AAV5-based vaccines against SARS-CoV-2

Abstract

This application relates to the fields of biotechnology, immunology, virology, genetics, and molecular biology. More specifically, the present invention relates to an isolated recombinant receptor-binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 (severe acute respiratory syndrome-associated coronavirus 2), a nucleic acid encoding the RBD-S of SARS-CoV-2, expression cassettes and vectors based thereon, and a recombinant AAV5 (adeno-associated virus serotype 5)-based virus for inducing SARS-CoV-2-specific immunity and / or preventing SARS-CoV-2-related coronavirus infection, an AAV5-based vaccine for inducing SARS-CoV-2-specific immunity and / or preventing SARS-CoV-2-related coronavirus infection, and uses thereof for inducing SARS-CoV-2-specific immunity and / or preventing SARS-CoV-2-related coronavirus infection.

Description

[Technical Field]

[0001] This application relates to the fields of biotechnology, immunology, virology, genetics, and molecular biology. More specifically, the present invention relates to an isolated recombinant receptor-binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 (severe acute respiratory syndrome-associated coronavirus 2), a nucleic acid encoding the RBD-S of SARS-CoV-2, expression cassettes and vectors based thereon, and a recombinant AAV5 (adeno-associated virus serotype 5)-based virus for inducing SARS-CoV-2-specific immunity and / or preventing SARS-CoV-2-related coronavirus infection, an AAV5-based vaccine for inducing SARS-CoV-2-specific immunity and / or preventing SARS-CoV-2-related coronavirus infection, and uses thereof for inducing SARS-CoV-2-specific immunity and / or preventing SARS-CoV-2-related coronavirus infection. [Background technology]

[0002] SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a member of the Sarbecovirus subgenus of the Betacoronavirus genus. SARS-CoV-2 was identified in December 2019 following analysis of samples taken from pneumonia patients. On December 31, 2019, the World Health Organization notified several cases of viral pneumonia caused by a previously unknown pathogen. The complete genome of the virus was sequenced for the first time in China.

[0003] Coronaviruses, including SARS-CoV-2, commonly cause acute respiratory illness. This family also includes SARS-CoV and MERS-CoV, which cause severe acute respiratory syndrome and Middle East respiratory syndrome, respectively.

[0004] SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic. In January 2020, the World Health Organization declared the SARS-CoV-2 outbreak a public health emergency of international concern, and on March 11, 2020, characterized the global spread of the disease as a pandemic.

[0005] The GenBank database, Wu F., Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome, 2020, GenBank:MN908947.3 (https: / / www.ncbi.nlm.nih.gov / nuccore / MN908947) and the article by Fan Wu et al., A new coronavirus associated with human respiratory disease in China, 2020, Nature, Issue 579, pp. 265-269 (https: / / www.nature.com / articles / s41586-0020-2008-3) provide information about the SARS-CoV-2 genome.

[0006] Patent document CN110951756(B) discloses nucleotide acid sequences encoding SARS-CoV-2 antigen peptides and suggests that these nucleotide acid sequences can be used to induce appropriate immune responses; they are expected to be used in vaccines against SARS-CoV-2.

[0007] Patent document CN110974950B discloses a vaccine for preventing SARS-CoV-2 infection, which includes an Ad5 adenovirus vector containing a nucleic acid sequence encoding a SARS-CoV-2 antigenic peptide.

[0008] Patent document RU2720614 C1 (NF Gamaleya National Research Centre for Epidemiology and Microbiology, Honorary Member of the Ministry of Health of the Russian Federation) discloses a vaccine based on 26 particles containing recombinant adenovirus serotype 5 and / or S protein genes of SARS-CoV-2.

[0009] As of the filing date of this application, SARS-CoV-2 cases have reached over 23,000,000, and SARS-CoV-2 deaths have reached over 800,000. Furthermore, the COVID-19 pandemic is still ongoing as of the filing date of this application. Therefore, there is an urgent global need for effective means to prevent and treat diseases caused by the severe acute respiratory syndrome virus SARS-CoV-2. Summary of the Invention [Problem to be solved by the invention]

[0010] The authors of the present invention have developed an isolated recombinant receptor-binding domain (RBD-S) of glycoprotein S of SARS-CoV-2, which can be used as an antigen for effective immunization in mammals by inducing SARS-CoV-2-specific immunity, thereby contributing to the prevention of SARS-CoV-2-related diseases. The authors of the present invention have also developed means for delivering said antigen to mammalian organisms, in particular an expression vector containing a nucleic acid encoding said antigen, a recombinant AAV5 (adeno-associated virus serotype 5)-based virus containing a nucleic acid encoding said antigen, a vaccine containing said object, and methods for its use to induce SARS-CoV-2-specific immunity and / or prevent infection with SARS-CoV-2-related coronaviruses. [Means for solving the problem]

[0011] In one aspect, the present invention relates to an isolated recombinant receptor-binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2, represented by the amino acid sequence of SEQ ID NO:1.

[0012] In one aspect, the present invention relates to an isolated recombinant receptor-binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 (Severe Acute Respiratory Syndrome-associated Coronavirus 2), represented by the amino acid sequence of SEQ ID NO:1.

[0013] In one aspect, the present invention relates to an isolated nucleic acid encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. In some embodiments, the isolated nucleic acid is DNA.

[0014] In some embodiments, the isolated nucleic acid is the nucleotide sequence of SEQ ID NO:2. In some embodiments, the isolated nucleic acid is a codon-optimized nucleotide sequence.

[0015] In one aspect, the present invention relates to an expression cassette comprising any one of the above nucleic acids encoding the isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2.

[0016] In some embodiments, the expression cassette comprises the following elements from the 5' to the 3' end: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; introns of the hBG1 gene (hemoglobin subunit gamma 1 gene); Any one of the foregoing nucleic acids encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Contains the right (second) ITR.

[0017] In some embodiments, the expression cassette comprises the nucleic acid of SEQ ID NO:3. In one aspect, the present invention relates to an expression vector comprising any one of the nucleic acids encoding the isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 or any one of the expression cassettes.

[0018] In one aspect, the invention relates to an isolated recombinant AAV5 (adeno-associated virus serotype 5)-based virus for inducing SARS-CoV-2-specific immunity and / or preventing infection with a SARS-CoV-2-related coronavirus, the virus comprising a capsid and any of the above expression cassettes.

[0019] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1. In some embodiments, the AAV5-based recombinant virus has a capsid that includes the AAV protein VP1, which has the amino acid sequence of SEQ ID NO:4.

[0020] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 containing one or more point mutations.

[0021] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:5 (S2A and T711S).

[0022] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 containing one or more point mutations, and the expression cassette comprises, from the 5' to the 3' end, the following elements: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; introns of the hBG1 gene (hemoglobin subunit gamma 1 gene); Any one of the foregoing nucleic acids encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Contains the right (second) ITR.

[0023] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 containing one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO:3.

[0024] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 containing one or more point mutations, wherein the AAV5 protein VP1 is the amino acid sequence of SEQ ID NO:5 (S2A and T711S).

[0025] In one aspect, the present invention relates to a pharmaceutical composition for preventing infection with a SARS-CoV-2 related coronavirus, the pharmaceutical composition comprising any of the above AAV5-based recombinant viruses in combination with one or more pharmaceutically acceptable excipients.

[0026] In one aspect, the present invention relates to a pharmaceutical composition for inducing SARS-CoV-2 specific immunity, the pharmaceutical composition comprising any of the above AAV5-based recombinant viruses in combination with one or more pharmaceutically acceptable excipients.

[0027] In one aspect, the invention relates to the use of any of said recombinant AAV5-based viruses or said pharmaceutical compositions to prevent infection with a SARS-CoV-2 related coronavirus.

