CN116583604A - Beta coronavirus temperature sensitive strain and vaccine - Google Patents

Abstract

The present invention provides an effective strain as an active ingredient of a vaccine against beta coronavirus. The SARS-CoV-2 virus comprises a nonstructural protein with the following responsible mutations: a mutation of the amino acid residue corresponding to L at position 445 of SEQ ID NO. 1 in NSP 3; mutations in NSP14 correspond to the G at position 248 and G at position 416 of SEQ ID NO. 2, and/or mutations in NSP16 correspond to the V at position 67 of SEQ ID NO. 3.

Description

Beta coronavirus temperature sensitive strain and vaccine

Technical Field

The present invention relates to a temperature-sensitive strain of beta coronavirus and a vaccine using the same.

Background

Currently, vaccine development against SARS-CoV-2 is rapidly advancing worldwide. The vaccine currently approved in 10 months in 2020 is only satellite-V (spotnik V) approved by russia (non-patent document 1).

Prior art literature

Non-patent literature

Non-patent document 1: THE LANCET, VOLUME 396, ISSUE 10255, P887-897, SEPTELBE 26, 2020

Disclosure of Invention

Technical problem to be solved by the invention

However, satellite-V is still the stage of clinical trials, and sound is increasing in terms of safety, effectiveness. Furthermore, even if vaccination is assumed to be initiated, there is still a risk of small effects or serious side effects being found, and it is unclear whether it is a decisive means for inhibiting the prevalence of infections. Thus, further options are desired for vaccines against SARS-CoV-2 virus.

Accordingly, it is an object of the present invention to provide at least an effective strain as an effective component of a vaccine against SARS-CoV-2 virus. In addition, since there are viruses that may exist in addition to SARS-CoV-2 virus among the beta coronaviruses such as SARS-CoV-2 virus, the present invention aims to provide an effective strain as an effective component of a vaccine against general beta coronaviruses.

Means for solving the technical problems

As a result of intensive studies, the present inventors have found that a predetermined mutation strain of SARS-CoV-2 virus causes a decrease in the proliferation of human body temperature (so-called lower respiratory tract temperature), and further, a back mutation test was conducted on the predetermined mutation strain, whereby it was found that a predetermined responsible mutation (Japanese patent publication No. ) causes a decrease in the proliferation of human body temperature (so-called lower respiratory tract temperature) in general beta-coronavirus. The present invention was completed based on this finding and repeated further studies.

In the present invention, "temperature sensitivity" is a property having a low temperature (i.e., the upper airway temperature of a human) specific proliferation ability, and is a property exerted by obtaining a property that proliferation ability at a high temperature (i.e., the lower airway temperature of a human) is limited. In the present specification, the term "cryo-tamed" is used in the sense of obtaining a property of having a low-temperature (i.e., the upper airway temperature of a human) specific proliferation ability, and the fact that this low-temperature specific proliferation ability is exerted by obtaining a property that the proliferation ability is limited at a high temperature (i.e., the lower airway temperature of a human) is used in the sense of "temperature sensitivity". In addition, a strain that is "temperature-sensitive" is referred to as a "temperature-sensitive strain". Therefore, in the present specification, "low temperature domesticated strain" and "temperature sensitive strain" have the same meaning. In the present invention, the "responsible mutation" means a mutation which causes a mutant phenotype (a phenotype showing a biological trait is changed by mutation) or has a causal relationship with the mutant phenotype, and the "responsible mutation for temperature sensitivity" means a mutation which causes a temperature sensitivity or has a causal relationship with a temperature sensitivity. The present invention provides the following aspects.

Item 1. A β coronavirus temperature sensitive strain comprising a non-structural protein having the following mutations of (b), (e) and (f) in combination, and/or (h) as responsible mutations for the temperature sensitive properties:

(b) Mutation of the amino acid residue corresponding to leucine at position 445 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3,

(e) Mutation of an amino acid residue corresponding to glycine 248 of 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 of 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 of the amino acid sequence shown in SEQ ID NO. 3 in NSP 16.

Specific examples of the invention according to item 1 above include the following inventions.

A β coronavirus temperature-sensitive strain comprising a nonstructural protein consisting of at least any one of the polypeptides of (I), (II) and (III) below:

(I) At least one polypeptide of the following (I-1) to (I-3):

(I-1) a polypeptide (NSP 3) comprising an amino acid sequence having a mutation (b') of leucine at position 445 in the amino acid sequence shown in SEQ ID NO. 1 as a responsible mutation,

(I-2) a polypeptide (NSP 14) comprising an amino acid sequence having, as responsible mutations, a mutation (e ') of glycine 248 and a mutation (f') of glycine 416 in the amino acid sequence represented by SEQ ID NO. 2,

(I-3) a polypeptide (NSP 16) comprising an amino acid sequence having a mutation (h') of valine at position 67 as a responsible mutation in the amino acid sequence represented by SEQ ID NO. 3;

(II) in the amino acid sequence of the polypeptide of (I), 1 or more amino acid residues other than the amino acid residues involved in the responsible mutation are substituted, added, inserted or deleted to form a polypeptide of the beta coronavirus which has obtained temperature sensitive properties;

(III) the polypeptide of (I) above, wherein the amino acid sequence identity of the amino acid sequence other than the amino acid residue involved in the responsible mutation is 50% or more, to form a polypeptide of beta-coronavirus which is temperature sensitive.

The virus temperature sensitive strain according to item 1, wherein the beta coronavirus is SARS-CoV-2 virus.

The virus temperature-sensitive strain according to item 1 or 2, wherein the proliferation potency of the human lower respiratory tract temperature is reduced as compared with the proliferation potency of a β coronavirus comprising a non-structural protein having no responsible mutation as described above.

The virus temperature sensitive strain according to item 3, wherein the human lower respiratory tract temperature is 36 to 38 ℃.

The virus temperature-sensitive strain according to any one of items 1 to 4, wherein the mutation of (b) is a substitution of phenylalanine, the mutation of (e) is a substitution of valine, the mutation of (f) is a substitution of serine, and the mutation of (h) is a substitution of isoleucine.

The virus temperature-sensitive strain according to any one of items 1 to 5, which comprises: the NSP3 having the mutation of the above (b) in the amino acid sequence shown in the above sequence No. 1,

the NSP14 having the mutation of the (e) and the mutation of the (f) in the amino acid sequence shown in the sequence No. 2, and/or

NSP16 having the mutation of (h) in the amino acid sequence shown in the sequence No. 3.

The virus temperature sensitive strain according to any one of items 1 to 6, which has the mutation of (e) and the mutation of (f).

The virus temperature sensitive strain according to any one of items 1 to 6, which has the mutation of (h) above.

The virus temperature sensitivity according to any one of items 1 to 6, which has the mutation of (b).

An attenuated live vaccine comprising the virus temperature sensitive strain of any one of claims 1-9.

Item 11. The live attenuated vaccine of item 10, which is administered nasally.

Item 12. The live attenuated vaccine of item 10, which is administered intramuscularly, subcutaneously or intradermally.

A beta coronavirus gene vaccine comprising a gene encoding a non-structural protein having the following mutations of (b), (e) and (f) in combination, and/or (h) as responsible mutations for temperature sensitive performance:

(b) Mutation of the amino acid residue corresponding to leucine at position 445 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3,

(e) Mutation of an amino acid residue corresponding to glycine 248 of 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 of 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 of the amino acid sequence shown in SEQ ID NO. 3 in NSP 16.

Item 14. The genetic vaccine of item 13, which is administered nasally, intramuscularly, subcutaneously or intradermally.

Effects of the invention

According to the present invention, an effective strain can be provided as an effective ingredient of a vaccine against a beta coronavirus.

Drawings

FIG. 1 shows a method for temperature sensitization (low temperature acclimation) of SARS-CoV-2.

FIG. 2 shows the results of confirmation of the temperature sensitivity (low temperature domestication) of SARS-CoV-2 (CPE image).

FIG. 3A shows the results of mutation analysis of each strain.

FIG. 3B shows the CPE image produced by a strain with the potential for back mutation of the temperature sensitive strain (low temperature acclimatized strain) (A50-18).

FIG. 3C shows the CPE image produced by a strain with the potential for back mutation of the temperature sensitive strain (low temperature acclimatized strain) (A50-18).

FIG. 3D shows the results of confirming the temperature sensitivity of recombinant viruses into which mutations have been introduced in temperature sensitive strains (low temperature domesticated strains) (A50-18).

FIG. 3E shows the results of confirming the temperature sensitivity of the recombinant virus into which the mutation was introduced in the temperature sensitive strain (low temperature domesticated strain) (A50-18).

FIG. 4A shows the results of proliferation assay of temperature sensitive strain (low temperature acclimatized strain) (A50-18).

FIG. 4B shows the results of proliferation assay of temperature sensitive strain (low temperature acclimatized strain) (A50-18).

FIG. 5 shows the body weight change of hamsters infected with SARS-CoV-2.

FIG. 6 shows weight fluctuation of hamsters infected with SARS-CoV-2.

FIG. 7 shows the viral load of the intrapulmonary or nasal wash.

FIG. 8 shows lung images of SARS-CoV-2 infected hamsters.

FIG. 9 shows the results of lung histology analysis of SARS-CoV-2 infected hamsters.

FIG. 10 shows lung histological analysis (HE staining and IHC staining) of SARS-CoV-2 infected hamsters.

FIG. 11 shows weight fluctuation of SARS-CoV-2 reinfected hamsters.

FIG. 12 shows weight change of hamsters after SARS-CoV-2 infection.

FIG. 13 shows recovery of neutralizing antibody titers in hamster serum following SARS-CoV-2 infection.

FIG. 14 shows a method for temperature sensitivity (low temperature acclimation) of SARS-CoV-2 (G.about.L50 system).

FIG. 15 shows the results of confirming the temperature sensitivity (low temperature domestication) of SARS-CoV-2 (CPE image).

FIG. 16A shows the results of mutation analysis of additional isolates (H50-11, L50-33, L50-40).

FIG. 16B shows an image of CPE produced by a strain that is temperature sensitive (low temperature acclimatized strain) (H50-11) and has the potential to revert to mutation.

FIG. 16C shows CPE images produced by strains potentially back mutated of temperature sensitive strains (low temperature acclimatized strains) (L50-33, L50-40).

FIG. 17 shows deletion of nucleotide sequences found in association with temperature-sensitive strains (low-temperature-domesticated strains) (H50-11, L50-33, L50-40).

FIG. 18 is a schematic diagram showing deletion of the nucleotide sequence shown in FIG. 17 and deletion of the encoded amino acid sequence.

FIG. 19 shows the results of proliferation assay of temperature-sensitive strains (low temperature domesticated strains) (H50-11, L50-33, L50-40).

FIG. 20 shows weight fluctuation of hamsters infected with SARS-CoV-2.

FIG. 21 shows lung weights of SARS-CoV-2 infected hamsters.

FIG. 22 shows the viral load of the intrapulmonary or nasal wash.

FIG. 23 shows weight fluctuation of SARS-CoV-2 reinfected hamsters.

FIG. 24 shows neutralizing antibody titers of hamster serum after SARS-CoV-2 infection.

FIG. 25 shows the evaluation of neutralization activity of a temperature-sensitive strain (low-temperature-domesticated strain) against SARS-CoV-2 mutant strain.

FIG. 26 shows a comparison of immunity induction ability based on the administration route of temperature sensitive strain (cryogenically domesticated strain).

FIG. 27 shows a comparison of immunity induction ability based on the amount of the temperature sensitive strain (low temperature domesticated strain) administered.

FIG. 28 shows the evaluation of neutralization activity of a temperature-sensitive strain (low-temperature-domesticated strain) against SARS-CoV-2 mutant strain.

FIG. 29 shows the evaluation of neutralization activity of a temperature-sensitive strain (low-temperature-domesticated strain) against SARS-CoV-2 mutant strain.

Detailed Description

1. Beta coronavirus temperature sensitive strain (Low temperature domesticated strain)

The strain (low temperature domesticated strain) of the present invention is characterized in that it is a temperature-sensitive strain of a beta coronavirus comprising a nonstructural protein having a predetermined mutation as a responsible mutation for temperature-sensitive performance.

Coronaviruses are morphologically spherical with a diameter of about 100 to 200nm, and have protrusions on the surface. Virologically, coronaviruses are classified as a subfamily of the order of the mantle viridae, the subfamily of the coronaviruses. The envelope of the lipid bilayer membrane includes a genome of a single-stranded RNA wound around a positive strand of a nucleocapsid protein (also referred to as a nucleocapsid), and spike protein (hereinafter also referred to as "spike") and envelope protein (hereinafter also referred to as "envelope") and membrane protein are disposed on the surface of the envelope. The size of the viral genome is the longest of about 30kb in RNA viruses.

