WO2023080246A1 - ベータコロナウイルス弱毒株 - Google Patents

ベータコロナウイルス弱毒株 Download PDF

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WO2023080246A1
WO2023080246A1 PCT/JP2022/041445 JP2022041445W WO2023080246A1 WO 2023080246 A1 WO2023080246 A1 WO 2023080246A1 JP 2022041445 W JP2022041445 W JP 2022041445W WO 2023080246 A1 WO2023080246 A1 WO 2023080246A1
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mutation
strain
amino acid
mutations
temperature
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French (fr)
Japanese (ja)
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志郎 竹河
博貴 蝦名
真弥 岡村
秋穂 柏原
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Research Foundation for Microbial Diseases of Osaka University BIKEN
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Research Foundation for Microbial Diseases of Osaka University BIKEN
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Priority to JP2023558098A priority Critical patent/JP7594686B2/ja
Priority to EP22890066.8A priority patent/EP4431599A4/en
Priority to AU2022380191A priority patent/AU2022380191A1/en
Priority to CN202280074172.6A priority patent/CN118660957A/zh
Priority to US18/708,189 priority patent/US20250002871A1/en
Priority to KR1020247015314A priority patent/KR20240099257A/ko
Publication of WO2023080246A1 publication Critical patent/WO2023080246A1/ja
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Definitions

  • the present invention relates to attenuated strains of betacoronavirus.
  • the infectious disease (COVID-19) caused by the new coronavirus (SARS-CoV-2) has caused a pandemic and is still a social problem.
  • genetic vaccines such as adenovirus vector vaccines and mRNA vaccines have been approved, starting with Sputnik V, an adenovirus vector vaccine approved in Russia (Non-Patent Document 1), and vaccination is now available worldwide. is underway.
  • genetic vaccines are next-generation vaccines that differ from conventional vaccines, and side effects such as fever and thrombosis have been reported. Therefore, it is considered important to continue to develop new vaccines.
  • the purpose of the present invention is to provide a strain useful as a new betacoronavirus vaccine.
  • the present inventors have found that a novel betacoronavirus having, as predetermined mutations related to attenuation, a combination of a predetermined substitution mutation related to temperature sensitivity and a predetermined deletion mutation related to growth reduction or other attenuation.
  • a novel betacoronavirus having, as predetermined mutations related to attenuation, a combination of a predetermined substitution mutation related to temperature sensitivity and a predetermined deletion mutation related to growth reduction or other attenuation.
  • it was found to be useful as a betacoronavirus vaccine strain with excellent attenuation.
  • the present invention is an invention completed by further studies based on this finding. That is, the present invention provides inventions in the following aspects.
  • Section 1 a nonstructural protein having the following mutations (b), a combination of mutations (e) and (f), and/or mutations (h); Structural proteins, accessory proteins and/or non-structural proteins having the following mutations (n), (o) and/or (r); Attenuated strains of betacoronavirus, including: (b) mutation of the amino acid residue corresponding to leucine at position 445 in the amino acid sequence shown in SEQ ID NO: 1 in NSP3; (e) mutation of the amino acid residue corresponding to glycine at position 248 in the amino acid sequence shown in SEQ ID NO: 2 in NSP14; (f) mutation of the amino acid residue corresponding to glycine at position 416 in the amino acid sequence shown in SEQ ID NO: 2 in NSP14; (h) mutation of the amino acid residue corresponding to valine at position 67 in the amino acid sequence shown in SEQ ID NO: 3 in NSP16; (n) a loss-of-function mutation of ORF8; (O) deletion
  • the attenuated virus strain of claim 1 wherein 4 mutations or combinations of mutations are selected among the species mutations or combinations of mutations.
  • Item 3. 1 to 2 mutations or a combination of mutations selected from the three types of mutations or combinations of mutations of the mutation (b), the combination of mutations (e) and (f), and the mutation (h) Item 3.
  • the attenuated virus strain according to any one of Items 1 to 3, wherein the structural protein further comprises the following (p) and/or (q) mutations: (p) mutations in the spike, including deletions of amino acid sequences corresponding to positions 679 to 680 and positions 685 to 686 of the amino acid sequence shown in SEQ ID NO: 4; (q) Mutation of the amino acid residue corresponding to valine at position 687 in the amino acid sequence shown in SEQ ID NO: 4 in the spike. Item 5. Item 5.
  • the virus-attenuated strain according to item 4 comprising a combination of the mutations (e) and (f) and the following mutation (g): (g) Mutation of an amino acid residue corresponding to alanine at position 504 in the amino acid sequence shown in SEQ ID NO: 2 in NSP14.
  • Item 6. Item 6.
  • the mutation (b) is a substitution to phenylalanine
  • the mutation (e) is a substitution to valine
  • the mutation (f) is a substitution to serine
  • the mutation (h) is isoleucine 7.
  • Item 9. The virus-attenuated strain of any of paragraphs 1-8, wherein the betacoronavirus is the SARS-CoV-2 virus.
  • Item 10 is a substitution to phenylalanine
  • the mutation (e) is a substitution to valine
  • the mutation (f) is a substitution to serine
  • the mutation (h) is isoleucine 7.
  • a live attenuated vaccine comprising the virus attenuated strain according to any one of Items 1 to 9.
  • Item 11. The live attenuated vaccine of Item 10, which is administered intranasally.
  • Item 12. The live attenuated vaccine of Item 10, administered intramuscularly, subcutaneously or intradermally.
  • strains useful as new betacoronavirus vaccines are provided.
  • FIG. 2 shows confirmation results (CPE images) of temperature sensitivity of SARS-CoV-2. Mutation analysis results for each virus strain are shown. CPE images of a temperature-sensitive strain (A50-18 [reference example]) with the possibility of reversion are shown. CPE images of a temperature-sensitive strain (A50-18 [reference example]) with the possibility of reversion are shown. Fig. 2 shows the results of confirming the temperature sensitivity of a recombinant virus into which a mutation was introduced in a temperature sensitive strain (A50-18 [reference example]). Fig.
  • FIG. 2 shows the results of confirming the temperature sensitivity of a recombinant virus into which a mutation was introduced in a temperature sensitive strain (A50-18 [reference example]).
  • the results of proliferative analysis of a temperature-sensitive strain (A50-18 [reference example]) are shown.
  • the results of proliferative analysis of a temperature-sensitive strain (A50-18 [reference example]) are shown.
  • Fig. 2 shows body weight fluctuations of hamsters infected with a temperature-sensitive strain (A50-18 [reference example]).
  • Fig. 2 shows body weight fluctuations of hamsters infected with a temperature-sensitive strain (A50-18 [reference example]).
  • Figure 2 shows the viral load in lung or nasal washes of hamsters infected with a temperature-sensitive strain (A50-18 [reference example]).
  • Lung images of a hamster infected with a temperature-sensitive strain (A50-18 [reference example]) are shown.
  • the results of lung histological analysis of hamsters infected with a temperature-sensitive strain (A50-18 [reference example]) are shown.
  • FIG. 1 shows lung histological analysis (HE staining and IHC staining) of temperature-sensitive strain (A50-18 [reference example]) infected hamsters.
  • Fig. 2 shows weight fluctuations of hamsters reinfected with a temperature-sensitive strain (A50-18 [reference example]).
  • FIG. 2 shows body weight changes of hamsters after infection with a temperature-sensitive strain (A50-18 [reference example]). Temperature-sensitive strain (A50-18 [reference example]) Neutralizing antibody titer of recovered hamster serum after infection is shown. SARS-CoV-2 temperature sensitization method (G-L50 series [Example]) is shown.
