Patent Application
20230381296
The present invention relates to temperature-sensitive strain of betacoronavirus and a vaccine using the same.
The infection with the novel coronavirus (SARS-CoV-2) as a pathogen that occurred in Wuhan, China in 2019 (COVID-19) has caused a pandemic worldwide and is a major social problem. For this reason, vaccine development for SARS-CoV-2 is progressing rapidly around the world. The only vaccine that has been approved as of October 2020 is Sputnik V that has been approved in Russia (Non-Patent Document 1).
Non-Patent Document 1: THE LANCET, VOLUME 396, ISSUE 10255, P887-897, SEP. 26, 2020
However, Sputnik V is still in the stage of conducting clinical trials, and there are raised questions about safety and efficacy. In addition, even if vaccination is started, there is still a risk that the effect is small or heavy side effects are found, and it is not clear whether the vaccination is a decisive blow to prevent the spread of an infection. Thus, additional options for vaccines against the SARS-CoV-2 are desired.
Therefore, an object of the present invention is to provide at least a strain that is effective as an active ingredient of a vaccine against SARS-CoV-2. In addition, since betacoronaviruses such as SARS-CoV-2 include viruses that may be present other than SARS-CoV-2, it is an object of the present invention to provide a strain that is effective as an active ingredient of a vaccine against betacoronaviruses in general. MEANS FOR SOLVING THE PROBLEM
As a result of intensive studies, the present inventors have found that proliferation of specific mutants strain of SARS-CoV-2 at a human body temperature (so-called lower respiratory tract temperature) is decreased, and further have found that specific responsible mutation(s) can cause a decrease in growth of betacoronaviruses in general at a human body temperature (so-called lower respiratory tract temperature) by performing a reverse mutation test on the predetermined mutant strain. The present invention has been completed by further conducting studies based on these findings.
In the present invention, “temperature sensitivity” is a property having a growth capability specific to a low temperature (i.e., human upper respiratory tract temperature), and is a property exhibited by acquiring a property in which a growth capability at a high temperature (i.e., human lower respiratory tract temperature) is limited. As used herein, the term “cold adaptation” is used in the meaning of acquiring a property having a growth capability specific to a low temperature (i.e., human upper respiratory tract temperature), and in its actual state, the term is used in the meaning of “temperature sensitization” since the growth capability specific to a low temperature is exhibited by acquiring a property in which a growth capability at a high temperature (i.e., human lower respiratory tract temperature) is limited. In addition, a “temperature-sensitized” strain is referred to as a “temperature-sensitive strain”. Therefore, as used herein, the “cold-adapted strain” and the “temperature-sensitive strain” have the same meaning. In addition, in the present invention, the “responsible mutations” refer to mutations that can cause a mutant phenotype (obtained as a result of a change by mutations in a phenotype representing a trait of an organism) or have a causal relationship with the mutant phenotype, and “responsible mutations for temperature-sensitive capability” refer to mutations that can cause acquisition of temperature sensitivity or has a causal relationship with acquisition of temperature sensitivity. The present invention provides inventions of the following embodiments.
Item 1. A betacoronavirus temperature-sensitive strain containing non-structural protein(s) having the following mutation of (b), a combination of the following mutations of (e) and (f), and/or the following mutation of (h) as responsible mutation(s) for temperature-sensitive capability:
Examples of the invention of the above item 1 include the following inventions.
A betacoronavirus temperature-sensitive strain containing non-structural protein(s) consisting of at least any one of the following polypeptides (I), (II), and (III):
Item 2. The virus temperature-sensitive strain according to item 1, wherein the betacoronavirus is SARS-CoV-2.
Item 3. The virus temperature-sensitive strain according to item 1 or 2, wherein a growth capability at a human lower respiratory tract temperature is decreased as compared with a growth capability of a betacoronavirus containing non-structural protein(s) not having the responsible mutations.
Item 4. The virus temperature-sensitive strain according to item 3, wherein the human lower respiratory tract temperature is 36 to 38° C.
Item 5. The virus temperature-sensitive strain according to any one of items 1 to 4, wherein the mutation of (b) is a substitution with phenylalanine, the mutation of (e) is a substitution with valine, the mutation of (f) is a substitution with serine, and the mutation of (h) is a substitution with isoleucine.
Item 6. The virus temperature-sensitive strain according to any one of items 1 to 5, containing:
Item 7. The virus temperature-sensitive strain according to any one of items 1 to 6, having the mutation of (e) and the mutation of (f).
Item 8. The virus temperature-sensitive strain according to any one of items 1 to 6, having the mutation of (h).
Item 9. The virus temperature sensitive according to any one of items 1 to 6, having the mutation of (b).
Item 10. A live attenuated vaccine containing the virus temperature-sensitive strain according to any one of items 1 to 9.
Item 11. The live attenuated vaccine according to claim 10, which is administered nasally.
Item 12. The live attenuated vaccine according to item 10, which is administered intramuscularly, subcutaneously, or intradermally.
Item 13. A betacoronavirus gene vaccine containing a gene encoding non-structural protein(s) having the following mutation of (b), a combination of the following mutations of (e) and (f), and/or the following mutation of (h) as responsible mutation(s) for temperature-sensitive capability:
Item 14. The gene vaccine according to item 13, which is administered nasally, intramuscularly, subcutaneously, or intradermally.
According to the present invention, there is provided a strain that is effective as an active ingredient of a vaccine against a betacoronavirus.
A betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention is a betacoronavirus containing non-structural protein(s) having predetermined mutations as responsible mutation(s) for temperature-sensitive capability, and is characterized by being temperature-sensitive.