Coronaviruses are classified into groups of α, β, γ, δ according to genetic characteristics. As coronaviruses infecting humans, 4 kinds of human coronaviruses 229E, OC, NL63, HKU-1, which are causative viruses of cold, and Severe Acute Respiratory Syndrome (SARS) coronavirus occurring in 2002 and Middle East Respiratory Syndrome (MERS) coronavirus occurring in 2012, which cause severe pneumonia, are known. Human coronaviruses 229E and NL63 are classified in the alpha genus coronavirus, and human coronaviruses OC43, HKU-1, SARS coronavirus and MERS coronavirus are classified in the beta genus coronavirus.

SARS-CoV-2 was repeatedly mutated from the initial NC045512 strain, and mutants such as strains detected in the United kingdom, strains detected in south Africa, and strains detected in India were found. The possibility of a mutant strain which has not yet been detected and a mutant strain newly occurring in the future can also be considered. In the present invention, the virus contained in the genus beta coronavirus is not limited to the strain of SARS-CoV-2, but includes all other beta coronaviruses (other SARS-CoV-2 mutant strains and other beta coronaviruses other than SARS-CoV-2 newly detected in the future).

The specific mutation of the beta coronavirus temperature sensitive strain (low temperature strain) of the present invention will be described based on the following table 1. The mutation (b), the combination of the mutation (e) and the mutation (f), and/or the mutation (h) shown as "responsible mutation" in table 1 are responsible mutations of temperature sensitivity (low temperature domestication ability) that the β coronavirus temperature sensitive strain (low temperature domestication strain) of the present invention must contain. The mutations (a), (c), (d), (g), (i) and (m) shown as "other mutations" in table 1 are mutations that may be optionally contained in the β -coronavirus temperature-sensitive strain (low-temperature-treated strain) of the present invention, and the β -coronavirus temperature-sensitive strain (low-temperature-treated strain) of the present invention may or may not contain at least any one of the other mutations.

TABLE 1

That is, the mutations responsible for the temperature-sensitive performance of the β -coronavirus temperature-sensitive strain (low-temperature-domesticated strain) of the present invention are the mutations of (b), (e) and (f) in combination, and/or (h) below.

(b) Mutation of the amino acid residue corresponding to leucine at position 445 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3,

(e) Mutation of an amino acid residue corresponding to glycine 248 of 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 of 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 of the amino acid sequence shown in SEQ ID NO. 3 in NSP 16.

The beta-coronavirus temperature-sensitive strain (low-temperature domesticated strain) of the present invention may contain, in addition to the above-described responsible mutation, at least any one of the following mutations (a), (c), (d), (g) and (i) to (m) as another mutation.

(a) A mutation of the amino acid residue corresponding to valine at position 404 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3,

(c) Mutation of the amino acid residue corresponding to lysine 1792 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3,

(d) Mutation of an amino acid residue of aspartic acid at 1832 nd position of the amino acid sequence shown in SEQ ID NO. 1 in NSP3,

(g) Mutation of an amino acid residue corresponding to alanine at position 504 of the amino acid sequence shown in SEQ ID NO. 2 in NSP14,

(i) Mutation of the amino acid residue in the spike corresponding to leucine at position 54 of the amino acid sequence shown in SEQ ID NO. 4,

(j) Mutation of the amino acid residue corresponding to threonine at position 739 of the amino acid sequence shown in SEQ ID NO. 4 in the spike,

(k) Mutation of the amino acid residue in the spike corresponding to alanine at position 879 of the amino acid sequence shown in SEQ ID NO. 4,

(l) Mutation of amino acid residue corresponding to leucine at position 28 of amino acid sequence shown in SEQ ID No. 5 in envelope, and

(m) mutation of an amino acid residue in the nucleocapsid corresponding to serine at position 2 of the amino acid sequence shown in SEQ ID NO. 6.

The amino acid sequence of NSP3 in SARS-CoV-2 having sequence number 1 of NC_045512 (NCBI); the amino acid sequence of NSP14 in SARS-CoV-2 having sequence number 2 of NC_045512 (NCBI); SEQ ID NO. 3 is the amino acid sequence of NSP16 in SARS-CoV-2 of NC_045512 (NCBI).

In addition, the amino acid sequence of spike in SARS-CoV-2 having sequence number 4 of NC_045512 (NCBI); the amino acid sequence of the envelope in SARS-CoV-2, SEQ ID NO. 5 being NC_045512 (NCBI); SEQ ID NO. 6 is the amino acid sequence of the nucleocapsid in SARS-CoV-2 of NC_045512 (NCBI).

"equivalent" means that when the strain of the present invention is a mutant strain of SARS-CoV-2 having NC-045512 (NCBI) as the strain of the present invention, a mutation is present at the above-mentioned predetermined position in the amino acid sequence of SEQ ID NO. 1-3 or SEQ ID NO. 1-6, and when the strain of the present invention is another strain of the present invention other than the above-mentioned mutant strain, a mutation is present at the above-mentioned predetermined position in the amino acid sequence of the polypeptide of the other strain of the present invention corresponding to SEQ ID NO. 1-3 or SEQ ID NO. 1-6. The corresponding position can be determined by aligning the amino acid sequences of the proteins of SEQ ID Nos. 1 to 3 or 1 to 6 of SARS-CoV-2 of NC_045512 (NCBI) and the proteins of other beta-coronavirus mutants corresponding to the proteins of SEQ ID Nos. 1 to 3 or 1 to 6.

The virus temperature-sensitive strain (low temperature-domesticated strain) of the present invention is not limited to the mutant strain of a specific SARS-CoV-2 that is recorded in NC_045512 (NCBI) and may include other beta-coronavirus mutants (i.e., any other mutant strain of SARS-CoV-2 and other than SARS-CoV-2 that is contained in the genus beta-coronavirus) because it is only required that the amino acid residue corresponding to the above-mentioned predetermined position in the amino acid sequence of SEQ ID Nos. 1 to 3 or 1 to 6 is mutated. The mutant strain of a specific SARS-CoV-2 incorporated in NC_045512 (NCBI) is a mutant strain in which at least one of the amino acid residues at the predetermined positions in the amino acid sequence represented by SEQ ID Nos. 1 to 3 or 1 to 6 in the specific SARS-CoV-2 has been mutated, and the other mutant strain of a beta-coronavirus is a mutant strain in which at least one of the amino acid residues at the predetermined positions in the amino acid sequence represented by SEQ ID Nos. 1 to 3 or 1 to 6 in the other SARS-CoV-2 has been mutated, or a mutant strain in which at least one of the amino acid residues at the predetermined positions in the amino acid sequence represented by SEQ ID Nos. 1 to 3 or 1 to 6 in the virus other than the SARS-CoV-2 in the beta-coronavirus has been mutated.

The amino acid sequences corresponding to SEQ ID Nos. 1 to 3 or 1 to 6 in other mutant betacoronavirus strains are allowed to differ from the amino acid sequences shown in SEQ ID Nos. 1 to 3 or 1 to 6, respectively, as long as they do not significantly affect the properties of the polypeptide. No significant effect on the properties of the polypeptide means that the state of the function as a non-structural protein, respectively, is maintained. Specifically, in the case of a site other than the amino acid corresponding to the responsible mutation in the above-mentioned sequence numbers 1 to 3 or in the case of further having another mutation, the site other than the amino acid residue corresponding to the responsible mutation and the other mutation in the above-mentioned sequence numbers 1 to 6 (the site other than the amino acid corresponding to these mutations is also referred to as "any different site" hereinafter), the difference from the sequence numbers 1 to 3 or 1 to 6 is allowed. The permissible differences may be 1 kind of differences (e.g., substitutions) selected from the group consisting of substitutions, additions, insertions, and deletions, or may include 2 or more kinds of differences (e.g., substitutions and insertions). The sequence identity calculated by comparing only any different part of the amino acid sequence corresponding to SEQ ID NO. 1 to 3 or 1 to 6 with the amino acid sequence shown in SEQ ID NO. 1 to 3 or 1 to 6 in any other SARS-CoV-2 may be 50% or more. In any other SARS-CoV-2, the 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, still more preferably 99% or more, particularly preferably 99.3% or more, 99.5% or more, 99.7% or more, 99.9% or more. In any of the remaining beta coronaviruses, 60% or more is preferable as the sequence identity. Here, "sequence identity" means based on BLASTAKAGE [ sgi32 bit edition, version 2.0.12; available from National Center for Biotechnology Information (NCBI, national center for Biotechnology information), b 2seq program (Tatiana A. Tatsusova, thomas L. Madden, FEMS Microbiol. Lett., vol.174, p247250, 1999). The parameters were set to Gap insertion Cost value: 11. gap extension Cost value: 1.

That is, the β -coronavirus temperature-sensitive strain (cryo-tamed strain) of the present invention is more specifically shown below:

a strain of beta coronavirus temperature sensitive (cryo-tamed) comprising a non-structural protein consisting of at least any one of the following polypeptides (I), (II) and (III):

(I) At least one of the following polypeptides (I-1) to (I-3):

(I-1) a polypeptide (NSP 3) comprising an amino acid sequence having a mutation (b') of leucine at position 445 in the amino acid sequence shown in SEQ ID NO. 1 as a responsible mutation,

(I-2) a polypeptide (NSP 14) comprising an amino acid sequence having, as responsible mutations, a mutation (e ') of glycine 248 and a mutation (f') of glycine 416 in the amino acid sequence represented by SEQ ID NO. 2,

(I-3) a polypeptide (NSP 16) comprising an amino acid sequence having a mutation (h') of valine at position 67 as a responsible mutation in the amino acid sequence represented by SEQ ID NO. 3;

(II) in the amino acid sequence of the polypeptide of (I), 1 or more amino acid residues other than the amino acid residues involved in the responsible mutation are substituted, added, inserted or deleted, thereby constituting a polypeptide of a beta coronavirus which has been obtained with the ability to be temperature sensitive (low temperature domestication);

(III) the polypeptide of (I) above, wherein the amino acid sequence identity of the amino acid sequence other than the amino acid residue involved in the responsible mutation is 50% or more, to constitute a polypeptide of a beta coronavirus which has acquired a temperature sensitivity (low temperature domestication) ability.

In the case where the above-mentioned more specific strain sensitive to temperature of beta coronavirus (low temperature-domesticated strain) contains other mutations in addition to the responsible mutation, the polypeptides (non-structural proteins) of the above-mentioned (I-1) and (I-2) may be the polypeptides of the following (I-1 a) and (I-2 a) having other mutations in addition to the responsible mutation, respectively, and the polypeptide of the above-mentioned (I) may further contain the polypeptides (structural proteins) of the following (I-4 a) to (I-6 a) having other mutations, as shown below.

A strain of beta coronavirus temperature sensitive (cryo-tamed) comprising a structural protein, or a structural protein and a non-structural protein, the structural protein, non-structural protein being comprised of at least any one of the polypeptides of (I), (II) and (III) below:

(I) At least one of the following (I-1 a), (I-2 a) and (I-3), or at least one of the following (I-4 a) to (I-6 a) may be added in addition to (or in addition to) the polypeptide:

(I-1 a) a polypeptide (NSP 3) comprising an amino acid sequence having at least one of a mutation (b ') of leucine 445 as a responsible mutation and a mutation (a') of valine 404, a mutation (c ') of lysine 1792 and a mutation (d') of aspartic acid 1832 as other mutations in the amino acid sequence shown in SEQ ID NO. 1,

(I-2 a) a polypeptide (NSP 14) comprising the amino acid sequence having, as the responsible mutation, a mutation (e ') of glycine 248 and a mutation (f ') of glycine 416 and a mutation (g ') of alanine 504 as another mutation in the amino acid sequence shown in SEQ ID NO. 2,

(I-3) a polypeptide (NSP 16) comprising an amino acid sequence having a mutation (h') of valine at position 67 as a responsible mutation in the amino acid sequence represented by SEQ ID NO. 3,

(I-4 a) a polypeptide (spike) comprising an amino acid sequence having at least one of a mutation (I ') of leucine at position 54, a mutation (j ') of threonine at position 739 and a mutation (k ') of alanine at position 879, which are other mutations, in the amino acid sequence represented by SEQ ID NO. 4,

(I-5 a) a polypeptide (envelope) comprising an amino acid sequence having a mutation (l') of leucine 28 as another mutation in the amino acid sequence shown in SEQ ID NO. 5,

(I-6 a) a polypeptide (nucleocapsid) composed of an amino acid sequence having a mutation (m') of serine at position 2 as another mutation in the amino acid sequence shown in SEQ ID NO. 6;

(II) in the amino acid sequence of the polypeptide of (I), 1 or more amino acid residues other than the amino acid residues involved in the responsible mutation and other mutations are replaced, added, inserted or deleted, to form a polypeptide of a beta coronavirus which has acquired a temperature sensitivity (low temperature domestication) ability;

(III) the polypeptide of (I) above, wherein the amino acid sequence identity of the amino acid sequence other than the amino acid residue involved in the responsible mutation is 50% or more, to constitute a polypeptide of a beta coronavirus which has acquired a temperature sensitivity (low temperature domestication) ability.

