Novel coronavirus vaccine based on Salmonella

Abstract

Provision of DNA vaccines for use in the prevention and / or treatment of coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection. [Solution] The present invention relates to a DNA vaccine comprising Salmonella typhi Ty21a strain, which comprises a DNA molecule containing a eukaryotic expression cassette encoding the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof. In particular, the present invention relates to a DNA vaccine for use in the prevention and / or treatment of coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection.

Description

[Technical Field]

[0001] The present invention relates to a DNA vaccine comprising Salmonella typhi Ty21a strain, which comprises a DNA molecule containing a eukaryotic expression cassette encoding the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof. In particular, the present invention relates to the said DNA vaccine for use in the prevention and / or treatment of coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection. [Background technology]

[0002] At the end of December 2019, Chinese public health authorities reported several cases of acute respiratory syndrome in Wuhan, Hubei Province, China. Chinese scientists quickly identified a novel coronavirus as the primary causative agent. This disease is now called coronavirus disease 2019 (COVID-19), and the causative virus is called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This is a new strain of coronavirus that had never been previously identified in humans.

[0003] The initial outbreak in Wuhan spread rapidly, affecting other parts of China. Soon, multiple cases were detected in several other countries. Since then, outbreaks and clusters of the disease have been observed in Asia, Europe, Australia, Africa, and the Americas.

[0004] At its initial emergency meeting, the WHO estimated the fatality rate for COVID-19 to be around 4%. While fatality rates vary by country and may not be accurate due to the unknown number of unreported cases, the spread of SARS-CoV-2 (originally called the 2019 novel coronavirus (2019-nCoV)) has become a global thread, and treatment and / or vaccination against COVID-19 are urgently needed to stop the virus from spreading further.

[0005] Coronaviruses are single-stranded positive-sense RNA viruses belonging to the Coronaviridae family. These viruses mostly infect animals (including birds and mammals). In humans, coronaviruses typically cause mild respiratory infections. Since 2003, two highly pathogenic human coronaviruses, namely Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), have caused global epidemics with high morbidity and mortality rates. Both epidemics were caused by zoonotic coronaviruses belonging to the Betacoronavirus genus within the Coronaviridae family.

[0006] Like SARS-CoV and MERS-CoV, the novel SARS-CoV-2 belongs to the genus Betacoronavirus. As reported by Zhou et al. (Cell Discovery (2020) 6:14), SARS-CoV-2 shows the greatest nucleotide sequence agreement with SARS-CoV (79.7%). Specifically, the envelope protein and nucleocapsid protein of SARS-CoV-2 are two evolutionarily conserved regions, with sequence agreements of 96% and 89.6% respectively compared to SARS-CoV. While the spike protein has been reported to show the least sequence conserved between SARS-CoV-2 and SARS-CoV (77% sequence agreement), the spike protein of SARS-CoV-2 has only a 31.9% sequence agreement with the spike protein of MERS-CoV.

[0007] Various reports on SARS-CoV suggest protective roles in both humoral and cell-mediated immune responses. The S protein is the most exposed protein, and antibody responses against the SARS-CoV S protein have been shown to protect against SARS-CoV infection in mouse models. Antibody responses are effective but may be short-lived. Conversely, T cell responses have been shown to provide long-term protection against SARS-CoV. Therefore, vaccines that can induce cell-mediated immune responses in addition to humoral responses are very promising.

[0008] Several domestic and international research groups are working on developing vaccines for the prevention and treatment of 2019-nCoV / SARS-CoV-2, but no effective vaccine is yet available. Therefore, there remains an urgent need for an effective therapeutic and / or preventive vaccine that can be developed and approved in a short period of time. [Overview of the project]

[0009] In light of the current understanding of the novel coronavirus and the global pandemic caused by SARS-CoV-2, one object of the present invention is to provide a novel oral DNA vaccine for the prevention and / or treatment of coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection. The DNA vaccine of the present invention comprises Salmonella Typhi Ty21a strain containing a DNA molecule comprising a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof. This vaccine is based on a bio-attenuated Salmonella strain called Salmonella Typhi Ty21a, which functions as a carrier and adjuvant for a DNA molecule encoding an immunogenic antigen to be expressed in host cells. This carrier, based on Salmonella and containing a DNA molecule encoding its antigen, can be developed in a short period of time and manufactured on a large scale, and can be adapted to any possible mutations that may occur in the virus if necessary.