The coronavirus is morphologically spherical with a diameter of about 100 to 200 nm and has protrusions on the surface. The coronavirus is virologically classified into Nidovirales, Coronavirinae, Coronaviridae. In the envelope of the lipid bilayer membrane, there is a genome of positive-stranded single-stranded RNA wound around a nucleocapsid protein (also referred to as a nucleocapsid), and a spike protein (hereinafter also referred to as a “spike”), an envelope protein (hereinafter also referred to as an “envelope”), and a membrane protein are arranged on the surface of the envelope. The size of the viral genome is about 30 kb, the longest among RNA viruses.
Coronaviruses are classified into groups of alpha, beta, gamma, and delta from genetic characteristics. As coronaviruses infecting humans, four types of human coronaviruses 229E, OC43, NL63, and HKU-1 as causative viruses of cold, and severe acute respiratory syndrome (SARS) coronavirus that occurred in 2002 and Middle East respiratory syndrome (MERS) coronavirus that occurred in 2012, which cause serious pneumonia, are known. Human coronaviruses 229E and NL63 are classified into Alphacoronavirus genus, and human coronaviruses OC43, HKU-1, SARS coronavirus, and MERS coronavirus are classified into the Betacoronavirus genus.
SARS-CoV-2 classified as SARS coronavirus has been isolated and identified as a causative virus of the novel coronavirus infection that occurred in Wuhan in 2019. SARS-CoV-2 has been mutated repeatedly from the early Wuhan strain, and variant strains such as a strain detected in the United Kingdom, a strain detected in South Africa, and a strain detected in India have been found. There is also a possibility that there is a variant strain that has not yet been detected or a new variant strain will occur in the future. In the present invention, viruses included in the Betacoronavirus genus are not limited to the above SARS-CoV-2 strain, and include all other betacoronaviruses (other SARS-CoV-2 variant strains that will be newly detected in the future and betacoronaviruses other than SARS-CoV-2).
The predetermined mutations possessed by the betacoronavirus temperature-sensitive strain (cold-adapted strains) of the present invention is described based on Table 1 below. Mutation (b), a combination of mutation (e) and mutation (f), and/or mutation (h) indicated as “Responsible mutation” in Table 1 are responsible mutations for temperature sensitivity (cold adaptation capability) essentially contained in the betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention. Mutations (a), (c), (d), (g), and (i) to (m) indicated as “Other mutation” in Table 1 are mutations that can be optionally contained in the betacoronavirus temperature-sensitive strains (cold-adapted strain) of the present invention, and the betacoronavirus temperature-sensitive strains (cold-adapted strain) of the present invention may or may not contain at least any one of other mutations.
In other words, the responsible mutation(s) for temperature-sensitive capability possessed by the betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention is/are the following mutation of (b), a combination of the following mutations of (e) and (f), and/or the following mutation of (h).
The betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention can further contain at least any one of the following mutations of (a), (c), (d), (g), and (i) to (m) as other mutation(s) in addition to the above responsible mutations.
SEQ ID NO: 1 is an amino acid sequence of NSP3 in SARS-CoV-2 of NC_045512 (NCBI); SEQ ID NO: 2 is an amino acid sequence of NSP14 in SARS-CoV-2 of NC_045512 (NCBI); and SEQ ID NO: 3 is an amino acid sequence of NSP16 in SARS-CoV-2 of NC_045512 (NCBI).
In addition, SEQ ID NO: 4 is an amino acid sequence of a spike in SARS-CoV-2 of NC_045512 (NCBI); SEQ ID NO: 5 is an amino acid sequence of an envelope in SARS-CoV-2 of NC_045512 (NCBI); and SEQ ID NO: 6 is an amino acid sequence of a nucleocapsid in SARS-CoV-2 of NC_045512 (NCBI).
“Corresponding” means that there is(are) mutation(s) at the above predetermined position(s) in the amino acid sequence of SEQ ID NOs: 1 to 3 or 1 to 6 when the betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention is a mutant strain of SARS-CoV-2 of NC_045512 (NCBI), and that there is(are) mutation(s) at position(s) corresponding to the above predetermined position(s) in the amino acid sequence corresponding to SEQ ID NOs: 1 to 3 or 1 to 6 of the polypeptide possessed by another betacoronavirus variant when the betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention is another betacoronavirus mutant strain other than the above variant. The corresponding position(s) can be identified by aligning amino acid sequences for proteins of SEQ ID NOs: 1 to 3 or 1 to 6 of SARS-CoV-2 of NC_045512 (NCBI) and proteins of another betacoronavirus mutant strain corresponding to the proteins of SEQ ID NOs: 1 to 3 or 1 to 6.
The virus temperature-sensitive strain (cold-adapted strain) of the present invention is not limited to a mutant strain of the SARS-CoV-2 listed in NC_045512 (NCBI) as long as an amino acid residue corresponding to the above predetermined positions in the amino acid sequence of SEQ ID NOs: 1 to 3 or 1 to 6 is mutated, and includes other betacoronavirus mutant strains (i.e., other any variants of SARS-CoV-2 and mutant strains of viruses other than SARS-CoV-2 included in the Betacoronavirus genus). The mutant strains of the SARS-CoV-2 listed in NC_045512 (NCBI) are defined as mutant strains in which at least any one of amino acid residues at the above determined positions in the amino acid sequence represented by SEQ ID NOs: 1 to 3 or 1 to 6 in the specific SARS-CoV-2 is mutated, and the other betacoronavirus mutant strains refer to both any other mutant strains of SARS-CoV-2 (i.e., mutant strains in which amino acid residues corresponding to the above predetermined positions in the amino acid sequence corresponding to SEQ ID NOs: 1 to 3 or 1 to 6 in any other SARS-CoV-2 are mutated) and mutant strains of viruses other than SARS-CoV-2 included in the Betacoronavirus genus (i.e., mutant strains in which amino acid residues corresponding to the above predetermined positions in the amino acid sequence corresponding to SEQ ID NOs: 1 to 3 or 1 to 6 in viruses other than SARS-CoV-2 included in the Betacoronavirus genus are mutated).