The mutations (a ') to (m') mentioned above refer to the case where the mutations (a) to (m) are specifically present in the amino acid sequences of SEQ ID Nos. 1 to 6, respectively. That is, the polypeptide of (I) above is a polypeptide comprising the amino acid sequences of SEQ ID Nos. 1 to 6 of SARS-CoV-2 of NC_045512 (NCBI) and a responsible mutation or other mutations. The polypeptides (II) and (III) are polypeptides in which a responsible mutation or other mutations are introduced into a polypeptide comprising an amino acid sequence corresponding to the amino acid sequences of SEQ ID Nos. 1 to 6 of other betacoronaviruses. Preferred ranges of sequence identity for the polypeptides of (II) and (III) above have been described above.

By having the above-described responsible mutation, the betacoronavirus can acquire temperature sensitivity (low temperature domestication) characteristics. The virus temperature-sensitive strain (low-temperature domesticated strain) of the present invention has at least the ability to proliferate at the temperature of the human lower respiratory tract Lower than the proliferation potency at a temperature lower than the temperature of the lower respiratory tract of the person, preferably does not have the proliferation potency at the temperature of the lower respiratory tract of the person. In the present invention, the temperature sensitivity (low temperature acclimation) ability is confirmed by the following means: after infection of Vero cells with a virus temperature-sensitive strain at the lower respiratory tract temperature of human MOI=0.01, the virus titer (TCID 50/mL) in the culture supernatant after 1 day of culture at the lower respiratory tract temperature of human is reduced by, for example, 10 compared with the virus titer in the culture supernatant after 1 day of culture at the upper respiratory tract temperature of human after infection of Vero cells with a virus temperature-sensitive strain at the upper respiratory tract temperature of human with MOI=0.01 ² Above, preferably reduced by 10 ³ The above.

Typically, the virus temperature-sensitive strain (low temperature domesticated strain) of the present invention has a lower proliferation potency at human lower respiratory tract temperature than that of a human without the above-mentioned mutation responsible for the disease. This can be confirmed by: the virus titer (TCID 50/mL) in the culture supernatant after 1 day of culture at human lower respiratory temperature after infection of Vero cells with a virus temperature-sensitive strain at MOI=0.01 at human lower respiratory temperature is reduced by, for example, 10 compared with the virus titer in the culture supernatant after 1 day of culture at human lower respiratory temperature after infection of Vero cells with a strain not having the above-mentioned responsible mutation at MOI=0.01 at human lower respiratory temperature ² Above, preferably reduced by 10 ³ The above.

Typical examples of the lower airway temperature of a person include about 37 ℃, specifically, a temperature higher than the upper airway temperature described later, preferably 36 to 38 ℃, more preferably 36.5 to 37.5 ℃ or 37 to 38 ℃. The virus temperature-sensitive strain (low-temperature domesticated strain) of the present invention may have a capability of proliferating at a temperature lower than the temperature of the lower respiratory tract of a human. For example, the temperature lower than the lower respiratory tract temperature of the human may include the upper respiratory tract temperature of the human (specifically, about 32 to 35.5 ℃), for example.

The responsible mutations described above are not present in the receptor binding domains of spike proteins present on the surface of the virus, which are important when the virus infects cells. Therefore, it is reasonable to expect that temperature sensitivity can be achieved by introducing the above-described responsible mutation not only in the specific SARS-CoV-2 recorded in NC_045512 (NCBI) but also in other beta coronaviruses. That is, even when a mutation such as a change in the immunogenicity of a virus occurs due to the prevalence of worldwide infectious diseases, it is reasonable to expect that the mutant virus can be given temperature sensitivity by introducing the above-described responsible mutation into the mutant virus.

In addition, regarding the responsible mutation, the mutation of (b) may be replaced with an amino acid residue other than leucine, the mutation of (e) may be replaced with an amino acid residue other than glycine, the mutation of (f) may be replaced with an amino acid residue other than glycine, and the mutation of (h) may be replaced with an amino acid residue other than valine. In the case of other mutations, the mutation of (a) may be replaced with an amino acid residue other than valine, the mutation of (c) may be replaced with an amino acid residue other than lysine, the mutation of (d) may be replaced with an amino acid residue other than aspartic acid, the mutation of (g) may be replaced with an amino acid residue other than alanine, the mutation of (i) may be replaced with an amino acid residue other than leucine, the mutation of (j) may be replaced with an amino acid residue other than threonine, the mutation of (k) may be replaced with an amino acid residue other than alanine, the mutation of (l) may be replaced with an amino acid residue other than leucine, and the mutation of (m) may be replaced with an amino acid residue other than serine.

In a preferred example of the virus temperature-sensitive strain (low temperature-domesticated strain) of the present invention, the mutation of (b) is substituted with phenylalanine, the mutation of (e) is substituted with valine and the mutation of (f) is substituted with serine, and/or the mutation of (h) is substituted with isoleucine. In the case where this preferred embodiment has another mutation, the mutation of (a) is replaced with alanine, the mutation of (c) is replaced with arginine, the mutation of (d) is replaced with asparagine, the mutation of (g) is replaced with valine, the mutation of (i) is replaced with tryptophan, the mutation of (j) is replaced with lysine, the mutation of (k) is replaced with valine, the mutation of (l) is replaced with proline, and/or the mutation of (m) is replaced with phenylalanine.

In another example of the virus temperature-sensitive strain (low-temperature domesticated strain) of the present invention, the substitution may be a so-called conservative substitution. Amino acid substitutions may be made with similar structure and/or properties, for example, as examples of conservative substitutions: if the amino acid before substitution is a nonpolar amino acid, the amino acid before substitution is replaced with another nonpolar amino acid, if the amino acid before substitution is a uncharged amino acid, the amino acid before substitution is replaced with another uncharged amino acid, if the amino acid before substitution is an acidic amino acid, the amino acid before substitution is replaced with another acidic amino acid, and if the amino acid before substitution is a basic amino acid, the amino acid before substitution is replaced with another basic amino acid. In general, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan, while "uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, while "acidic amino acids" include aspartic acid and glutamic acid, and "basic amino acids" include lysine, arginine, and histidine.

More preferable examples of the virus temperature-sensitive strain (low-temperature domesticated strain) of the present invention include: for the mutant strain of SARS-CoV-2 recorded in NC_045512 (NCBI), the mutation of (b) (i.e., the mutation of (b')) was a substitution of phenylalanine (L445F) for leucine at position 445 of the amino acid sequence shown in SEQ ID NO. 1 in NSP 3; the mutation (i.e., mutation (e ') described above) is a substitution of valine (G248V) for glycine at position 248 of the amino acid sequence shown in sequence No. 2 in NSP14, and the mutation (i.e., mutation (f') described above) is a substitution of serine (G416S) for glycine at position 416 of the amino acid sequence shown in sequence No. 2 in NSP 14; and/or the mutation of (h) (i.e., the mutation of (h') above) is a substitution of valine at position 67 of the amino acid sequence shown in SEQ ID NO. 3 in NSP16 with isoleucine (V67I). Further, as an example of the case where the above-mentioned more preferable example further has another mutation, there may be mentioned a case where the mutation of (a) (i.e., mutation of (a')) is a substitution of valine at position 404 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3 with alanine (V404A); the mutation of (c) (i.e., the mutation of (c') is a substitution of arginine (K1792R) for lysine at position 1792 of the amino acid sequence shown in sequence number 1 in NSP 3; the mutation of (D) (i.e., the mutation of (D') is an aspartic acid substitution at position 1832 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3 with asparagine (D1832N); the mutation of (g) (i.e., the mutation of (g') is a substitution of valine for alanine at position 504 of the amino acid sequence shown in SEQ ID NO. 2 in NSP14 (A504V); the mutation of (i) (i.e., the mutation of (i')) is a substitution of leucine at position 54 of the amino acid sequence shown in SEQ ID NO. 4 in the spike with tryptophan (L54W); the mutation of (j) (i.e., the mutation of (j') is a substitution of threonine at position 739 of the amino acid sequence shown in SEQ ID NO. 4 in the spike with lysine (T739K); the mutation of (k) (i.e., the mutation of (k')) is a substitution of valine for alanine at position 879 of the amino acid sequence shown in SEQ ID NO. 4 in the spike (A879V); the mutation of (L) (i.e., the mutation of (L') is a substitution of leucine at position 28 of the amino acid sequence shown in SEQ ID No. 5 in the envelope with proline (L28P); and/or the mutation of (m) (i.e., the mutation of (m') is a substitution of serine at position 2 of the amino acid sequence shown in SEQ ID NO. 6 in the nucleocapsid with phenylalanine (S2F).

The strain of the present invention may also have a deletion of an amino acid sequence encoded by the base sequence represented by SEQ ID No. 7. The base sequence shown in SEQ ID NO. 7 is part of the open reading frame of SARS-CoV-2 of NC_045512 (NCBI).

Particularly preferred examples of the virus temperature-sensitive strain (low-temperature domesticated strain) of the present invention include the following strains.

Strains having the mutation of (e) above (preferably the mutation of (e ') and/or G248V) and the mutation of (f) above (preferably the mutation of (f') and/or G416S) as responsible mutations; or further as other mutations, a strain having a mutation of (g) (preferably a mutation of (g ') and/or A504V), a mutation of (k) (preferably a mutation of (k') and/or A879V), a mutation of (L) (preferably a mutation of (L ') and/or L28P), and a mutation of (m) (preferably a mutation of (m') and/or S2F).

A strain having the mutation (preferably the mutation of (h') and/or V67I) as the responsible mutation; or further as other mutations, a strain having a mutation of (a) above (preferably a mutation of (a ') and/or V404A), a mutation of (D) above (preferably a mutation of (D ') and/or D1832N), a mutation of (j) above (preferably a mutation of (j ') and/or T739K); or a strain further having a deletion of the amino acid sequence encoded by the base sequence shown in SEQ ID NO. 7.

Strains having the mutation(s) of (b) above (preferably the mutation(s) of (b') and/or L445F) as responsible mutations; or further as other mutations, a strain having the mutation of (c) above (preferably the mutation of (c') and/or K1792R); or a strain further comprising a mutation of (i) above (preferably a mutation of (i') and/or L54W); or a strain further having a deletion of the amino acid sequence encoded by the base sequence shown in SEQ ID NO. 7.

2. Vaccine

2-1 active ingredient of attenuated vaccine

As described above, the β coronavirus temperature-sensitive strain (low temperature-treated strain) described in "1.β coronavirus temperature-sensitive strain (low temperature-treated strain)" can only proliferate efficiently at a temperature lower than the temperature of the lower respiratory tract of a human, and therefore cannot proliferate efficiently at least in the deep part of a living body, particularly in the lower respiratory tract including the lung causing a significant injury, and a significant reduction in pathogenicity can be expected. Therefore, the virus temperature-sensitive strain (low-temperature-domesticated strain) can be used as a live attenuated vaccine by infecting an organism as a virus that is attenuated by itself. Accordingly, the present invention also provides a vaccine comprising the above-mentioned strain sensitive to temperature of beta coronavirus (low temperature domesticated strain) as an active ingredient. Details of the active ingredient are described in "1. Beta. Coronavirus temperature sensitive strain (low temperature domesticated strain)".

2-2 active ingredient of Gene vaccine

As described in the above "1. Beta. Coronavirus temperature-sensitive strain (low temperature domesticated strain)", the predetermined mutation contributes to the imparting of the temperature-sensitive (low temperature domestication) ability. Thus, the present invention also provides a beta coronavirus gene vaccine comprising a gene encoding a non-structural protein having the responsible mutation of the above temperature sensitive property as an active ingredient. Details of the mutation responsible for the temperature-sensitive performance contained in the active ingredient are described in "1. Beta. Coronavirus temperature-sensitive strain (low temperature domesticated strain)".

2-3 virus as target

The vaccine of the present invention is expected to be effective not only against the initial NC045512 strain of SARS-CoV-2 virus but also against a wide range of SARS-CoV-2 virus-related strains and viruses other than SARS-CoV-2 contained in the genus beta coronavirus, including the mutant strain detected in the United kingdom at 9 and the other known mutant strain and other unknown mutant strain not yet detected in the south Africa at 10 in 2020. Thus, the vaccine of the present invention targets the beta coronavirus.

2-4 other ingredients

The vaccine of the present invention may contain, in addition to the above-mentioned active ingredients, other ingredients such as adjuvants, buffers, isotonic agents, analgesics, preservatives, antioxidants, corrigents, light absorbing dyes, stabilizers, carbohydrates, casein digests, and various vitamins, depending on the purpose and use.

Examples of the adjuvant include animal oils (squalene and the like) and solidified oils thereof; vegetable oils (palm oil, castor oil, etc.) or their solidified oils; oily adjuvants comprising dehydrated mannitol/oleate, liquid paraffin, polybutene, octanoic acid, oleic acid, higher fatty acid esters, and the like; water-soluble adjuvants such as PCPP, saponins, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymers, methacrylic acid copolymers, maleic anhydride copolymers, alkenyl derivative polymers, oil-in-water emulsions, cationic lipids containing quaternary ammonium salts, and the like; aluminum salts such as aluminum hydroxide (alum), aluminum phosphate, aluminum sulfate, or combinations thereof, and a precipitative adjuvant such as sodium hydroxide; toxin components derived from microorganisms such as cholera toxin and escherichia coli heat-labile toxin; other components (bentonite, muramyl dipeptide derivatives, interleukins, etc.).

