the S1 and RBD proteins was observed in all vaccine administration groups (see Figure 9). BC-PIV is known to be poorly permissive in mice, resulting in low transcription and translation efficiency. However, secreted BC-PIV / S1 induced antibody titers against the S1 protein similar to those of membrane-associated BC-PIV / S1M, and slightly higher antibody titers against the RBD protein. However, the antibody titers were lower than those of the full-length Spike protein vaccine group. This suggests that full-length Spike protein is more effective than S1 region protein in inducing antibodies as a SARS-COV-2 vaccine antigen. BC-PIV / S2PM, which contains the full-length Spike protein, was confirmed to be the most suitable vaccine.

[0052] [Example 10] Evaluation of IgG and IgA antibody induction against Spike / RBD protein in mouse serum Mice were intranasally administered the BC-PIV / S2PM vaccine once or twice, and serum IgG antibody titers and intranasal IgA antibody titers against S1 and RBD were evaluated. Five- to seven-week-old BALB / c mice were administered 2 × 10 7 TCID 50Mice were intranasally administered BC-PIV / S2PM or BC-PIV. Two doses were administered 4 weeks later. Thirty-five days after the first dose, blood was collected from the mice and centrifuged. Serum was collected and heat-inactivated. Nasal washes were collected in PBS, and antibody titers were determined by ELISA. S1 protein (SinoBiological Inc. Cat. 40591-V02H) or RBD protein (SinoBiological Inc. Cat. 40592-V02H) was immobilized on a 96-well ELISA plate. After blocking, diluted serum or nasal washes were reacted with HRP-conjugated anti-mouse IgG antibody (Biolegend Cat. 405306) or HRP-conjugated anti-mouse IgA antibody (Southern Biotech Cat. 1040-05). After reaction with TMB reagent, absorbance at 450 nm was measured. This study was performed on 5-6 mice. The serum IgG antibody titers against S1 and RBD were elevated in the single-dose group, but the antibody titers increased further in the double-dose group (see Figure 10). The IgA antibody titers against S1 and RBD in the nasal wash were elevated in the single-dose group. In the double-dose group, even higher IgA antibody titers were observed one week after the second dose. Although the half-life of IgA is known to be short, the presence of Spike protein-specific IgA against S1 or RBD was confirmed in the nasal cavity even five weeks after the single dose (see Figure 10).

[0053] [Example 11] Confirmation of neutralizing antibodies Mice were vaccinated twice, and the induction of neutralizing antibodies was examined using a commercially available kit. 5-7 week old BALB / c mice were inoculated with 2 × 10 7 TCID 50The mice were intranasally administered two doses at 4-week intervals. This study consisted of four mice. Seven days after the final dose, blood was collected from the mice and centrifuged. Serum was then inactivated and neutralizing antibodies were measured using the SARS-CoV-2 Surrogate Virus Neutralization Test Kit (cPass kit) (GenScript Cat. L00847). Each serum was diluted 20, 60, 180, 540, 1620, or 4860 times and incubated with HRP-conjugated RBD protein at 37°C for 30 minutes. The mixture was then applied to a plate coated with hACE2 protein and incubated at 37°C for 15 minutes. After washing, the plate was irradiated with TMB and the absorbance at 450 nm was measured. The inhibition rate (1 - (sample OD / negative control OD)) × 100% was calculated from the obtained values. (Note: The negative control value is a 20-fold dilution of the nonspecific antibody provided with the kit.) According to the manual, a 20% inhibition rate or higher is considered to indicate neutralizing antibody activity. In the BC-PIV / S2PM group, all individuals exceeded 20% at a 540-fold dilution, and even at a 1620-fold dilution, some individuals were confirmed to have an inhibition rate of more than 20% (see Figure 11). However, in the BC-PIV group, even at a 20-fold dilution, the inhibition rate was below 20% in all individuals, so neutralizing antibodies could not be confirmed. Two intranasal administrations of BC-PIV / S2PM were able to induce high levels of neutralizing antibodies.