Each amino acid sequence corresponding to SEQ ID NOs: 1 to 3 or 1 to 6 in other betacoronavirus mutant strains is allowed to differ from the amino acid sequence set forth in SEQ ID NOs: 1 to 3 or 1 to 6, unless it significantly affects the properties of the polypeptide. The phrase “does not significantly affect the properties of the polypeptide” refers to a state in which a function as a non-structural protein or a structural protein of each polypeptide is maintained. Specifically, at a site other than an amino acid corresponding to the responsible mutations in SEQ ID NOs: 1 to 3 described above, or in the case of further having other mutations, at site(s) other than amino acid residue(s) corresponding to the responsible mutation(s) and another mutation in SEQ ID NOs: 1 to 6 described above (hereinafter, a site other than amino acids corresponding to these mutations is also referred to as an “any difference site”), a difference from SEQ ID NOs: 1 to 3 or 1 to 6 is acceptable. The acceptable difference may be one type of difference selected from substitution, addition, insertion, and deletion (e.g., substitution), or may include two or more types of difference (e.g., substitution and insertion). A sequence identity calculated by comparing only any difference sites of the amino acid sequences corresponding to SEQ ID NOs: 1 to 3 or 1 to 6 described above in any other SARS-CoV-2 and the amino acid sequences set forth in SEQ ID NOs: 1 to 3 or 1 to 6 may be 50% or more. In the any other SARS-CoV-2, the sequence identity is preferably 60% or more or 70% or more, more preferably 80% or more, further 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, and particularly preferably 99.3% or more, 99.5% or more, 99.7% or more, or 99.9% or more. In any other betacoronaviruses, the sequence identity preferably includes 60% or more. Here, the “sequence identity” refers to a value of identity of an amino acid sequence obtained by bl2seq program of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247?250, 1999). Parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
In other words, the betacoronavirus temperature-sensitive strain (cold-adapted strain) of the present invention is more specifically as follows:
A betacoronavirus temperature-sensitive (cold-adapted) strain containing non-structural proteins consisting of at least any one of the following polypeptides (I), (II), and (III):
When the more specific betacoronavirus temperature-sensitive strain (cold-adapted strain) described above contains other mutations in addition to responsible mutation(s), as shown below, the polypeptides (non-structural proteins) (I-1) and (I-2) described above may be the polypeptides (I-1a) and (I-2a) described below that also have other mutation in addition to responsible mutation(s), respectively, and the polypeptide (I) described above may further contain the polypeptides (structural proteins) (I-4a) to (I-6a) described below that have other mutations.
A betacoronavirus temperature-sensitive (cold-adapted) strain containing structural protein(s), or structural protein(s) and non-structural protein(s) consisting of at least any one of the following polypeptides (I), (II), and (III):
The above mutations of (a′) to (m′) refer to mutations when the mutations of (a) to (m) are specifically present in the amino acid sequences of SEQ ID NOs: 1 to 6, respectively. In other words, the above polypeptide (I) is a polypeptide obtained by introducing responsible mutations, or in addition thereto, other mutation into polypeptides consisting of the amino acid sequence of SEQ ID NOs: 1 to 6 possessed by SARS-CoV-2 of NC_045512 (NCBI). In addition, the above polypeptides (II) and (III) are obtained by introducing responsible mutations, or in addition thereto, other mutations into polypeptides consisting of amino acid sequences corresponding to the amino acid sequence of SEQ ID NOs: 1 to 6 possessed by another betacoronavirus. Preferred ranges of the sequence identity of the above polypeptides (II) and (III) are as described above.
Having the above responsible mutations, the betacoronavirus can acquire a temperature-sensitive (cold-adapted) property. In the virus temperature-sensitive strain (cold-adapted strain) of the present invention, a growth capability at a human lower respiratory tract temperature is at least decreased as compared with a growth capability at a temperature lower than a human lower respiratory tract temperature, and preferably, the virus temperature-sensitive strain (cold-adapted strain) of the present invention does not have a growth capability at a human lower respiratory tract temperature. In the present invention, the temperature-sensitive (cold adaptation) capability can be confirmed by the fact that a virus titer (TCID50/mL) in culture supernatant after Vero cells are infected with the virus temperature-sensitive strain at MOI=0.01 at a human lower respiratory tract temperature and then the virus temperature-sensitive strain is cultured for 1 day at a human lower respiratory tract temperature is decreased, for example, by 102 or more, preferably by 103 or more, as compared with a virus titer in culture supernatant after Vero cells are infected with the virus temperature-sensitive strain at MOI=0.01 at a human upper respiratory tract temperature and then the virus temperature-sensitive strain is cultured for 1 day at a human upper respiratory tract temperature.
Typically, in the virus temperature-sensitive strain (cold-adapted strain) of the present invention, a growth capability at a human lower respiratory tract temperature is decreased as compared with a growth capability at a human lower respiratory tract temperature in the case of not having the above responsible mutations. This can be confirmed by the fact that a virus titer (TCID50/mL) in culture supernatant after Vero cells are infected with the virus temperature-sensitive strain at MOT=0.01 at a human lower respiratory tract temperature and then the virus temperature-sensitive strain is cultured for 1 day at a human lower respiratory tract temperature is decreased, for example, by 102 or more, preferably by 103 or more, as compared with a virus titer in culture supernatant after Vero cells are infected with a strain not having the above responsible mutations at MOT=0.01 at a human lower respiratory tract temperature and then the strain is cultured for 1 day at a human lower respiratory tract temperature.
Representative examples of the human lower respiratory tract temperature include about 37° C., and specifically include a temperature higher than the upper respiratory tract temperature described below, preferably 36 to 38° C., and more preferably 36.5 to 37.5° C. or 37 to 38° C. In addition, the virus temperature-sensitive strain (cold-adapted strain) of the present invention may have a growth capability at a temperature lower than a human lower respiratory tract temperature. For example, the temperature lower than the human lower respiratory tract temperature may include, for example, a human upper respiratory tract temperature (as a specific example, about 32° C. to 35.5° C.).