  • FIG. 2 shows confirmation results (CPE images) of temperature sensitivity of SARS-CoV-2 (G to L50 series [Examples]). Mutation analysis results of additional isolates (H50-11, L50-33, L50-40 [Example]) are shown. CPE images of a temperature-sensitive strain (H50-11 [Example]) with potential for reversion are shown.
  • FIG. 17 shows a schematic diagram of the deletion of the nucleotide sequence shown in FIG. 17 and the deletion of the amino acid sequence encoded by it.
  • the growth analysis results of temperature-sensitive strains (H50-11, L50-33, L50-40 [Example]) are shown.
  • Temperature-sensitive strains (A50-18 [reference example], and H50-11, L50-33, L50-40 [example]) show body weight changes of infected hamsters.
  • Lung weights of hamsters infected with temperature sensitive strains are shown.
  • Figure 2 shows viral loads in lung or nasal washes of hamsters infected with temperature-sensitive strains (A50-18 [reference example] and H50-11, L50-33, L50-40 [example]).
  • Temperature-sensitive strains show weight changes of re-infected hamsters.
  • Temperature-sensitive strains show neutralizing antibody titers of post-infection hamster sera.
  • Fig. 2 shows the evaluation of the neutralizing activity of a temperature-sensitive strain (A50-18 strain [reference example]) against SARS-CoV-2 mutants.
  • 1 shows a comparison of the ability of a temperature-sensitive strain (A50-18 strain [reference example]) to induce immunity depending on the route of administration.
  • a comparison of the dose-dependent immune induction of the temperature-sensitive strain is shown.
  • FIG. 2 shows the evaluation of the neutralizing activity of a temperature-sensitive strain (A50-18 strain [reference example]) against SARS-CoV-2 mutants.
  • Fig. 2 shows the evaluation of the neutralizing activity of a temperature-sensitive strain (A50-18 strain [reference example]) against SARS-CoV-2 mutants.
  • Vaccine candidate strains 1 to 7 Shown are CPE images during recovery culture after production. Temperature sensitivity evaluation of vaccine candidate strains 1-7 [Example] is shown. Temperature sensitivity evaluation of the rTs-all strain [Example] is shown. The ability of vaccine candidate strains 1, 3, 4, 6, and 7 [Examples] to induce neutralizing antibodies when administered at low titers and low doses is shown.
  • the results of the infection protection test after immunization with low titer and low dose administration of vaccine candidate strain 7 [Example] are shown. It shows the ability of vaccine candidate strains 2 and 5 [Example] to induce neutralizing antibodies when administered at a high titer and a high dose.
  • the growth evaluation of vaccine candidate strain 2 [Example] at each temperature is shown.
  • Fig. 2 shows results of persistence test of humoral immunity induced by administration of vaccine candidate strain 2 [Example].
  • FIG. 2 shows the results of an infection prevention test (body weight change of infected hamsters) by administration of vaccine candidate strain 2 [Example].
  • FIG. 10 shows the results of examining the restoration of virulence (presence or absence of CPE) during in vivo passage of vaccine candidate strain 2 [Example].
  • FIG. 10 shows the results of examining the reversion to virulence of vaccine candidate strain 2 [Example] during in vivo passage (sequence of viral RNA extracted from each nasal wash).
  • Fig. 10 shows the results (body weight change) of examination of reversion to virulence during in vivo passage of vaccine candidate strain 2 [Example].
  • Fig. 2 shows tissue damage evaluation (nasal cavity level 1) by vaccine candidate strain 2 [Example].
  • Fig. 2 shows tissue damage evaluation (nasal cavity level 2) by vaccine candidate strain 2 [Example].
  • FIG. 2 shows tissue damage evaluation (nasal cavity level 3) by vaccine candidate strain 2 [Example].
  • Fig. 2 shows tissue damage evaluation (lung) by vaccine candidate strain 2 [Example].
  • Fig. 2 shows neutralizing antibody titers induced by administration of vaccine candidate strain 2 [Example].
  • FIG. 2 shows the results of an infection prevention test (body weight change of infected hamsters) by administration of vaccine candidate strain 2 [Example].
  • Fig. 2 shows neutralizing antibody titers induced by administration of vaccine candidate strain 2 [Example].
  • Attenuated Betacoronavirus Strains comprise, as predetermined mutations for attenuation, nonstructural proteins having predetermined substitution mutations for temperature sensitivity, and predetermined deletion mutations for growth-reducing or other attenuation.
  • a betacoronavirus comprising in combination a structural protein, ancillary protein and/or a non-structural protein having
  • a given substitution mutation for temperature sensitivity is also referred to as a "temperature-sensitive mutation”
  • a given deletion mutation for growth-reducing is also referred to as a "proliferation-reducing mutation”
  • a given deletion other than a growth-reducing mutation Mutations are also described as "other attenuating mutations”.
  • Attenuation refers to the property of attenuating host pathogenicity of a virus.
  • temperature sensitivity refers to the characteristic of having limited proliferation at human body temperature (so-called lower respiratory tract temperature) and specific proliferation at low temperatures (typically lower than human upper respiratory tract temperature).
  • proliferation-reducing property refers to the property of restricted proliferation and the property of which is not temperature-specific.
  • the attenuated betacoronavirus strain of the present invention not only exhibits efficacy as a vaccine by having the above-described predetermined mutations related to attenuation, but also has a combination of substitution mutations and deletion mutations that are difficult to revert. Therefore, the possibility of reversal of toxicity is extremely low. In this respect, the usefulness is remarkably increased when the application to humans is assumed.
  • coronaviruses are spherical with a diameter of about 100 to 200 nm, and have projections on the surface. Coronaviruses are virologically classified into the order Nidoviridae, the subfamily Coronavirinae, and the family Coronaviridae.
  • nucleocapsid protein
  • Nucleocapsid nucleocapsid
  • spike protein protein
  • envelope protein envelope protein
  • membrane protein membrane protein
  • Coronaviruses are classified into alpha, beta, gamma, and delta groups based on their genetic characteristics. As coronaviruses that infect humans, there are four types of human coronavirus 229E, OC43, NL63, and HKU-1 as viruses that cause colds, and severe acute respiratory syndrome (SARS) that occurred in 2002 causing severe pneumonia. ) coronaviruses and the 2012 Middle East Respiratory Syndrome (MERS) coronavirus.
  • the Alphacoronavirus genus includes human coronaviruses 229E and NL63
  • the Betacoronavirus genus includes human coronaviruses OC43, HKU-1, SARS and MERS coronaviruses.
  • SARS-CoV-2 which is classified as a SARS coronavirus, has been isolated and identified as the causative virus of the new coronavirus infection that occurred in Wuhan in 2019.
  • SARS-CoV-2 has repeatedly mutated from the early Wuhan strain, and mutant strains such as the strain detected in the UK, the strain detected in South Africa, and the strain detected in India have been found. Mutants that have not yet been detected and new mutant strains may occur in the future.
  • viruses included in the genus Betacoronavirus are not limited to the strains of SARS-CoV-2 described above, and all other betacoronaviruses (e.g., other SARS-CoVs that will be newly detected in the future).
  • Mutation (b), a combination of mutation (e) and mutation (f), and/or mutation (h), indicated as "temperature-sensitive mutation” in Table 1, are substitution mutations and are betacoronaviruses of the invention. It is a responsible mutation that contributes to conferring temperature sensitivity, which is essential for attenuated strains. That is, in the present invention, the temperature-sensitive mutation includes three types of "mutation (b)", "a combination of mutation (e) and mutation (f)", and “mutation (h)”.