[0028] In one aspect, the invention relates to the use of any of said recombinant AAV5-based viruses or said pharmaceutical compositions for inducing SARS-CoV-2 specific immunity. In one aspect, the invention relates to a vaccine for preventing infection with a SARS-CoV-2 related coronavirus, the vaccine comprising an effective amount of any of the above recombinant AAV5-based viruses.

[0029] In one aspect, the invention relates to a vaccine for inducing SARS-CoV-2 specific immunity, the vaccine comprising an effective amount of any of the above recombinant AAV5-based viruses.

[0030] In one aspect, the invention relates to a method for inducing SARS-CoV-2 specific immunity, the method comprising administering to a mammalian organism an effective amount of any one of said recombinant AAV5-based virus, said composition or said vaccine to induce SARS-CoV-2 specific immunity.

[0031] In one aspect, the present invention relates to a method for preventing infection with a SARS-CoV-2 related coronavirus, the method comprising administering to a mammalian organism an effective amount of any one of the recombinant AAV5-based viruses, the composition or the vaccine to prevent infection with a SARS-CoV-2 related coronavirus. [Brief explanation of the drawings]

[0032] [Figure 1]Figure 1 shows a schematic diagram of the plasmid pAAV RBD-S, which is intended to generate an AAV vector containing an expression cassette containing the RBD-S gene sequence of the recombinant receptor-binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. The leader peptide is a peptide that enables secretion of the target protein. RBD-S is the recombinant receptor-binding domain of the S glycoprotein of SARS-CoV-2. AmpR is the beta-lactamase gene that confers resistance to ampicillin. The pUC origin is the bacterial pUC origin of replication. The ITRs are inverted terminal repeats. The CMV enhancer is the cytomegalovirus enhancer. The CMV promoter is the promoter of the cytomegalovirus early gene. The polyA is a polyadenylation signal sequence to increase mRNA stability. The HBG intron is the human beta-globin intron. [Figure 2] FIG. 2 is a graph showing the concentration of RBD-S protein in CHO-K1-S cell cultures 3 days after cell transduction with viral AAV5-RBD-S product. [Figure 3] Figure 3 is a graph showing the content of antibodies against the RBD-S protein in the plasma of study animals after immunization with the AAV5-RBD-S product (intramuscular injection at 1 x 10 VG / mouse; injection volume 200 μL). Figure 3 shows the individual scores for each animal in the study. VG is the viral genome. [Figure 4] Figure 4 is a graph showing the content of antibodies against the RBD-S protein in the plasma of study animals after immunization with the AAV5-RBD-S product (intramuscular injection at 1 x 10 VG / mouse; injection volume 200 μL). Figure 4 shows the mean ± standard deviation (n = 8). VG is the viral genome. [Figure 5] Figure 5 is a graph showing the content of antibodies against the RBD-S protein in the plasma of study animals after immunization with the AAV5-RBD-S product (intramuscular injection at 4 x 10 VG / mouse; injection volume 200 μL). Figure 5 shows the individual scores for each animal in the study. VG is the viral genome. [Figure 6]Figure 6 is a graph showing the content of antibodies against the RBD-S protein in the plasma of study animals after immunization with the AAV5-RBD-S product (intramuscular injection at 4 x 10 VG / mouse; injection volume 200 μL). Figure 6 shows the mean ± standard deviation (n = 8). VG is the viral genome. [Figure 7] Figure 7 is a graph showing the content of antibodies against the RBD-S protein in the plasma of study animals after immunization with an AAV5 product whose genome does not contain an expression cassette containing the RBD-S gene (a product containing an empty AAV5 capsid). 8.6 x 10 CP / mouse intramuscularly injected; injection volume 200 μL. The graph shows the individual scores for each animal in the study. CP is the viral capsid. [Figure 8] Figure 8 is a graph showing the content of antibodies against RBD-S protein in the plasma of study animals after immunization with purified recombinant RBD-S protein product (20 μg / mouse intramuscularly).Figure 8 shows the individual scores for each animal in the study. [Figure 9] Figure 9 is a graph showing the content of antibodies against RBD-S protein in the plasma of study animals after immunization with purified recombinant RBD-S protein product (20 μg / mouse intramuscularly). Figure 9 shows the mean ± standard deviation (n=7). [Figure 10] Figure 10 is a graph showing the content of antibodies against the RBD-S protein in the plasma of study animals after immunization with a control AAV-free product. The graph shows the individual scores for each animal in the study. Definitions and General Methods Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0033] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the present classifications and methods of cell culture, molecular biology, immunology, bacteriology, genetics, analytical chemistry, organic synthetic chemistry, medical and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known by and widely used in the art. Enzymatic reactions and purification methods are performed according to manufacturer's guidelines or as described herein, as is common to those skilled in the art.

[0034] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in an animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a genetically modified cell.

[0035] The terms "naturally occurring," "native," or "wild-type" are used to describe something that can be found in nature, as opposed to something that has been artificially created. For example, a protein or nucleotide sequence that is present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man in the laboratory is naturally occurring.

[0036] The term "genome" refers to the complete genetic material of an organism. As used in this description and the claims that follow, unless the context dictates otherwise, the words "include" and "comprise," or variations thereof such as "having," "includes," "including," "comprises," or "comprising," will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Proteins (peptides) As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no restriction on the maximum number of amino acids that a protein or peptide sequence may contain. A polypeptide includes any peptide or protein containing two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to by those skilled in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to by those skilled in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. nucleic acid molecule The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in this description, refer to the exact sequence of nucleotides, modified or not, that defines a fragment or region of a nucleic acid, that may or may not contain non-naturally occurring nucleotides, and that is either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0037] Those skilled in the art have the general understanding that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used in this description, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available to those skilled in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes, using conventional cloning techniques and PCR, etc., as well as synthetic means.

[0038] It should also be noted here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. The sequences of the present invention are isolated and / or purified, i.e., sampled directly or indirectly, for example by copying, and their environment is at least partially modified. Thus, isolated nucleic acids obtained by means of recombinant genetics, e.g., host cells, or obtained by chemical synthesis, should also be mentioned here.

[0039] Unless otherwise specified, the term nucleotide sequence includes its complement. Thus, a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

[0040] The terms "transformation," "transfection," and "transduction" refer to any method or means by which nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, infection, PEG fusion, etc. Adeno-associated virus (AAV) Viruses in the Parvoviridae family are small DNA-containing animal viruses. The Parvoviridae family can be divided into two subfamilies: the Parvovirinae, whose members infect vertebrates, and the Densovirinae, whose members infect insects. As of 2006, 11 serotypes of adeno-associated viruses have been described [Mori, S. et al., 2004, "Two novel adeno-associated viruses from cynomolgus monkeys: pseudotyping characterization of capsid protein," Virology, T. 330(2):375-83]. All known serotypes can infect cells from multiple tissue types. Tissue specificity is determined by the serotype of the capsid protein; therefore, adeno-associated virus-based vectors are constructed by assigning the desired serotype. Further information about parvoviruses and other members of the Parvoviridae family can be found in the literature [Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 of Fields Virology (3rd ed. 1996)].

[0041] The genome organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for replicating nonstructural proteins (Rep) and structural proteins (Cap). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) that form the capsid. The terminal 145 nucleotides are self-complementary and organized to allow the formation of an energetically stable intramolecular duplex that forms a T-shaped hairpin. Such a hairpin structure serves as an origin of replication for viral DNA and as a primer for the cellular DNA polymerase complex. After wild-type AAV (wtAAV) infection in mammalian cells, Rep genes (e.g., Rep78 and Rep52) are expressed using the P5 and P19 promoters, respectively, and both Rep proteins have specific functions in viral genome replication. Splicing events in the Rep open reading frame (Rep ORF) actually result in the expression of four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that the unspliced ​​mRNAs encoding the Rep78 and Rep52 proteins are sufficient for the production of AAV vectors in mammalian cells. vector As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0042] The terms "infectious unit (iu)," "infectious particle," or "replication unit" as used in reference to viral titer refer to the number of infectious recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al., J. Virol. (1988) 62:1963-1973.