Examples of the buffer include buffers such as phosphate, acetate, carbonate, and citrate. Examples of the isotonic agent include sodium chloride, glycerin, and D-mannitol. Examples of the analgesic include benzyl alcohol and the like. Examples of the preservative include thimerosal, parabens, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, antibiotics, and synthetic antibacterial agents. Examples of the antioxidant include sulfite and ascorbic acid.

Examples of the light absorbing dye include riboflavin, adenine, and adenosine. Examples of the stabilizer include a chelating agent and a reducing agent. Examples of the carbohydrate include sorbitol, lactose, mannitol, starch, sucrose, glucose, and dextran.

The vaccine of the present invention may further comprise 1 or more other vaccines against viruses or bacteria that cause diseases other than the infection with beta coronavirus such as covd-19. That is, the vaccine of the present invention may be prepared as a mixed vaccine containing other vaccines.

2-5 dosage forms

The dosage form of the vaccine of the present invention is not particularly limited, and may be appropriately determined depending on the administration method, storage conditions, and the like. Specific examples of the dosage form include liquid preparations, solid preparations, and the like, and more specifically, oral administration preparations such as tablets, capsules, powders, granules, pills, liquid preparations, syrups, and the like; parenteral administration such as injection and spray.

2-6 methods of administration

The method of administration of the vaccine of the present invention is not particularly limited, and any of intramuscular, intraperitoneal, intradermal, subcutaneous and the like administration, inhalation administration from the nasal cavity and oral cavity, oral administration and the like can be used, but preferable examples include intramuscular, intradermal, subcutaneous and the like administration (intramuscular administration, intradermal administration and subcutaneous administration), inhalation administration from the nasal cavity (nasal administration), and more preferable examples include nasal administration.

2-7 applicable object

The vaccine of the present invention is not particularly limited as long as it is applicable to a subject capable of causing various symptoms caused by infection with beta coronavirus (preferably a subject capable of causing symptoms of covd-19 by infection with SARS-CoV-2 virus), and examples thereof include mammals, more specifically, humans; pets such as dogs and cats; and laboratory animals such as mice, mice and hamsters.

2-8 dose

The dose of the vaccine of the present invention is not particularly limited, and may be appropriately determined depending on the type of the active ingredient, the administration method, and the administration subject (conditions such as age, weight, sex, and presence or absence of underlying diseases). For example, as a dose to a human, there may be mentioned 1×10 ¹⁰ TCID50/kg or less, preferably 1X 10 ⁸ TCID50/kg or less.

3. Method for producing beta-coronavirus temperature-sensitive strain (low-temperature domesticated strain)

The method for producing the β -coronavirus temperature-sensitive strain (low-temperature domesticated strain) of the present invention is not particularly limited, and can be appropriately determined by one skilled in the art based on the above-mentioned amino acid sequence information. For example, from the viewpoint of producing a relatively inexpensive vaccine with a small number of differences between batches, an inverse genetics method using an artificial chromosome such as Bacterial Artificial Chromosome (BAC) or Yeast Artificial Chromosome (YAC), or a CPER method using a genomic fragment of a β -coronavirus is preferable.

In the method of reconstructing a virus by reverse genetics, first, the genome of a strain (mother strain) having no responsible mutation of a temperature-sensitive strain (low-temperature domesticated strain) of a β coronavirus is cloned. The stock strain used in this case may be any strain as long as it is a β coronavirus, and specifically may be selected from a specific SARS-CoV-2 recorded in NC_045512 (NCBI) described above, any of the other SARS-CoV-2 described above, and viruses other than SARS-CoV-2 contained in the genus β coronavirus.

Furthermore, when artificial chromosomes are used in the reverse genetics method, the full-length DNA of the viral genome is cloned into BAC DNA, YAC DNA, or the like, and a transcription promoter sequence for eukaryotic cells is inserted upstream of the viral sequence. Examples of the promoter sequence include a CMV promoter and a CAG promoter. A ribozyme sequence and a poly a (poly a) sequence are inserted downstream of the viral sequence. Examples of the ribozyme sequence include a hepatitis D virus ribozyme and a hammerhead ribozyme. Examples of the poly-A sequence include poly-A of Simian Virus 40 (Simian Virus 40).

On the other hand, when the CPER method is used in the reverse genetics method, the full-length DNA of the viral genome is cloned by dividing it into a plurality of fragment fragments. As a method for obtaining the fragment, there may be mentioned an artificial synthesis method of a nucleic acid, a PCR method using a plasmid cloned with the above artificial chromosome or fragment as a template, and the like.

For introducing at least any of the above-mentioned responsible mutations into the viral genome cloned by the above-mentioned method, known point mutation introduction methods such as double crossover and lambda/RED recombination homologous recombination methods, overlap PCR methods, CRISPR/Cas9 methods and the like can be used.

Then, the artificial chromosome into which the responsible mutation is introduced is transfected into a host cell, and the recombinant virus is reconstituted. In the case of the reverse genetics method using the CPER method, a recombinant virus is reconstituted by ligating fragments into which responsible mutations have been introduced by a reaction using DNA polymerase and then transfecting the fragments into host cells. The method of transfection is not particularly limited, and known methods can be used. The host is not particularly limited, and known cells can be used.

Then, the recombinant virus after reconstitution is added to the cultured cells, and subculture is performed on the recombinant virus. The cultured cells to be used in this case are not particularly limited, and examples thereof include: vero cells, veroE6 cells, vero cells supplemented with TMPRESS2 expression, veroE6 cells supplemented with TMPRESS2 expression, calu-3 cells, 293T cells supplemented with ACE2 expression, BHK cells, 104C1 cells, NA cells derived from mouse neuroblastoma, vero cells, etc. The virus can be recovered by a known method such as centrifugation or membrane filtration. In addition, the recovered virus is further added to the cultured cells, whereby a recombinant virus can be mass-produced.

Examples

The present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Test example 1]

Test example 1-1]Isolation of temperature-sensitive strain (Low temperature domesticated strain) A50-18 of SARS-CoV-2

Based on the method of FIG. 1, 2 kinds of mutation inducers 5-fluorouracil (hereinafter, 5-FU) and 5-azacytidine (hereinafter, 5-AZA) were added to a clinical isolate of SARS-CoV-2 (hereinafter, B-1 strain), thereby obtaining an A to F50 strain and an A to F500 strain virus group which were domesticated at 32 ℃. Further, from 406 candidate strains obtained by passaging each virus group several times, strains (A50-18 strain, hereinafter sometimes referred to as Ts strain) having significantly reduced proliferation performance at 37℃were found, isolated and selected, and the strains were allowed to proliferate at 32 ℃.

Test examples 1 to 2]Analysis of temperature-sensitive strain (Low temperature domesticated strain) A50-18 strain by second-generation sequencing

(1-2-1) mutation analysis of individual viral strains

Mutation analysis of the following strains was performed using a second generation sequencer. The analysis was performed by extracting RNA from the culture supernatant of Vero cells infected with SARS-CoV-2. As a reference (reference), NC045512 strain was used.

B-1: wild strain (clinical isolate)

A50-18: temperature sensitive strain (Low temperature domesticated strain)

F50-37: non-temperature sensitive strain

C500-1: non-temperature sensitive strain

F500-53: non-temperature sensitive strain

F500-40: non-temperature sensitive strain

F500-2: non-temperature sensitive strain

(for viruses subjected to mutation induction except B-1)

(1-2-2) mutation of temperature-sensitive Strain (Low temperature domesticated Strain)

According to (1-2-1), the analysis result of FIG. 3A was obtained. The point mutation of D614G was also found in B-1 strain, and thus was not a characteristic point mutation in the temperature sensitive strain (low temperature domesticated strain), but as a characteristic point mutation in the temperature sensitive strain (low temperature domesticated strain) (A50-18), G248V, G416S, A V of NSP14, A879V of Spike (Spike), L28P of Envelope (Envelope) and S2F of Nucleocapsid (nucleoapsid) were found.

(1-2-3) analysis of revertant 1

"back mutation" refers to the return to the same phenotype as the original virus prior to mutation by adding further mutations to the mutated virus. In the present specification, "back mutation" refers to a property of losing temperature sensitivity by adding a further mutation to a temperature sensitive strain. Further mutations include the return of the amino acid at the mutation site added at the time of temperature sensitivity to the amino acid before mutation.

A Vero cell was infected with strain B-1 or strain A50-18 at MOI=0.01, and the proliferation at 32℃and 34℃and 37℃was evaluated, whereby a sample (hereinafter referred to as "revertant") having recovered proliferation ability at 37℃was found from strain A50-18. Among the back mutant strains, it is considered that, among the mutations possessed by the temperature-sensitive strain (strain A50-18) having acquired temperature sensitivity, a part of amino acid residues are back mutated (hereinafter, simply referred to as "back mutation") to amino acids before mutation, whereby the temperature sensitivity is lowered and the proliferation potency at 37℃is recovered. A CPE image showing this situation is shown in fig. 3B. The sequence of the obtained sample was confirmed, and as a result, the mutation of G248V in NSP14 was back mutated to wild-type G, while the mutation of G416S, A V in NSP14 and L28P in the envelope was maintained. Thus, it was suggested that the G248V mutation of NSP14 is a responsible mutation contributing to temperature sensitivity.

Analysis of the back mutations of the isolate (1-2-4) 2

Vero cells were infected with strain a50-18 with moi=1, and proliferation at 37 ℃ and 38 ℃ was evaluated. As a result, a sample (revertant) having a proliferative recovery at 37℃and 38℃was found. CPE images obtained after 3 days of incubation of the resulting revertant strain at 37℃are shown in FIG. 3C. The sequences of the resulting revertants were confirmed, and as a result, the following 2 revertants of <1> and <2> were found.

<1> mutation of G248V in nsp14 reverted to wild-type G, on the other hand, mutation of G416S, A V was maintained.

<2> mutation of G416S in nsp14 reverted to wild-type G, on the other hand, mutation maintenance of G248V, A V.

In G248V and G416S of NSP14, if at least 1 back mutation, temperature sensitivity is lost, thus suggesting that the combination of the G248V mutation and G416S mutation of NSP14 is a responsible mutation contributing to temperature sensitivity.

(1-2-5) analysis of wild-type mutant-introduced recombinant Virus

For BAC DNA having the whole genome of wild-type SARS-CoV-2, NSP14 derived from strain A50-18, spike (Spike), nucleocapsid (Nucleocapsid), envelope (Envelope) were introduced by homologous recombination. The virus was reconstituted by transfection (transfection) of the resulting recombinant BAC DNA into 293T cells. The temperature sensitivity was evaluated by infecting Vero cells with the recombinant virus and observing CPE at 37 ℃ and 32 ℃. The result is shown in fig. 3D. By introducing NSP14 derived from strain A50-18, a temperature-sensitive strain showing no CPE was obtained when cultured at 37℃and thus NSP14 was known to be a responsible mutation contributing to temperature sensitivity. On the other hand, even when the envelope derived from the A50-18 strain is introduced, the temperature sensitivity does not occur, and therefore, it is considered that mutation of the envelope does not contribute to the temperature sensitivity.

(1-2-6) analysis of wild-type mutant-introduced recombinant Virus 2

The virus into which the NSP14 mutation was introduced was reconstituted by CPER method. 3 recombinant viruses having the following mutations of each NSP14 were reconstituted.

G248A only

G416S only

G248V and G416S (hereinafter also referred to as "double mutant").

The respective recombinant viruses were allowed to infect Vero cells, and CPE after 3 days of culture at 37 ℃ or 32 ℃ was observed. As is clear from FIG. 3-E, in the viruses having only the G248V mutation and the viruses having only the G416S mutation, CPE was observed similarly to the B-1 strain at 37℃and 32℃and thus was not temperature-sensitized. On the other hand, in the virus of the double mutant strain having mutations of G248V and G416S, CPE was observed at 32 ℃, but at 37 ℃, CPE was slightly observed, and CPE was significantly weakened compared to 32 ℃. From the above results, it was found that by having the mutations of G248V and G416S, the virus proliferation at 37℃became slow and the temperature was sensitive. From this, it is clear that the combination of the G248V mutation and the G416S mutation of NSP14 is a responsible mutation contributing to temperature sensitivity.

(1-2-6) analysis of temperature-sensitive strains (Low temperature domesticated strains) by Sanger sequencing

Mutation analysis of strain A50-18 was performed using Sanger sequencing. The analysis was performed by extracting RNA from the culture supernatant of Vero cells infected with SARS-CoV-2. As a result, the absence of the test piece (2-2-5) in test example 2-2 described later was not found.