[0010] Furthermore, the live attenuated Salmonella typhi strain Ty21a used as a carrier is the active ingredient of Typhoral L (also known as Vivotif), the only live oral vaccine approved against typhoid fever (manufactured by Berna Biotech Ltd., a Crucell Company, Switzerland). This vaccine has been extensively investigated for patient toxicity and transmission to third parties and has been proven to be safe (Wahdan et al., J. Infectious Diseases 1982, 145: 292-295). This vaccine is approved in over 40 countries and has been used in millions of individuals, including thousands of children, for preventive vaccination against typhoid fever. It has an unparalleled record of safety. Therefore, the carrier used in the DNA vaccine of the present invention is suitable for obtaining approval and launching products on the market in a short period of time.

[0011] Therefore, the DNA vaccine of the present invention has several advantages that are particularly suitable for the challenge of providing an effective vaccine against COVID-19 and / or SARS-CoV-2 infection.

[0012] There is provided a DNA vaccine comprising a Salmonella typhi strain Ty21a comprising a DNA molecule comprising a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof. In certain embodiments, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof comprises (a) the SARS-CoV-2 full-length S protein; (b) the SARS-CoV-2 S protein ectodomain; (c) the SARS-CoV-2 S protein subunit S1; (d) the SARS-CoV-2 S protein receptor binding domain (RBD); or (d) at least three immunodominant epitopes of the SARS-CoV-2 S protein.

[0013] In one embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein is the SARS-CoV-2 full-length S protein. The SARS-CoV-2 full-length S protein can include the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 95% identity with SEQ ID NO: 1. As the SARS-CoV-2 full-length S protein, the full-length S proteins of variants of SARS-CoV-2 (such as lineage B.1.1.7, B.1.351, or B.1.1.28 (renamed P.1), etc.) are also possible. As the SARS-CoV-2 full-length S protein, a pre-fusion stabilized form of the SARS-CoV-2 full-length S protein (including two or more stabilized forms, etc.) is also possible. In one embodiment, the pre-fusion stabilized form of the SARS-CoV-2 full-length S protein includes two stabilizing mutations to proline corresponding to amino acid positions K986 and V987 in the amino acid sequence of SEQ ID NO: 1.

[0014] In certain embodiments, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof includes the SARS-CoV-2 S protein ectodomain. The SARS-CoV-2 S protein ectodomain has the amino acid sequence of amino acid residues 1 to 1208 of SEQ ID NO: 1, or an amino acid sequence having at least 95% identity with amino acid residues 1 to 1208 of SEQ ID NO: 1. As the SARS-CoV-2 S protein ectodomain, the S protein ectodomains of variants of SARS-CoV-2 (such as lineage B.1.1.7, B.1.351, or P.1, etc.) are also possible. The SARS-CoV-2 S protein or a portion thereof can also include those containing two or more stabilizing mutations in a pre-fusion stabilized form of the SARS-CoV-2 S protein ectodomain. In one embodiment, the pre-fusion stabilized form of the SARS-CoV-2 S protein ectodomain includes two stabilizing mutations to proline corresponding to amino acid positions K986 and V987 in the amino acid sequence of amino acid residues 1 to 1208 of SEQ ID NO: 1.

[0015] In one embodiment, the SARS-CoV-2 S protein or a portion thereof has an amino acid sequence of SEQ ID NO: 1 containing two stabilizing mutants K986P and V987P, or an amino acid sequence having a sequence that is at least 95% identical to SEQ ID NO: 1. In an alternative embodiment, the SARS-CoV-2 S protein or a portion thereof has an amino acid sequence of amino acid residues 1-1208 of SEQ ID NO: 1 containing two stabilizing mutants K986P and V987P, or an amino acid sequence having a sequence that is at least 95% identical to amino acid residues 1-1208 of SEQ ID NO: 1.

[0016] In one embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof includes the SARS-CoV-2 S protein subunit S1. The SARS-CoV-2 protein subunit S1 may include the amino acid sequence of amino acid residues 1-681 of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with amino acid residues 1-681 of SEQ ID NO: 1. The SARS-CoV-2 S protein subunit S1 may also be the S protein subunit S1 of a variant of SARS-CoV-2 (e.g., lineage B.1.1.7, B.1.351, or P.1).