  • Typical attenuated betacoronavirus strains of the invention have one or two of these three temperature sensitive mutations.
  • Mutation (n), Mutation (O) and/or Mutation (r), indicated as “proliferation-reducing mutation” and “other attenuating mutation” in Table 1 are deletion mutations and are beta-coronaviruses of the present invention. It is thought to contribute to conferment of growth-reducing property and other attenuation, which is essential for virus-attenuated strains (in particular, mutation (r) is thought to contribute to conferring growth-reducing property), and is excellent in combination with temperature-sensitive mutations. It is a mutation that expresses attenuated toxicity.
  • Mutations (a), (c), (d), (g), (i) to (m), (p), and (q), indicated as "other mutations” in Table 1, are the beta Mutations that attenuated strains of coronavirus may optionally contain, and attenuated betacoronavirus strains of the invention may or may not contain at least one of the other mutations.
  • the essential attenuating mutation of the betacoronavirus of the present invention is a temperature-sensitive mutation, the following mutation (b), a combination of mutations (e) and (f), and / or (h) and mutations of (n), (o) and/or (r) below, which are growth-reducing or other attenuating mutations.
  • the above temperature-sensitive mutations that is, the combination of (b) mutations, (e) and (f) mutations, (h) 1 to 2 mutations or combinations of mutations are selected from among the 3 mutations or combinations of mutations of the above.
  • the (O) mutation and the (r) mutation are mentioned.
  • mutations related to attenuation that is, mutation (b), (e) and Combination of mutations of (f), mutations of (h), mutations of (n), mutations of (O), and mutations of (r)), 4 types of mutations or mutations A combination is selected.
  • the attenuated betacoronavirus strain of the present invention has the following (a), (c), (d), (g), (i) to (m), At least one of (p) and (q) may be mutated.
  • the betacoronavirus-attenuated strain of the present invention has other mutations, it preferably has the mutation (g) among the above other mutations from the viewpoint of enhancing temperature sensitivity.
  • the mutation (g) is used in combination with the mutations (e) and (f) from the viewpoint of enhancing temperature sensitivity. preferably.
  • SEQ ID NO: 1 is the amino acid sequence of NSP3 in SARS-CoV-2 of NC_045512 (NCBI);
  • SEQ ID NO: 2 is the amino acid sequence of NSP14 in SARS-CoV-2 of NC_045512 (NCBI);
  • NC_045512 (NCBI) is the amino acid sequence of NSP16 in SARS-CoV-2.
  • SEQ ID NO: 4 is the amino acid sequence of the spike in SARS-CoV-2 of NC_045512 (NCBI);
  • SEQ ID NO: 5 is the amino acid sequence of the envelope in SARS-CoV-2 of NC_045512 (NCBI);
  • 6 is the amino acid sequence of the nucleocapsid in SARS-CoV-2 from NC_045512 (NCBI).
  • SEQ ID NO: 7 is a nucleotide sequence spanning part of the open reading frame of SARS-CoV-2 of NC_045512 (NCBI), specifically part of ORF7a, all of ORF7b, and most of ORF8; SEQ ID NO:8 is the amino acid sequence of NSP1 in SARS-CoV-2 from NC_045512 (NCBI).
  • Corresponding amino acid residues are SEQ ID NOs: 1-4 (or 1-6), 8 or the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 7, the amino acid residue present at the above-mentioned predetermined position, and the attenuated beta-coronavirus strain of the present invention is a beta-coronavirus other than the above mutant strain
  • the amino acid sequence encoded by the amino acid sequence of SEQ ID NOS: 1 to 4 (or 1 to 6) refers to an amino acid residue present at a position corresponding to the above-mentioned predetermined position.
  • the corresponding position is the amino acid sequence encoded by the protein having the amino acid sequence of SEQ ID NOS: 1 to 4 (or 1 to 6) or 8 of SARS-CoV-2 of NC_045512 (NCBI) or the nucleotide sequence shown in SEQ ID NO: 7 It can be identified by aligning the amino acid sequences of the protein having and other betacoronavirus proteins corresponding to the protein.
  • the virus-attenuated strain of the present invention is an amino acid residue corresponding to the predetermined position in the amino acid sequence encoded by the amino acid sequence of SEQ ID NOS: 1 to 4 (or 1 to 6), 8 or the nucleotide sequence of SEQ ID NO: 7, or the amino acid
  • the sequence is mutated, it is not limited to the specific SARS-CoV-2 variants listed in NC_045512 (NCBI), but other betacoronavirus variants [i.e., any other and variants of viruses other than SARS-CoV-2 included in the genus Betacoronavirus].
  • the specific SARS-CoV-2 mutant listed in NC_045512 is the amino acid sequence of SEQ ID NOs: 1 to 4 (or 1 to 6), 8 in the specific SARS-CoV-2 or SEQ ID NO: A mutant strain in which at least one of the amino acid residues or the amino acid sequence at the predetermined position in the amino acid sequence encoded by the nucleotide sequence shown in 7 is mutated, and other beta coronavirus mutant strains are any other SARS - Mutant strain of CoV-2 [that is, any other SARS-CoV-2 above SEQ ID NOS: 1 to 4 (or 1 to 6), the amino acid sequence of 8 or the amino acid encoded by the nucleotide sequence shown in SEQ ID NO: 7 A mutant strain in which the amino acid residue or amino acid sequence corresponding to the predetermined position in the sequence is mutated] and a mutant strain of a virus other than SARS-CoV-2 included in the betacoronavirus genus [that is, in the betacoronavirus genus Cor
  • Betacoronavirus any other variants of SARS-CoV-2 and variants of viruses other than SARS-CoV-2 included in the genus Betacoronavirus include SARS-CoV-2 or a betacoronavirus other than SARS-CoV-2 Recombinant virus mutants in which the spike protein is replaced with at least one spike protein of other SARS-CoV-2 and betacoronaviruses other than SARS-CoV-2 (including viruses that will be newly detected in the future) is also included.
  • “Not significantly affecting the properties of the polypeptide” means a state in which the functions of each nonstructural protein, structural protein, and accessory protein are maintained.
  • Permissible differences may be one type of difference (e.g., substitution) selected from among substitutions, additions, insertions, and deletions, or two or more types of difference (e.g., substitutions and insertions). may contain. Any other SARS-CoV-2 amino acid sequence corresponding to SEQ ID NOS: 1 to 4 (or 1 to 6), 8 or the base sequence shown in SEQ ID NO: 7, and SEQ ID NOS: 1 to 4 (or 1 to 6) , 8 or the base sequence shown in SEQ ID NO: 7, and the sequence identity calculated by comparing only the arbitrary different sites may be 50% or more.
  • sequence identity is preferably 60% or more or 70% or more, more preferably 80% or more, still more preferably 85% or more or 90% or more, still more preferably 95% or more, 96% or more, 97% or more, or 98% or more, more preferably 99% or more, particularly preferably 99.3% or more, 99.5% or more, 99.7% or more, 99.9% The above are mentioned.
  • sequence identity preferably includes 60% or more.
  • sequence identity means BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] bl2seq program (Tatiana A. Tats Usova, Thomas L. Madden, FEMS Microbiol.Lett., Vol.174, p247-250, 1999).