Summary of mutations in temperature-sensitive strains (Low temperature domesticated strains) (1-2-7)

In the temperature-sensitive strain (low temperature domesticated strain) A50-18, a mutation of the tag pair hook was found in the amino acid sequence of the displayed sequence number, as shown in Table 2 below, wherein the mutation of the tag pair hook was found as a responsible mutation. In addition, as shown in table 2 below, it was found that the double mutant strain having only the responsible mutation of NSP14 was also a temperature sensitive strain (low temperature domesticated strain).

TABLE 2

[ test ]Examples 1 to 3]Proliferation assay of temperature sensitive strain (Low temperature domesticated strain) A50-18 strain

(1-3-1) analysis at 32℃and 37 ℃

Clinical isolates (B-1 strain) and temperature sensitive strains (cryo-acclimated strains) (a 50-18 strain) were infected with Vero cells (n=3) using 6-well plates under moi=0.01 or 0.1. After culturing at 37℃or 32℃the respective culture supernatants were recovered at 0 to 5 dpi. The virus titer of the culture supernatant was measured at TCID50/mL using Vero cells at 0-5 dpi. The results are shown in fig. 4A.

As can be seen from FIG. 4A, the virus titer of strain A50-18 at 37℃on the third day after infection was below the limit of detection, and the proliferation at 37℃was significantly reduced.

(1-3-2) analysis at 32 ℃, 34 ℃ and 37 DEG C

Clinical isolates (B-1 strain) and temperature sensitive strains (cryo-acclimated strains) (a 50-18 strain) were infected with Vero cells (n=3) using 6-well plates at moi=0.01. After culturing at 37℃and 34℃or 32℃the respective culture supernatants were recovered at 0 to 5 dpi. The virus titer of the culture supernatant was measured at TCID50/mL using Vero cells at 0-5 dpi. The results are shown in fig. 4B.

As is clear from FIG. 4B, the A50-18 strain proliferated to the same extent as the clinical isolate at 32℃and 34℃and the proliferation at 37℃was markedly deficient.

Test examples 1 to 4]Pathogenic analysis of temperature-sensitive strain (Low temperature domesticated strain) A50-18 strain

(1-4-1) weight change in hamsters infected with SARS-CoV-2

After raising 4-week-old male Syrian hamsters (n=4) for one week, the clinical isolates (B-1 strain) and temperature sensitive strains (low temperature acclimatized strains) (a 50-18 strain) (1×10) ⁴ Or 1x10 ⁶ TCID 50) was nasally administered at a volume of 100 μl, and body weight fluctuation was observed for 10 days. The same volume of D-MEM medium was used as a non-infectious control (MOCK). The results are shown in FIG. 5. Suggesting that no weight loss was observed when A50-18 strains were infected, and the pathogenicity was significantly low.

(1-4-2) viral load in the lung or nasal cavity of hamsters infected with SARS-CoV-2

After raising 4-week-old male syrian hamsters (n=3) for one week, the clinical isolates (B-1 strain) and the temperature sensitive strains (low temperature domesticated strains) (A50-18 strain) (1X 10) ⁶ TCID 50) was nasally administered in a volume of 100 μl. The results of observing weight fluctuation for 3 days are shown in fig. 6. After euthanizing hamsters at 3dpi, nasal washes were recovered with 1mL of D-PBS. Further, hamster lungs were removed, right lungs were crushed, suspended with 1mL of D-MEM, and then the supernatant was collected as a lung crushing solution by centrifugation. The amounts of viruses in these nasal wash and lung disruption solution were evaluated by plaque formation assay in Vero cells, and the results are shown in fig. 7. Further, the left lung thus removed was fixed with 10% formalin, and the result of the photographing is shown in fig. 8.

As is clear from FIG. 7, in the nasal wash, there was no difference in the viral load between strain B-1 and strain A50-18, while in the lung, the amount of virus in strain A50-18 was significantly reduced. Further, as is clear from FIG. 8, the hamsters infected with strain B-1 and having reduced body weight were observed to swell and blacken in the lung, while the hamsters infected with strain A50-18 were not observed to significantly change in body weight and in the lung. From the above results, it was estimated that the temperature-sensitive strain (low temperature domesticated strain) was a strain which proliferated in the upper respiratory tract, but failed to proliferate in the lower respiratory tract.

(1-4-3) histological analysis of SARS-CoV-2 infected hamsters

Sections were prepared from formalin-fixed lungs obtained by conducting an infection experiment on hamsters in (1-4-2), and HE staining was performed, thereby analyzing histologic pathogenicity of lungs based on SARS-CoV-2 infection. The results are shown in fig. 9.

As shown in fig. 9, infiltration of erythrocytes and disintegration of alveolar structure were observed in the lung obtained from hamsters infected with the clinical isolate (strain B-1). On the other hand, in the lungs infected with the temperature-sensitive strain (low-temperature domesticated strain) (strain a 50-18), these severe symptoms were not observed. It is also strongly suggested that strain A50-18 does not induce severe inflammation in the lung at the time of infection, and is of low pathogenicity.

(1-4-4) histological analysis of hamster infection with SARS-CoV-2 Using immunochromatography

For the histological pathogenicity observed in (1-4-3), in order to evaluate the correlation between the proliferation and pathogenicity of the virus, the viral proteins were detected by immunochemical staining. After raising 4-week-old male syrian hamsters (B-1, a50.about.18:n=5, mock:n=3) for one week, the clinical isolates (B-1 strain) and the temperature sensitive strain (cryogenically domesticated strain) (a 50.about.18 strain) (1×10) were bred ⁶ TCID 50) was nasally administered in a volume of 100 μl. After euthanizing hamsters at 3dpi, the extracted left lung was fixed with 10% formalin, and serial sections were prepared. The obtained serial sections were subjected to HE staining and immunochemical staining (also referred to as Immunohistochemistry (IHC) staining). Rabbit anti-spike polyclonal antibodies (Sinobiological: 40589-T62) were used in immunochemical staining. The HE staining image and the immunochemical staining image are shown in fig. 10. As in (1-4-3), infiltration of erythrocytes and disintegration of alveolar structures were found in B-1 infected hamsters, and spike proteins were detected in a large scale in immunochemical staining. On the other hand, in hamsters infected with strain A50-18, no such tissue damage was found, and spike proteins were also detected only in locally limited regions. From these results, it was found that B-1 strain significantly proliferated in lung tissue to exhibit tissue injury, while A50-18 strain failed to efficiently proliferate in lung tissue and had low lung tissue injury.

Test examples 1 to 5]Immunogenicity analysis of temperature-sensitive strain (Low temperature domesticated strain) A50-18 strain(1-5-1) wild strain challenge test against hamster infection with temperature-sensitive strain (Low temperature domesticated strain)

An challenge test was performed on wild strains (clinical isolates) of hamsters infected with temperature sensitive strains (low temperature domesticated strains) according to the following procedure.

After raising 4-week-old male syrian hamsters (n=4) for one week, the clinical isolate (B-1 strain) or the temperature sensitive strain (low temperature domesticated strain) (A50-18 strain) (1X 10) ⁴ Or 1x10 ⁶ TCID 50) was nasally administered in a volume of 100 μl. After 21 days, the clinical isolate (B-1 strain) (1X 10 ⁶ TCID 50) was nasally administered at a volume of 100 μl, and body weight fluctuation was observed for 10 days. At this time, the same week age of the heart will not be usedInfected hamsters (n=3) were used as a blank control (Naive control). The results are shown in fig. 11.

As shown in FIG. 11, the weight loss of hamsters infected with B-1 strain was observed in the blank hamsters, while the weight of hamsters infected with B-1 strain or A50-18 strain once was not reduced. From these results, it was found that not only the B-1 strain, which is a wild-type strain, but also immunity contributing to infection defense can be induced even in infection by the A50-18 strain having low pathogenicity.

(1-5-2) neutralizing antibody induction analysis of hamster infection by temperature-sensitive strain (cryo-domesticated strain)

After raising 4-week-old male syrian hamsters (n=5) for one week, the clinical isolate (B-1 strain) or the temperature sensitive strain (low temperature domesticated strain) (A50-18 strain) (1X 10) ⁶ TCID 50) was nasally administered in a volume of 100 μl. The body weight changes after infection are shown in fig. 12. As with the previous results, the weight loss was observed due to infection with strain B-1, while no weight loss was observed in infection with strain A50-18.

Whole blood was collected 21 days after infection, serum was separated, and inactivation with hot serum was performed at 56℃for 30 minutes. The B-1 strain of 100TCID50 was mixed with the inactivated serum subjected to the stage dilution and reacted at 37℃for 1 hour. The culture broth after the reaction was inoculated into Vero cells, and after culturing at 37 ℃, the virus neutralization activity was evaluated by observing CPE. The highest dilution ratio that did not produce CPE was taken as neutralizing antibody titer (Neutralizing antibody titer). The results are shown in FIG. 13. It was clarified that serum from a non-infected hamster does not exhibit neutralizing activity against strain B-1, and that serum from a strain B-1 or strain A50-18 was able to induce neutralizing antibodies.

Test example 2]

Test example 2-1]SARS-CoV-2 temperature sensitive strain (low temperature acclimatized strain) H50-11 strain, L50-33 strain, L50-40 Additional isolation of plants

For the purpose of further isolation of candidate strains, isolation of temperature sensitive strains (low temperature domesticated strains) was performed by the method of FIG. 14. A clinical isolate of SARS-CoV-2 (hereinafter, B-1 strain) was used to infect Vero cells, and a G.about.L50 virus group which had been acclimatized at 32℃was obtained in a state where a mutation inducer 5-FU was added. Further, from 253 strains obtained by passaging each virus group several times, virus strains (H50-11 strain, L50-33 strain, L50-40 strain) having significantly reduced proliferation at 37℃were found, isolated and selected (FIG. 15).

Test example 2-2]Additional isolates H50-11, L50- Analysis of 33 strains, L50-40 strains

(2-2-1) mutation analysis method of additional isolate

Mutation analysis of the following strains was performed using a second generation sequencer. The analysis was performed by extracting RNA from the culture supernatant of Vero cells infected with SARS-CoV-2. For reference, NC045512 strain was used.

H50-11 strain: temperature sensitive strain (Low temperature domesticated strain)

L50-33 strain: temperature sensitive strain (Low temperature domesticated strain)

L50-40 strain: temperature sensitive strain (Low temperature domesticated strain)

(2-2-2) mutation analysis results of temperature-sensitive strain (Low temperature domesticated strain)

The analysis result of FIG. 16A was obtained by (2-2-1). V404A and D1832N, NSP of NSP3 and T739K of spike were found as characteristic point mutations of H50-11 strain. In addition, as characteristic point mutations of the L50-33 strain, L445F, K1792R of NSP3 was found, as characteristic point mutations of the L50-40 strain, L445F, K1792R of NSP3 and spike L54W were found.

(2-2-3) analysis of the back mutant of the additional isolate (H50-11 strain)

H50-11 strain was infected with Vero cells at moi=1, and proliferation at 37 ℃ and 38 ℃ was evaluated. As a result, a sample (revertant) having a proliferative recovery at 37℃and 38℃was found. CPE images obtained after the back mutant strain was cultured at 38℃for 3 days are shown in FIG. 16B. As a result of confirming the sequence of the obtained sample, the mutation of NSP 16V 67I was back mutated to wild-type V, while other amino acid mutations were maintained. Thus suggesting that the V67I mutation of NSP16 is a responsible mutation contributing to temperature sensitivity.

(2-2-4) analysis of the back mutant strains of the additional isolates (L50-33 strain and L50-40 strain)

The L50-33 strain and the L50-40 strain were infected with Vero cells at MOI=0.01, and proliferation at 32℃and 34℃and 37℃was evaluated. As a result, samples (hereinafter, revertants) having recovered proliferation at 37℃were found from the L50-33 strain and the L50-40 strain. CPE images of the individual strains after 3 days incubation at 37 ℃ are shown in fig. 16C. The back mutants of the L50-33 strain and the L50-40 strain are denoted as L50-33 strain Rev1, 2 and L50-40 strain Rev1, 2, respectively. As a result of confirming the sequence of the obtained sample, it was found that the mutation of L445F in NSP3 was changed to wild-type L or C, while the mutation of K1792R in NSP3 was maintained. Thus suggesting that the L445F mutation of NSP3 is a responsible mutation contributing to temperature sensitivity.

(2-2-5) analysis of temperature-sensitive strains (Low temperature domesticated strains) sequenced by Morganella

Mutation analysis of H50-11 strain, L50-33 strain and L50-40 strain was performed using Sanger sequencing. The analysis was performed by extracting RNA from the culture supernatant of Vero cells infected with SARS-CoV-2.

As a result, deletion of the nucleotide sequence (SEQ ID NO: 7) at positions 27549 to 28251 as shown in FIG. 17 was found in all of 3 strains. A schematic diagram of the deletion of the nucleotide sequence at positions 27549-28251 and the deletion of the amino acid sequence encoded thereby is shown in FIG. 18. In FIG. 18, ORF7a is a base sequence of 27394 to 27759, ORF7b is a base sequence of 27756 to 27887, and ORF8 is a base sequence of 27894 to 28259.