[0043] The term "heterologous" with respect to nucleic acid sequences, such as coding sequences and regulatory sequences, refers to sequences that are not normally linked together and / or not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid that is within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct can include a coding sequence flanked by sequences that are not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from the native gene).

[0044] As used in this description, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter. use The terms "prophylaxis" or "prevention" and the like mean slowing or preventing the onset of symptoms of a disease, disorder or infection.

[0045] The term "induction of an immune response" as used in the present invention refers to the specific control of or effect on the activity of an immune response, and includes activation of an immune response, stimulation of an immune response, and enhancement of an immune response.

[0046] The term "specific immunity" as used in the present invention refers to a state of immunity to a disease after induction of an immune response. The term "disorder" means any condition that would benefit from treatment according to the present invention, including chronic and acute disorders or diseases, and includes pathological conditions that predispose a mammal to the disorder in question.

[0047] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis, and if the disease is not ameliorated, the animal's health will continue to deteriorate thereafter. In this description, the terms "subject," "patient," "individual," etc. are used interchangeably and refer to any animal suitable for the methods described in this description. In certain non-limiting embodiments, the subject, patient, or individual is a human. The subject may be male or female of any age.

[0048] A "therapeutically effective amount" or "effective amount" refers to that amount of the therapeutic agent being administered that will relieve to some extent one or more symptoms of the disease being prevented. DETAILED DESCRIPTION OF THE INVENTION

[0049] Peptide antigens In one aspect, the present invention provides a method for producing a medicament comprising the amino acid sequence

[0050] [ka]

[0051] Regarding the isolated recombinant receptor-binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 (severe acute respiratory syndrome-associated coronavirus 2), represented by The RBD-S of SARS-CoV-2 is obtained from the full-length S glycoprotein of SARS-CoV-2, which is described in the GenBank database: Wu F., Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome, 2020, GenBank: MN908947.3 (https: / / www.ncbi.nlm.nih.gov / nuccore / MN908947) and Fan Wu et al., A new coronavirus associated with human respiratory disease in China, 2020, Nature, 579, 265-269 (https: / / www.nature.com / articles / s41586-0020-2008-3), and has the following amino acid sequence:

[0052] [ka]

[0053] It has. The receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 was selected from the above sequence of the S glycoprotein of SARS-CoV-2 based on an analysis of the structure of this glycoprotein (see Example 1), and the receptor binding domain of the S glycoprotein of SARS-CoV-2 has the following amino acid sequence:

[0054] [ka]

[0055] It has. Next, further stability of the RBD-S protein of SARS-CoV-2 is achieved by introducing the point amino acid substitution C272S into the amino acid sequence of SEQ ID NO:7, thereby generating the recombinant receptor binding domain of the S glycoprotein of SARS-CoV-2 (RBD-S) having the amino acid sequence of SEQ ID NO:1. nucleic acid In one aspect, the present invention relates to an isolated nucleic acid encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2.

[0056] An "isolated" nucleic acid molecule is one that is identified and separated from at least one nucleic acid molecule impurity, with which the former is generally associated in the natural source of the nuclease nucleic acid. An isolated nucleic acid molecule differs from the form or set found under natural conditions. Thus, an isolated nucleic acid molecule differs from the nucleic acid molecule present in cells under natural conditions. However, an isolated nucleic acid molecule includes nucleic acid molecules located in cells where the nuclease is normally expressed, for example, when the nucleic acid molecule has a chromosomal location that differs from its location in cells under natural conditions.

[0057] In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence

[0058] [ka]

[0059] is. In some embodiments, the isolated nucleic acid is a codon-optimized nucleotide sequence. Expression cassette. Expression vector.

[0060] In one aspect, the present invention relates to an expression cassette comprising any one of the above nucleic acids encoding the isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2.

[0061] As used herein, the term "expression cassette" particularly refers to a DNA fragment that, under appropriate settings, can induce the expression of the polynucleotide encoding the polypeptide of interest contained in the expression cassette.When introduced into host cells, the expression cassette is particularly capable of participating in the cellular machinery for transcribing the polynucleotide encoding the polypeptide of interest into RNA, and then the RNA is generally further processed and ultimately translated into the polypeptide of interest.The expression cassette can be contained in an expression vector.

[0062] The expression cassette of the present invention includes a promoter as an element. As used herein, the term "promoter" specifically refers to a DNA element that promotes transcription of a polynucleotide to which it is operably linked. A promoter may also form part of a promoter / enhancer element. While the physical boundary between a "promoter" and an "enhancer" element is not always clear, the term "promoter" generally refers to a site on a nucleic acid molecule to which RNA polymerase and / or any associated factors bind and transcription is initiated. Enhancers temporally and spatially enhance promoter activity. Many promoters are known to those skilled in the art to be transcriptionally active in a variety of cell types. Promoters can be divided into two classes: those that function constitutively and those that are regulated by induction or derepression. Both classes are suitable for protein expression. Promoters used for high-level production of polypeptides in eukaryotic cells, particularly mammalian cells, should be strong, and preferably active, in a variety of cell types. Strong constitutive promoters capable of driving expression in many cell types are well known to those skilled in the art, and therefore need not be described in detail herein. According to the concept of the present invention, it is preferred to use a cytomegalovirus (CMV) promoter. A promoter or promoter / enhancer obtained from the immediate-early (IE) region of human cytomegalovirus (hCMV) is particularly suitable as a promoter in the expression cassette of the present invention. The immediate-early (IE) region of human cytomegalovirus (hCMV) and functional expression-inducing and / or expression-enhancing fragments obtained therefrom are described, for example, in European Patent Nos. 0173177 and 0323997, and are well known to those skilled in the art. Thus, several fragments of the immediate-early (IE) region of hCMV can be used as a promoter and / or promoter / enhancer. According to one embodiment of the present invention, a human CMV promoter is used in the expression cassette of the present invention.

[0063] In some embodiments, the expression cassette comprises the following elements from the 5' to the 3' end: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; introns of the hBG1 gene (hemoglobin subunit gamma 1 gene); Any one of the foregoing nucleic acids encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Contains the right (second) ITR.

[0064] In some embodiments, the left (first) ITR (inverted terminal repeat) has the following nucleic acid sequence:

[0065] [ka]

[0066] It has. In some embodiments, the CMV (cytomegalovirus) enhancer comprises the following nucleic acid sequence:

[0067] [ka]

[0068] It has. In some embodiments, the CMV (cytomegalovirus) promoter comprises the following nucleic acid sequence:

[0069] [ka]

[0070] It has. In some embodiments, the intron of the hBG1 (hemoglobin subunit gamma 1) gene has the following nucleic acid sequence:

[0071] [ka]

[0072] It has. In some embodiments, the hGH1 (human growth hormone 1 gene) polyadenylation signal comprises the following nucleic acid sequence:

[0073] [ka]

[0074] It has. In some embodiments, the right (second) ITR has the following nucleic acid sequence:

[0075] [ka]

[0076] It has. In some embodiments, the expression cassette comprises the nucleotide sequence:

[0077] [ka]

[0078] The nucleic acid includes: In one aspect, the present invention relates to an expression vector comprising any one of the nucleic acids encoding the isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 or any one of the expression cassettes.

[0079] In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector and additional DNA segments may be ligated into the viral genome.

