Patent Application
20250002871
The present invention relates to a betacoronavirus attenuated strain.
An infectious disease (COVID-19) with the novel coronavirus (SARS-CoV-2) has caused a pandemic and is still a social problem. As vaccines against this infectious disease, gene vaccines such as adenovirus vector vaccine and mRNA vaccines have been approved, and inoculation has been advanced all over the world, starting from Sputnik V (Non-Patent Document 1), which is an adenovirus vector vaccine approved in Russia.
However, gene vaccines are next-generation vaccines different from conventional vaccines, and side reactions such as fever and thrombosis have been reported. Therefore, it is considered that development of new vaccines is still important.
Therefore, it is an object of the present invention to provide a strain useful as a new betacoronavirus vaccine.
As a result of intensive studies, the present inventors have found that a novel betacoronavirus having a combination of a prescribed substitution mutation related to temperature sensitivity and a prescribed deletion mutation related to growth reduction or other attenuation as prescribed mutations related to attenuation is useful as a vaccine strain of the betacoronavirus having excellent attenuation. The present invention has been completed by further studies based on this knowledge. That is, the present invention provides the inventions of the following modes.
Item 1. A betacoronavirus attenuated strain containing:
According to the present invention, a strain useful as a novel betacoronavirus vaccine is provided.
The betacoronavirus attenuated strain of the present invention is characterized by being a betacoronavirus having, non-structural protein(s) having prescribed substitution mutation(s) related to temperature sensitivity, in combination with structural protein(s), accessory protein(s), and/or non-structural protein(s) having prescribed deletion mutation(s) related to growth reduction or other attenuation, as prescribed mutations related to attenuation. Hereinafter, a prescribed substitution mutation related to temperature sensitivity is also referred to as a “temperature-sensitive mutation”, a prescribed deletion mutation related to growth reduction is also described as a “growth reducing mutation”, and a prescribed deletion mutation other than the growth reducing mutations is also described as a “other attenuating mutation”.
In the present invention, the “attenuation” refers to a characteristic of attenuating pathogenicity of a virus against host. In addition, in the present invention, the “temperature sensitivity” refers to a characteristic in which growth at a human body temperature (so-called a lower respiratory tract temperature) is limited and a characteristic having a growth capability specifically at a low temperature (typically, not higher than a human upper respiratory tract temperature). Moreover, in the present invention, the “growth reducing” refers to a characteristic in which the growth is limited and the characteristic is not temperature-specific.
The betacoronavirus attenuated strain of the present invention not only exhibits efficacy as a vaccine by having the prescribed mutation(s) related to attenuation described above, but also has a combination of the substitution mutation(s) and deletion mutation(s) that is less likely to revert to mutation, so that the possibility of causing reversion of virulence is extremely low. In this respect, the usefulness in the case of assuming application to humans is remarkably increased.
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. There is a genome of positive-stranded single-stranded RNA wound around a nucleocapsid protein (hereinafter, also referred to as a “nucleocapsid” or “Nucleocapsid”) in the envelope of the lipid bilayer membrane, and a spike protein (hereinafter also referred to as a “spike” or “Spike”), an envelope protein (hereinafter also referred to as an “envelope” or “Envelope”), and a membrane protein (hereinafter also referred to as a “membrane” or “Membrane”) are arranged on the surface of the envelope. The size of the viral genome is about 30 kb, the longest among RNA viruses. The nucleocapsid, spike, envelope, and membrane are structural proteins of coronaviruses. NSP1 to NSP16 are non-structural proteins of coronavirus. In addition, ORF7a, ORF7b, ORF8, and the like are accessory proteins of coronavirus. The accessory protein can also be referred to as an accessory protein.
Coronaviruses are classified into groups of alpha, beta, gamma, and delta from genetic characteristics. As coronaviruses infecting humans, there are known 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, both of which cause serious pneumonia. 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 mutant 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 are also possibilities that there is a mutant strain that has not yet been detected and that a new mutant strain will occur in the future. In the present invention, the virus included in the genus Betacoronavirus is not limited to the strain of SARS-CoV-2 described above, and includes all other betacoronaviruses (for example, other SARS-CoV-2 mutant strains that will be newly detected in the future and betacoronaviruses other than SARS-CoV-2, and recombinant viruses in which the spike protein of SARS-CoV-2 or betacoronavirus other than SARS-CoV-2 is replaced with a spike protein of at least one of other SARS-CoV-2 and betacoronavirus other than SARS-CoV-2 (including viruses that will be newly detected in the future), and the like).
The prescribed mutations related to attenuation possessed by the betacoronavirus attenuated strain of the present invention will be described based on Table 1 below. Mutation (b), a combination of mutation (e) and mutation (f), and/or mutation (h) indicated as “Temperature-sensitive mutation” in Table 1 are substitution mutations and are responsible mutations that contribute to providing a temperature-sensitive capability essentially held by the betacoronavirus attenuated strain of the present invention. In other words, in the present invention, examples of the temperature-sensitive mutation include three types: “mutation (b)”. “combination of mutation (e) and mutation (f)”, and “mutation (h)”. Typical betacoronavirus attenuated strains of the present invention have one or two types of these three types of temperature-sensitive mutations. Mutation (n), mutation (o) and/or mutation (r) indicated as “Growth reducing mutation” and “Other attenuating mutation” in Table 1 are deletion mutations, and are mutations essentially held by the betacoronavirus attenuated strain of the present invention that are considered to contribute to providing growth reduction or other attenuation (in particular, it is considered that the mutation (r) contributes to providing growth reduction), and expresses excellent attenuation in combination with temperature-sensitive mutation(s). Mutations (a), (c), (d). (g), (i) to (m), (p), and (q) indicated as “Other mutation” in Table 1 are mutations that can be optionally held by the betacoronavirus attenuated strains of the present invention, and the betacoronavirus attenuated strains of the present invention may or may not hold at least any of the other mutations.
That is, the essential mutations related to attenuation possessed by the betacoronavirus of the present invention are the following mutation of (b), a combination of the following mutations of (e) and (f), and/or the following mutation of (h), which are temperature-sensitive mutations; and the following mutations of (n), (o) and/or (r), which are growth reducing or other attenuating mutations.
The above mutation of (n) is acceptable as long as it is a mutation by which the function of ORF8 is lost, but preferably includes a deletion of an amino acid sequence corresponding to the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 7.
In a preferred embodiment of the present invention, from the viewpoint of controlling the level of temperature sensitivity to the preferred degree, one to two types of mutations or combinations of mutations are selected from the above temperature-sensitive mutations (that is, three types of mutations or combinations of mutations: the mutation of (b), the combination of mutations of (e) and (f), and the mutation of (h)).