  • NCBI National Center for Biotechnology Information
  • betacoronavirus attenuated strains of the present invention are more specifically as follows: An attenuated strain of betacoronavirus comprising nonstructural, accessory and structural proteins consisting of at least one of the following polypeptides (I), (II) and (III): (I) at least one of the following (I-1) to (I-3) polypeptides and at least one of the following (1-4) to (I-6) polypeptides: (I-1) A polypeptide (NSP3) consisting of an amino acid sequence represented by SEQ ID NO: 1 and having a substitution mutation (b') of leucine at position 445 as a temperature-sensitive mutation; (I-2) A polypeptide consisting of an amino acid sequence having a substitution mutation (e') at position 248 glycine and a substitution mutation (f') at position 416 glycine in the amino acid sequence shown in SEQ ID NO: 2 as temperature-sensitive mutations ( NSP14), (I-3) A polypeptide (NSP16) consisting of
  • the above (I-1) and (I-) contains other mutations in addition to temperature-sensitive mutations and growth-reducing or other attenuating mutations, as shown below, the above (I-1) and (I- The following ( It may be a polypeptide of I-1a) and (I-2a) and (I-5a), and the polypeptide of (I) above further has other mutations below (I-7a), ( It may also include polypeptides (structural proteins) of I-8a).
  • An attenuated strain of betacoronavirus comprising structural, accessory and nonstructural proteins consisting of at least one of the following polypeptides (I), (II) and (III): (I) At least one of the following (I-1a), (I-2a), and (I-3) and the following (I-4), (I-5a), and (I-6) polypeptides At least one, or in addition to at least one of the following (I-7a) and (I-8a): (I-1a) In the amino acid sequence shown in SEQ ID NO: 1, leucine mutation (b') at position 445 as a temperature-sensitive mutation, and valine mutation (a') at position 404 and lysine at position 1792 as other mutations A polypeptide (NSP3) consisting of an amino acid sequence having at least one of a mutation (c′) at position 1832 and a mutation (d′) at position 1832 aspartic acid; (I-2a) In the amino acid sequence shown in SEQ ID NO: 2, a mutation (e')
  • polypeptide a polypeptide (spike) consisting of an amino acid sequence having at least one of a sequence deletion mutation (p') and a mutation (q') at position 687 valine;
  • a polypeptide (NSP1) consisting of an amino acid sequence shown in SEQ ID NO: 8 and having deletion mutations (r') at positions 32 to 39 as growth-reducing mutations
  • I-7a a polypeptide (envelope) consisting of an amino acid sequence shown in SEQ ID NO: 5 and having a mutation (l') of leucine at position 28 as another mutation
  • I-8a A polypeptide (nucleocapsid) consisting of an amino acid sequence shown in SEQ ID NO: 6 and having a mutation (m') at position 2 serine as another mutation;
  • II) In the amino acid sequence of the polypeptide of (I), one other than the amino acid sequence related to the temperature-sensitive mutation, the growth-reducing or other attenuating mutation, or the amino acid residue or amino acid sequence related to other mutations
  • the above (a') to (r') mutations are specifically the amino acid sequences of SEQ ID NOS: 1 to 6, the nucleotide sequences of SEQ ID NOS: 7, and SEQ ID NOS: 8. Refers to mutations when present in an amino acid sequence. That is, the polypeptide of (I) above includes the amino acid sequences of SEQ ID NOS: 1 to 6 of SARS-CoV-2 of NC_045512 (NCBI), the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO: 8 A polypeptide consisting of an amino acid sequence has been introduced with temperature-sensitive mutations, growth-reducing or other attenuating mutations, or other mutations in addition thereto.
  • polypeptides of (II) and (III) above include the amino acid sequences of SEQ ID NOS: 1 to 6 possessed by other betacoronaviruses, the amino acid sequences encoded by the nucleotide sequences of SEQ ID NO: 7, and the amino acid sequences of SEQ ID NO: 8.
  • a polypeptide consisting of an amino acid sequence corresponding to a sequence has been introduced with temperature-sensitive mutations and growth-reducing or other attenuating mutations, or other mutations in addition thereto.
  • the preferred ranges of sequence identity for the polypeptides (II) and (III) above are as described above.
  • Betacoronavirus can acquire temperature sensitivity by having the above temperature-sensitive mutation, and can acquire excellent attenuation by having the above-mentioned growth-reducing and other attenuating mutations together with the above temperature-sensitive mutation.
  • the attenuated virus strain of the present invention has at least a reduced ability to grow at human lower respiratory tract temperatures than it does at temperatures below human lower respiratory tract temperatures, preferably at human lower respiratory tract temperatures. No proliferative capacity.
  • the virus-attenuated strain of the present invention has reduced ability to grow at human lower respiratory tract temperatures compared to the ability to grow at human lower respiratory tract temperatures in the absence of the temperature sensitive mutation.
  • a representative example of the human lower respiratory tract temperature is about 37°C, specifically a temperature higher than the upper respiratory tract temperature described below, preferably 36 to 38°C, more preferably 36.5 to 37.5°C or 37°C. ⁇ 38°C.
  • the attenuated strains of the virus of the invention may have the ability to grow at temperatures below the human lower respiratory tract temperature.
  • temperatures below the human lower respiratory tract temperature may include, for example, the human upper respiratory tract temperature (eg, about 32° C. to 35.5° C.).
  • the above temperature-sensitive mutation does not exist on the receptor-binding domain of the spike protein present on the virus surface, which is important when the virus infects cells. Therefore, it is rational that not only the specific SARS-CoV-2 listed in NC_045512 (NCBI) but also other betacoronaviruses can be made temperature sensitive by introducing the above temperature-sensitive mutations. reasonably expected. In other words, even if a mutation that changes the immunogenicity of the virus occurs due to a worldwide epidemic of an infectious disease, by further introducing the above temperature-sensitive mutation into the mutant virus, It is reasonably expected that temperature sensitivity can be imparted by
  • the above mutation (b) may be a substitution with an amino acid residue other than leucine
  • the above mutation (e) may be a substitution with an amino acid residue other than glycine
  • the mutation (f) may be substitution with an amino acid residue other than glycine
  • the mutation (h) above may be substitution with an amino acid residue other than valine.
  • the above mutation (a) may be substitution with an amino acid residue other than valine
  • the above mutation (c) may be substitution with an amino acid residue other than lysine
  • the above (d ) mutation may be a substitution to an amino acid residue other than aspartic acid
  • the mutation in (g) may be a substitution to an amino acid residue other than alanine
  • the mutation in (i) is a substitution other than leucine It may be a substitution to an amino acid residue
  • the above mutation (j) may be a substitution to an amino acid residue other than threonine
  • the above mutation (k) may be a substitution to an amino acid residue other than alanine.
  • the above mutation (l) may be a substitution with an amino acid residue other than leucine
  • the above mutation (m) may be a substitution with an amino acid residue other than serine
  • the above (q) A mutation may be a substitution to an amino acid residue other than valine.
  • the temperature-sensitive mutation is a substitution of phenylalanine for the mutation (b), a substitution for valine for the mutation (e), and a substitution for valine for the mutation (f). It is a substitution to serine and/or the mutation in (h) is a substitution to isoleucine.
  • the other mutation is that the mutation in (a) is a substitution to alanine, the mutation in (c) is a substitution to arginine, and the (d ) is a substitution of asparagine, the mutation of (g) is a substitution of valine, the mutation of (i) is a substitution of tryptophan, and the mutation of (j) is a substitution of lysine wherein the mutation in (k) is a substitution to valine, the mutation in (l) is a substitution to proline, the mutation in (m) is a substitution to phenylalanine, and/or the Mutation of (q) is a substitution to isoleucine.
  • the substitution may be a so-called conservative substitution.