As shown in FIG. 18, the nucleotide sequence regions at positions 27549 to 28251 correspond to the amino acid sequences of a part of ORF7a (amino acid sequence from position 53 to the end, the same applies hereinafter), the entire ORF7b and a large part of ORF 8. The deletion of this region is accompanied by a frame shift, and thus it is thought that a protein is produced by fusing the amino acid sequences of the 1 st to 52 th amino acids of ORF7a and the amino acid sequence encoded by the 3' -terminal 8 bases of ORF8, the intergenic region and the nucleocapsid base sequence. In addition, ORF7b is deleted entirely, and the original sequence of ORF8 is also deleted entirely.

(2-2-6) summary of mutations in temperature-sensitive strains (Low temperature domesticated strains)

Among the temperature-sensitive strains (low temperature domesticated strains) H50-11, L50-33 and L50-40, mutations were found in which double-hooked mutations were marked as responsible mutations in the amino acid sequences of the sequence numbers shown below as shown in Table 3.

TABLE 3

Test example 3]Proliferation assay of additional isolates H50-11, L50-33 and L50-40

The additional isolate was allowed to infect Vero cells (n=3) at moi=0.01. After culturing at 37℃and 34℃or 32℃the respective culture supernatants were recovered in 0 to 5d.p.i. By TCID ₅₀ The viral titers of these culture supernatants were determined using Vero cells. The results are shown in FIG. 19. Thus, the obtained additional isolate showed proliferation at 32℃and 34℃and showed a delay and decrease in proliferation at 37 ℃.

Test example 4]Pathogen analysis of each temperature-sensitive strain

(4-1) weight variation in hamster infected with temperature-sensitive strain

After raising 4-week-old male syrian hamsters (n=5) for one week, the clinical isolates (B-1 strain) or temperature sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, H50-11 strain) (3X 10) ⁵ TCID 50) was nasally administered at a volume of 100 μl, and body weight fluctuation was observed for 10 days. The same volume of D-MEM medium was used as a non-infectious control (MOCK). The results are shown in FIG. 20. In contrast to the weight loss of about 20% observed in hamsters infected with strain B-1 on day 7, no significant weight loss was observed in all groups for hamsters infected with temperature sensitive strain, suggesting a significantly lower pathogenicity.

(4-2) viral load in the lung or nasal cavity of hamsters infected with temperature-sensitive strains

After raising 4-week-old male syrian hamsters (n=5) for one week, the clinical isolates (B-1 strain) or temperature sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, H50-11 strain) (3X 10) ⁵ TCID 50) was nasally administered in a volume of 100 μl. After euthanizing hamsters at 3dpi, nasal washes were recovered using 1mL of D-PBS. Further, hamster lungs were removed, the weight of the lungs was measured, the right lung was crushed, suspended with 1mL of D-MEM, and the supernatant was collected as a lung crushing solution by centrifugation. The lung weight per total body weight of hamsters is shown in figure 21. The amounts of viruses in these nasal wash and lung-breaking fluid were evaluated by plaque formation assay in Vero cells, and the results are shown in fig. 22.

The comparison of lung weights per total weight of hamsters revealed that strain B-1 infected hamsters had an increased lung weight, which strongly suggests that lung swelling was caused by inflammation or the like. On the other hand, in hamsters infected with the temperature-sensitive strain, such an increase in lung weight was not observed. In addition, the comparison of the viral load in nasal wash revealed that there was no significant difference between hamsters infected with strain B-1 and hamsters infected with temperature-sensitive strain other than strain H50-11, and that the viral load in nasal wash was small in hamsters infected with strain H50-11. Furthermore, it was found that infection of hamsters with temperature-sensitive strains significantly reduced the amount of intrapulmonary virus compared with B-1 strain. From these results, it was estimated that each temperature-sensitive strain was a weak strain which could not proliferate in the lower respiratory tract, similar to the A50-18 strain in test example 1.

Test example 5]Immunogenicity analysis of each temperature-sensitive strain

(5-1) wild strain challenge test for hamster infection with temperature-sensitive strain

After raising 4-week-old male syrian hamsters (n=5) for one week, the clinical isolates (B-1 strain) or temperature sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, H50-11 strain) (3X 10) ⁵ TCID 50) was nasally administered in a volume of 100 μl. After 21 days, the clinical isolate (B-1 strain) (3X 10 ⁵ TCID 50) was nasally administered at a volume of 100 μl, and weight change was observed for 9 days. At this time, uninfected hamsters of the same week (n=5) were used as a blank. The result is shownFig. 23 shows the structure. A decrease in body weight was observed in the hamsters infected with B-1 strain, while the hamsters infected with B-1 strain and each temperature-sensitive strain were not decreased in body weight once. From these results, it was found that not only the wild-type B-1 strain but also the infection with each temperature-sensitive strain having low pathogenicity can induce immunity contributing to infection defense.

(5-2) neutralizing antibody induction assay for hamster infection by temperature sensitive strains

After raising 4-week-old male syrian hamsters (n=5) for one week, the clinical isolates (B-1 strain) or temperature sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, H50-11 strain) (3X 10) ⁵ TCID 50) was nasally administered in a volume of 100 μl. After 20 days, partial blood collection was performed, and the neutralizing activity against the clinical isolate (strain B-1) was measured using the obtained serum. As a method for measuring the neutralizing activity, the same method as (1-5-2) was used. The measurement results are shown in fig. 24. It was found that antibodies having neutralizing activity were induced not only in B-1 strain but also in hamsters infected with each temperature-sensitive strain.

Test example 6]Analysis of the effectiveness of SARS-CoV-2 mutant

(6-1) evaluation of neutralizing Activity of SARS-CoV-2 mutant strain infected with hamster serum by temperature-sensitive strain

After raising 4-week-old male syrian hamsters (n=3 or 5) for one week, the clinical isolate (B-1 strain) or the temperature sensitive strain (A50-18 strain) (3X 10) ⁵ TCID 50) was nasally administered in a volume of 100 μl. The results of partial blood collection from hamsters 3 weeks after infection and measurement of the neutralization activity of live viruses against SARS-CoV-2 European clinical isolate (B-1) and Brazilian mutant (hCoV-19/Japan/TY 7-503/2021) using the obtained serum are shown in FIG. 25. As a method for measuring the neutralizing activity, the same method as (1-5-2) was used. Hamsters infected with the B-1 strain and the temperature-sensitive strain were found to have a neutralizing activity against Brazilian mutant strains. Thus, it is believed that the present attenuated live vaccine may be effective against SARS-CoV-2 mutant strain as well.

Test example 7]Comparative study experiments on route of administration and dosage

(7-1) comparison of immune Induction ability based on administration route

After raising 4-week-old male syrian hamsters (n=5) for one week, the clinical isolate (B-1 strain) or the temperature sensitive strain (A50-18 strain) (3X 10) ⁵ TCID 50) is administered nasally or subcutaneously in a volume of 100 μl. Untreated groups were set as blank (naive). After 3 weeks, the neutralizing activity against SARS-CoV-2 Brazilian mutant strain (hCoV-19/Japan/TY 7-503/2021 strain) was evaluated using serum obtained by partial blood collection from hamsters. As a method for measuring the neutralizing activity, the same method as (1-5-2) was used. The result of the neutralization activity is shown in FIG. 26.i.n administration via the nose and s.c subcutaneous administration. In the nasal administration of strain B-1 and strain A50-18, neutralizing antibodies can be induced against live viruses. Regarding subcutaneous administration, neutralizing antibodies were hardly induced at the tested dose, but in view of the results of nasal administration, it is considered that even subcutaneous administration can induce neutralizing antibodies if the dose is increased.

(7-2) comparison of the Immunity-inducing Capacity based on the amount administered

After raising 4-week-old male syrian hamsters (n=5) for one week, the temperature sensitive strains (a 50-18 strains) were administered nasally or subcutaneously. The dosage is shown in Table 4.

TABLE 4

The results of partial blood collection from hamsters 3 weeks after infection and measurement of neutralization activity of live viruses against SARS-CoV-2 Brazilian mutant strain (hCoV-19/Japan/TY 7-503/2021 strain) using the serum obtained are shown in FIG. 27.i.n administration via the nose and s.c subcutaneous administration. As a method for measuring the neutralizing activity, the same method as (1-5-2) was used. As with (7-1), in nasal administration, even 1X10 ² An increase in neutralizing antibody titers was also observed in the low dose group with TCID 50/10. Mu.L. Thus, it was suggested that even a small amount of nasal administration, the temperature sensitive strain could induce sufficient immunity. With respect to subcutaneous administration, neutralizing antibodies were hardly induced at the doses tested, but in view of the knots of nasal administrationAs a result, it is considered that even subcutaneous administration can induce neutralizing antibodies if the dosage is increased.

Test example 8]Analysis of the effectiveness of SARS-CoV-2 mutant

After raising 4 week old male syrian hamsters (n=4) for one week, 1x10 ⁴ TCID50 or 1x10 ² The temperature sensitive strain of TCID50 (strain A50-18) was nasally administered in a volume of 10. Mu.L. The results of partial blood collection from hamsters 3 weeks after infection and measurement of neutralization activities against live viruses of SARS-CoV-2 European wild-type strain (B-1 strain), indian-type mutant strain (autoisolate) and Brazilian mutant strain (hCoV-19/Japan/TY 7-503/2021 strain) using the serum obtained are shown in FIG. 28. As a method for measuring the neutralizing activity, the same method as (1-5-2) was used. It was found that even a small amount of the A50-18 strain was administered nasally, not only the B-1 strain, which was the mother strain and the wild type, but also neutralizing antibodies against the Indian type mutant strain and the Brazilian mutant strain were induced in a dose-dependent manner.

The results of comparing the neutralizing antibody titers of the respective strains with the serum of the respective individuals are shown in fig. 29. It was found that, in the brazilian mutant, the neutralizing antibody was maintained in all individuals although a decrease in the neutralizing antibody titer was observed in some individuals. These results suggest the possibility that immunity generated by nasal administration of temperature sensitive strains shows cross defenses.

Sequence listing

<110> general financial group legal person, institute of microbiological diseases, sakada (BIKEN Co., ltd.)