[0080] In some embodiments, vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In further embodiments, the vector (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genes. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0081] Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. DNA molecules can be ligated into vectors such that transcriptional and translational control sequences within the vector perform their intended function of regulating DNA transcription and translation. Expression vectors and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules can be introduced into expression vectors by standard methods (e.g., ligation of complementary restriction sites, or blunt-end ligation if no restriction sites are present).

[0082] The recombinant expression vector may also encode a leader peptide (or signal peptide) that facilitates secretion of the protein of interest from the host cell. The gene for the protein of interest may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the protein of interest. The leader peptide (or signal peptide) may be an immunoglobulin leader peptide or a heterologous leader peptide (i.e., a leader peptide of a non-immunoglobulin protein).

[0083] In addition to the SARS-CoV-2 RBD-S gene described in the present invention, recombinant expression vectors described in the present invention may carry regulatory sequences that control the expression of the SARS-CoV-2 RBD-S gene in host cells. Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, and other factors. Preferred regulatory sequences for mammalian expression host cells include viral elements that ensure high levels of protein expression in mammalian cells, such as promoters and / or enhancers from retroviral LTRs, cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the major late promoter adenovirus (AdMLP)), and polyomavirus, as well as strong mammalian promoters such as the native immunoglobulin promoter or actin promoter.

[0084] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0085] The term "promoter" or "transcriptional regulatory sequence" or "regulatory sequence" as used in this description refers to a nucleic acid fragment that controls the transcription of one or more coding sequences, is located upstream in the direction of reading compared to the direction of transcription from the transcription start site of the coding sequence, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, as well as any other nucleotide sequences known to those skilled in the art that directly or indirectly regulate the level of transcription by the promoter. A "constitutive" promoter is a promoter that is active in most tissues under typical physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, under the influence of a chemical inducer. A "tissue-specific" promoter is only active in specific types of tissues or cells.

[0086] As used herein, the term "enhancers" or "enhancer" can refer to a DNA sequence located adjacent to a DNA sequence that encodes a recombinant product. Enhancer elements are generally located 5' from a promoter element, or can be located downstream of or within a coding DNA sequence (e.g., a DNA sequence that is transcribed or translated into a recombinant product or products). Thus, an enhancer element can be located 100, 200, or 300 or more base pairs upstream of or downstream of a DNA sequence that encodes a recombinant product. An enhancer element can increase the amount of recombinant product expressed from a DNA sequence above the expression level associated with a single promoter element. Multiple enhancer elements are readily available to those of skill in the art.

[0087] In addition to the above-mentioned genes and regulatory sequences, the recombinant expression vector of the present invention may carry additional sequences, such as sequences regulating replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. Selectable marker genes facilitate selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells during methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0088] The term "expression control sequence" as used in this description refers to a polynucleotide sequence necessary to achieve the expression and processing of a ligated coding sequence. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a ribosome binding site, promoter, and transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0089] As used herein, the term "operably linked" refers to the linking of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is present in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. The term "operably linked" means that the DNA sequences being linked are generally contiguous, and, where necessary to join two protein-coding regions, also contiguous and in reading frame.

[0090] In one embodiment of the present invention, an "expression vector" relates to a vector comprising one or more polynucleotide sequences of interest, a gene of interest, or a "transgene" flanked by parvoviral sequences or inverted terminal repeat (ITR) sequences.

[0091] None of the cassettes or vectors of the present invention contain nucleotide sequences of genes encoding the nonstructural (Rep) and structural (Cap) proteins of adeno-associated virus. AAV5 (adeno-associated virus serotype 5)-based recombinant virus In one aspect, the invention relates to an isolated recombinant AAV5 (adeno-associated virus serotype 5)-based virus for inducing SARS-CoV-2-specific immunity and / or preventing infection with a SARS-CoV-2-related coronavirus, the virus comprising a capsid and any of the above expression cassettes.

[0092] As used in this description, the term "AAV-based recombinant virus" (or "AAV-based virus-like particle," or "AAV recombinant virus strain," or "AAV recombinant vector," or "rAAV vector") refers to the expression cassette (or the expression vector) encapsulated in an AAV capsid.

[0093] The Cap gene encodes three capsid proteins (VP1, VP2, and VP3), among other alternative products. VP1, VP2, and VP3 exist in a 1:1:10 ratio to form an icosahedral capsid [Xie Q. et al., The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002;99:10405-10410]. Transcription of these genes is initiated by a single promoter, p40. The molecular weights of the corresponding proteins (VP1, VP2, and VP3) are 87, 72, and 62 kDa, respectively. All three proteins are translated from a single mRNA. After transcription, the precursor mRNA can be spliced ​​in two different ways, resulting in the excision of either longer or shorter introns, resulting in the formation of mRNAs of various nucleotide lengths.

[0094] In the generation of recombinant AAV (rAAV)-based viruses, an expression cassette flanked by ITRs is packaged into an AAV capsid. Genes required for AAV replication are not included in the cassette, as described above.

[0095] The expression cassette DNA is packaged into the viral capsid in the form of a single-stranded DNA molecule (ssDNA) approximately 3000 nucleotides in length. When a cell is infected with the virus, the single-stranded DNA is converted into a double-stranded DNA (dsDNA) form. The dsDNA can only be used by cellular proteins to transcribe the present gene or genes into RNA.

[0096] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1. In some embodiments, the AAV5-based recombinant virus comprises the amino acid sequence

[0097] [ka]

[0098] It has a capsid containing the AAV5 protein VP1. In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP2.

[0099] In some embodiments, the AAV5-based recombinant virus comprises the following amino acid sequence:

[0100] [ka]

[0101] It has a capsid containing the AAV5 protein VP2. In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP3.

[0102] In some embodiments, the AAV5-based recombinant virus has the amino acid sequence

[0103] [ka]

[0104] It has a capsid containing the AAV5 protein VP3. In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 proteins VP1, VP2 and VP3.

[0105] In some embodiments, the AAV5-based recombinant virus has a capsid comprising a VP1 protein having the amino acid sequence of SEQ ID NO:4, a VP2 protein having the amino acid sequence of SEQ ID NO:14, and a VP3 protein having the amino acid sequence of SEQ ID NO:15.

[0106] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 containing one or more point mutations.

[0107] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having an amino acid sequence comprising amino acid substitutions at positions S2A and T711S of wild-type AAV5 VP1 (SEQ ID NO: 4), and the amino acid sequence

[0108] [ka]

[0109] It has. In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP2 having the amino acid sequence of SEQ ID NO: 14, which contains one or more point mutations.

[0110] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP2 having an amino acid sequence comprising an amino acid substitution at position T575S of wild-type AAV5 VP2 (SEQ ID NO: 14), and the amino acid sequence

[0111] [ka]

[0112] It has. In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP3 having the amino acid sequence of SEQ ID NO: 15, which contains one or more point mutations.

[0113] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP3 having an amino acid sequence comprising an amino acid substitution at position T519S of wild-type AAV5 VP3 (SEQ ID NO: 15), and the amino acid sequence

[0114] [ka]

[0115] It has. In some embodiments, the AAV5-based recombinant virus has a capsid comprising a VP1 protein of the amino acid sequence of SEQ ID NO: 4 with one or more point mutations, a VP2 protein of the amino acid sequence of SEQ ID NO: 14 with one or more point mutations, and a VP3 protein of the amino acid sequence of SEQ ID NO: 15 with one or more point mutations.

[0116] In some embodiments, the AAV5-based recombinant virus has a capsid comprising a VP1 protein of the amino acid sequence of SEQ ID NO:5, a VP2 protein of the amino acid sequence of SEQ ID NO:16, and a VP3 protein of the amino acid sequence of SEQ ID NO:17.