In a preferred embodiment of the present invention, among the above growth reducing or other attenuating mutations, the mutation of (o) and the mutation of (r) are preferable.
In a preferred embodiment of the present invention, four types of mutations or combinations of mutations are selected from six types of the prescribed mutations related to attenuation (that is, six types: the mutation of (b), the combination of mutations of (e) and (f), the mutation of (h), the mutation of (n), the mutation of (o), and the mutation of (r)) from the viewpoint of exhibiting preferred immunogenicity as well as preferred attenuation.
In addition to the above essential mutations, the betacoronavirus attenuated strain of the present invention can further hold at least any one of the following mutations (a), (c), (d), (g), (i) to (m), (p), and (q) as other mutation(s).
When the betacoronavirus attenuated strain of the present invention has other mutation, it is preferable to have the mutation of (g) among the other mutations from the viewpoint of enhancing temperature sensitivity. When the betacoronavirus attenuated strain of the present invention has the mutation of (g) as other mutation, the mutation of (g) is preferably used together with the combination of the mutations of (e) and (f) from the viewpoint of enhancing temperature sensitivity.
SEQ ID NO: 1 is the amino acid sequence of NSP3 in SARS-CoV-2 of NC_045512 (NCBI); SEQ ID NO: 2 is the amino acid sequence of NSP14 in SARS-CoV-2 of NC_045512 (NCBI); and SEQ ID NO: 3 is the amino acid sequence of NSP16 in SARS-CoV-2 of NC_045512 (NCBI).
In addition, SEQ ID NO: 4 is the amino acid sequence of the spike in SARS-CoV-2 of NC_045512 (NCBI); SEQ ID NO: 5 is the amino acid sequence of the envelope in SARS-CoV-2 of NC_045512 (NCBI); and SEQ ID NO: 6 is the amino acid sequence of the nucleocapsid in SARS-CoV-2 of NC_045512 (NCBI).
Further, SEQ ID NO: 7 is the base sequence of the part of open reading frames of SARS-CoV-2 of NC_045512 (NCBI), specifically, across a portion of ORF7a, the entire ORF7b, and most of ORF8; and SEQ ID NO: 8 is the amino acid sequence of NSP1 in SARS-CoV-2 of NC_045512 (NCBI).
The “amino acid residue corresponding” refers to an amino acid residue present at the above prescribed position in the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6), 8, or the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 7 when the betacoronavirus attenuated strain of the present invention is a mutant strain of SARS-CoV-2 of NC_045512 (NCBI), and refers to an amino acid residue present at a position corresponding to the above prescribed position in the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6), 8 of the polypeptide possessed by another betacoronavirus, or the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 7 when the betacoronavirus attenuated strain of the present invention is another betacoronavirus mutant strain other than the above mutant strain. The corresponding position can be identified by aligning amino acid sequences between proteins having the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6) and 8 or proteins having the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 7 of SARS-CoV-2 of NC_045512 (NCBI) and proteins of other betacoronaviruses corresponding to the proteins.
The virus attenuated strain of the present invention is not limited to a mutant strain of specific SARS-CoV-2 listed in NC_045512 (NCBI) as long as an amino acid residue or amino acid sequence corresponding to the above prescribed positions in the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6), 8, or the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 7 is mutated, but includes other betacoronavirus mutant strains [i.e., any other mutant strains of SARS-CoV-2 and mutant strains of viruses other than SARS-CoV-2 which are included in the Betacoronavirus genus]. The mutant strains of specific SARS-CoV-2 listed in NC_045512 (NCBI) are defined as mutant strains in which at least any one of amino acid residues or amino acid sequences at the above specific positions in the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6), 8, or the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 7 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 or amino acid sequences corresponding to the above prescribed positions in the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6), 8, or the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 7 in any other SARS-CoV-2 are mutated] and mutant strains of viruses other than SARS-CoV-2 which are included in the Betacoronavirus genus [i.e., mutant strains in which amino acid residues or amino acid sequences corresponding to the above prescribed positions in the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6), 8, or the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 7 in viruses other than SARS-CoV-2 which are included in the Betacoronavirus genus are mutated]. Any other mutant strains of SARS-CoV-2 and mutant strains of viruses other than SARS-CoV-2 which are included in the Betacoronavirus genus also include mutant strains of recombinant viruses in which the spike protein of SARS-CoV-2 or betacoronavirus other than SARS-CoV-2 is replaced with a spike protein of at least one of other SARS-CoV-2 and betacoronaviruses other than SARS-CoV-2 (including viruses that will be newly detected in the future).
Each of the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6), 8, or the sequence corresponding to the base sequence set forth in SEQ ID NO: 7 in other betacoronavirus mutant strains is allowed to differ from the amino acid sequences of SEQ ID NOs: 1 to 4 (or 1 to 6), 8, or the base sequence set forth in SEQ ID NO: 7, as long as it does not significantly affect the characteristics of the polypeptide. The phrase “does not significantly affect the characteristics of the polypeptide” refers to a state in which a function as a non-structural protein, a structural protein, or an accessory protein of each polypeptide is maintained. Specifically, at a site other than an amino acid or base sequence corresponding to the prescribed mutations related to attenuation in the amino acid sequences of SEQ ID NOs: 1 to 4, 8, or the base sequence set forth in SEQ ID NO: 7, or in the case of further having other mutations, at site(s) other than amino acid residue(s) corresponding to the other mutations in SEQ ID NOs: 5 and 6 described above (hereinafter, a site other than amino acid residues or bases corresponding to these mutations is also referred to as an “any different site”), a difference from SEQ ID NOs: 1 to 4, 7, 8 (or 1 to 8) 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 differences (e.g., substitution and insertion). A sequence identity calculated by comparing only any different sites of amino acid sequences corresponding to SEQ ID NOs: 1 to 4 (or 1 to 6), 8 or the base sequence set forth in SEQ ID NO: 7 in any other SARS-CoV-2 and the amino acid sequences set forth in SEQ ID NOs: 1 to 4 (or 1 to 6), 8 or the base sequence set forth in SEQ ID NO: 7 may be not less than 50%. In any other SARS-CoV-2, the sequence identity is preferably not less than 60% or not less than 70%, more preferably not less than 80%, further preferably not less than 85% or not less than 90%, still more preferably not less than 95%, not less than 96%, not less than 97%, or not less than 98%, still more preferably not less than 99%, and particularly preferably not less than 99.3%, not less than 99.5%, not less than 99.7%, or not less than 99.9%. In any remaining betacoronavirus, the sequence identity is preferably not less than 60%. Here, the “sequence identity” shows an identity value of an amino acid sequence obtained by BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999). Parameters may be set to Gap insertion Cost value: II and Gap extension Cost value: 1.