The above responsible mutations are not present on receptor-binding domains of a spike protein present on a surface of a virus, which is important when the virus infects cells. Therefore, it is reasonably expected that not only the SARS-CoV-2 listed in NC_045512 (NCBI) but also other betacoronaviruses can be made temperature-sensitive by introducing the above responsible mutations. In other words, even if a mutation occurs that alters the immunogenicity of the virus due to worldwide infection, it is reasonably expected that temperature sensitivity can be imparted to the mutant virus by further introducing the above responsible mutations into the mutant virus.
Regarding the responsible mutations, the above mutation of (b) may be a substitution with an amino acid residue other than leucine, the above mutation of (e) may be a substitution with an amino acid residue other than glycine, the above mutation of (f) may be a substitution with an amino acid residue other than glycine, and the above mutation of (h) may be a substitution with an amino acid residue other than valine. Regarding other mutations, the above mutation of (a) may be a substitution with an amino acid residue other than valine, the above mutation of (c) may be a substitution with an amino acid residue other than lysine, the above mutation of (d) may be a substitution with an amino acid residue other than aspartic acid, the above mutation of (g) may be a substitution with an amino acid residue other than alanine, the above mutation of (i) may be a substitution with an amino acid residue other than leucine, the above mutation of (j) may be a substitution with an amino acid residue other than threonine, the above mutation of (k) may be a substitution with an amino acid residue other than alanine, the above mutation of (l) may be a substitution with an amino acid residue other than leucine, and the above mutation of (m) may be a substitution with an amino acid residue other than serine.
In a preferred example of the virus temperature-sensitive strain (cold-adapted strain) of the present invention, regarding the responsible mutation, the mutation of (b) is a substitution with phenylalanine, the mutation of (e) is a substitution with valine and the mutation of (f) is a substitution with serine, and/or the mutation of (h) is a substitution with isoleucine. When the preferred example further has other mutations, regarding the other mutations, the mutation of (a) is a substitution with alanine, the mutation of (c) is a substitution with arginine, the mutation of (d) is a substitution with asparagine, the mutation of (g) is a substitution with valine, the mutation of (i) is a substitution with tryptophan, the mutation of (j) is a substitution with lysine, the mutation of (k) is a substitution with valine, the mutation of (l) is a substitution with proline, and/or the mutation of (m) is a substitution with phenylalanine.
In another example of the virus temperature-sensitive strain (cold-adapted strain) of the present invention, the substitution may be a so-called conservative replacement. The conservative substitution refers to a substitution with an amino acid having a similar structure and/or property, and examples of the conservative substitution include a substitution with another non-polar amino acid if the amino acid before substitution is a non-polar amino acid, a substitution with another non-charged amino acid if the amino acid before substitution is a non-charged amino acid, a substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and a substitution with another basic amino acid if the amino acid before substitution is a basic amino acid. In general, the “non-polar amino acid” includes alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan, the “non-charged amino acid” includes glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, the “acidic amino acid” includes aspartic acid and glutamic acid, and the “basic amino acid” includes lysine, arginine, and histidine.
A more preferred example of the virus temperature-sensitive strain (cold-adapted strain) of the present invention is a mutant strain of the SARS-CoV-2 listed in NC_045512 (NCBI), wherein the mutation of (b) (i.e., the mutation of (b′)) is a substitution of leucine at position 445 of an amino acid sequence set forth in SEQ ID NO: 1 with phenylalanine in NSP3 (L445F); the mutation of (e) (i.e., the mutation of (e′)) is a substitution of glycine at position 248 of an amino acid sequence set forth in SEQ ID NO: 2 with valine in NSP14 (G248V), and the mutation of (f) (i.e., the mutation of (f′)) is a substitution of glycine at position 416 of an amino acid sequence set forth in SEQ ID NO: 2 with serine in NSP14 (G416S); and/or the mutation of (h) (i.e., the mutation of (h′)) is a substitution of valine at position 67 of an amino acid sequence set forth in SEQ ID NO: 3 with isoleucine in NSP16 (V67I). An example when the more preferred example also has other mutations is the mutant strain, wherein the mutation of (a) (i.e., the mutation of (a′)) is a substitution of valine at position 404 of an amino acid sequence set forth in SEQ ID NO: 1 with alanine in NSP3 (V404A); the mutation of (c) (i.e., the mutation of (c′)) is a substitution of lysine at position 1792 in an amino acid sequence set forth in SEQ ID NO: 1 with arginine in NSP3 (K1792R); the mutation of (d) (i.e., the mutation of (d′)) is a substitution of aspartic acid at position 1832 of an amino acid sequence set forth in SEQ ID NO: 1 with asparagine in NSP3 (D1832N); the mutation of (g) (i.e., the mutation of (g′)) is a substitution of alanine at position 504 of an amino acid sequence set forth in SEQ ID NO: 2 with valine in NSP14 (A504V); the mutation of (i) (i.e., the mutation of (i′)) is a substitution of leucine at position 54 of an amino acid sequence set forth in SEQ ID NO: 4 with tryptophan in a spike (L54W); the mutation of (j) (i.e., the mutation of (j′)) is a substitution of threonine at position 739 of an amino acid sequence set forth in SEQ ID NO: 4 with lysine in a spike (T739K); the mutation of (k) (i.e., the mutation of (k′)) is a substitution of alanine at position 879 of an amino acid sequence set forth in SEQ ID NO: 4 with valine in a spike (A879V); the mutation of (l) (i.e., the mutation of (l′)) is a substitution of leucine at position 28 of an amino acid sequence set forth in SEQ ID NO: 5 with proline in an envelope (L28P); and/or the mutation of (m) (i.e., the mutation of (m′)) is a substitution of serine at position 2 of an amino acid sequence set forth in SEQ ID NO: 6 with phenylalanine in a nucleocapsid (S2F).