[0017] In one embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof includes a SARS-CoV-2 S protein receptor-binding domain (RBD). The SARS-CoV-2 protein RBD may include the amino acid sequence of amino acid residues 319-541 of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with amino acid residues 319-541 of SEQ ID NO: 1. The SARS-CoV-2 S protein RBD may also be the S protein RBD of a variant of SARS-CoV-2 (e.g., lineage B.1.1.7, B.1.351, or P.1).

[0018] The DNA vaccine of the present invention may include a DNA molecule that encodes the SARS-CoV-2 S protein or a portion thereof, and optionally further encodes another SARS-CoV-2 protein or a portion thereof (preferably the SARS-CoV-2 N protein). In one embodiment, a eukaryotic expression cassette encodes the SARS-CoV-2 S protein or a portion thereof, and further encodes another SARS-CoV-2 protein or a portion thereof (such as the SARS-CoV-2 N protein or a portion thereof).

[0019] The DNA vaccine of the present invention may further comprise one or more pharmaceutically acceptable excipients. In some embodiments, the DNA vaccine is in oral dosage form (such as enteric-coated capsules, lyophilized powder, or suspension). The DNA vaccine of the present invention may further comprise one or more adjuvants.

[0020] This specification also provides the DNA vaccine of the present invention for use in the treatment and / or prevention of coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection.

[0021] This specification also provides a method comprising administering the DNA vaccine of the present invention to a patient in need as a method for treating and / or preventing coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection. In preferred embodiments, the DNA vaccine is administered orally. In some embodiments, a single dose of the DNA vaccine is approximately 1 × 10⁻⁶ 6 ~Approx. 1×10 9 The DNA vaccine, containing colony-forming units (CFUs) of the Salmonella typhi Ty21a strain, and / or this DNA vaccine, is administered 2 to 4 times per week for priming, optionally followed by at least one boosting dose. In one embodiment, the DNA vaccine is administered 2 to 4 times within the first week, followed by one or more single-dose boostings at least 2 weeks (preferably at least 4 weeks) after each. [Brief explanation of the drawing]

[0022] [Figure 1] Figure 1: This is the amino acid sequence (SEQ ID NO: 1) of the SARS-CoV-2 spike protein, with amino acid residues 1-1208 underlined and K986, V987, R682G, R683S, and R685S in bold. [Figure 2] Figure 2: Plasmid map of pVAX10.SCV-1. [Figure 3] Figure 3: SARS-CoV-2 construct for cloning into pVAX10. X indicates the presence of domains in order from N-terminus (left) to C-terminus (right), using the following abbreviations: S FL (full-length S protein, SEQ ID NO: 1; * indicates the signal domain (Met1-SER12 in SEQ ID NO: 1) substituted with the invariant signal domain (Met1-Arg29 in SEQ ID NO: 15), S ecto: (S protein ectodomain), S1 (S protein S1 subunit), RBD (receptor-binding domain), T4 trimer (T4 fibrin trimerization motif), 3C3d (enhancer sequence containing three copies of C3d protein), 2A (2A peptide (T2a or P2a, etc.)), Ubi. (ubiquitin), N (N protein), S2 (S protein S2 subunit), and SV40 DTS (SV40 DNA nuclear target sequence). [Figure 4] Figure 4: Immune response induced by VXM-SCV-3 in healthy mice. Serum from vaccinated mice was analyzed for antibodies against the SARS-CoV spike protein (see Example 5). The assay background is located at the 400 endpoint titer, as indicated by the straight dotted line. [Figure 5] Figure 5: Immune response induced by VXM-SCV-30 in healthy mice. Serum from vaccinated mice was analyzed for antibodies against the SARS-CoV spike protein (see Example 6). The assay background is located at the 400 endpoint titer, as indicated by the straight dotted line. [Figure 6]Figure 6: Immune response induced by VXM-SCV-42 in healthy mice. Serum from vaccinated mice was analyzed for antibodies against the SARS-CoV spike protein (see Example 7). The assay background is located at the 400 endpoint titer, as indicated by the straight dotted line. [Figure 7] Figure 7: Immune response induced by VXM-SCV-53 in healthy mice. Serum from vaccinated mice was analyzed for antibodies against the SARS-CoV spike protein (see Example 8). The assay background is located at the 400 endpoint titer, as indicated by the straight dotted line. [Modes for carrying out the invention]

[0023] Detailed description of the invention This specification provides a DNA vaccine comprising the Salmonella Typhi Ty21a strain, which comprises a DNA molecule containing a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof.