That is, the betacoronavirus attenuated strain of the present invention is more specifically as follows:
A betacoronavirus attenuated strain containing non-structural protein(s), accessory protein(s), and structural protein(s) consisting of at least any one of the following polypeptides (I), (II) and (III):
(I) at least any one of the following polypeptides (I-1) to (I-3) and at least any one of the following polypeptides (1-4) to (I-6):
When the more specific betacoronavirus attenuated strain described above contains other mutation(s) in addition to the temperature-sensitive mutation(s) and growth reducing or other attenuating mutation(s), as shown below, the polypeptides (I-1) and (I-2) described above (non-structural proteins) and the polypeptide (I-5) described above (structural protein) may be the polypeptides (I-1a) and (I-2a) and (I-5a) described below that also have other mutation(s) in addition to temperature-sensitive mutation(s) and growth reducing or other attenuating mutation(s), respectively, and the polypeptide (I) described above may further contain the polypeptides (I-7a) and (I-8a) described below (structural proteins) that have other mutation(s).
A betacoronavirus attenuated strain containing structural protein(s), accessory protein(s) and non-structural protein(s) consisting of at least any one of the following polypeptides (I), (II), and (III):
(I) at least any one of the following polypeptides (I-1a), (I-2a), and (I-3); and at least any one of the following polypeptides (I-4), (I-5a), and (I-6), or in addition thereto, at least one of the following polypeptides (I-7a) and (I-8a):
The above mutations of (a′) to (r′) refer to mutations when the mutations of (a) to (r) are specifically present in the amino acid sequences of SEQ ID NOs: 1 to 6, the base sequence of SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO: 8, respectively. In other words, the above polypeptide (1) is a polypeptide obtained by introducing temperature-sensitive mutation(s), growth reducing or other attenuating mutation(s), or in addition thereto, other mutation(s) into a polypeptide consisting of the amino acid sequences of SEQ ID NOs: 1 to 6, the amino acid sequence encoded by the base sequence of SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO: 8 possessed by SARS-CoV-2 of NC_045512 (NCBI). In addition, the above polypeptides (II) and (III) are obtained by introducing temperature-sensitive mutation(s), growth reducing or other attenuating mutation(s), or in addition thereto, other mutation(s) into a polypeptide consisting of amino acid sequences corresponding to the amino acid sequences of SEQ ID NOs: 1 to 6, the amino acid sequence encoded by the base sequence of SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO: 8, which are possessed by another betacoronavirus. Preferred ranges of the sequence identity of the above polypeptides (II) and (III) are as described above.
The betacoronavirus can acquire temperature sensitivity by having the above temperature-sensitive mutation, and can acquire excellent attenuation by having the above growth reducing or other attenuating mutation together with the above temperature-sensitive mutation. In the virus attenuated 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 attenuated 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 capability can be confirmed by the fact that after Vero cells are infected with the virus attenuated strain at MOI=0.01 at a human lower respiratory tract temperature and then the virus attenuated strain is cultured at a human lower respiratory tract temperature for 1 day a virus titer (TCID50/mL) in culture supernatant is decreased, for example, by not less than 102, preferably by not less than 103, as compared with a virus titer in culture superatant after Vero cells are infected with the virus attenuated strain at MOI=0.01 at a human upper respiratory tract temperature and then the virus attenuated strain is cultured at a human upper respiratory tract temperature for 1 day.
Typically, in the virus attenuated 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 temperature-sensitive mutation. This can be confirmed by the fact that, after Vero cells are infected with the virus attenuated strain at MOI=0.01 at a human lower respiratory tract temperature and then the virus attenuated strain is cultured at a human lower respiratory tract temperature for 1 day, a virus titer (TCID50/mL) in culture supernatant is decreased, for example, by not less than 102, preferably by not less than 103, as compared with a virus titer in culture supernatant after Vero cells are infected with a strain not having the above temperature-sensitive mutation at MOI=0.01 at a human lower respiratory tract temperature and then the strain is cultured at a human lower respiratory tract temperature for 1 day.
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 attenuated 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 temperature-sensitive mutations are not present on receptor-binding domains of a spike protein present on a surface of the 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 temperature-sensitive mutation. In other words, it is reasonably expected that, even if a mutation occurs so as to alter the immunogenicity of the virus due to worldwide infection, temperature sensitivity can be provided for the mutant virus by further introducing the above temperature-sensitive mutation into the mutant virus.
Regarding the temperature-sensitive 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, the above mutation of (m) may be a substitution with an amino acid residue other than serine, and the above mutation of (q) may be a substitution with an amino acid residue other than valine.
In a preferred example of the virus attenuated strain of the present invention, regarding the temperature-sensitive mutations, 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 those 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, the mutation of (m) is a substitution with phenylalanine, and/or the mutation (q) is a substitution with isoleucine.
In another example of the virus attenuated strain of the present invention, the substitution may be a so-called conservative substitution. The conservative substitution refers to a substitution with an amino acid having a similar structure and/or characteristic, 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 attenuated strain of the present invention includes a mutant strain of the SARS-CoV-2 listed in NC_045512 (NCBI), in which the mutation of (b) (i.e., the mutation of (b′)) is a substitution of leucine at the 445th position of the 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 the 248th position of the 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 the 416th position of the 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 the 67th position of the 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 mutation(s) includes the mutant strains, in which the mutation of (a) (i.e., the mutation of (a′)) is the substitution of valine at the 404th position of the 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 the 1792nd position in the 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 the 1832nd position of the 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 the 504th position of the 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 the 54th position of the amino acid sequence set forth in SEQ ID NO: 4 with tryptophan in Spike (L54W): the mutation of (j) (i.e., the mutation of (j′)) is a substitution of threonine at the 739th position of the amino acid sequence set forth in SEQ ID NO: 4 with lysine in Spike (T739K); the mutation of (k) (i.e., the mutation of (k′)) is a substitution of alanine at the 879th position of the amino acid sequence set forth in SEQ ID NO: 4 with valine in Spike (A879V); the mutation of (1) (i.e., the mutation of (l′)) is a substitution of leucine at the 28th position of the amino acid sequence set forth in SEQ ID NO: 5 with proline in Envelope (L28P); the mutation of (m) (i.e., the mutation of (m′)) is a substitution of serine at the 2nd position of the amino acid sequence set forth in SEQ ID NO: 6 with phenylalanine in a nucleocapsid (S2F); and/or the mutation of (q) (i.e., the mutation of (q′)) is a substitution of valine at the 687th position of the amino acid sequence set forth in SEQ ID NO: 4 with isoleucine in Spike (V687I).