[0117] The phrase "more point mutations" refers to 2, 3, 4, 5, 6, 7, 8, 9 or 10 point substitutions. Particularly preferred embodiments include naturally conservative substitutions (mutations), i.e., substitutions that occur within a family of amino acids connected at the side chain.In particular, amino acids are generally divided into four families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.For example, it is reasonably predictable that the single substitution of leucine for isoleucine or valine, aspartic acid for glutamic acid, and threonine for serine, or similar conservative substitutions of amino acids for structurally related amino acids, will not have a significant effect on biological activity. For example, a subject polypeptide can contain up to about 5-10 conservative or non-conservative amino acid substitutions, as long as the desired function of the molecule remains intact.

[0118] The embodiment of point mutation within the sequence of the AAV5 protein VP1, VP2 or VP3 using amino acid substitution is the substitution of at least one amino acid residue within the AAV5 protein VP1, VP2 or VP3 by another amino acid residue.

[0119] Conservative substitutions are shown in Table A under "preferred substitutions."

[0120] [Table 1]

[0121] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 containing one or more point mutations, and the expression cassette comprises, from the 5' to the 3' end, the following elements: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; introns of the hBG1 gene (hemoglobin subunit gamma 1 gene); Any one of the foregoing nucleic acids encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal); Contains the right (second) ITR.

[0122] In some embodiments, the AAV5-based recombinant virus has a capsid comprising a VP1 protein having the amino acid sequence of SEQ ID NO:4, a VP2 protein having the amino acid sequence of SEQ ID NO:14, and a VP3 protein having the amino acid sequence of SEQ ID NO:15, and the expression cassette comprises, from the 5' end to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene (hemoglobin subunit gamma 1 gene); Any one of the foregoing nucleic acids encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. hGH1 polyadenylation signal; Includes the ITR on the right.

[0123] In some embodiments, the AAV5-based recombinant virus has a capsid comprising a VP1 protein of the amino acid sequence of SEQ ID NO:4 with one or more point mutations, a VP2 protein of the amino acid sequence of SEQ ID NO:14 with one or more point mutations, and a VP3 protein of the amino acid sequence of SEQ ID NO:15 with one or more point mutations, and the expression cassette comprises, from the 5' end to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene; Any one of the foregoing nucleic acids encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. hGH1 polyadenylation signal; Includes the ITR on the right.

[0124] In some embodiments, the AAV5-based recombinant virus has a capsid comprising a VP1 protein having the amino acid sequence of SEQ ID NO:5, a VP2 protein having the amino acid sequence of SEQ ID NO:16, and a VP3 protein having the amino acid sequence of SEQ ID NO:17, and the expression cassette comprises, from the 5' end to the 3' end, the following elements: CMV enhancer; CMV promoter; introns of the hBG1 gene; Any one of the foregoing nucleic acids encoding said isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2. hGH1 polyadenylation signal; Includes the ITR on the right.

[0125] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 containing one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO:3.

[0126] In some embodiments, the AAV5-based recombinant virus has a capsid comprising the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 containing one or more point mutations, wherein the AAV5 protein VP1 is the amino acid sequence of SEQ ID NO:5 (S2A and T711S).

[0127] In some embodiments, the AAV5-based recombinant virus has a capsid comprising VP1 having the amino acid sequence of SEQ ID NO:4, VP2 having the amino acid sequence of SEQ ID NO:14, and VP3 having the amino acid sequence of SEQ ID NO:15, and the expression cassette comprises the nucleic acid of SEQ ID NO:3.

[0128] In some embodiments, the AAV5-based recombinant virus has a capsid comprising a VP1 protein of the amino acid sequence of SEQ ID NO: 4 with one or more point mutations, a VP2 protein of the amino acid sequence of SEQ ID NO: 14 with one or more point mutations, and a VP3 protein of the amino acid sequence of SEQ ID NO: 15 with one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO: 3.

[0129] In some embodiments, the AAV5-based recombinant virus has a capsid comprising a VP1 protein having the amino acid sequence of SEQ ID NO:5, a VP2 protein having the amino acid sequence of SEQ ID NO:16, and a VP3 protein having the amino acid sequence of SEQ ID NO:17, and the expression cassette comprises the nucleic acid of SEQ ID NO:3. Pharmaceutical Compositions / Vaccines In one aspect, the present invention relates to a pharmaceutical composition for preventing infection with a SARS-CoV-2 related coronavirus, the pharmaceutical composition comprising any of the above AAV5-based recombinant viruses in combination with one or more pharmaceutically acceptable excipients.

[0130] In one aspect, the present invention relates to a pharmaceutical composition for inducing SARS-CoV-2 specific immunity, the pharmaceutical composition comprising any of the above AAV5-based recombinant viruses in combination with one or more pharmaceutically acceptable excipients.

[0131] In certain embodiments, the present invention relates to pharmaceutical compositions comprising the AAV5-based recombinant viruses of the present invention in a pharmaceutically acceptable carrier or other medicinal agent, adjuvant, diluent, etc. For injection, the carrier will generally be a liquid carrier. For other modes of administration, the carrier can be either a solid or a liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline. For inhalation administration, the carrier is respirable and preferably in solid or liquid granular form. As the injection medium, it is preferred to use water containing stabilizers, additives commonly used in injection solutions, such as salts or saline, and / or buffers.

[0132] The term "pharmaceutical composition" refers to a composition comprising the above-described AAV5-based recombinant virus of the present invention and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, auxiliary agents, dispersing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, and long-term delivery control agents, the selection and proportion of which will depend on the type and route of administration and dosage. The pharmaceutical composition of the present invention and methods for its preparation will be readily apparent to those skilled in the art. Pharmaceutical compositions should preferably be manufactured in accordance with Good Manufacturing Practice (GMP) requirements. The composition may also contain a buffer composition, an isotonicity agent, a stabilizer, and a solubilizer.

[0133] "Pharmaceutically acceptable" means a material that is free of biological or other negative side effects, e.g., the material can be administered to a subject without causing any undesired biological effects. Thus, such pharmaceutical compositions can be used, for example, for transfection of cells ex vivo or for direct administration of the AAV5-based recombinant viruses of the invention to a subject in vivo.

[0134] The term "excipient" is used herein to describe any other component of the present invention. These are inorganic or organic natural substances used in pharmaceutical manufacturing to obtain the necessary physicochemical properties for the formulation.

[0135] "Stabilizer" refers to an excipient or a mixture of two or more excipients that provides physical and / or chemical stability to an active agent. The terms "buffer," "buffer composition," and "buffering agent" refer to a solution capable of resisting pH changes due to the action of acid-base conjugate components, allowing the rAAV5 vector product to resist pH changes. Generally, pharmaceutical compositions preferably have a pH of 4.0 to 8.0. Examples of buffers that can be used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, and the like.

[0136] A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical stability and / or biological activity at a storage temperature, e.g., 2-8°C, for a specified shelf life. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The shelf life is adjusted based on the results of stability testing under accelerated or natural degradation conditions.

[0137] The pharmaceutical composition of the present invention can be manufactured, packaged, or widely sold in the form of a ready-to-use preparation in the form of a single unit dose or a plurality of single unit doses.As used herein, the term "single unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dose of active ingredient administered to a subject, or a convenient fraction of such a dose, for example, half or one-third of such a dose.

[0138] In one aspect, the invention relates to a vaccine for preventing infection with a SARS-CoV-2 related coronavirus, the vaccine comprising an effective amount of any of the above recombinant AAV5-based viruses.

[0139] In one aspect, the invention relates to a vaccine for inducing SARS-CoV-2 specific immunity, the vaccine comprising an effective amount of any of the above recombinant AAV5-based viruses.