The betacoronavirus temperature-sensitive (cold-adapted) strain of the present invention may further have a deletion of an amino acid sequence encoded by a base sequence set forth in SEQ ID NO: 7. The base sequence set forth in SEQ ID NO: 7 is a part of an open reading frame of the SARS-CoV-2, NC_045512 (NCBI).
Particularly preferred examples of the virus temperature-sensitive strain (cold-adapted strain) of the present invention include the following strains.
As described above, since the betacoronavirus temperature-sensitive strain (cold-adapted strain) described in “1. Betacoronavirus temperature-sensitive strain (cold-adapted strain)” can efficiently proliferate only at a temperature lower than a human lower respiratory tract temperature, it can be expected that it cannot efficiently proliferate at least in a deep part of a living body, especially in the lower respiratory tract including the lung that causes serious disorders, and pathogenicity is significantly decreased. Therefore, the virus temperature-sensitive strain (cold-adapted strain) can be used as a live attenuated vaccine by infecting a living body as an attenuated virus itself. Therefore, the present invention also provides a vaccine containing the above betacoronavirus temperature-sensitive strain (cold-adapted strain) as an active ingredient. Details of the active ingredient are as described in “1. Betacoronavirus temperature-sensitive strain (cold-adapted strain)”.
As described in the above “1. Betacoronavirus temperature-sensitive strain (cold-adapted strain)”, the predetermined mutations contribute to imparting a temperature-sensitive (cold adaptation) capability. Therefore, the present invention also provides a betacoronavirus gene-based vaccine containing, as an active ingredient, a gene encoding non-structural protein(s) having the above responsible mutation(s) for a temperature-sensitive capability. Details of the responsible mutation(s) for a temperature-sensitive capability contained in the active ingredient are as described in “1. Betacoronavirus temperature-sensitive strain (cold-adapted strain)”.
The vaccine of the present invention can be reasonably expected to be effective against not only the early Wuhan strain of SARS-CoV-2 but also a wide range of SARS-CoV-2 virus-associated strains including the variants detected in the United Kingdom in September 2020 and detected in South Africa in October 2020, and other known variants, as well as unknown mutant strains yet to be detected, and viruses other than SARS-CoV-2 included in the Betacoronavirus genus. Therefore, the vaccine of the present invention targets betacoronaviruses.
The vaccine of the present invention can contain other components such as adjuvants, buffer, tonicity agents, soothing agents, preservatives, antioxidants, flavoring agents, light-absorbing dye, stabilizers, carbohydrate, casein digest, and various vitamins, in addition to the above active ingredients, according to the purpose, use, and the like.
Examples of the adjuvants include animal oils (squalene and the like) or hardened oils thereof; vegetable oils (palm oil, castor oil, and the like) or hardened oils thereof; oily adjuvants including anhydrous mannitol/oleic acid ester, liquid paraffin, polybutene, caprylic acid, oleic acid, higher fatty acid ester, and the like; water-soluble adjuvants such as PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymer, methacrylic acid copolymer, maleic anhydride copolymer, alkenyl derivative polymer, oil-in-water emulsion, and cationic lipid containing quaternary ammonium salt; precipitating adjuvants including aluminum salts such as aluminum hydroxide (alum), aluminum phosphate, and aluminum sulfate or combinations thereof, sodium hydroxide, and the like; microorganism-derived toxin components such as cholera toxin and E. coli heat-labile toxin; and other components (bentonite, muramyl dipeptide derivative, interleukin, and the like).
Examples of the buffers include buffers such as phosphate, acetate, carbonate, and citrate. Examples of the isotonizing agents include sodium chloride, glycerin, and D-mannitol. Examples of the soothing agents include benzyl alcohol. Examples of the preservatives include thimerosal, para-hydroxybenzoates, 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 stabilizers include chelating agents and reducing agents. Examples of the carbohydrate include sorbitol, lactose, mannitol, starch, sucrose, glucose, and dextran.
Furthermore, the vaccine of the present invention may contain one or more other vaccines against viruses or bacteria that cause other diseases other than betacoronavirus infection, such as COVID-19. In other words, the vaccine of the present invention may be prepared as a combination vaccine containing other vaccines.
A dosage form of the vaccine of the present invention is not particularly limited, and can be appropriately determined based on an administration method, storage conditions, and the like. Specific examples of the dosage form include liquid preparations and solid preparations, and more specifically, oral administration agents such as tablets, capsules, powders, granules, pills, solutions, and syrups; and parenteral administration agents such as injections and sprays.
A method for administering the vaccine of the present invention is not particularly limited, and may be any of injection administration such as intramuscular, intraperitoneal, intradermal, and subcutaneous administration; inhalation administration from the nasal cavity and the oral cavity; oral administration, and the like, but injection administration such as intramuscular, intradermal, and subcutaneous administration (intramuscular administration, intradermal administration, and subcutaneous administration) and inhalation administration from the nasal cavity (nasal administration) are preferable, and nasal administration is more preferable.
A subject to which the vaccine of the present invention is applied is not particularly limited as long as the subject that can develop various symptoms by betacoronavirus infection (preferably a subject that can develop COVID-19 symptoms by SARS-CoV-2 infection), and examples thereof include mammals, and more specifically, humans; pet animals such as dogs and cats; and experimental animals such as rats, mice, and hamsters.
A dose of the vaccine of the present invention is not particularly limited, and can be appropriately determined according to the type of an active ingredient, an administration method, and a subject receiving administration (conditions such as age, weight, sex, and presence or absence of underlying disease). For example, a dose for a human includes 1×1010 TCID50/kg or less, preferably 1×108 TCID50/kg or less.