Particularly preferred examples of the virus attenuated strain of the present invention include the following strains:
Most preferred examples of the virus attenuated strain of the present invention include the following strain:
A strain having, as temperature-sensitive mutations, the mutation of (e) (preferably the mutation of (e′) and/or G248V) and the mutation of (f) (preferably the mutation of (f) and/or G416S); and, as growth reducing or other attenuating mutations, the mutation of (n) (preferably the mutation of (n′)), the mutation of (o), and the mutation of (r); and a strain further having, as other mutations, the mutation of (g) (preferably the mutation of (g′) and/or A504V), the mutation of (p), and the mutation of (q) (preferably the mutation of (q′) and/or V687I)
As described above, since the betacoronavirus attenuated strain described in “1. Betacoronavirus attenuated strain” can efficiently grow only at a temperature lower than a human lower respiratory tract temperature by having a temperature-sensitive mutation, it can be expected that it cannot efficiently grow at least in a deep part of a living body, especially in the lower respiratory tract including lungs, which cause serious disorders, so that the pathogenicity is significantly decreased. In addition, the betacoronavirus attenuated strain shows limited growth regardless of temperature by having growth reducing or other attenuating mutations, and has excellent attenuation in combination with the above temperature-sensitive mutations. By taking such a combined form, the betacoronavirus attenuated strain has a characteristic that, when a live attenuated vaccine is accompanied by growth in a host's body, even when the temperature-sensitive mutation is lost, the deletion mutation, which is a growth reducing or other attenuating mutation, is less likely to revert to mutation, and thus it can be expected that the attenuation can be maintained.
Therefore, the virus attenuated 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 attenuated strain as an active ingredient. Details of the active ingredient are as described in “1. Betacoronavirus attenuated strain”.
As described in the above “1. Betacoronavirus attenuated strain”, the prescribed mutations contribute to providing attenuation. Therefore, the present invention also provides a betacoronavirus gene vaccine containing, as an active ingredient, a gene encoding non-structural protein(s), accessory protein(s), and structural protein(s) having the prescribed mutation(s) as described in the above. Details of the prescribed mutation(s) contained in the active ingredient are as described in “1. Betacoronavirus attenuated strain”.
It can be reasonably expected that the vaccine of the present invention is effective against not only the early Wuhan strain of SARS-CoV-2 but also a wide range of SARS-CoV-2 virus-associated strains and viruses included in the Betacoronavirus genus other than SARS-CoV-2, 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. Therefore, the vaccine of the present invention targets betacoronaviruses.
The vaccine of the present invention can contain another ingredient such as an adjuvant, a buffer, an isotonizing agent, a soothing agent, a preservative, an antioxidant, a deodorant, a light-absorbing dye, a stabilizer, a carbohydrate, a casein digest, any sort of vitamin or the like, in addition to the above active ingredient, according to the purpose, use, and the like.
Examples of the adjuvant 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 such as aluminum salts such as aluminum hydroxide (alum), aluminum phosphate, and aluminum sulfate or combinations thereof, and sodium hydroxide: microorganism-derived toxin components such as cholera toxin and E. coli heat-labile toxin; and other ingredients (bentonite, muramyl dipeptide derivatives, interleukin, and the like).
Examples of the buffer include buffers such as phosphate, acetate, carbonate, and citrate. Examples of the isotonizing agent include sodium chloride, glycerol. D-mannitol, and the like. Examples of the soothing agent include benzyl alcohol and the like. Examples of the preservative include thimerosal, para-hydroxybenzoic acid esters, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, antibiotics, synthetic antibacterial agents, and the like. Examples of the antioxidant include sulfite, ascorbic acid, and the like.
Examples of the light-absorbing dye include riboflavin, adenine, adenosine, and the like. Examples of the stabilizer include chelating agents, reducing agents, and the like. Examples of the carbohydrate include sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, and the like.
Furthermore, the vaccine of the present invention may contain one or more other vaccines against viruses or bacteria that cause 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 another vaccine.
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 formulations and solid formulations, and the like, and more specifically, oral administration agents such as tablets, capsules, powders, granules, pills, liquids, and syrups; parenteral administration agents such as dried formulations including freeze-dried formulations, injections, sprays, and patches (concretely, intramuscular administration agents, intradermal administration agents, subcutaneous administration agents, nasal administration agents, transdermal administration agents, and the like).
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 nasal cavity and oral cavity; oral administration, and the like, but injection administration such as intramuscular, intradermal, and subcutaneous administration (intramuscular administration, intradermal administration, and subcutaneous administration), inhalation administration from nasal cavity (nasal administration), and absorption administration from the skin (transdermal 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 a type of an active ingredient, an administration method, and an applicable subject (conditions such as age, weight, sex, and presence or absence of underlying disease).
In addition, the amount per dose of the vaccine of the present invention for a human is not less than 1×10 PFU/body, preferably not less than 1×102 PFU/body, more preferably not less than 2×102 PFU/body, still more preferably not less than 1×103 PFU/body, and still more preferably not less than 2×103 PFU/body. Moreover, the amount per dose of the vaccine of the present invention for a human is also not more than 6×1011 PFU/body, preferably not more than 1×1011 PFU/body, more preferably not more than 6×1010 PFU/body, and still more preferably not more than 1×1010 PFU/body.
Further, the amount per dose of the vaccine of the present invention for a human is not less than 1×10 TCID50/body, preferably not less than 1×102 TCID50/body, more preferably not less than 2×102 TCID50/body, more preferably not less than 1×103 TCID50/body, and more preferably not less than 2×103 TCID50/body. Furthermore, the amount per dose of the vaccine of the present invention for a human is also not more than 6×1011 TCID50/body, preferably not more than 1×1011 TCID50/body, more preferably not more than 6×1010 TCID50/body, and still more preferably not more than 1×1010 TCID50/body.
A method for producing the betacoronavirus attenuated strain 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, the production method preferably includes 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.
In a method for reconstructing the virus by the reverse genetics method, first, a genome of a strain (parent strain) having no temperature-sensitive mutation and no growth reducing or other attenuating mutation of the betacoronavirus attenuated strain is cloned. The parent strain used at this time may be a betacoronavirus, and concretely, 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 which are included in the 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 poly A sequence are inserted downstream of a sequence of the virus. Examples of a ribozyme sequence include a hepatitis D virus ribozyme and a hammerhead 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 obtaining fragments include a method for artificial synthesis of a nucleic acid and a PCR method using a plasmid obtained by cloning the above artificial chromosome or fragments as a template.
In order to introduce at least any one of the above temperature-sensitive mutation(s), growth reducing or other attenuating mutation(s), and other mutation(s) as necessary 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 W/RED recombination, an overlap PCR method, or a CRISPR/Cas9 method can be used.