[0140] The term "vaccine" refers to an immunogenic composition comprising antigens obtained from a pathogen that is used to induce an immune response against the pathogen that confers protective immunity (e.g., immunity that protects a subject from infection caused by the pathogen and / or reduces the severity of disease or symptoms resulting from infection by the pathogen). Protective immunity can include the production of antibodies and / or a cell-mediated response.

[0141] Depending on the context, the term "vaccine" can also refer to an antigenic suspension or solution that is administered to a vertebrate to confer protective immunity. The vaccine comprises a recombinant virus based on AAV5, and preferably the virus is present in the vaccine in a biologically effective amount. A "biologically effective" amount of a recombinant virus is an amount sufficient to cause infection (or transduction) and expression of a heterologous nucleic acid sequence in a cell. When the virus is administered to a cell in vivo (e.g., the virus is administered to a subject as described below), a "biologically effective" amount of a viral vector is an amount sufficient to cause transduction and expression of a heterologous nucleic acid sequence in a target cell.

[0142] All definitions and explanations relating to pharmaceutical compositions also apply to vaccines. use In one aspect, the invention relates to the use of any of said recombinant AAV5-based viruses or said pharmaceutical compositions to prevent infection with a SARS-CoV-2 related coronavirus.

[0143] In one aspect, the invention relates to the use of any of said recombinant AAV5-based viruses or said pharmaceutical compositions for inducing SARS-CoV-2 specific immunity. In one aspect, the present invention relates to a method for inducing SARS-CoV-2 specific immunity, the method comprising administering to a mammalian organism an effective amount of any one of said recombinant AAV5-based virus, said composition or said vaccine to induce SARS-CoV-2 specific immunity.

[0144] In one aspect, the present invention relates to a method for preventing SARS-CoV-2 related coronavirus infection, the method comprising administering to a mammalian organism an effective amount of any one of the recombinant AAV5-based viruses, the compositions or the vaccines to prevent infection with a SARS-CoV-2 related coronavirus.

[0145] Any method of administering an AAV5-based recombinant virus recognized by one of skill in the art may be suitably used for the above-described AAV5-based recombinant viruses of the present invention. Typical modes of administration include topical application, intranasal, inhalation, transmucosal, transdermal, enteral (e.g., oral, rectal), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular) administration, and direct injection into a tissue or organ.

[0146] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, the AAV5-based recombinant viruses of the invention can be administered in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0147] The AAV5-based recombinant virus is introduced into an organism in an effective amount. Preferably, the AAV5-based recombinant virus is introduced into an organism in a biologically effective amount. A "biologically effective" amount of the recombinant virus is an amount sufficient to cause infection (or transduction) and expression of a heterologous nucleic acid sequence in a cell. When the virus is administered to a cell in vivo (for example, when the virus is administered to a subject as described below), a "biologically effective" amount of the viral vector is an amount sufficient to cause transduction and expression of a heterologous nucleic acid sequence in a target cell.

[0148] The dosage of the AAV5-based recombinant virus of the present invention will depend on the mode of administration and the particular viral vector, which can be determined in a conventional manner. A typical dose to achieve a therapeutic effect is at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 transducing units or higher, preferably about 10 8 ~10 13 transducing units, even more preferably 10 12is the viral titer in transducing units.

[0149] Cells for administering the AAV5-based recombinant viruses of the present invention can be any type of cell, including, but not limited to, epithelial cells (e.g., skin, respiratory and intestinal epithelial cells), liver cells, muscle cells, pancreatic cells (including islet cells), liver cells, spleen cells, fibroblasts, endothelial cells, etc.

[0150] The AAV5-based recombinant viruses are not used to modify the genetic integrity of human germline cells. The present invention includes, but is not limited to, the following aspects. [Aspect 1] An isolated recombinant receptor-binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2, represented by the amino acid sequence of SEQ ID NO:1. [Aspect 2] 2. An isolated nucleic acid encoding an isolated recombinant receptor-binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 according to embodiment 1. [Aspect 3] 3. The isolated nucleic acid of embodiment 2, wherein the nucleic acid is DNA. [Aspect 4] 3. The isolated nucleic acid of embodiment 2, which has the nucleotide sequence of SEQ ID NO:2. [Aspect 5] 3. The isolated nucleic acid of embodiment 2, wherein the nucleic acid is a codon-optimized nucleotide sequence. [Aspect 6] 6. An expression cassette comprising a nucleic acid according to any one of aspects 2 to 5. [Aspect 7] From the 5' to the 3' end, the following elements: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; introns of the hBG1 gene (hemoglobin subunit gamma 1 gene); A nucleic acid according to any one of aspects 2 to 5; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Right (second) ITR 7. The expression cassette of embodiment 6, comprising: [Aspect 8] 8. The expression cassette of embodiment 7, comprising the nucleic acid of SEQ ID NO:3. [Aspect 9] 9. An expression vector comprising the nucleic acid of any one of aspects 2 to 5 or the cassette of any one of aspects 6 to 8. [Aspect 10] 9. An isolated recombinant AAV5 (adeno-associated virus serotype 5) based virus for inducing SARS-CoV-2 specific immunity and / or preventing infection with a SARS-CoV-2 related coronavirus, the virus comprising a capsid and an expression cassette according to any one of aspects 6 to 8. [Aspect 11] 11. The AAV5-based recombinant virus of embodiment 10, wherein the capsid comprises the AAV5 protein VP1. [Aspect 12] 12. The AAV5-based recombinant virus of embodiment 11, wherein the capsid comprises the AAV protein VP1 having the amino acid sequence of SEQ ID NO:4. [Aspect 13] 11. The AAV5-based recombinant virus of embodiment 10, wherein the capsid comprises the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4, comprising one or more point mutations. [Aspect 14] 14. The AAV5-based recombinant virus of embodiment 13, wherein the capsid comprises the AAV protein VP1 having the amino acid sequence of SEQ ID NO: 5 (S2A and T711S). [Aspect 15] The capsid comprises the amino acid sequence of SEQ ID NO: 4 or one or more point mutations. and an AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4, said expression cassette comprising, in a 5' to 3' direction, the following elements: left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; introns of the hBG1 gene (hemoglobin subunit gamma 1 gene); A nucleic acid according to any one of aspects 2 to 5; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Right (second) ITR 14. The AAV5-based recombinant virus of any one of embodiments 10 to 13, comprising: [Aspect 16] 16. The AAV5-based recombinant virus of embodiment 15, wherein the capsid comprises the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 comprising one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO: 3. [Aspect 17] 17. The AAV5-based recombinant virus of any one of aspects 15 or 16, wherein the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO: 4 comprising one or more point mutations is the amino acid sequence of SEQ ID NO: 5 (S2A and T711S). [Aspect 18] 18. A pharmaceutical composition comprising an AAV5-based recombinant virus according to any one of aspects 10 to 17 in combination with one or more pharmaceutically acceptable excipients. [Aspect 19] 20. The pharmaceutical composition of embodiment 18 for preventing infection with a SARS-CoV-2 related coronavirus. [Aspect 20] 19. The pharmaceutical composition according to aspect 18, for inducing immunity specific to SARS-CoV-2. [Aspect 21] 19. Use of a recombinant AAV5-based virus according to any one of aspects 10 to 17 or a composition according to aspect 18 for preventing infection with a SARS-CoV-2 related coronavirus. [Aspect 22] 19. Use of a recombinant AAV5-based virus according to any one of aspects 10 to 17 or a composition according to aspect 18 for inducing SARS-CoV-2 specific immunity. [Aspect 23] 18. A vaccine for preventing SARS-CoV-2 associated coronavirus infection, comprising an effective amount of a recombinant AAV5-based virus according to any one of aspects 10 to 17. [Aspect 24] 18. A vaccine for inducing SARS-CoV-2 specific immunity comprising an effective amount of a recombinant AAV5-based virus according to any one of aspects 10 to 17. [Aspect 25] A method for inducing immunity specific to SARS-CoV-2, comprising: A recombinant AAV5-based virus according to any one of aspects 10 to 17; The composition of embodiment 18; or the vaccine of embodiment 24; A method for inducing immunity specific to SARS-CoV-2, comprising administering to a mammalian organism an effective amount of [Aspect 26] 1. A method for preventing infection with a SARS-CoV-2 related coronavirus, comprising: A recombinant AAV5-based virus according to any one of aspects 10 to 17; The composition of embodiment 18; or the vaccine of embodiment 23; A method for preventing infection with a SARS-CoV-2 related coronavirus, comprising administering to a mammalian organism an effective amount of [Example] [Example]

[0151] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. All publications, patents, and patent applications cited herein are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the accompanying embodiments.