[0054] [Example 12] Confirmation of infection protection test using hamsters To conduct animal protection tests, vaccine efficacy was examined using hamsters susceptible to BC-PIV and SARS-CoV-2. Four-week-old Syrian hamsters were given a single intranasal dose of BC-PIV or BC-PIV / S2PM. Two additional groups were given two intranasal doses of BC-PIV / S2PM, each at a 9-week interval. Serum samples were collected from the tail 10 weeks (single-dose group) and 9 weeks (double-dose group) after the first dose, and the induction of neutralizing antibodies was confirmed using the cPass kit. The study was performed with n=4. Evaluation of neutralizing antibodies was performed in the same manner as in Example 11, except that the dilution ratio was 30 times. In the BC-PIV / S2PM-administered group, both the first and second administrations yielded high inhibition rates equivalent to that of the positive control (note: values ​​obtained at a 20-fold dilution of the anti-RBD-specific positive antibody provided with the kit) (see Figure 12). On the other hand, in the BC-PIV-administered group, the inhibition rate was below 20%, so the presence of neutralizing antibodies could not be confirmed.

[0055] Using the same hamsters that had been used for neutralizing antibody evaluation, 11 weeks after the first vaccination, 10 3 PFU of SARS-CoV-2 was administered intranasally, and lung and nasal turbinate tissues were collected 3 days later. The number of SARS-CoV-2 viruses per gram of tissue was assessed by plaque assay using TMPRSS2-recombinant VeroE6 cells. In the BC-PIV-administered group, 10 8 However, no SARS-CoV-2 particles were detected in the groups receiving BC-PIV / S2PM once or twice. BC-PIV / S2PM administration induced high levels of neutralizing antibodies and strongly inhibited viral replication (see Figure 13). In addition, in the nasal turbinate tissue (per g) BC-PIV administration group, 10 8 SARS-CoV-2 particles were detected in the single-dose group, whereas in the single-dose group, 10 6 -10 7 In the two-dose group, virus counts were reduced in two animals, and in the remaining two animals, virus was not detected (see Figure 13). These results show that the effect of inhibiting viral proliferation in the lungs persists for a long period, even with a single dose. In the nasal turbinates, a protective effect against infection, thought to be mainly mediated by IgA, was confirmed, but it was also revealed that due to the characteristics of IgA, protection from infection cannot be maintained for as long as IgG antibodies (see Figure 13).

[0056] [Example 13] Construction of BC-PIV vaccines against mutant strains of SARS-CoV-2 (UK type (alpha): N501Y or N501Y / D614G, South African type (beta): E484K / N501Y or E484K / N501Y / D614G, Japanese type: E484K or E484K / D614G, Indian type (kappa): L452R / E484Q or L452R / E484Q / D614G)

[0057] Since the Wuhan strain of SARS-CoV-2 spread worldwide, numerous mutant strains have been reported, which may reduce the efficacy of vaccines using the Spike protein derived from the Wuhan strain against these mutant strains. Therefore, we created BC-PIVs containing Spike genes carrying 2P mutations corresponding to the mutant strains (British type (alpha): N501Y or N501Y / D614G, South African type (beta): E484K / N501Y or E484K / N501Y / D614G, Japanese type: E484K or E484K / D614G, Indian type (kappa): L452R / E484Q or L452R / E484Q / D614G). The nucleotide sequence of the Spike gene of the British type (alpha) N501Y / D614G is shown in SEQ ID NO: 3, the nucleotide sequence of the Spike gene of the South African type (beta) E484K / N501Y / D614G is shown in SEQ ID NO: 4, the nucleotide sequence of the Spike gene of the Japanese type E484K / D614G is shown in SEQ ID NO: 5, and the nucleotide sequence of the Spike gene of the Indian type (kappa) L452R / E484Q / D614G is shown in SEQ ID NO: 6.

[0058] The mutation sites are shown in Figure 14. These viruses were recovered by the reverse genetic analysis method similar to that used in Example 3. Western blots performed using the recovered virus particles in the same manner as in Example 3 are shown in Figures 15A and 15B. Spike protein was expressed in cells infected with all viruses, and mutant Spike protein was detected on BC-PIV. BC-PIV against this mutant virus is thought to be effective against the mutant strain as well, given the high vaccine effectiveness of BC-PIV against the Wuhan type SARS-CoV-2 to date.

[0059] [Example 14] Construction of BC-PIV vaccines against mutant SARS-CoV-2 strains (Indian type (delta): L452R / T478K or L452R / T478K / D614G, Indian type (delta plus): K417N / L452R / T478K or K417N / L452R / T478K / D614G, South American type (lambda): L452Q / F490S or L452Q / F490S / D614G).