  • Conservative substitution refers to substitution with an amino acid similar in structure and / or properties. For example, as an example of conservative substitution, if the amino acid before substitution is a nonpolar amino acid, it is substituted with another nonpolar amino acid. , if the amino acid before substitution is a non-charged amino acid, it is substituted with another non-charged amino acid, if the amino acid before substitution is an acidic amino acid, it is substituted with another acidic amino acid, and if the amino acid before substitution is a basic amino acid, it is substituted with another non-charged amino acid is substituted with a basic amino acid.
  • non-polar amino acids include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan
  • non-charged amino acids include glycine, serine, threonine, cysteine, Tyrosine, asparagine, and glutamine
  • acidic amino acids include aspartic acid and glutamic acid
  • basic amino acids include lysine, arginine, and histidine.
  • a more preferred example of the virus-attenuated strain of the present invention is a mutant strain of SARS-CoV-2 listed in NC_045512 (NCBI), wherein the mutation (b) (that is, the mutation (b')) is , NSP3, a substitution of phenylalanine for leucine at position 445 of the amino acid sequence shown in SEQ ID NO: 1 (L445F); 2 substitution of glycine with valine at position 248 of the amino acid sequence shown in SEQ ID NO: 2 (G248V), and the mutation of (f) (that is, mutation of (f')) is the amino acid sequence shown in SEQ ID NO: 2 in NSP14 substitution of glycine to serine at position 416 (G416S); and/or the mutation of (h) (that is, mutation of (h')) is the 67th amino acid sequence shown in SEQ ID NO: 3 in NSP16 valine to isoleucine substitution (V67I).
  • NCBI NC_045512
  • the mutation (a) (that is, the mutation (a')) is the amino acid sequence shown in SEQ ID NO: 1 in NSP3 at position 404 A substitution of valine with alanine (V404A);
  • the mutation of (c) (that is, the mutation of (c′)) is a substitution of arginine for lysine at position 1792 of the amino acid sequence shown in SEQ ID NO: 1 in NSP3.
  • the mutation (d) (that is, the mutation (d')) is a substitution of asparagine for aspartic acid at position 1832 in the amino acid sequence shown in SEQ ID NO: 1 in NSP3 (D1832N)
  • the mutation of (g) (that is, the mutation of (g')) is a substitution of alanine to valine at position 504 of the amino acid sequence shown in SEQ ID NO: 2 in NSP14 (A504V);
  • the mutation (that is, (i') mutation is substitution of tryptophan for leucine at position 54 of the amino acid sequence shown in SEQ ID NO: 4 in the spike (L54W);
  • the above (j) mutation (that is, (j' ) mutation) is a substitution of lysine for threonine at position 739 of the amino acid sequence shown in SEQ ID NO: 4 in the spike (T739K); Substitution of alanine to valine (A879V) at position 879 of the amino acid sequence shown in SEQ ID
  • virus-attenuated strains of the invention include the following strains. - As temperature-sensitive mutations, the mutation (e) (preferably the mutation (e') and / or G248V) and the mutation (f) (preferably the mutation (f') and / or G416S), and (h) mutation (preferably (h') mutation and / or V67I); and growth-reducing or other attenuating mutation, the (n) mutation (preferably (n') mutation), the ( A strain having the mutation of o) and the mutation of (r); Mutation, and a strain having the mutation (q) (preferably the mutation (q') and / or V687I)-as a temperature-sensitive mutation, the mutation (e) (preferably the mutation (e') and / or G248V) and the mutation of (f) (preferably the mutation of (f′) and/or G416S); ), a strain having the mutation (O), and the mutation (r); A strain having the mutation (p) and the mutation (q
  • virus-attenuated strains of the invention include the following strains.
  • the mutation (e) preferably the mutation (e') and/or G248V) and the mutation (f) (preferably the mutation (f') and/or G416S); Strains having the (n) mutation (preferably (n') mutation, the (O) mutation, and the (r) mutation as sexual and other attenuating mutations; and further other mutations , the mutation (g) (preferably the mutation (g') and / or A504V), the mutation (p), and the mutation (q) (preferably the mutation (q') and / or V687I) stocks with
  • the attenuated beta coronavirus strain described in "1. Attenuated beta coronavirus strain” has a temperature-sensitive mutation, so it is effective only at temperatures lower than the human lower respiratory tract temperature. Therefore, it cannot grow efficiently at least in the deep part of the body, especially in the lower respiratory tract including the lung, which causes serious damage, and it can be expected that the pathogenicity will be remarkably reduced.
  • the attenuated betacoronavirus strain has growth-reducing and other attenuating mutations that limit its growth regardless of temperature. have By adopting such a complex form, the attenuated betacoronavirus strain will have growth-reducing or other Since deletion mutations, which are attenuating mutations, are resistant to reversion, it is expected that attenuation can be maintained.
  • the attenuated strain of the virus can be used as a live attenuated vaccine by infecting a living body with the attenuated virus itself. Accordingly, the present invention also provides a vaccine comprising the attenuated strain of betacoronavirus as an active ingredient.
  • the details of the active ingredient are as described in "1. Attenuated Betacoronavirus".
  • Attenuated Betacoronavirus Strains certain mutations contribute to conferring attenuation. Accordingly, the present invention also provides a betacoronavirus gene vaccine comprising, as active ingredients, genes encoding nonstructural proteins, accessory proteins, and structural proteins having the predetermined mutations described above. The details of the predetermined mutation contained in the active ingredient are as described in "1. Attenuated betacoronavirus strain”.
  • the vaccine of the present invention not only targets the early Wuhan strain of the SARS-CoV-2 virus, but also the variants detected in the UK in September 2020 and in South Africa in October 2020, and others.
  • the vaccine of the present invention targets betacoronavirus.
  • the vaccine of the present invention may contain adjuvants, buffers, tonicity agents, soothing agents, preservatives, antioxidants, corrigents, Other ingredients such as light absorbing pigments, stabilizers, carbohydrates, casein digests, various vitamins, and the like can be included.
  • Adjuvants include, for example, animal oils (squalene, etc.) or their hardened oils; vegetable oils (palm oil, castor oil, etc.) or their hardened oils; mannitol/oleic anhydride, liquid paraffin, polybutene, caprylic acid, oleic acid, Oily adjuvants containing higher fatty acid esters; PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymer, methacrylic acid copolymer, maleic anhydride copolymer, alkenyl derivative polymer, water water-soluble adjuvants such as oil emulsions, cationic lipids containing quaternary ammonium salts; aluminum salts such as aluminum hydroxide (alum), aluminum phosphate, aluminum sulfate or combinations thereof, precipitating adjuvants such as sodium hydroxide; Microorganism-
  • buffers include buffers such as phosphate, acetate, carbonate, and citrate.
  • tonicity agents include sodium chloride, glycerin, D-mannitol and the like.
  • soothing agents include benzyl alcohol and the like.
  • antiseptics include thimerosal, paraoxybenzoic acid esters, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, antibiotics, synthetic antibacterial agents, and the like.
  • antioxidants include sulfites, ascorbic acid, and the like.
  • Examples of light-absorbing pigments include riboflavin, adenine, and adenosine.
  • stabilizers include chelating agents, reducing agents, and the like.
  • Examples of carbohydrates include sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, and the like.
  • the vaccine of the present invention may contain one or more other vaccines against viruses or bacteria that cause diseases other than betacoronavirus infections, such as COVID-19. That is, the vaccine of the present invention may be prepared as a combination vaccine containing other vaccines.
  • Dosage form The dosage form of the vaccine of the present invention is not particularly limited, and can be appropriately determined based on the administration method, storage conditions, and the like.