A method for producing the betacoronavirus temperature-sensitive strain (cold-adapted strains) of the present invention is not particularly limited, and can be appropriately determined by those skilled in the art based on the above amino acid sequence information. For example, from the viewpoint of producing a vaccine that is relatively inexpensive and has little lot difference, a reverse genetics method using an artificial chromosome such as a bacterial artificial chromosome (BAC) or a yeast artificial chromosome (YAC), or CPER method or the like using genomic fragments of a betacoronavirus is preferable.
In a method for reconstituting a virus by the reverse genetics method, first, a genome of a strain (parent strain) having no responsible mutation of the betacoronavirus temperature-sensitive strain (cold-adapted strain) is cloned. The parent strain used at this time may be a betacoronavirus, and specifically, it can be selected from the group consisting of the above SARS-CoV-2 listed in NC_045512 (NCBI), the above any other SARS-CoV-2, and viruses other than SARS-CoV-2 included in Betacoronavirus genus.
Furthermore, when an artificial chromosome is used in the reverse genetics method, a full-length DNA of a viral genome is cloned into BAC DNA, YAC DNA, or the like, and a transcription promoter sequence for eukaryotic cells is inserted upstream of a sequence of the virus. Examples of the promoter sequence include CMV promoter and CAG promoter. A ribozyme sequence and a polyA sequence are inserted downstream of a sequence of the virus. Examples of the ribozyme sequence include a hepatitis D virus ribozyme and a hammer head ribozyme. Examples of the polyA sequence include polyA of Simian 40 virus.
On the other hand, when using the CPER method in the reverse genetics method, full-length DNA of a viral genome is divided into several fragments and cloned. Examples of a method for acquiring fragments include a method for artificially synthesizing a nucleic acid and a PCR method using a plasmid obtained by cloning the artificial chromosome or fragments as a template.
In order to introduce at least any one of the above responsible mutations into the viral genome cloned by the methods described above, a known point mutation introduction method such as a homologous recombination method such as double crossover or 2IRED recombination, an overlap PCR method, or a CRISPR/Cas9 method can be used.
Subsequently, the artificial chromosome into which responsible mutation(s) has(have) been introduced is transfected into host cells to reconstitute recombinant viruses. In the case of the reverse genetics method by the CPER method, fragments into which responsible mutation(s) has(have) been introduced are assembled by reactions using DNA polymerase, and then transfected into host cells to reconstitute recombinant viruses. The method for transfection is not particularly limited, and a known method can be used. The host is also not particularly limited, and known cells can be used.
Subsequently, the reconstituted recombinant virus is infected to cultured cells to subculture the recombinant virus. The cultured cells used at that time are not particularly limited, and examples thereof include Vero cells, VeroE6 cells, Vero cells supplementing expression of TMPRESS2, VeroE6 cells supplementing expression of TMPRESS2, Calu-3 cells, 293T cells supplementing expression of ACE2, BHK cells, 104C1 cells, mouse neuroblastoma-derived NA cells, and Vero cells. The virus can be recovered by a known method such as centrifugation or membrane filtration. In addition, mass production of recombinant viruses become possible by further adding the recovered viruses to cultured cells.
Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
Based on the schematic diagram in
Mutation analysis of the following virus strains was performed using a next-generation sequencer. The analysis was performed by extracting RNA from culture supernatants of Vero cells infected with SARS-CoV-2. As a reference, Wuhan-Hu-1 (NC045512), a Wuhan clinical isolate, was used.
The term “reverse mutation” refers to changing back to the same phenotype as that of the parent virus before mutation by occurring further mutations into mutated viruses. As used herein, the term “reverse mutation” means that additional mutations occurred into a temperature-sensitive strain, so that a temperature-sensitive property is lost. The additional mutations include the situation that an amino acid at a site of mutation which is responsible for temperature sensitivity changes back to the wild-type amino acid.
As a result of infecting Vero cells with the B-1 strain or the A50-18 strain at MOI of 0.01 and evaluating growth at 32° C., 34° C., and 37° C., samples in which growth at 37° C. were recovered (hereinafter referred to as “revertants”) were found out from the A50-18 strain. In the revertants, it is considered that among the mutations possessed by the temperature-sensitive strain (A50-18 strain) that had acquired temperature sensitivity, some amino acid residues changed back to the wild-type amino acid (hereinafter simply referred to as “reverse mutation”), so that the temperature sensitivity decreased, and the growth at 37° C. was recovered. CPE images showing this are shown in
Vero cells were infected with the A50-18 strain at moi=1, and growth at 37° C. and 38° C. were evaluated. As a result, samples in which the growth at 37° C. and 38° C. was recovered (revertant) were found. CPE images after culturing the obtained revertants at 37° C. for 3 days are shown in
Since temperature sensitivity was lost when at least one of G248V and G416S in NSP14 changed back to the wild-type amino acid, it was suggested that a combination of the G248V and the G416S in NSP14 is a responsible mutation contributing to temperature sensitivity.
An NSP14, a spike, a nucleocapsid, and an envelope derived from the A50-18 strain were introduced into BAC DNA having a complete genome of wild-type SARS-CoV-2 by homologous recombination. The obtained recombinant BAC DNA was transfected into 293T cells to reconstruct viruses. Temperature sensitivity was evaluated by infecting Vero cells with the recombinant viruses and observing CPE at 37° C. and 32° C. The results are shown in
We reconstruct recombinant viruses which have mutations in NSP14 by the CPER method. Three types of recombinant viruses having the following respective substitutions in NSP14 were reconstructed:
Vero cells were infected with each recombinant virus, and CPE after culture at 37° C. or 32° C. for 3 days was observed. From
The analysis was performed by extracting RNA from culture supernatants of Vero cells infected with the SARS-CoV-2. As a result, no deletion as observed in (2-2-5) of Test Example 2-2 described later was found.