Subsequently, the artificial chromosome into which temperature-sensitive mutation(s), growth reducing or other attenuating mutation(s), and other mutation(s) as necessary have been introduced is transfected into host cells to reconstruct recombinant viruses. In the case of the reverse genetics method by the CPER method, fragments into which temperature-sensitive mutation(s), growth reducing or other attenuating mutation(s), and other mutation(s) as necessary have been introduced are assembled by reactions using DNA polymerase, and then transfected into host cells to reconstruct recombinant viruses. The method for transfection is not particularly limited, and a known method can be used. The hosts are also not particularly limited, and known cells can be used.
Subsequently, the reconstructed 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, and the like. The virus can be recovered by a known method such as centrifugation or membrane filtration. In addition, mass production of recombinant viruses can be made 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 method of
Mutation analysis of the following virus strains was carried out using a next-generation sequencer. The analysis was carried out 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 (1-2-1), the analysis results in
The “reverse mutation” refers to changing back to the same phenotype as that of the parent virus, which is yet to be mutated, by occurring further mutations into mutated viruses. As used herein, the “reverse mutation” means that an additional mutation occurs into a temperature-sensitive strain, so that a temperature-sensitive characteristic is lost. The additional mutation includes a situation that an amino acid at a site of mutation which is responsible for temperature sensitivity changes back to the amino acid which is yet to be mutated.
As a result of infecting Vero cells with B-1 strain or A50-18 strain at MOI=0.01 and evaluating growth at 32° C., 34° C. and 37° C., samples in which the growth at 37° C. was recovered (hereinafter referred to as “revertants”) were found out from 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 amino acids which is yet to be mutated (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 A50-18 strain at moi=1, and growth at 37° C. and 38° C. was evaluated. As a result, samples in which the growth at 37° C. and 38° C. was recovered (revertants) were found. CPE images after culturing the obtained revertants at 37° C. for 3 days are shown in
<1> G248V mutation in NSP14 reverted to mutation to the wild-type G, whereas G416S and A504V mutations were maintained.
<2> G416S mutation in NSP14 reverted to mutation to the wild-type G, whereas G248V and A504V mutations were maintained.
Since temperature sensitivity was lost when at least one of G248V and G416S in NSP14 reverted to mutation, it was suggested that a combination of the G248V mutation and the G416S mutation in NSP14 was a responsible mutation that contributed to temperature sensitivity (temperature-sensitive mutation).
(1-2-5) Analysis of Recombinant Viruses Whose Mutations were Introduced into Wild Type Strain
NSP14, Spike, Nucleocapsid or Envelope derived from A50-18 strain was introduced into BAC DNA having the whole 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 CPEs at 37° C. and 32° C. The results are shown in
(1-2-6) Analysis of Recombinant Viruses Whose Mutations were Introduced into Wild-Type Strain 2
Viruses into which the mutations in NSP14 were introduced were reconstructed by the CPER method. Three types of recombinant viruses having the following respective mutations in NSP14 were reconstructed.
Vero cells were infected with each recombinant virus, and CPEs after culture at 37° C. or 32° C. for 3 days was observed. From
Mutation analysis of A50-18 strain was carried out using Sanger sequencing. The analysis was carried out by extracting RNA from culture supernatants of Vero cells infected with SARS-CoV-2. As a result, such a deletion as observed in (2-2-5) of Test Example 2-2 described later was not found.
In the temperature-sensitive strain, A50-18 strain, as shown in Table 2 below, mutations with check marks in the amino acid sequences of the indicated SEQ ID NOs were found, and among them, mutations with double check marks were found as temperature-sensitive mutations. Also, as shown in Table 2 below, it was found that a double mutant strain which only had temperature-sensitive mutations in NSP14 was also a temperature-sensitive strain.
Vero cells were infected with the clinical isolate (B-1 strain [Comparative Example]) and the temperature-sensitive strain (A50-18 strain) using 6-well plates under conditions of MOI=0.01 or 0.1 (N=3). After culturing at 37° C. or 32° C., each culture supernatant was collected on 0 to 5 dpi. Virus titers of culture supernatants on 0 to 5 dpi were measured by TCID50/mL using the Vero cells. The results are shown in
From
Vero cells were infected with the clinical isolate (B-1 strain [Comparative Example]) and the temperature-sensitive strain (A50-18 strain) using 6-well plates under conditions of MOI=0.01 (N=3). After culturing at 37° C., 34° C., or 32° C., each culture supernatant was collected on 0 to 5 dpi. Virus titers of culture supernatants on 0 to 5 dpi were measured by TCID50/mL using the Vero cells. The results are shown in
From
After 4-week-old male Syrian hamsters (n=4) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) and the temperature-sensitive strain (A50-18 strain) (1×104 or 1×106 TCID50) were nasally administered at a dose of 100 μL. and weight changes for 10 days were observed. A group to which the same dose of D-MEM medium was nasally administered was defined as a non-infected control (MOCK). The results are shown in
From
After 4-week-old male Syrian hamsters (n=3) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) and the temperature-sensitive strain (A50-18 strain) (1×106 TCID50) were nasally administered at a dose of 100 μL. The results of observing the weight changes for 3 days are shown in
From
Sections were prepared from the formalin-fixed lungs obtained by the infection experiment to the hamsters carried out in (1-4-2), and HE staining was carried out to analyze histological pathogenicity of the lungs due to SARS-CoV-2 infection. The results are shown in
As shown in
In order to evaluate relationships between virus growth and pathogenicity as to the histological pathogenicity observed in (1-4-3), viral proteins were detected by immunochemical staining. After 4-week-old male Syrian hamsters (B-1. A50-18: n=5, MOCK: n=3) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) and the temperature-sensitive strain (A50-18 strain) (1×106 TCID50) were nasally administered at a dose of 100 μL. The hamsters were euthanized on 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 carried out using the obtained serial sections. For immunochemical staining, rabbit anti-spike polyclonal antibodies (Sino Biological: 40589-T62) were used. HE staining images and immunochemical staining images are shown in
According to the following procedure, temperature-sensitive strain-infected hamsters were challenged with a wild-type strain (clinical isolate).