<120> beta coronavirus temperature sensitive strain and vaccine

<130> 21058WO

<150> JP2020-173494

<151> 2020-10-14

<150> JP2020-180524

<151> 2020-10-28

<150> JP2020-210564

<151> 2020-12-18

<150> JP2021-017633

<151> 2021-02-05

<150> JP2021-051107

<151> 2021-03-25

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 1945

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

<400> 1

Ala Pro Thr Lys Val Thr Phe Gly Asp Asp Thr Val Ile Glu Val Gln

1 5 10 15

Gly Tyr Lys Ser Val Asn Ile Thr Phe Glu Leu Asp Glu Arg Ile Asp

20 25 30

Lys Val Leu Asn Glu Lys Cys Ser Ala Tyr Thr Val Glu Leu Gly Thr

35 40 45

Glu Val Asn Glu Phe Ala Cys Val Val Ala Asp Ala Val Ile Lys Thr

50 55 60

Leu Gln Pro Val Ser Glu Leu Leu Thr Pro Leu Gly Ile Asp Leu Asp

65 70 75 80

Glu Trp Ser Met Ala Thr Tyr Tyr Leu Phe Asp Glu Ser Gly Glu Phe

85 90 95

Lys Leu Ala Ser His Met Tyr Cys Ser Phe Tyr Pro Pro Asp Glu Asp

100 105 110

Glu Glu Glu Gly Asp Cys Glu Glu Glu Glu Phe Glu Pro Ser Thr Gln

115 120 125

Tyr Glu Tyr Gly Thr Glu Asp Asp Tyr Gln Gly Lys Pro Leu Glu Phe

130 135 140

Gly Ala Thr Ser Ala Ala Leu Gln Pro Glu Glu Glu Gln Glu Glu Asp

145 150 155 160

Trp Leu Asp Asp Asp Ser Gln Gln Thr Val Gly Gln Gln Asp Gly Ser

165 170 175

Glu Asp Asn Gln Thr Thr Thr Ile Gln Thr Ile Val Glu Val Gln Pro

180 185 190

Gln Leu Glu Met Glu Leu Thr Pro Val Val Gln Thr Ile Glu Val Asn

195 200 205

Ser Phe Ser Gly Tyr Leu Lys Leu Thr Asp Asn Val Tyr Ile Lys Asn

210 215 220

Ala Asp Ile Val Glu Glu Ala Lys Lys Val Lys Pro Thr Val Val Val

225 230 235 240

Asn Ala Ala Asn Val Tyr Leu Lys His Gly Gly Gly Val Ala Gly Ala

245 250 255

Leu Asn Lys Ala Thr Asn Asn Ala Met Gln Val Glu Ser Asp Asp Tyr

260 265 270

Ile Ala Thr Asn Gly Pro Leu Lys Val Gly Gly Ser Cys Val Leu Ser

275 280 285

Gly His Asn Leu Ala Lys His Cys Leu His Val Val Gly Pro Asn Val

290 295 300

Asn Lys Gly Glu Asp Ile Gln Leu Leu Lys Ser Ala Tyr Glu Asn Phe

305 310 315 320

Asn Gln His Glu Val Leu Leu Ala Pro Leu Leu Ser Ala Gly Ile Phe

325 330 335

Gly Ala Asp Pro Ile His Ser Leu Arg Val Cys Val Asp Thr Val Arg

340 345 350

Thr Asn Val Tyr Leu Ala Val Phe Asp Lys Asn Leu Tyr Asp Lys Leu

355 360 365

Val Ser Ser Phe Leu Glu Met Lys Ser Glu Lys Gln Val Glu Gln Lys

370 375 380

Ile Ala Glu Ile Pro Lys Glu Glu Val Lys Pro Phe Ile Thr Glu Ser

385 390 395 400

Lys Pro Ser Val Glu Gln Arg Lys Gln Asp Asp Lys Lys Ile Lys Ala

405 410 415

Cys Val Glu Glu Val Thr Thr Thr Leu Glu Glu Thr Lys Phe Leu Thr

420 425 430

Glu Asn Leu Leu Leu Tyr Ile Asp Ile Asn Gly Asn Leu His Pro Asp

435 440 445

Ser Ala Thr Leu Val Ser Asp Ile Asp Ile Thr Phe Leu Lys Lys Asp

450 455 460

Ala Pro Tyr Ile Val Gly Asp Val Val Gln Glu Gly Val Leu Thr Ala

465 470 475 480

Val Val Ile Pro Thr Lys Lys Ala Gly Gly Thr Thr Glu Met Leu Ala

485 490 495

Lys Ala Leu Arg Lys Val Pro Thr Asp Asn Tyr Ile Thr Thr Tyr Pro

500 505 510

Gly Gln Gly Leu Asn Gly Tyr Thr Val Glu Glu Ala Lys Thr Val Leu

515 520 525

Lys Lys Cys Lys Ser Ala Phe Tyr Ile Leu Pro Ser Ile Ile Ser Asn

530 535 540

Glu Lys Gln Glu Ile Leu Gly Thr Val Ser Trp Asn Leu Arg Glu Met

545 550 555 560

Leu Ala His Ala Glu Glu Thr Arg Lys Leu Met Pro Val Cys Val Glu

565 570 575

Thr Lys Ala Ile Val Ser Thr Ile Gln Arg Lys Tyr Lys Gly Ile Lys

580 585 590

Ile Gln Glu Gly Val Val Asp Tyr Gly Ala Arg Phe Tyr Phe Tyr Thr

595 600 605

Ser Lys Thr Thr Val Ala Ser Leu Ile Asn Thr Leu Asn Asp Leu Asn

610 615 620

Glu Thr Leu Val Thr Met Pro Leu Gly Tyr Val Thr His Gly Leu Asn

625 630 635 640

Leu Glu Glu Ala Ala Arg Tyr Met Arg Ser Leu Lys Val Pro Ala Thr

645 650 655

Val Ser Val Ser Ser Pro Asp Ala Val Thr Ala Tyr Asn Gly Tyr Leu

660 665 670

Thr Ser Ser Ser Lys Thr Pro Glu Glu His Phe Ile Glu Thr Ile Ser

675 680 685

Leu Ala Gly Ser Tyr Lys Asp Trp Ser Tyr Ser Gly Gln Ser Thr Gln

690 695 700

Leu Gly Ile Glu Phe Leu Lys Arg Gly Asp Lys Ser Val Tyr Tyr Thr

705 710 715 720

Ser Asn Pro Thr Thr Phe His Leu Asp Gly Glu Val Ile Thr Phe Asp

725 730 735

Asn Leu Lys Thr Leu Leu Ser Leu Arg Glu Val Arg Thr Ile Lys Val

740 745 750

Phe Thr Thr Val Asp Asn Ile Asn Leu His Thr Gln Val Val Asp Met

755 760 765

Ser Met Thr Tyr Gly Gln Gln Phe Gly Pro Thr Tyr Leu Asp Gly Ala

770 775 780

Asp Val Thr Lys Ile Lys Pro His Asn Ser His Glu Gly Lys Thr Phe

785 790 795 800

Tyr Val Leu Pro Asn Asp Asp Thr Leu Arg Val Glu Ala Phe Glu Tyr

805 810 815

Tyr His Thr Thr Asp Pro Ser Phe Leu Gly Arg Tyr Met Ser Ala Leu

820 825 830

Asn His Thr Lys Lys Trp Lys Tyr Pro Gln Val Asn Gly Leu Thr Ser

835 840 845

Ile Lys Trp Ala Asp Asn Asn Cys Tyr Leu Ala Thr Ala Leu Leu Thr

850 855 860

Leu Gln Gln Ile Glu Leu Lys Phe Asn Pro Pro Ala Leu Gln Asp Ala

865 870 875 880

Tyr Tyr Arg Ala Arg Ala Gly Glu Ala Ala Asn Phe Cys Ala Leu Ile

885 890 895

Leu Ala Tyr Cys Asn Lys Thr Val Gly Glu Leu Gly Asp Val Arg Glu

900 905 910

Thr Met Ser Tyr Leu Phe Gln His Ala Asn Leu Asp Ser Cys Lys Arg

915 920 925

Val Leu Asn Val Val Cys Lys Thr Cys Gly Gln Gln Gln Thr Thr Leu

930 935 940

Lys Gly Val Glu Ala Val Met Tyr Met Gly Thr Leu Ser Tyr Glu Gln

945 950 955 960

Phe Lys Lys Gly Val Gln Ile Pro Cys Thr Cys Gly Lys Gln Ala Thr

965 970 975

Lys Tyr Leu Val Gln Gln Glu Ser Pro Phe Val Met Met Ser Ala Pro

980 985 990

Pro Ala Gln Tyr Glu Leu Lys His Gly Thr Phe Thr Cys Ala Ser Glu

995 1000 1005

Tyr Thr Gly Asn Tyr Gln Cys Gly His Tyr Lys His Ile Thr Ser

1010 1015 1020

Lys Glu Thr Leu Tyr Cys Ile Asp Gly Ala Leu Leu Thr Lys Ser

1025 1030 1035

Ser Glu Tyr Lys Gly Pro Ile Thr Asp Val Phe Tyr Lys Glu Asn

1040 1045 1050

Ser Tyr Thr Thr Thr Ile Lys Pro Val Thr Tyr Lys Leu Asp Gly

1055 1060 1065

Val Val Cys Thr Glu Ile Asp Pro Lys Leu Asp Asn Tyr Tyr Lys

1070 1075 1080

Lys Asp Asn Ser Tyr Phe Thr Glu Gln Pro Ile Asp Leu Val Pro

1085 1090 1095

Asn Gln Pro Tyr Pro Asn Ala Ser Phe Asp Asn Phe Lys Phe Val

1100 1105 1110

Cys Asp Asn Ile Lys Phe Ala Asp Asp Leu Asn Gln Leu Thr Gly

1115 1120 1125

Tyr Lys Lys Pro Ala Ser Arg Glu Leu Lys Val Thr Phe Phe Pro

1130 1135 1140

Asp Leu Asn Gly Asp Val Val Ala Ile Asp Tyr Lys His Tyr Thr

1145 1150 1155

Pro Ser Phe Lys Lys Gly Ala Lys Leu Leu His Lys Pro Ile Val

1160 1165 1170

Trp His Val Asn Asn Ala Thr Asn Lys Ala Thr Tyr Lys Pro Asn

1175 1180 1185

Thr Trp Cys Ile Arg Cys Leu Trp Ser Thr Lys Pro Val Glu Thr

1190 1195 1200

Ser Asn Ser Phe Asp Val Leu Lys Ser Glu Asp Ala Gln Gly Met

1205 1210 1215

Asp Asn Leu Ala Cys Glu Asp Leu Lys Pro Val Ser Glu Glu Val

1220 1225 1230

Val Glu Asn Pro Thr Ile Gln Lys Asp Val Leu Glu Cys Asn Val

1235 1240 1245

Lys Thr Thr Glu Val Val Gly Asp Ile Ile Leu Lys Pro Ala Asn

1250 1255 1260

Asn Ser Leu Lys Ile Thr Glu Glu Val Gly His Thr Asp Leu Met

1265 1270 1275

Ala Ala Tyr Val Asp Asn Ser Ser Leu Thr Ile Lys Lys Pro Asn

1280 1285 1290

Glu Leu Ser Arg Val Leu Gly Leu Lys Thr Leu Ala Thr His Gly

1295 1300 1305

Leu Ala Ala Val Asn Ser Val Pro Trp Asp Thr Ile Ala Asn Tyr

1310 1315 1320

Ala Lys Pro Phe Leu Asn Lys Val Val Ser Thr Thr Thr Asn Ile

1325 1330 1335

Val Thr Arg Cys Leu Asn Arg Val Cys Thr Asn Tyr Met Pro Tyr

1340 1345 1350

Phe Phe Thr Leu Leu Leu Gln Leu Cys Thr Phe Thr Arg Ser Thr

1355 1360 1365

Asn Ser Arg Ile Lys Ala Ser Met Pro Thr Thr Ile Ala Lys Asn

1370 1375 1380

Thr Val Lys Ser Val Gly Lys Phe Cys Leu Glu Ala Ser Phe Asn

1385 1390 1395

Tyr Leu Lys Ser Pro Asn Phe Ser Lys Leu Ile Asn Ile Ile Ile

1400 1405 1410

Trp Phe Leu Leu Leu Ser Val Cys Leu Gly Ser Leu Ile Tyr Ser

1415 1420 1425

Thr Ala Ala Leu Gly Val Leu Met Ser Asn Leu Gly Met Pro Ser

1430 1435 1440

Tyr Cys Thr Gly Tyr Arg Glu Gly Tyr Leu Asn Ser Thr Asn Val

1445 1450 1455

Thr Ile Ala Thr Tyr Cys Thr Gly Ser Ile Pro Cys Ser Val Cys

1460 1465 1470

Leu Ser Gly Leu Asp Ser Leu Asp Thr Tyr Pro Ser Leu Glu Thr

1475 1480 1485

Ile Gln Ile Thr Ile Ser Ser Phe Lys Trp Asp Leu Thr Ala Phe

1490 1495 1500

Gly Leu Val Ala Glu Trp Phe Leu Ala Tyr Ile Leu Phe Thr Arg

1505 1510 1515

Phe Phe Tyr Val Leu Gly Leu Ala Ala Ile Met Gln Leu Phe Phe

1520 1525 1530

Ser Tyr Phe Ala Val His Phe Ile Ser Asn Ser Trp Leu Met Trp

1535 1540 1545

Leu Ile Ile Asn Leu Val Gln Met Ala Pro Ile Ser Ala Met Val

1550 1555 1560

Arg Met Tyr Ile Phe Phe Ala Ser Phe Tyr Tyr Val Trp Lys Ser

1565 1570 1575

Tyr Val His Val Val Asp Gly Cys Asn Ser Ser Thr Cys Met Met

1580 1585 1590

Cys Tyr Lys Arg Asn Arg Ala Thr Arg Val Glu Cys Thr Thr Ile

1595 1600 1605

Val Asn Gly Val Arg Arg Ser Phe Tyr Val Tyr Ala Asn Gly Gly

1610 1615 1620

Lys Gly Phe Cys Lys Leu His Asn Trp Asn Cys Val Asn Cys Asp

1625 1630 1635

Thr Phe Cys Ala Gly Ser Thr Phe Ile Ser Asp Glu Val Ala Arg

1640 1645 1650

Asp Leu Ser Leu Gln Phe Lys Arg Pro Ile Asn Pro Thr Asp Gln

1655 1660 1665

Ser Ser Tyr Ile Val Asp Ser Val Thr Val Lys Asn Gly Ser Ile

1670 1675 1680

His Leu Tyr Phe Asp Lys Ala Gly Gln Lys Thr Tyr Glu Arg His

1685 1690 1695

Ser Leu Ser His Phe Val Asn Leu Asp Asn Leu Arg Ala Asn Asn

1700 1705 1710

Thr Lys Gly Ser Leu Pro Ile Asn Val Ile Val Phe Asp Gly Lys

1715 1720 1725

Ser Lys Cys Glu Glu Ser Ser Ala Lys Ser Ala Ser Val Tyr Tyr

1730 1735 1740

Ser Gln Leu Met Cys Gln Pro Ile Leu Leu Leu Asp Gln Ala Leu

1745 1750 1755

Val Ser Asp Val Gly Asp Ser Ala Glu Val Ala Val Lys Met Phe

1760 1765 1770

Asp Ala Tyr Val Asn Thr Phe Ser Ser Thr Phe Asn Val Pro Met

1775 1780 1785

Glu Lys Leu Lys Thr Leu Val Ala Thr Ala Glu Ala Glu Leu Ala

1790 1795 1800

Lys Asn Val Ser Leu Asp Asn Val Leu Ser Thr Phe Ile Ser Ala

1805 1810 1815

Ala Arg Gln Gly Phe Val Asp Ser Asp Val Glu Thr Lys Asp Val

1820 1825 1830

Val Glu Cys Leu Lys Leu Ser His Gln Ser Asp Ile Glu Val Thr

1835 1840 1845

Gly Asp Ser Cys Asn Asn Tyr Met Leu Thr Tyr Asn Lys Val Glu

1850 1855 1860

Asn Met Thr Pro Arg Asp Leu Gly Ala Cys Ile Asp Cys Ser Ala

1865 1870 1875

Arg His Ile Asn Ala Gln Val Ala Lys Ser His Asn Ile Ala Leu

1880 1885 1890

Ile Trp Asn Val Lys Asp Phe Met Ser Leu Ser Glu Gln Leu Arg

1895 1900 1905

Lys Gln Ile Arg Ser Ala Ala Lys Lys Asn Asn Leu Pro Phe Lys

1910 1915 1920

Leu Thr Cys Ala Thr Thr Arg Gln Val Val Asn Val Val Thr Thr

1925 1930 1935

Lys Ile Ala Leu Lys Gly Gly

1940 1945

<210> 2

<211> 527

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

<400> 2

Ala Glu Asn Val Thr Gly Leu Phe Lys Asp Cys Ser Lys Val Ile Thr

1 5 10 15

Gly Leu His Pro Thr Gln Ala Pro Thr His Leu Ser Val Asp Thr Lys

20 25 30

Phe Lys Thr Glu Gly Leu Cys Val Asp Ile Pro Gly Ile Pro Lys Asp

35 40 45

Met Thr Tyr Arg Arg Leu Ile Ser Met Met Gly Phe Lys Met Asn Tyr

50 55 60