[0060] Furthermore, mutant strains (Indian type (delta) L452R / T478K (CTC / ACA → cgg / aaa) or L452R / T478K / D614G (CTC / ACA / GAT → cgg / aaa / ggt) and Indian type (delta We constructed BC-PIVs containing Spike genes carrying 2P mutations corresponding to the following: (lambda): K417N / L452R / T478K (AAG / CTC / ACA → aac / cgg / aaa) or K417N / L452R / T478K / D614G (AAG / CTC / ACA / GAT → aac / cgg / aaa / ggt), and (lambda): L452Q / F490S (CTC / TTT → cag / tct) or L452Q / F490S / D614G (CTC / TTT / GAT → cag / tct / ggt). The nucleotide sequence of the Spike gene of the Indian type (delta): L452R / T478K / D614G is shown in SEQ ID NO: 7, the nucleotide sequence of the Spike gene of the Indian type (delta plus): K417N / L452R / T478K / D614G is shown in SEQ ID NO: 8, and the nucleotide sequence of the Spike gene of the South American type (lambda): L452Q / F490S / D614G is shown in SEQ ID NO: 12. Each mutation site is shown in Figure 16. BC-PIV against the Indian variant virus is thought to be effective against variants that are expected to emerge in the future, given the high vaccine effectiveness of BC-PIV against the Wuhan variant of SARS-CoV-2 to date.

[0061] [Example 15] Construction of BC-PIV vaccines against anticipated future variants of SARS-CoV-2 (UK type + Indian type (alpha + kappa): L452R / E484Q / N501Y or L452R / E484Q / N501Y / D614G, UK type + Indian type (alpha + delta): L452R / T478K / N501Y or L452R / T478K / N501Y / D614G, UK type + Indian type (alpha + delta plus): K417N / L452R / T478K / N501Y or K417N / L452R / T478K / N501Y / D614G)

[0062] Furthermore, the following variants are expected in the future: British + Indian (alpha + kappa): L452R / E484Q / N501Y (CTC / GAG / AAT → cgg / cag / tat) or L452R / E484Q / N501Y / D614G (CTC / GAG / AAT / GAT → cgg / cag / tat / ggt), British + Indian (alpha + delta): L452R / T478K / N501Y (CTC / ACA / AAT → cgg / aaa / tat) or L452R / T478K / N501Y / D614G (CTC / ACA / AAT / GAT → cgg / aaa / tat / ggt), British + Indian (alpha + delta): We constructed BC-PIVs containing Spike genes carrying 2P mutations corresponding to either K417N / L452R / T478K / N501Y (AAG / CTC / ACA / AAT → aac / cgg / aaa / tat) or K417N / L452R / T478K / N501Y / D614G (AAG / CTC / ACA / AAT / GAT → aac / cgg / aaa / tat / ggt). The nucleotide sequence of the Spike gene of British type + Indian type (alpha + kappa) L452R / E484Q / N501Y / D614G is shown in SEQ ID NO: 9, the nucleotide sequence of the Spike gene of British type + Indian type (alpha + delta) L452R / T478K / N501Y / D614G is shown in SEQ ID NO: 10, and the nucleotide sequence of the Spike gene of British type + Indian type (alpha + delta plus) K417N / L452R / T478K / N501Y / D614G is shown in SEQ ID NO: 11. The mutation sites are shown in Figure 17. The viruses were produced using the reverse genetics method described in Example 3. BC-PIV is expected to be effective against mutant viruses expected to emerge in the future, given the high vaccine effect of BC-PIV against Wuhan-type SARS-CoV-2 to date. [Industrial Applicability]

[0063] According to the present invention, a SARS-CoV-2 vaccine, which is a new modality, can be provided.

Claims

1. (a) A vaccine comprising, as an active ingredient, a viral vector that displays on the viral particle envelope a protein that inhibits the binding of the receptor binding domain (RBD) of SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2) and induces neutralizing antibodies, wherein the viral vector is human parainfluenza type 2 virus, and the vaccine is for nasal spray administration.

2. 2. The vaccine according to claim 1, wherein the human parainfluenza type 2 virus used as the viral vector is a non-replicating virus in which the F gene is deleted from the genome.

3. The vaccine according to claim 1 or 2, wherein the genome of the human parainfluenza type 2 virus used as the viral vector is inactivated.

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