After 4-week-old male Syrian hamsters (n=4) were bred for 1 week, the clinical isolate (B-1 strain, [Comparative Example]) or the temperature-sensitive strain (A50-18 strain) (1×104 or 1×106 TCID50) was nasally administered at a dose of 100 μL. After 21 days, the clinical isolate (B-1 strain) (1×106 TCID50) was nasally administered again at a dose of 100 μL, and weight changes for 10 days were observed. At this time, non-infected hamsters of the same age (n=3) were used as a naive control. The results were shown in
As shown in
After 4-week-old male Syrian hamsters (n=5) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) or the temperature-sensitive strain (A50-18 strain) (1×106 TCID50) was nasally administered at a dose of 100 μL. Weight changes after infection are shown in
21 days after infection, whole blood was collected, and serum was separated, and then the serum was inactivated by heating at 56° C. for 30 minutes. B-1 strain of 100 TCID50 was mixed with inactivated serum which had been serially diluted, and the mixture was reacted at 37° C. for 1 hour. After the reaction, the culture mixture was seeded on Vero cells, and after culturing at 37° C., neutralizing activity of the virus was evaluated by observing CPE. The highest dilution rate 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 were isolated by the method of
Mutation analysis of the following virus strains was carried out using a next-generation sequencer. The analysis was carried out 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 in
Vero cells were infected with 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 (revertants) were found. CPE images after culturing the obtained revertants at 38° C. for 3 days are shown in
Vero cells were infected with L50-33 strain and L50-40 strain at MOI=0.01, and growth at 32° C., 34° C. and 37° C. was evaluated. As a result, from among L50-33 strain and L50-40 strain, samples in which the growth at 37° C. was recovered (hereinafter, revertants) were found. CPE images after culturing each strain at 37° C. for 3 days are shown in
Mutation analysis of H50-11 strain, L50-33 strain, and L50-40 strain was carried out using Sanger sequencing. The analysis was carried out 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, H50-11 strain, L50-33 strain, and L50-40 strain, as shown in Table 3 below, mutations with check marks in the amino acid sequences of the indicated SEQ ID NOs were found, and among them, mutations with double check marks were found as temperature-sensitive mutations.
Vero cells were infected with additional isolates under the condition of MOI=0.01 (N=3). After culturing at 37° C., 34° C. or 32° C., each culture supernatant was collected on 0 to 5 d.p.i. Virus titers of these culture supernatants were measured by TCID50/mL using the Vero cells. The results are shown in
After 4-week-old male Syrian hamsters (n=5) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) or the temperature-sensitive strains (A50-18 strain [Reference Example], and L50-33 strain, L50-40 strain, and H50-11 strain [Examples]) (3×105 TCID50) were nasally administered at a dose of 100 μL, and weight changes for 10 days were observed. A group to which the same dose of D-MEM medium was nasally administered was defined as a non-infected control (MOCK). The results are shown in
After 4-week-old male Syrian hamsters (n=5) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) or the temperature-sensitive strains (A50-18 strain [Reference Example], and L50-33 strain, L50-40 strain, and H50-11 strain [Examples]) (3×105 TCID50) were nasally administered at a dose of 100 μL. The hamsters were euthanized on 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. Thereafter, the right lungs were disrupted, 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 is shown in
As a result of comparing lung weight per total weight of hamsters, the lung weight of B-1 strain-infected hamsters 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 temperature-sensitive strain-infected hamsters. In addition, as a result of comparing viral amounts in the nasal wash, there was no significant difference between B-1 strain-infected hamsters and the hamsters infected with the temperature-sensitive strains except H50-11 strain, but H50-11 strain-infected hamsters had a small viral amount in the nasal wash. Furthermore, it was revealed that the intrapulmonary viral amounts of the temperature-sensitive strain-infected hamsters were significantly lower than those of B-1 strain-infected hamsters. From these results, it was speculated that each temperature-sensitive strain was an attenuated strain that could not grow in the lower respiratory tract, similarly to A50-18 strain in Test Example 1.
After 4-week-old male Syrian hamsters (n=5) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) or the temperature-sensitive strains (A50-18 strain [Reference Example], and L50-33 strain, L50-40 strain, and H50-11 strain [Examples]) (3×105 TCID50) were nasally administered at a dose of 100 μL. After 21 days, the clinical isolate (B-1 strain) (3×103 TCID50) was nasally administered again at a dose of 100 μL. and weight changes for 9 days were observed. At this time, non-infected hamsters of the same age (n=5) were used as a naive control. The results are shown in
After 4-week-old male Syrian hamsters (n=S) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) or the temperature-sensitive strains (A50-18 strain [Reference Example], and L50-33 strain, L50-40 strain, and H50-11 strain [Examples]) (3×105 TCID50) were nasally administered at a dose of 100 μL. After 20 days, blood was partially collected, and the obtained serum was used to measure neutralizing activity against the clinical isolate (B-1 strain). The neutralizing activity was measured in the same method as in (1-5-2). The measurement results are shown in
Evaluation of neutralizing activity of serum of temperature-sensitive strain-infected hamsters against SARS-CoV-2 mutant strains
After 4-week-old male Syrian hamsters (n=3 or 5) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) or the temperature-sensitive strain (A50-18 strain [Reference Example]) (3×105 TCID50) was nasally administered at a dose of 100 μL. Blood was partially collected from hamsters at 3 weeks after infection, and the obtained serum was used to measure the neutralizing activity against live viruses of the SARS-CoV-2 European clinical isolate (B-1) and a Brazilian variant (hCoV-19/Japan/TY7-503/2021 strain). The results are shown in
After 4-week-old male Syrian hamsters (n=5) were bred for 1 week, the clinical isolate (B-1 strain [Comparative Example]) or the temperature-sensitive strain (A50-18 strain [Reference Example]) (3×105 TCID50) was nasally or subcutaneously administered at a dose of 100 μL. The untreated group was defined as a naive control. After 3 weeks, the serum obtained by partial blood collection from the hamsters was used to evaluate neutralizing activity against the SARS-CoV-2 Brazilian variant (hCoV-19/Japan/TY7-503/2021 strain). The neutralizing activity was measured in the same method as in (1-5-2). The results of the neutralizing activity are shown in
After 4-week-old male Syrian hamsters (n=5) were bred for 1 week, the temperature-sensitive strain (A50-18 strain) was administered nasally or subcutaneously. Doses are shown in Table 4.
Blood was partially collected from hamsters at 3 weeks after infection, and the obtained serum was used to measure the neutralizing activity against live viruses of the SARS-CoV-2 Brazilian variant (hCoV-19/Japan/TY7-503/2021 strain). The results are shown in
After 4-week-old male Syrian hamsters (n=4) were bred for 1 week, the temperature-sensitive strain (A50-18 strain [Reference Example]) of 1×104 TCID50 or 1×102 TCID50 was nasally administered at a dose of 10 μL. Blood was partially collected from hamsters at 3 weeks after infection, and the obtained serum was used to measure the neutralizing activity against live viruses of the SARS-CoV-2 European wild-type strain (B-1 strain), an Indian variant (self-isolated strain), and the Brazilian variant (hCoV-19/Japan/TY7-503/2021 strain). The results are shown in
Moreover, the results of comparing neutralizing antibody titers against each strain of the serum of each individual are shown in
Since live attenuated vaccines inherently involve growth in a host's body, there is a possibility that a mutation that occurs during nucleic acid replication results in the development of a virulent wild-type strain. In order to reduce the possibility, strains were constructed in which temperature-sensitive mutations and growth reducing or other attenuating mutations were combined. Such strains were constructed so as to be able to maintain their attenuation even when the temperature-sensitive mutations were lost. As temperature-sensitive mutations, growth reducing or other attenuating mutations, and other mutations, mutations checked in Table 5 [i.e., L445F in NSP3, G248V and G416S in NSP14, and V67I in NSP16; deletion of eight amino acids at positions 32 to 39 in NSP1, deletion of furin cleavage site (FCS) in Spike (specifically, the deletion at the 679th to 686th positions of the spike and V687I), and loss of function in ORF8; any of the other mutations found in the temperature-sensitive strains (NSP3 K1792R and NSP14 A504V)] were used.