  • Specific examples of dosage forms include liquid formulations and solid formulations, and more specifically oral administration formulations such as tablets, capsules, powders, granules, pills, liquid formulations, and syrups; freeze-dried formulations.
  • parenteral administration agents such as dry formulations such as injections, sprays, patches (specifically, intramuscular administration agents, intradermal administration agents, subcutaneous administration agents, nasal administration agents, transdermal administration agents, etc.) is mentioned.
  • the administration method of the vaccine of the present invention is not particularly limited, and may be any of injection administration such as intramuscular, intraperitoneal, intradermal and subcutaneous administration, inhalation administration from nasal and oral cavities, and oral administration. , preferably intramuscular, intradermal and subcutaneous injection administration (intramuscular administration, intradermal administration and subcutaneous administration), inhalation administration through the nasal cavity (nasal administration), and absorption administration through the skin (transdermal administration). , and more preferably nasal administration.
  • the subject to which the vaccine of the present invention is applied is not particularly limited as long as it is a subject that can cause various symptoms due to betacoronavirus infection (preferably a subject that can cause COVID-19 symptoms due to SARS-CoV-2 virus infection).
  • mammals more specifically, humans; pet animals such as dogs and cats; laboratory animals such as rats, mice and hamsters.
  • dose of the vaccine of the present invention is not particularly limited, and can be appropriately determined according to the type of active ingredient, administration method, and administration subject (age, body weight, sex, presence or absence of underlying disease, etc.).
  • the dose per human of the vaccine of the present invention is 1 ⁇ 10 PFU/body or more, preferably 1 ⁇ 10 2 PFU/body or more, more preferably 2 ⁇ 10 2 PFU/body or more, more preferably 1 x10 3 PFU/body or more, more preferably 2 x 10 3 PFU/body or more.
  • the dose of the vaccine of the present invention per human is 6 ⁇ 10 11 PFU/body or less, preferably 1 ⁇ 10 11 PFU/body or less, more preferably 6 ⁇ 10 10 PFU/body or less, and further Preferably, it is 1 ⁇ 10 10 PFU/body or less.
  • the dose of the vaccine of the present invention to humans per dose is 1 ⁇ 10 TCID50/body or more, preferably 1 ⁇ 10 2 TCID50/body or more, more preferably 2 ⁇ 10 2 TCID50/body or more, more preferably 1 ⁇ 10 TCID50/body or more. ⁇ 10 3 TCID50/body or more, more preferably 2 ⁇ 10 3 TCID50/body or more.
  • the dose per human of the vaccine of the present invention is 6 ⁇ 10 11 TCID50/body or less, preferably 1 ⁇ 10 11 TCID50/body or less, more preferably 6 ⁇ 10 10 TCID50/body or less, and further Preferably 1*10 ⁇ 10> TCID50/body or less is also mentioned.
  • the method for producing an attenuated betacoronavirus strain of the present invention is not particularly limited, and can be appropriately determined by those skilled in the art based on the amino acid sequence information described above.
  • CPER using an artificial chromosome such as a bacterial artificial chromosome (BAC) or a yeast artificial chromosome (YAC), or a betacoronavirus genome fragment
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • the genome of the attenuated betacoronavirus strain (parent strain) that does not have any temperature-sensitive mutations, growth-reducing mutations, or other attenuating mutations is cloned.
  • the parent strain used at this time may be a betacoronavirus, specifically, the specific SARS-CoV-2 listed in NC_045512 (NCBI) above, any other SARS-CoV above -2, and viruses other than SARS-CoV-2 within the genus Betacoronavirus.
  • the full-length DNA of the viral genome is cloned into BAC DNA or YAC DNA, etc., and a transcription promoter sequence for eukaryotic cells is inserted upstream of the viral sequence.
  • Promoter sequences include CMV promoter and CAG promoter.
  • a ribozyme sequence as well as a poly A sequence are inserted downstream of the viral sequence. Ribozyme sequences include hepatitis D virus ribozymes and hammerhead ribozymes. Examples of poly A sequences include the poly A of simian 40 virus.
  • the full-length DNA of the viral genome is divided into multiple fragments and cloned.
  • methods for obtaining fragments include artificial synthesis of nucleic acids, PCR using the above-mentioned artificial chromosomes or plasmids cloned with fragments as templates, and the like.
  • double crossover and ⁇ / Homologous recombination methods such as RED recombination, overlap PCR method, CRISPR/Cas9 method, and other known point mutation introduction methods can be used.
  • the host cells are transfected with artificial chromosomes introduced with temperature-sensitive mutations, growth-reducing or other attenuating mutations, and other mutations as necessary to reconstruct the recombinant virus.
  • fragments introduced with temperature-sensitive mutations, growth-reducing or other attenuating mutations, and, if necessary, other mutations are ligated by a reaction using DNA polymerase, and then transferred to host cells.
  • Recombinant viruses are reconstituted by transfection.
  • the transfection method is also not particularly limited, and known methods can be used.
  • the host is not particularly limited, and known cells can be used.
  • the reconstructed recombinant virus is added to the cultured cells, and the recombinant virus is subcultured.
  • the cultured cells used at that time are not particularly limited, but for example, Vero cells, VeroE6 cells, Vero cells supplemented with TMPRESS2 expression, VeroE6 cells supplemented with TMPRESS2 expression, Calu-3 cells, supplemented with ACE2 expression. 293T cells, BHK cells, 104C1 cells, mouse neuroblastoma-derived NA cells, Vero cells and the like.
  • Viruses can be recovered by known methods such as centrifugation and membrane filtration. Furthermore, by adding the recovered virus to the cultured cells, it becomes possible to mass-produce the recombinant virus.
  • Test Example 1 SARS-CoV-2 temperature-sensitive strain A50-18 strain (reference example)]
  • Test Example 1-1 Isolation of SARS-CoV-2 temperature-sensitive strain A50-18 strain Based on the method of Figure 1, SARS-CoV-2 clinical isolate (B-1 strain [comparative example]) (LC603286 , In this example, B-1 strain, wild strain (clinical isolate), European wild strain (B-1 strain), B-1 strain (D614G type: pre-alpha European strain)), 2 A to F50 series and A to F500 series conditioned at 32 ° C. by adding mutagens 5-fluorouracil (hereinafter, 5-FU) and 5-azacytidine (hereinafter, 5-AZA) A virus population was obtained.
  • 5-FU mutagens 5-fluorouracil
  • 5-AZA 5-azacytidine
  • each virus population was passaged multiple times, and among the 406 candidate strains obtained, a virus strain that can grow at 32°C but has significantly reduced growth at 37°C (strain A50-18 ., sometimes referred to as Ts strain below.) was found, isolated, and selected (Fig. 2).
  • Revertive mutation refers to a return to the same phenotype as the original virus before mutation due to further mutation in the mutated virus.
  • reverse mutation refers to loss of temperature-sensitive properties due to further mutation in a temperature-sensitive strain. Further mutation includes reverting the amino acid at the mutation site that was entered when the temperature was sensitized to the amino acid before the mutation.
  • a sample (hereinafter referred to as "revertant strain") in which the proliferation of the strain was recovered was found.
  • revertant strain among the mutations of the temperature-sensitive strain (A50-18 strain) that has acquired temperature sensitivity, some amino acid residues are reverted to the amino acid before mutation (hereinafter simply referred to as "revertant mutation”). It is thought that the temperature sensitivity decreased and the growth at 37°C was restored. A CPE image showing this is shown in FIG. 3B.