In the temperature-sensitive strain (cold-adapted strain), the A50-18 strain, as shown in Table 2 below, mutations with check marks in the amino acid sequences of the SEQ ID NO indicated were found, and among them, mutations with double check marks were found as responsible for temperature sensitivity. As shown in Table 2 below, it was found that a double mutant strain which have responsible mutations only in NSP14 was also a temperature-sensitive strain (cold-adapted strain).
Vero cells were infected with a clinical isolate (B-1 strain) and a temperature-sensitive strain (cold-adapted strain) (A50-18 strain) using 6-well plates under conditions of MOT=0.01 or 0.1 (N=3). The viruses cultured at 37° C. or 32° C., each culture supernatant was collected at 0 to 5 dpi. Virus titer of culture supernatants at 0 to 5 dpi were evaluated by TCID50/mL using the Vero cells. The results were shown in
From
Vero cells were infected with a clinical isolate (B-1 strain) and a temperature-sensitive strain (cold-adapted strain) (A50-18 strain) using 6-well plates under conditions of MOI=0.01 (N=3). The viruses cultured at 37° C., 34° C., or 32° C., each culture supernatant was collected at 0 to 5 dpi. Virus titer of culture supernatant at 0 to dpi were evaluated by TCID50/mL using the Vero cells. The results are shown in
From
4-week-old male Syrian hamsters (n=4) were bred for 1 week, and then a clinical isolate (B-1 strain) and a temperature-sensitive strain (cold-adapted strain) (A50-18 strain) (1×104 or 1×106 TCID50) were nasally administered in a volume of 100 μL, and weight changes for 10 days were observed. A group to which the same volume of D-MEM medium was nasally administered was defined as a non-infected control (MOCK). The results were shown in
4-week-old male Syrian hamsters (n=3) were bred for 1 week, and then a clinical isolate (B-1 strain) and a temperature-sensitive strain (cold-adapted strain) (A50-18 strain) (1×106 TCID50) were nasally administered in a volume of 100 μL. The results of observing the weight changes for 3 days were shown in
From
Lung sections were prepared from the formalin-fixed lungs obtained by the infection experiment to hamsters performed in (1-4-2), and HE staining was performed to analyze histological pathogenicity of the lungs by SARS-CoV-2 infection. The results were shown in
As shown in
In order to evaluate relationships between virus growth and pathogenicity for the histological pathogenicity observed in (1-4-3), viral proteins were detected by immunochemical staining. 4-week-old male Syrian hamsters (B-1, A50-18: n=5, MOCK: n=3) were bred for 1 week, and then a clinical isolate (B-1 strain) and a temperature-sensitive strain (cold-adapted strain) (A50-18 strain) (1×106 TCID50) were nasally administered in a volume of 100 μL. The hamsters were euthanized at 3 dpi, and then the extracted left lungs were fixed with 10% formalin to prepare serial sections. HE staining and immunochemical staining (also referred to as immunohistochemistry (IHC) staining) were performed on the obtained serial sections. For immunochemical staining, rabbit anti-spike polyclonal antibody (Sino Biological, Inc.: 40589-T62) was used. HE staining images and immunochemical staining images are shown in
According to the following procedure, hamsters which infected with a temperature-sensitive strain (cold-adapted strain) were challenged with a wild-type strain (clinical isolate).
4-week-old male Syrian hamsters (n=4) were bred for 1 week, and then a clinical isolate (B-1 strain) or a temperature-sensitive strain (cold-adapted strain) (A50-18 strain) (1×104 or 1×106 TCID50) were nasally administered in a volume of 100 μL. After 21 days, the clinical isolate (B-1 strain) (1×106 TCID50) was nasally administered again in a volume of 100 μL, and weight changes for 10 days were monitored. At this time, naive hamsters (n=3) of the same age were used as a naive control. The results were shown in
As shown in
4-week-old male Syrian hamsters (n=5) were bred for 1 week, and then a clinical isolate (B-1 strain) or a temperature-sensitive strain (cold-adapted strain) (A50-18 strain) (1×106 TCID50) were nasally administered in a volume of 100 μL. Weight changes after infection are shown in
At 21 days post infection, whole blood was collected, serum was separated, and then serum was inactivated by heat at 56° C. for 30 minutes. The B-1 strain of 100 TCID50 was mixed with serially diluted inactivated serum, and the mixture was incubated at 37° C. for 1 hour. The culture solution after incubation was added on Vero cells, neutralizing activity of the virus was evaluated by observing CPE after culturing at 37° C. The highest dilution rate of serum which did not cause CPE, was defined as a neutralizing antibody titer. The results are shown in
For the purpose of isolation of further candidate strains, temperature-sensitive strains (cold-adapted strains) were isolated by the method of
Mutation analysis of the following virus strains was performed using a next-generation sequencer. The analysis was performed by extracting RNA from culture supernatants of Vero cells infected with SARS-CoV-2. As a reference, Wuhan-Hu-1 (NC045512), a Wuhan clinical isolate, was used.
From (2-2-1), the analysis results of
Vero cells were infected with the H50-11 strain at moi=1, and growth at 37° C. and 38° C. were evaluated. As a result, samples in which the growth at 37° C. and 38° C. was recovered (revertant) were found. CPE images after culturing the obtained revertant at 38° C. for 3 days are shown in
Vero cells were infected with the L50-33 strain and the L50-40 strain at MOI=and growth at 32° C., 34° C., and 37° C. was evaluated. As a result, from among the L50-33 strain and the L50-40 strain, samples in which the growth at 37 ° C. was recovered (hereinafter, revertant) were found. CPE images after culturing each strain at 37° C. for 3 days are shown in
Mutation analysis of the H50-11 strain, the L50-33 strain, and the L50-40 strain was performed using Sanger sequencing. The analysis was performed by extracting RNA from culture supernatants of Vero cells infected with SARS-CoV-2.