By a reverse genetics method using CPER, strains having the above temperature-sensitive mutations and growth reducing or other attenuating mutations together with other mutations were constructed (Torii et al. cell report 2020). The genome of SARS-CoV-2 B-1 strain was fragmented and cloned into a plasmid. Inverse PCR was used to introduce the mutations of interest into the cloned fragments. Using the plasmid obtained by cloning the wild-type fragment as a template, a SARS-CoV-2 wild-type genomic fragment was obtained by PCR. In addition, PCR or RT-PCR was carried out using the plasmid into which the mutations were introduced or the genome of the SARS-CoV-2 mutant strains having the mutations of interest as a template, so as to obtain SARS-CoV-2 mutant genomic fragments.
The obtained fragments and linker fragments containing a CMV promoter were mixed, and CPER was carried out using PrimeStar GXL polymerase to circularize a plurality of fragments. The reaction mixture was transfected into BHK/hACE2 cells, and the cells were cultured at 34° C. to reconstruct target viruses. The culture supernatant of the obtained cells was added to VeroE6/TMPRSS II cells and cultured at 34° C. to recover the reconstructed viruses. The temperature-sensitive strain mutation(s) and/or growth reducing or other attenuating mutation(s) introduced into each candidate strain and CPE images are shown in
In order to evaluate the temperature sensitivity of candidate strains 1 to 7 (Examples) obtained in Test Example 9, Vero cells were infected with 2 μL of supernatant of recovery culture of each candidate strain, and growth at 34° C. and 37° C. was compared. CPE images after culturing for 3 days are shown in
A strain reconstructed by introducing all temperature-sensitive mutations (rTs-all strain [Example]) was obtained. In rTs-all strain, three types: a mutation of (b), a combination of mutations of (e) and (f), and a mutation of (h), are introduced as temperature-sensitive mutations, and only a mutation of (n) is introduced as other attenuating mutation, and none of other mutations is introduced.
Vero cells were infected with rTs-all strain at MOI=0.01, cultured at 32, 37 or 39° C. for 5 days, and culture supernatants were sampled over time (n=3). Virus titers at each time point were measured by TCID50 method. For comparison, this B-1 strain (rB-1 strain) prepared by the CPER method was used. The resulting growth curves are shown in
In order to evaluate the immunogenicity of the candidate strains 1, 3, 4, 6 and 7 (Examples) among the candidate strains obtained in Test Example 9, 10 μL of each candidate strain of 100 TCID50 was nasally administered to five S-week-old male hamsters. Also, as a positive control, the SARS-CoV-2 temperature-sensitive strain, A50-18 strain, was nasally administered at the same titer and the same dose. After 3 weeks, the serum obtained by partially collecting blood was evaluated for neutralizing activity against SARS-CoV-2 B-1 strain. The neutralizing activity was evaluated by a method of determining the presence or absence of infectious viruses by mixing serially diluted serum and SARS-CoV-2 of 100 TCID50, reacting the mixture for 1 hour, then adding the mixture to Vero cells, and observing CPE after culturing for 4 days. The maximum dilution ratio of the serum in which CPE was not observed and the infectivity of the virus could be neutralized was defined as a neutralizing antibody titer. The results are shown in
Seroconversions of neutralizing activities were observed in all individual infected hamsters of A50-18 strain (Reference Example) as a positive control group. In the candidate strain 1 and the candidate strain 3, seroconversion of neutralizing activity was not observed under the administration conditions in the present test example, and thus it is suggested that administration at a higher dose is necessary in order to exhibit immunogenicity. On the other hand, in all of the candidate strain 4, 6 and 7, seroconversions of neutralizing activities were observed in some individuals or all individuals, and it was found that these candidate strains had immunogenicity even at a low dose as in the present test example. In particular, in the hamster infected with the candidate strain 7 (Example), seroconversions of neutralizing activities were observed in all the individuals as with A50-18 strain, and thus the degree of immunogenicity was particularly excellent. That is, among the candidate strains in which seroconversions of neutralizing activities were observed under the administration conditions in the present test example, the candidate strain 4 and 6 were constructed by combining temperature-sensitive mutations and other attenuating mutations, so as to improve the safety, and in particular, the candidate strain 7 was constructed by combining temperature-sensitive mutations and growth reducing or other attenuating mutations, so as to maintain excellent immunogenicity even though the growth in the body was remarkably reduced.
In order to evaluate whether the immunity induced in the candidate strain 7 contributes to the protection against infection, hamsters (male, 8 weeks old) after immunization were challenged by nasally administering 100 μL of 3×105 TCID50 SARS-CoV-2 B-1 strain. The weight changes of the hamsters at that time are shown in
The immunogenicity of the candidate strains 2 and 5 (Examples) when tested at high titer and high dose was evaluated. To five 5-week-old male hamsters, 20 μL of the candidate strains of 1×103 or 1×104 TCID50 were nasally administered. In addition, as a positive subject, the SARS-CoV-2 temperature-sensitive strain, A50-18 strain (Reference Example) of 1×103 TCID50 was nasally administered at the same dose. Blood was partially collected from hamsters recovered from infection after 3 weeks, and neutralizing activity of the obtained serum was evaluated. The neutralizing activity was measured by the same method as in Test Example 3. The results are shown in
In the candidate strain 2, seroconversion of neutralizing antibodies were observed in four out of five individuals by administration of 1×103 TCID50, and seroconversion of neutralizing antibodies were observed in all individuals when 1×104 TCID50 was administered. In the candidate strain 5, the neutralizing activity of the serum was confirmed only in one out of five individuals by the administration of 1×103 TCID50, but induction of neutralizing antibodies was observed in three out of five individuals by administering the virus of 1×104 TCID50. From these results, it was revealed that the vaccine candidate strains in which the temperature-sensitive mutation(s) and the growth reducing or other attenuating mutation(s) were combined had low immunogenicity at the same dose as compared with the strains having only the temperature-sensitive mutation(s), but exhibited immunogenicity necessary for induction of neutralizing antibodies by increasing the dose. In addition, it was found that the candidate strain 2 showed immune induction since neutralizing activity was confirmed also in monkeys.