  • FIG. 3C shows a CPE image after the obtained revertant strain was cultured at 37° C. for 3 days.
  • G248V mutation in NSP14 is reverted to wild-type G, while G416S and A504V mutations are maintained ⁇ 2> G416S mutation in NSP14 is reverted to wild-type G, while G248V and A504V mutation maintained
  • Test Example 1-3 Proliferative analysis of temperature-sensitive strain A50-18 strain (1-3-1) Analysis at 32 ° C. and 37 ° C.
  • FIG. 6 shows the results of observing body weight fluctuations for three days. After euthanizing the hamsters at 3 dpi, nasal washes were collected with 1 mL of D-PBS. In addition, the hamster lung was removed, the right lung was crushed, suspended in 1 mL of D-MEM, and then centrifuged to recover the supernatant as a lung lysate.
  • FIG. 7 shows the results of evaluation of the amount of virus in these nasal washes and lung lysates by plaque formation assay using Vero cells. Further, the excised left lung was fixed with 10% formalin and photographed, as shown in FIG.
  • FIG. 10 shows the HE-stained image and the immunochemically-stained image.
  • IHC immunohistochemistry
  • each virus population was passaged multiple times, and among the 253 strains obtained, a virus strain that could grow at 32°C but had significantly reduced growth at 37°C (strain H50-11 , L50-33 strain, L50-40 strain) were found, isolated and selected (Fig. 15).
  • FIG. 16B shows a CPE image after the obtained revertant strain was cultured at 38° C. for 3 days.
  • FIG. 16C shows CPE images after culturing each strain at 37° C. for 3 days.
  • the revertants of strain L50-33 and strain L50-40 are designated as strain L50-33 Rev1,2 and strain L50-40 Rev1,2, respectively.
  • FIG. 18 shows a schematic diagram of the deletion of the nucleotide sequence at positions 27549-28251 and the deletion of the amino acid sequence encoded by it.
  • ORF7a is the base sequence of positions 27394-27759
  • ORF7b is the base sequence of positions 27756-27887
  • ORF8 is the base sequence of positions 27894-28259.
  • the base sequence region from positions 27549 to 28251 corresponds to a portion of ORF7a (amino acid sequence from 53rd position to the end; the same applies hereinafter), the entire ORF7b, and most of the amino acid sequence of ORF8. Since the deletion of this region is accompanied by a frameshift, a protein is produced in which the 1-52nd amino acid sequence of ORF7a is fused with the amino acid sequence encoded by the 8 bases at the 3' end of ORF8, the intergenic region, and the base sequence of the nucleocapsid. was thought to have been In addition, ORF7b was deleted in its entirety, and the original sequence of ORF8 was also deleted in its entirety.
  • FIG. 21 shows the lung weight per total body weight of hamsters.
  • the viral load in these nasal lavages and lung lysates was evaluated by plaque formation assay using Vero cells, and the results are shown in FIG.
  • n nasal administration
  • C indicates subcutaneous administration.
  • Nasal administration of B-1 and A50-18 strains induced neutralizing antibodies against the live virus.
  • Subcutaneous administration could hardly induce neutralizing antibodies at the tested doses, but considering the results of intranasal administration, it was thought that subcutaneous administration could also induce neutralizing antibodies at higher doses.
  • Partial blood collection was performed from hamsters 3 weeks after infection, and the obtained serum was used to neutralize the live virus of SARS-CoV-2 Brazilian mutant strain (hCoV-19/Japan/TY7-503/2021 strain). is shown in FIG. i. n is nasal administration; C indicates subcutaneous administration. The same method as in (1-5-2) was used to measure the neutralizing activity. Similar to (7-1), an increase in neutralizing antibody titer was observed in the intranasal administration group even in the low-dose administration group of 1 ⁇ 10 2 TCID50/10 ⁇ L. This suggests the possibility that the temperature-sensitive strain can induce sufficient immunity even with a small amount of intranasal administration. Subcutaneous administration could hardly induce neutralizing antibodies at the tested doses, but considering the results of intranasal administration, it was thought that subcutaneous administration could also induce neutralizing antibodies at higher doses.
  • FIG. 29 shows the results of comparing the neutralizing antibody titers against each strain in the sera of each individual. Although some individuals showed a decrease in neutralizing antibody titers against the Brazilian mutant strain, all individuals possessed neutralizing antibodies.
  • Temperature-sensitive mutations and growth-reducing and other attenuating mutations, as well as other mutations, are checked in Table 5 [that is, L445F of NSP3, G248V and G416S of NSP14, and V67I of NSP16; 8 amino acid deletion of spike furin clearance site (FCS) deletion (specifically spike 679-686 deletion and V687I), functional deletion of ORF8; found in temperature sensitive strains any of the other mutations (NSP3 K1792R and NSP14 A504V)] were used.
  • a strain was constructed that has the above-mentioned temperature-sensitive mutation and growth-reducing and other attenuating mutations in combination with other mutations (Torii et al. cell report 2020).
  • the genome of SARS-CoV-2 strain B-1 was fragmented and cloned into a plasmid.
  • the desired mutation was introduced into the cloned fragment using inverse PCR.
  • a SARS-CoV-2 wild-type genome fragment was obtained by PCR using the plasmid cloned with the wild-type fragment as a template.
  • a SARS-CoV-2 mutant genome fragment was obtained by performing PCR or RT-PCR using the mutated plasmid or the genome of the SARS-CoV-2 mutant strain having the desired mutation as a template. .
  • FIG. 30 shows temperature-sensitive strain mutations and/or growth-reducing or other attenuating mutations introduced into each candidate strain, as well as CPE images. Observation of CPE in VeroE6/TMPRSS II cells revealed that the virus was reconstituted.
  • Test Example 10 Evaluation of temperature sensitivity of candidate strains (Example) (10-1) Temperature sensitivity of candidate strains 1 to 7 In order to evaluate the temperature sensitivity of candidate strains 1 to 7 obtained in Test Example 9 (Example), 2 ⁇ L of the supernatant of the recovery culture of each candidate strain was Vero. Cells were infected and the proliferative properties at 34°C and 37°C were compared. A CPE image after 3 days of culture is shown in FIG. 31A. All strains showed CPE at 34°C, whereas candidate strains 1, 2, 3, 6, 7 did not show CPE at 37°C.
  • Candidate strains 4 and 5 produced slight CPE, but to a lesser extent than 34°C. This confirmed that the vaccine candidate strain exhibited temperature sensitivity.
  • the rTs-all strain was slightly delayed in growth at 32 ° C. compared to the rB-1 strain, so the level of temperature sensitivity was preferable (that is, the growth at 32 ° C. was rB-1 strain). From this result, in order to control the level of temperature sensitivity to a preferred degree, three types of temperature-sensitive mutations (that is, mutation (b), a combination of mutations (e) and (f), and mutation (h)) (mutation or combination of mutations), it was found that it is more preferable to select one or two types than to select three types.
  • Test Example 11 Immunogenicity evaluation of candidate strains 1, 3, 4, 6, and 7 at low titer and low dose (neutralizing antibody titer induction) (Example)
  • 100 TCID50 of each candidate strain was applied to five 5-week-old male hamsters. was administered intranasally at 10 ⁇ L.
  • SARS-CoV-2 temperature-sensitive strain A50-18 was intranasally administered at the same titer and dose.
  • the neutralizing activity against SARS-CoV-2 B-1 strain of the serum obtained by partial blood sampling after 3 weeks was evaluated.