Gln Val Asn Gly Tyr Pro Asn Met Phe Ile Thr Arg Glu Glu Ala Ile

65 70 75 80

Arg His Val Arg Ala Trp Ile Gly Phe Asp Val Glu Gly Cys His Ala

85 90 95

Thr Arg Glu Ala Val Gly Thr Asn Leu Pro Leu Gln Leu Gly Phe Ser

100 105 110

Thr Gly Val Asn Leu Val Ala Val Pro Thr Gly Tyr Val Asp Thr Pro

115 120 125

Asn Asn Thr Asp Phe Ser Arg Val Ser Ala Lys Pro Pro Pro Gly Asp

130 135 140

Gln Phe Lys His Leu Ile Pro Leu Met Tyr Lys Gly Leu Pro Trp Asn

145 150 155 160

Val Val Arg Ile Lys Ile Val Gln Met Leu Ser Asp Thr Leu Lys Asn

165 170 175

Leu Ser Asp Arg Val Val Phe Val Leu Trp Ala His Gly Phe Glu Leu

180 185 190

Thr Ser Met Lys Tyr Phe Val Lys Ile Gly Pro Glu Arg Thr Cys Cys

195 200 205

Leu Cys Asp Arg Arg Ala Thr Cys Phe Ser Thr Ala Ser Asp Thr Tyr

210 215 220

Ala Cys Trp His His Ser Ile Gly Phe Asp Tyr Val Tyr Asn Pro Phe

225 230 235 240

Met Ile Asp Val Gln Gln Trp Gly Phe Thr Gly Asn Leu Gln Ser Asn

245 250 255

His Asp Leu Tyr Cys Gln Val His Gly Asn Ala His Val Ala Ser Cys

260 265 270

Asp Ala Ile Met Thr Arg Cys Leu Ala Val His Glu Cys Phe Val Lys

275 280 285

Arg Val Asp Trp Thr Ile Glu Tyr Pro Ile Ile Gly Asp Glu Leu Lys

290 295 300

Ile Asn Ala Ala Cys Arg Lys Val Gln His Met Val Val Lys Ala Ala

305 310 315 320

Leu Leu Ala Asp Lys Phe Pro Val Leu His Asp Ile Gly Asn Pro Lys

325 330 335

Ala Ile Lys Cys Val Pro Gln Ala Asp Val Glu Trp Lys Phe Tyr Asp

340 345 350

Ala Gln Pro Cys Ser Asp Lys Ala Tyr Lys Ile Glu Glu Leu Phe Tyr

355 360 365

Ser Tyr Ala Thr His Ser Asp Lys Phe Thr Asp Gly Val Cys Leu Phe

370 375 380

Trp Asn Cys Asn Val Asp Arg Tyr Pro Ala Asn Ser Ile Val Cys Arg

385 390 395 400

Phe Asp Thr Arg Val Leu Ser Asn Leu Asn Leu Pro Gly Cys Asp Gly

405 410 415

Gly Ser Leu Tyr Val Asn Lys His Ala Phe His Thr Pro Ala Phe Asp

420 425 430

Lys Ser Ala Phe Val Asn Leu Lys Gln Leu Pro Phe Phe Tyr Tyr Ser

435 440 445

Asp Ser Pro Cys Glu Ser His Gly Lys Gln Val Val Ser Asp Ile Asp

450 455 460

Tyr Val Pro Leu Lys Ser Ala Thr Cys Ile Thr Arg Cys Asn Leu Gly

465 470 475 480

Gly Ala Val Cys Arg His His Ala Asn Glu Tyr Arg Leu Tyr Leu Asp

485 490 495

Ala Tyr Asn Met Met Ile Ser Ala Gly Phe Ser Leu Trp Val Tyr Lys

500 505 510

Gln Phe Asp Thr Tyr Asn Leu Trp Asn Thr Phe Thr Arg Leu Gln

515 520 525

<210> 3

<211> 298

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

<400> 3

Ser Ser Gln Ala Trp Gln Pro Gly Val Ala Met Pro Asn Leu Tyr Lys

1 5 10 15

Met Gln Arg Met Leu Leu Glu Lys Cys Asp Leu Gln Asn Tyr Gly Asp

20 25 30

Ser Ala Thr Leu Pro Lys Gly Ile Met Met Asn Val Ala Lys Tyr Thr

35 40 45

Gln Leu Cys Gln Tyr Leu Asn Thr Leu Thr Leu Ala Val Pro Tyr Asn

50 55 60

Met Arg Val Ile His Phe Gly Ala Gly Ser Asp Lys Gly Val Ala Pro

65 70 75 80

Gly Thr Ala Val Leu Arg Gln Trp Leu Pro Thr Gly Thr Leu Leu Val

85 90 95

Asp Ser Asp Leu Asn Asp Phe Val Ser Asp Ala Asp Ser Thr Leu Ile

100 105 110

Gly Asp Cys Ala Thr Val His Thr Ala Asn Lys Trp Asp Leu Ile Ile

115 120 125

Ser Asp Met Tyr Asp Pro Lys Thr Lys Asn Val Thr Lys Glu Asn Asp

130 135 140

Ser Lys Glu Gly Phe Phe Thr Tyr Ile Cys Gly Phe Ile Gln Gln Lys

145 150 155 160

Leu Ala Leu Gly Gly Ser Val Ala Ile Lys Ile Thr Glu His Ser Trp

165 170 175

Asn Ala Asp Leu Tyr Lys Leu Met Gly His Phe Ala Trp Trp Thr Ala

180 185 190

Phe Val Thr Asn Val Asn Ala Ser Ser Ser Glu Ala Phe Leu Ile Gly

195 200 205

Cys Asn Tyr Leu Gly Lys Pro Arg Glu Gln Ile Asp Gly Tyr Val Met

210 215 220

His Ala Asn Tyr Ile Phe Trp Arg Asn Thr Asn Pro Ile Gln Leu Ser

225 230 235 240

Ser Tyr Ser Leu Phe Asp Met Ser Lys Phe Pro Leu Lys Leu Arg Gly

245 250 255

Thr Ala Val Met Ser Leu Lys Glu Gly Gln Ile Asn Asp Met Ile Leu

260 265 270

Ser Leu Leu Ser Lys Gly Arg Leu Ile Ile Arg Glu Asn Asn Arg Val

275 280 285

Val Ile Ser Ser Asp Val Leu Val Asn Asn

290 295

<210> 4

<211> 1273

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

<400> 4

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu

1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 5

<211> 75

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

<400> 5

Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser

1 5 10 15

Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala

20 25 30

Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

35 40 45

Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

50 55 60

Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val

65 70 75

<210> 6

<211> 419

<212> PRT

<213> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

<400> 6

Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr

1 5 10 15

Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg

20 25 30

Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

35 40 45

Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

50 55 60

Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

65 70 75 80

Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

85 90 95

Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

100 105 110

Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

115 120 125

Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

130 135 140

His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

145 150 155 160

Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

165 170 175

Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

180 185 190

Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

195 200 205

Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

210 215 220

Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

225 230 235 240

Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

245 250 255

Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

275 280 285

Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

290 295 300

Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

305 310 315 320

Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala

325 330 335

Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

340 345 350

Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

370 375 380

Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

385 390 395 400

Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser

405 410 415

Thr Gln Ala

<210> 7

<211> 703

<212> DNA

<213> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

<400> 7

caaatttgca ctgacttgct ttagcactca atttgctttt gcttgtcctg acggcgtaaa 60

acacgtctat cagttacgtg ccagatcagt ttcacctaaa ctgttcatca gacaagagga 120

agttcaagaa ctttactctc caatttttct tattgttgcg gcaatagtgt ttataacact 180

ttgcttcaca ctcaaaagaa agacagaatg attgaacttt cattaattga cttctatttg 240

tgctttttag cctttctgct attccttgtt ttaattatgc ttattatctt ttggttctca 300

cttgaactgc aagatcataa tgaaacttgt cacgcctaaa cgaacatgaa atttcttgtt 360

ttcttaggaa tcatcacaac tgtagctgca tttcaccaag aatgtagttt acagtcatgt 420

actcaacatc aaccatatgt agttgatgac ccgtgtccta ttcacttcta ttctaaatgg 480

tatattagag taggagctag aaaatcagca cctttaattg aattgtgcgt ggatgaggct 540

ggttctaaat cacccattca gtacatcgat atcggtaatt atacagtttc ctgtttacct 600

tttacaatta attgccagga acctaaattg ggtagtcttg tagtgcgttg ttcgttctat 660

gaagactttt tagagtatca tgacgttcgt gttgttttag att 703

Claims (14)

1. A β -coronavirus temperature-sensitive strain comprising a non-structural protein having a mutation of (b), (e) a combination of mutations of (f), and/or (h) as responsible mutation for temperature-sensitive performance:

(b) Mutation of the amino acid residue corresponding to leucine at position 445 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3,

(e) Mutation of an amino acid residue corresponding to glycine 248 of 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 of 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 of the amino acid sequence shown in SEQ ID NO. 3 in NSP 16.

2. The virus temperature sensitive strain of claim 1, wherein the beta coronavirus is SARS-CoV-2 virus.

3. The virus temperature-sensitive strain according to claim 1 or 2, wherein the proliferation potency at the lower respiratory tract temperature of a human is reduced compared to the proliferation potency of a β -coronavirus comprising a non-structural protein without the liability mutation.

4. A strain according to claim 3, wherein the human lower respiratory tract temperature is 36-38 ℃.

5. The virus temperature-sensitive strain according to any one of claims 1 to 4, wherein the mutation of (b) is a substitution to phenylalanine, the mutation of (e) is a substitution to valine, the mutation of (f) is a substitution to serine, and the mutation of (h) is a substitution to isoleucine.

6. The virus temperature-sensitive strain according to any one of claims 1 to 5, wherein the virus temperature-sensitive strain comprises:

said NSP3 having said mutation of (b) in the amino acid sequence shown in said SEQ ID NO. 1,

said NSP14 having said mutation of (e) and said mutation of (f) in the amino acid sequence shown in said SEQ ID NO. 2, and/or

NSP16 having the mutation of (h) in the amino acid sequence shown in the sequence No. 3.

7. The virus temperature-sensitive strain according to any one of claims 1 to 6, wherein the virus temperature-sensitive strain has the mutation of (e) and the mutation of (f).

8. The virus temperature-sensitive strain according to any one of claims 1 to 6, wherein the virus temperature-sensitive strain has the mutation of (h).

9. The virus temperature-sensitive strain according to any one of claims 1 to 6, wherein the virus temperature-sensitive strain has the mutation of (b).

10. An attenuated live vaccine comprising the virus temperature sensitive strain of any one of claims 1-9.

11. The live attenuated vaccine of claim 10, wherein the live attenuated vaccine is capable of nasal administration.

12. The live attenuated vaccine of claim 10, wherein the live attenuated vaccine is capable of intramuscular, subcutaneous, or intradermal administration.

13. A beta coronavirus gene vaccine comprising a gene encoding a non-structural protein having the following mutations of (b), (e) and (f) in combination, and/or (h) as responsible mutations for temperature sensitive performance:

(b) Mutation of the amino acid residue corresponding to leucine at position 445 of the amino acid sequence shown in SEQ ID NO. 1 in NSP3,

(e) Mutation of an amino acid residue corresponding to glycine 248 of 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 of 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 of the amino acid sequence shown in SEQ ID NO. 3 in NSP 16.

14. The genetic vaccine of claim 13, wherein the genetic vaccine is capable of nasal, intramuscular, subcutaneous, or intradermal administration.