[0152] Materials and general methods Recombinant DNA Technology DNA manipulations were performed according to standard techniques described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. Briefly, plasmid DNA was prepared and further manipulated in Escherichia coli (E. coli) cells grown under antibiotic selection pressure to prevent the plasmid from being lost in the cell population. Plasmid DNA was isolated from the cells using a commercially available kit, the concentration was measured, and it was used for cloning by restriction enzyme digestion or PCR amplification. DNA fragments were ligated together using ligase, transformed into bacterial cells, and clones were selected and further manipulated. All of the resulting genetic constructs were confirmed by restriction patterns and complete Sanger sequencing.

[0153] Gene synthesis The desired gene segments were prepared from chemically synthesized oligonucleotides. Gene segments of 300-1000 bp in length flanked by unique restriction sites were obtained by renaturing the oligonucleotides on top of each other and then PCR-amplified from flanking primers. The resulting mixture of fragments contained the desired fragments. The fragments were cloned into the restriction sites of an intermediate vector, and the DNA sequences of the subcloned fragments were then confirmed by DNA sequencing.

[0154] DNA sequencing DNA sequences were determined by Sanger sequencing. DNA and protein sequences were analyzed and sequence data were processed with SnapGene Viewer version 4.2 or later for sequence generation, mapping, analysis, annotation, and illustration.

[0155] Cell culture incubation Experiments used HEK293 (human embryonic kidney clone 293) and CHO-K1-S (Chinese hamster ovary cell) cell lines. Suspension HEK293 cells used to produce AAV were cultured at standard conditions of 37°C and 5% CO2 in complete culture medium without FBS and antibiotics. Adherent CHO-K1-S cells used to test the efficacy of AAV products were cultured at standard conditions of 37°C and 5% CO2 in complete DMEM / F12 medium supplemented with 5% FBS and antibiotics / antimycotics. CHO-K1-S cells were passaged when they reached 80-90% confluence. Cell viability was assessed using either trypan blue staining and a hemocytometer or PI staining and flow cytometry.

[0156] Determination of levels of RBD-S protein and specific antibodies against RBD-S protein The content of RBD-S protein in transduced cells and in the plasma of immunized animals was assessed by enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase as the indicator enzyme. Briefly, wells of a 96-well plate were pre-immunized with a primary antibody against RBD-S protein, followed by overlaying with test samples. When detecting antibodies against RBD-S protein using ELISA, the plate was pre-immunized with RBD-S protein, followed by the addition of animal plasma. The samples were then supplemented with a secondary antibody against RBD-S labeled with biotin and HRP-conjugated streptavidin (to analyze the protein itself) or a secondary antibody against the immunoglobulins of the study animals (to analyze the presence of antibodies against RBD-S). TMB solution was then added to visualize the enzyme reaction, followed by a stop solution to stop the reaction.

[0157] To determine the concentration of RBD-S / antibody to RBD-S in the test samples, a calibration curve showing the dependence of the optical density of the solution on the concentration of RBD-S / antibody to RBD-S in the standard samples was plotted, and the optical density was used to determine the concentration of the test samples.

[0158] Assembly and purification of recombinant AAV vector viral particles To assemble AAV particles containing the RBD-S gene, HEK293 producer cells were used, into which three plasmids were inserted: the pAAV-RBD-S plasmid containing the AAV genome with the RBD-S transgene expression cassette (Fig. 1 ); a plasmid for expressing the AAV5 serotype Cap gene and the AAV2 serotype Rep gene, each encoding several protein products using alternative reading frames; Plasmids for the expression of Ad5 (adenovirus serotype 5) genes required for AAV capsid assembly and packaging was transfected as follows.

[0159] After 72 hours, cells were lysed, and viral particles were purified and concentrated using filtration, chromatography and ultracentrifugation methods.The titer of viral particles was determined by quantitative PCR using primers specific to the region of the recombinant viral genome and sample, and expressed as the number of copies of viral genome per mL.

[0160] Cell culture transduction Cell lines were plated at a seeding density of 10,000 cells / cm. 2 The wells of a 6-well plate were pre-seeded with 100,000 vg / cell and 500,000 vg / cell of virion product at an MOI of 100,000 vg / cell, followed by addition of viral particle products at an MOI of 100,000 vg / cell and an MOI of 500,000 vg / cell. On day 3, the RBD-S protein content was determined by ELISA as described above. Transduction efficiency was estimated by measuring the percentage of GFP+ cells.

[0161] After transduction, CHO-K1-S cells were removed from the culture plates using TrypLE and washed in PBS; protein expression was analyzed as described above. All measurements were performed in three independent experiments. Intact cells were used as a negative control.

[0162] In vivo studies in laboratory animals The experiments were carried out in BALB / c mice (male and female, 6-8 weeks old). Immunization was performed by a single intramuscular injection of the product into the pelvic limb. A negative control group of animals was injected with buffer, and a positive control group was injected with a mixture of RBD-S protein, Freund's complete adjuvant, and saline.

[0163] Plasma was collected before administration of the product on the day of injection, and then on days 14, 21, 27, 42 and 56 after immunization. Example 1 Selection of RBD-S sequences of SARS-CoV-2 The development of the SARS-CoV-2 RBD-S antigen involved the analysis of the 5WRG structure of the SARS-CoV spike glycoprotein, provided by [End Page 119] et al., "Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding," Cell Res. 27, pp. 119–129, 2017. Analysis of the SARS-CoV spike glycoprotein structure indicated that both the conserved RBD-S and its extended portion could be used for immunization. The analysis revealed that increasing the length of the RBD-S should contribute to stabilizing the RBD-S protein structure by retaining secondary structure, which increases the likelihood of maintaining a stable protein conformation without the need for unwinding. Furthermore, a small increase in RBD-S length should not affect the outcome of immunization. The above analysis of the SARS-CoV spike glycoprotein structure was applied to the structure of the SARS-CoV-2 spike glycoprotein. Furthermore, to further stabilize the RBD-S protein of SARS-CoV-2, a substitution of the unpaired cysteine ​​closest to the domain with a serine (amino acid substitution at position C272S) was introduced into the structure of the RBD-S protein of SARS-CoV-2.

[0164] Therefore, the following amino acid sequence of SEQ ID NO: 1 was selected as the SARS-CoV-2 RBD-S antigen: This antigen will be used to effectively immunize mammals (see Example 5).

[0165] Example 2 Assembly of a genetic construct containing an AAV expression cassette containing a recombinant RBD-S gene The target plasmid pAAV-RBD-S (Figure 1), intended for generating an AAV5 viral vector carrying an expression cassette containing the RBD-S gene (SEQ ID NO: 1), was created by replacing the sequence of a modified green fluorescent protein in the original construct, pAAV-GFP control plasmid (VPK-402) from CellBiolab (USA), with the RBD-S sequence containing a signal peptide using the restriction enzyme ligase method for cloning at the ClaI / BamHI sites. The RBD-S sequence was synthesized de novo from chemically generated oligonucleotides with the addition of a ClaI restriction site at the 5' end and a BamHI restriction site at the 3' end.