  • Neutralizing activity is determined by mixing serially diluted serum and 100 TCID50 of SARS-CoV-2, reacting for 1 hour, adding to Vero cells, and observing CPE after 4 days of culture to determine the presence or absence of infectious virus. evaluated by the method.
  • the neutralizing antibody titer was defined as the maximum serum dilution rate at which CPE was not observed and virus infectivity could be neutralized. The results are shown in FIG.
  • candidate strains 4 and 6 were constructed by combining temperature-sensitive mutations and other attenuating mutations to ensure safety.
  • candidate strain 7 was constructed by combining temperature-sensitive mutations and growth-reducing or other attenuating mutations, despite the fact that the growth in the body was significantly reduced. It maintained excellent immunogenicity.
  • candidate strain 2 seroconversion of neutralizing antibodies was observed in 4 out of 5 individuals by administration of 1 ⁇ 10 3 TCID50, and seroconversion of neutralizing antibodies was observed in all individuals when 1 ⁇ 10 4 TCID50 was administered. observed.
  • candidate strain 2 seroconversion of neutralizing antibodies was observed in 4 out of 5 individuals by administration of 1 ⁇ 10 3 TCID50, and seroconversion of neutralizing antibodies was observed in all individuals when 1 ⁇ 10 4 TCID50 was administered. observed.
  • Candidate Strain 5 only 1 out of 5 mice was confirmed to have serum neutralizing activity when 1 ⁇ 10 3 TCID50 was administered, but neutralization activity was observed in 3 out of 5 mice when 1 ⁇ 10 4 TCID50 was administered. Induction of antibody was observed.
  • candidate strain 2 proliferated at 32°C to the same extent as the clinical isolate (B-1 strain [Comparative example]), but its growth at 37°C was significantly reduced.
  • candidate strain 2 increased the serum neutralizing antibody titer to 6-12.
  • 4 weeks after the first administration no increase in neutralizing antibody titer was observed when the second administration was performed, suggesting that a single administration induces sufficient humoral immunity. was done.
  • the serum neutralizing antibody thus obtained did not significantly decrease even 4 months after administration.
  • candidate strain 2 induces sufficient humoral immunity in a hamster model with a single administration of 1 ⁇ 10 3 TCID50, and that humoral immunity persists for at least 4 months after administration.
  • antigen-specific production of IFN- ⁇ was induced by stimulating spleen cells of candidate strain 2-infected hamsters with the spike antigen peptide. From these results, it was revealed that administration of candidate strain 2 induces antigen-specific Th1 cells, and that candidate strain 2 also induces cell-mediated immunity.
  • Test Example 16 Infection protection test by administration of candidate strain 2 (Example) In Test Example 13, 1 ⁇ 10 3 , 1 ⁇ 10 4 TCID50 candidate strain 2, or 1 ⁇ 10 3 TCID50 SARS-CoV-2 temperature-sensitive strain A50-18 was intranasally administered to 20 ⁇ L of hamsters for the first time. A challenge test was performed by intranasal administration of the SARS-CoV-2 strain B-1 (3 ⁇ 10 5 TCID50) at a dose of 100 ⁇ L under anesthesia conditions 3 weeks after . The body weight of the hamsters after the challenge test was measured up to 6 days after administration. The results are shown in FIG.
  • Fig. 38 shows the presence or absence of CPE in Vero cells inoculated with nasal washings after each passage;
  • FIG. 40 shows the results of hamster body weight changes during passage.
  • the A50-18 strain is a temperature-sensitive strain, it does not cause CPE when cultured at 37 ° C. or 39 ° C. after infecting Vero cells, but from FIG. CPE was observed to occur when nasal washes were added to Vero cells and cultured at 37°C and 39°C.
  • FIG. 39 as a result of confirming the sequence of the viral RNA contained in the nasal wash after in vivo passage, among the mutations of NSP14 that cause temperature sensitivity, the G416S and G248V mutations are lost. It became clear. Furthermore, from FIG.
  • the attenuated betacoronavirus strain of the present invention is a combination of a predetermined temperature-sensitive mutation (substitution mutation) and a predetermined growth-reducing or other attenuating mutation (deletion mutation). Even when the virus is inoculated to humans as a live vaccine, it can be speculated that the usefulness as a vaccine has remarkably improved in that the possibility of transmission of the virus that has returned to virulence is low.
  • SARS-CoV-2 strain B-1 (1 ⁇ 10 5 TCID50) was intranasally administered at a dose of 100 ⁇ L under anesthesia.
  • the virus fluid reached the upper respiratory tract of the hamster, and by nasal administration of 100 ⁇ L, the virus fluid reached the lower respiratory tract of the hamster.
  • the head and lungs were fixed with formalin after euthanasia, and tissue toxicity was evaluated by HE staining, and Rabbit anti-spike RBD antibody (Sinobiological (40592-T62)) was used. Viral antigens were detected by IHC staining.
  • Table 6 shows the scores for lesions in the nasal cavities and lungs of each individual and the detection of viral antigens by IHC.
  • Level 1 indicates the tip of the nasal cavity
  • Level 2 indicates the middle part of the nasal cavity
  • Level 3 indicates the deep part of the nasal cavity.
  • Representative examples of each site of each virus-infected hamster are shown in FIG. 41 for Level 1, FIG. 42 for Level 2, FIG. 43 for Level 3, and FIG. 44 for lungs.
  • candidate strain 2 (Example) and temperature-sensitive strain A50-18 strain (Reference example) cause viral proliferation and accompanying tissue damage at the tip of the nasal cavity close to the outside temperature, while the nasal cavity close to body temperature Virus multiplication and accompanying tissue damage are suppressed in deep areas and lungs, candidate strain 2 (Example) has a higher inhibitory effect on viral multiplication and accompanying tissue damage, and olfactory impairment is observed. It was strongly suggested that the risk of occurrence was low.
  • a challenge test was conducted by intranasally administering TY41-702 strain (3 ⁇ 10 5 PFU) at a dose of 100 ⁇ L under anesthesia to candidate strain 2-administered hamsters and naive hamsters. carried out. Body weight was measured over time and infectious virus was quantified in lung and nasal washes after euthanasia of the hamsters at 4 days after the challenge test was performed.
  • FIG. 46 shows the weight fluctuation at that time.
  • Test Example 20 Evaluation of immunogenicity by candidate strain 2 (Evaluation of neutralizing activity by heterozygous strain) It was evaluated whether immunity induced with candidate strain 2 was effective against SARS-CoV-2 delta and gamma variants.
  • the neutralizing activity of the candidate strain 2 immune serum obtained in Test Example 13 against gamma and delta variants was measured. 100 TCID50 of BK325 strain (delta variant) or TY7-501 strain (gamma variant) was mixed with serially diluted inactivated serum and allowed to react at 37° C. for 1 hour. The culture medium after the reaction was inoculated on Vero cells, and after culturing at 37° C., CPE was observed to evaluate the virus neutralizing activity.
  • the lowest dilution rate that does not cause CPE was defined as the neutralizing antibody titer.
  • the results are shown in FIG. 47 together with the evaluation of neutralizing activity against the wild strain in Test Example 13. In FIG. 47, Low indicates the result of nasal administration of 20 ⁇ L of the candidate strain of 1 ⁇ 10 3 TCID50, and High indicates the result of nasal administration of 20 ⁇ L of the candidate strain of 1 ⁇ 10 4 TCID50.
  • Fig. 47 shows that the immunity induced by candidate strain 2 exhibited neutralizing activity against gamma and delta variants and was effective. These results suggested that the immunity induced by administration of candidate strain 2 was also effective against heterozygous strain variants.

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