As a result, a deletion of a base sequence (SEQ ID NO: 7) at positions 27549 to 28251 as shown in
As shown in
In the temperature-sensitive strains (cold-adapted strains), the H50-11 strain, the L50-33 strain, and the L50-40 strain, as shown in Table 3 below, mutations with check marks in the amino acid sequences of the SEQ ID NO indicated were found, and among them, mutations with double check marks were found as responsible for temperature sensitivity.
Vero cells were infected with additional isolates under conditions of MOT=0.01 (N=3). The viruses cultured at 37° C., 34° C., or 32° C., and each culture supernatant was collected at 0 to 5 d.p.i. Virus titers of culture supernatants were measured at TCID50/mL using the Vero cells. The results are shown in
4-week-old male Syrian hamsters (n=5) were bred for 1 week, and then a clinical isolate (B-1 strain) or temperature-sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, or H50-11 strain) (3×105 TCID50) were nasally administered in a volume of 100 μL, and their weight changes for 10 days were observed. A group to which the same volume of D-MEM medium was nasally administered was defined as a non-infected control (MOCK). The results were shown in
4-week-old male Syrian hamsters (n=5) were bred for 1 week, and then a clinical isolate (B-1 strain) or a temperature-sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, or H50-11 strain) (3×105 TCID50) were nasally administered in a volume of 100 μL. The hamsters were euthanized at 3 dpi, and then nasal wash was collected with 1 mL of D-PBS. In addition, the lungs of the hamsters were extracted, and the lung weight was measured. The right lungs were homogenized and suspended with 1 mL of D-MEM, and then the supernatants were recovered as lung homogenates by centrifugation. The lung weight per total weight of the hamsters were shown in
As a result of comparing lung weight per total weight of hamsters, the lung weight of the hamsters infected with the B-1 strain increased, and it was strongly suggested that the lungs swelled due to inflammation or the like. On the other hand, such an increase in lung weight was not observed in the hamsters infected with the temperature-sensitive strains. In addition, as a result of comparing viral loads in the nasal wash, there was no significant difference between the hamsters infected with the B-1 strain and the hamsters infected with the temperature-sensitive strains except the H50-11 strain, and the hamsters infected with the H50-11 strain had a small viral load in the nasal wash. Furthermore, it was revealed that the intrapulmonary viral loads of the hamsters infected with the temperature-sensitive strains were significantly lower than that of the hamsters infected with the B-1 strain. From these results, it was suggested that each temperature-sensitive strain is an attenuated strain that cannot proliferate in the lower respiratory tract, similarly to the A50-18 strain in Test Example 1.
4-week-old male Syrian hamsters (n=5) were bred for 1 week, and then a clinical isolate (B-1 strain) or temperature-sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, or H50-11 strain) (3×105 TCID50) were nasally administered in a volume of 100 μL. After 21 days, the clinical isolate (B-1 strain) (3×105 TCID50) was nasally administered again in a volume of 100 μL, and weight changes for 9 days were observed. At this time, naive hamsters (n=5) of the same age were used as a naive control. The results were shown in
4-week-old male Syrian hamsters (n=5) were bred for 1 week, and then a clinical isolate (B-1 strain) or temperature-sensitive strains (A50-18 strain, L50-33 strain, L50-40 strain, or H50-11 strain) (3×105 TCID50) were nasally administered in a volume of 100 μL. After 20 days, blood was collected partially, and the obtained serum was used to evaluate neutralizing activity against the clinical isolate (B-1 strain). The neutralizing activity was measured in the same protocol as in (1-5 -2). The measurement results are shown in
4-week-old male Syrian hamsters (n=3 or 5) were bred for 1 week, and then a clinical isolate (B-1 strain) or a temperature-sensitive strain (A50-18 strain) (3×105 TCID50) were nasally administered in a volume of 100 μL. Partial blood was collected from hamsters at 3 weeks after infection, and the neutralizing antibody titers against the SARS-CoV-2 European clinical isolate (B-1) and the Brazilian variant (hCoV-19/Japan/TY7-503/2021 strain) using the obtained serum are shown in
4-week-old male Syrian hamsters (n=5) were bred for 1 week, and then a clinical isolate (B-1 strain) or a temperature-sensitive strain (A50-18 strain) (3×105 TCID50) were nasally or subcutaneously administered in a volume of 100 μL. The untreated group was defined as a naive control (naive). After 3 weeks, the serum obtained by partial blood collection from the hamsters was used to evaluate neutralizing activity against a SARS-CoV-2 Brazilian variant (hCoV-19/Japan/TY7-503/2021 strain). The neutralizing activity was measured in the same protocol as in (1-5-2). Neutralizing antibody titers were shown in
4-week-old male Syrian hamsters (n=5) were bred for 1 week, and then a temperature-sensitive strain (A50-18 strain) were administered nasally or subcutaneously. Doses are shown in Table 4.
Partial blood collection was performed from hamsters at 3 weeks post infection, and the results of measuring neutralizing activity against live viruses of a SARS-CoV-2 Brazilian type variant (hCoV-19/Japan/TY7-503/2021 strain) using the obtained serum are shown in
4-week-old male Syrian hamsters (n=4) were bred for 1 week, and then a temperature-sensitive strain (A50-18 strain) of 1×104 TCID50 or 1×102 TCID50 were nasally administered in a volume of 10 μL. Partial blood collection was performed from hamsters at 3 weeks after infection, and the results of measuring neutralizing activity against authentic viruses of a SARS-CoV-2 European wild strain (B-1 strain), an Indian variant (autologous isolate), and a Brazilian variant (hCoV-19/Japan/TY7-503/2021 strain) using the obtained serum are shown in
The results of comparing serum neutralizing antibody titers against each strain of each individual are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-173494 | Oct 2020 | JP | national |
| 2020-180524 | Oct 2020 | JP | national |
| 2020-210564 | Dec 2020 | JP | national |
| 2021-017633 | Feb 2021 | JP | national |
| 2021-051107 | Mar 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/037903 | 10/13/2021 | WO |