Vero cells were infected with the clinical isolate (B-1 strain [Comparative Example]) and the candidate strain 2 ([Example]) using 6-well plates under conditions of MOI=0.01 (N=3). The cells were cultured at 37° C. or 32° C., and each culture supernatant was collected on 0 to 5 dpi. Virus titers of culture supernatants on 0 to 5 dpi were measured by TCID50/mL using the Vero cells. The results are shown in
From
After 4-week-old male Syrian hamsters (n=10) were bred for 1 week, the candidate strain 2 (Example) (1×103 TCID50 or 1×104 TCID50) was nasally administered at a dose of 20 μL under anesthetic conditions. In the group of twice administration, at the time point of 4 weeks after the first administration, the candidate strain 2 (1×103 TCID50 or 1×104 TCID50) was nasally administered again at a dose of 20 μL under anesthetic conditions. Blood was partially collected over time, and the obtained serum was heat-treated at 56° C. for 30 minutes to be inactivated. The inactivated serum was serially diluted and mixed with B-1 strain (D614G type: pre-alpha European strain) or TY38-873 strain (Omicron variant) of 100 TCID50, and the mixture was reacted at 37° C. for 1 hour. After the reaction, the culture mixture was seeded on Vero cells, and after culturing at 37° C., neutralizing activity of the virus was evaluated by observing CPE. The lowest dilution rate which did not cause CPE was defined as a neutralizing antibody titer. The results are shown in
From
The hamsters to which 20 μL of the candidate strain 2 of 1×103 or 1×104 TCID50 or the SARS-CoV-2 temperature-sensitive strain, A50-18 strain, of 1×103 TCID50 was nasally administered in Test Example 13 were challenged by nasally administering SARS-CoV-2 B-1 strain (3×105 TCID50) at a dose of 100 μL under anesthetic conditions 3 weeks after the first administration. The weight of the hamsters after challenge were measured up to 6 days after administration. The results are shown in
From
After 4-week-old male Syrian hamsters (n=5) were bred for 1 week, the temperature-sensitive strain, A50-18 strain, or the candidate strain 2 (1×105 TCID50) was nasally administered at a dose of 100 μL under anesthetic conditions. This dose is a human equivalent dose with a divisor of 30 extrapolating the human no adverse effect level from the hamster no adverse effect level, and corresponds to 2×105 PFU/dose. The weight changes were observed, and the hamsters were euthanized 3 days after administration, and then nasal wash was collected using 500 μL of PBS. The obtained nasal wash was filtered and sterilized with a 0.22 μm filter, and then 100 μL of the nasal wash was nasally administered to next generation hamsters under anesthetic conditions. By performing the same operation three times, nasal wash when one to four in vivo passages were carried out was obtained. The obtained nasal wash was seeded on Vero cells and cultured at each temperature to evaluate the presence or absence of infectious viruses and the temperature sensitivity of the viruses. In addition, viral RNA was extracted from the nasal wash, and the base sequence of the target site was confirmed by Sanger sequencing method. The presence or absence of CPE in the Vero cells seeded with the nasal wash after each passage is shown in
Since A50-18 strain is a temperature-sensitive strain, CPE was not caused when cultured at 37° C. or 39° C. after infected to the Vero cells, but from
On the other hand, as to the candidate strain 2, from
After 4-week-old male Syrian hamsters (n=4) were bred for 1 week, SARS-CoV-2 B-1 strain [Comparative Example], the temperature-sensitive strain, A50-18 strain [Reference Example], or the candidate strain 2 [Example] (1×105 TCID50) was nasally administered with a dose of 20 μL under anesthetic conditions. This dose is a human equivalent dose with a divisor of 30 extrapolating the human no adverse effect level from the hamster no adverse effect level, and corresponds to 4×104 PFU/dose. The non-infected group (naive) was nasally administered with the same dose of a culture medium under anesthetic conditions. Also, as a positive control, SARS-CoV-2 B-1 strain (1×105 TCID50) was nasally administered at a dose of 100 μL under anesthetic conditions. The virus fluid reached the upper respiratory tract of the hamster by nasal administration at a dose of 20 μL, and the virus fluid reached the lower respiratory tract of the hamster by nasal administration at a dose of 100 μL. Three days after administration, the hamsters were euthanized, and then the head and lungs were fixed with formalin. Tissue damages were evaluated by HE staining, and viral antigens were detected by IHC staining using Rabbit anti-spike RBD antibodies (Sino Biological (40592-T62)). Lesions in the nasal cavity and lung part of each individual, and scores for detection of viral antigens by IHC are shown in Table 6. In the table, Level 1 shows the tip of the nasal cavity, Level 2 shows the middle of the nasal cavity, and Level 3 shows the back of the nasal cavity. In addition, for representative examples in each site of each virus-infected hamster. Level 1 is shown in
From
[Test Example 19] Evaluation of Immunogenicity by the Candidate Strain 2 (Challenge with Hetero Strain)
It was evaluated whether the immunity induced by the candidate strain 2 contributed to the protection against infection of a SARS-CoV-2 Omicron variant. After 4-week-old male Syrian hamsters were bred for 1 week, the candidate strain 2 (1×103 PFU) was nasally administered at a dose of 20 μL under anesthetic conditions. At the time point of 4 weeks after the administration, the serum was collected by partial blood collection. The obtained serum was heat-treated at 56° C. for 30 minutes to be inactivated. The inactivated serum was serially diluted and mixed with B-1 strain (D614G type: pre-alpha European strain) or TY41-702 strain (Omicron variant: BA.5) of 100 TCID50, and the mixture was reacted at 37° C. for 1 hour. After the reaction, the culture mixture was seeded on Vero cells, and after culturing at 37° C., neutralizing activity of the virus was evaluated by observing CPE. The lowest dilution rate which did not cause CPE was defined as a neutralizing antibody titer. The results are shown in
From
It was evaluated whether the immunity induced by the candidate strain 2 was effective against a SARS-CoV-2 delta variant and a gamma variant. Neutralizing activities against the gamma variant and the delta variant of the candidate strain 2 immune serum obtained in Test Example 13 were measured. The inactivated serum was serially diluted and mixed with BK325 strain (a delta variant) or TY7-501 strain (a gamma variant) of 100 TCID50, and the mixture was reacted at 37° C. for 1 hour. After the reaction, the culture mixture was seeded on Vero cells, and after culturing at 37° C., neutralizing activity of the virus was evaluated by observing CPE. The lowest dilution rate which did not cause CPE was defined as a neutralizing antibody titer. The results are shown in
From
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-182051 | Nov 2021 | JP | national |
| 2022-133080 | Aug 2022 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/041445 | 11/7/2022 | WO |