[0166] The final vector contains the elements necessary for gene expression and assembly as part of the recombinant AAV genome: 1) ITRs at the ends of the sequences encapsidated within the viral capsid; 2) elements for expression of the target gene (promoter, enhancer, intron, Kozak sequence, transgene, polyadenylation site); 3) A bacterial replication origin and antibiotic resistance gene for producing plasmid DNA within bacterial cells Contains everything.

[0167] Example 3: Creation of a viral product expressing RBD-S The target plasmid pAAV-RBD-S (Figure 1) was used together with the remaining plasmids required to generate recombinant AAV viral particles (see above) to generate the AAV5-RBD-S product. Bioprocessing resulted in recombinant AAV5-RBD-S viral particles containing an expression cassette containing the RBD-S gene. The purified AAV5-RBD-S product used for in vitro and in vivo studies was prepared using standard buffers and excipients that are safe and do not alter AAV properties. The concentration of the purified AAV5-RBD-S product was 4.6 x 10 11 ~1.8×10 12 It was VG / mL.

[0168] Example 4 In vitro testing of AAV5-RBD-S products The purified AAV5-RBD-S product was tested in vitro prior to animal studies. These experiments were performed using the CHO-K1-S adherent cell line (Figure 2). CHO-K1-S cells were plated into wells of a 6-well plate. Seeding was performed in the following growth medium: DMEM / F12 supplemented with glutamine, glucose content 4.5 g / L, and 5% fetal bovine serum (FBS).

[0169] The cell seeding density was 10,000 cells / cm 2 During the transduction process, pre-prepared cells were transduced at an MOI of 100,000 vg / cell and an MOI of 500,000 vg / cell. All samples were performed in triplicate. Intact cells were used as a negative control. After successful transduction, CHO-K1-S cells were removed from the culture medium and washed in phosphate buffer, and the expression of RBD-S protein was analyzed by enzyme-linked immunosorbent assay (ELISA) as described above. The developed product was shown to be able to efficiently deliver the RBD-S transgene into cells and reliably produce the target protein, as confirmed by the ELISA data (Figure 2).

[0170] Example 5 In vivo testing of AAV5-RBD-S products BALB / c laboratory mice were used for in vivo studies of the AAV5-RBD-S product. Two different doses of the AAV5-RBD-S product were used in the study: low dose (1 × 10 11 VG / mouse) and high dose (4 × 10 11VG / mouse). A control solution without AAV and an AAV5 product without an expression cassette containing the RBD-S gene (empty AAV5 capsid) were used as negative controls. Purified recombinant RBD-S protein was used as a positive control. Animals were immunized by a single intramuscular injection into the pelvic limb. On days 0, 14, 21, 27, 42, and 56 after immunization, the titers of antibodies against the RBD-S protein in plasma were determined by ELISA as described above. In vivo studies showed that immunization with the AAV5-RBD-S product resulted in the production of specific antibodies against RBD-S (Figures 3, 4, 5, and 6). Furthermore, the levels of antibodies against RBD-S were similar to those in animals immunized with the recombinant RBD-S protein (Figures 8 and 9). At the same time, no production of antibodies against RBD-S was observed in the groups of animals injected with the AAV-free control solution or the AAV5 product (empty AAV5 capsid) that does not contain the expression cassette containing the RBD-S gene (Figures 7 and 10).

[0171] Therefore, the recombinant AAV5-based viruses and vaccines based thereon described in this invention have high potential for inducing SARS-CoV-2-specific immunity and can be used to prevent infection with SARS-CoV-2-related coronaviruses. Furthermore, the ability of AAV vectors to achieve long-term antigen expression makes them advantageous over traditional systems based on recombinant protein antigens.

Claims

1. An isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2, represented by the amino acid sequence of SEQ ID NO:

1.

2. 2. An isolated nucleic acid encoding the isolated recombinant receptor binding domain (RBD-S) of the S glycoprotein of SARS-CoV-2 according to claim 1.

3. The isolated nucleic acid of claim 2 , wherein the nucleic acid is DNA.

4. 3. The isolated nucleic acid of claim 2, which is the nucleotide sequence of SEQ ID NO:

2.

5. 3. The isolated nucleic acid of claim 2, which is a codon-optimized nucleotide sequence.

6. An expression cassette comprising a nucleic acid according to any one of claims 2 to 5.

7. From the 5' to the 3' end, the following elements: the left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; an intron of the hBG1 gene (hemoglobin subunit gamma 1 gene); A nucleic acid according to any one of claims 2 to 5; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Right (second) ITR The expression cassette of claim 6, comprising:

8. 8. The expression cassette of claim 7, comprising the nucleic acid of SEQ ID NO:

3.

9. An expression vector comprising a nucleic acid according to any one of claims 2 to 5 or a cassette according to any one of claims 6 to 8.

10. 9. An isolated recombinant AAV5 (adeno-associated virus serotype 5)-based virus for inducing SARS-CoV-2 specific immunity and / or preventing infection with a SARS-CoV-2 related coronavirus, the virus comprising a capsid and an expression cassette of any one of claims 6 to 8.

11. 11. The AAV5-based recombinant virus of claim 10, wherein the capsid comprises the AAV5 protein VP1.

12. 12. The AAV5-based recombinant virus of claim 11, wherein the capsid comprises the AAV protein VP1 having the amino acid sequence of SEQ ID NO:

4.

13. 11. The AAV5-based recombinant virus of claim 10, wherein the capsid comprises AAV5 protein VP1 having the amino acid sequence of SEQ ID NO: 4 containing one or more point mutations.

14. 14. The AAV5-based recombinant virus of claim 13, wherein the capsid comprises the AAV protein VP1 having the amino acid sequence of SEQ ID NO: 5 (S2A and T711S).

15. The capsid comprises the amino acid sequence of SEQ ID NO: 4 or one or more point mutations. and an AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4, said expression cassette comprising, from the 5' end to the 3' end, the following elements: the left (first) ITR (inverted terminal repeat); CMV (cytomegalovirus) enhancer; CMV (cytomegalovirus) promoter; an intron of the hBG1 gene (hemoglobin subunit gamma 1 gene); A nucleic acid according to any one of claims 2 to 5; hGH1 polyadenylation signal (human growth hormone gene polyadenylation signal) Right (second) ITR 14. The AAV5-based recombinant virus of any one of claims 10 to 13, comprising:

16. 16. The AAV5-based recombinant virus of claim 15, wherein the capsid comprises the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 containing one or more point mutations, and the expression cassette comprises the nucleic acid of SEQ ID NO:

3.

17. 17. A recombinant virus based on AAV5 according to any one of claims 15 or 16, wherein the AAV5 protein VP1 having the amino acid sequence of SEQ ID NO: 4 containing one or more point mutations is the amino acid sequence of SEQ ID NO: 5 (S2A and T711S).

18. 18. A pharmaceutical composition comprising an AAV5-based recombinant virus according to any one of claims 10 to 17 in combination with one or more pharmaceutically acceptable excipients.

19. The pharmaceutical composition of claim 18 for preventing infection with SARS-CoV-2 related coronavirus.

20. The pharmaceutical composition of claim 18 for inducing immunity specific to SARS-CoV-2.

21. 20. A vaccine for preventing SARS-CoV-2 related coronavirus infection, comprising an effective amount of a recombinant AAV5-based virus according to any one of claims 10 to 17.

22. A vaccine for inducing SARS-CoV-2 specific immunity, comprising an effective amount of a recombinant AAV5-based virus according to any one of claims 10 to 17.

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