[0024] According to the present invention, the Salmonella Typhi Ty21a strain functions as a bacterial carrier for a DNA molecule containing a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof, and delivers the DNA molecule into a target cell. Thus, the DNA molecule is delivered to the host cell, and the S protein or a portion thereof is expressed by the host cell. The Salmonella Typhi Ty21a strain is an attenuated Salmonella strain, and the DNA vaccine of the present invention contains this live attenuated Salmonella Typhi strain Ty21a.

[0025] In the context of this invention, "attenuated" means that the virulence of the bacterial strain is reduced compared to the parent bacterial strain that does not have an attenuating mutation. Preferably, the attenuated bacterial strain has lost its virulence but retains the ability to induce protective immunity. Attenuation can be achieved by the deletion of various genes (including genes for virulence, regulation, and metabolism). Attenuated bacteria can be found in nature, or artificially produced in the laboratory, for example by adapting them to a new culture medium or cell culture, or by recombinant DNA technology. The attenuated Salmonella strain of this invention is approximately 10 11 It is preferable that the incidence of salmonellosis occurring in the target population after CFU administration is less than 5%, more preferably less than 1%, and most preferably less than 1‰.

[0026] The terms “contains” or “contains” mean “contains, but not limited to.” These terms are intended to be non-restrictive, identifying the presence of any feature, element, integer, process, or component described, but not excluding the presence or addition of one or more other features, elements, integers, processes, components, or groups. Thus, the term “contains” includes the more restrictive expressions “consist of” and “essentially consist of.” In one embodiment, the term “contains” can be individually replaced by the expression “consist of.” With respect to sequences, the expressions “having the amino acid sequence of” and “containing the amino acids of” are used interchangeably and include embodiments of “consisting of the amino acid sequence of.” The term “one” can include multiple in this specification, and therefore includes, but is not limited to “one.”

[0027] The terms “SARS-CoV-2 S protein or part thereof” or “another SARS-CoV-2 protein or part thereof” mean, as herein, the SARS-CoV-2 S protein or its immunogenic moiety, or another SARS-CoV-2 protein and its immunogenic moiety. An immunogenic moiety of a protein may contain one or more domains of that immunogenic protein. However, the invention also includes cases where the immunogenic moiety contains only one immunogenic moiety (such as a receptor-binding domain or an ectodomain). The term “immunogenic” means, as herein, a moiety of a protein that induces an immune response (such as a B-cell response and / or a T-cell response).

[0028] A DNA molecule containing at least one eukaryotic expression cassette can also be called a recombinant DNA molecule, i.e., an engineered DNA construct, and preferably consists of DNA fragments from different sources. The DNA molecule can be a linear or circular nucleic acid. The DNA molecule is preferably a plasmid, and more preferably an expression plasmid. This plasmid can be generated by introducing an open reading frame encoding at least the SARS-CoV-2 S protein or a portion thereof into a single plasmid eukaryotic expression cassette. A plasmid containing a eukaryotic expression cassette can also be called a eukaryotic expression plasmid.

[0029] In the context of this invention, the term “expression cassette” means a nucleic acid unit that includes at least one open reading frame (ORF) and is under the control of a regulatory sequence that controls its expression. Preferably, the expression cassette also includes a transcription termination signal. Preferably, the expression cassette can mediate the transcription of an open reading frame contained therein that encodes at least the SARS-CoV-2 S protein or a portion thereof in target cells. A eukaryotic expression cassette typically includes a promoter, at least one open reading frame, and a transcription termination signal, which enable expression in eukaryotic target cells.

[0030] Coronaviruses are single-stranded positive-sense RNA viruses belonging to the Coronaviridae family. These viruses mostly infect animals (including birds and mammals). In humans, coronaviruses typically cause mild respiratory infections. Since 2003, two highly pathogenic human coronaviruses, namely Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), have caused global epidemics with high morbidity and mortality rates. Both epidemics were caused by zoonotic coronaviruses belonging to the Betacoronavirus genus within the Coronaviridae family.

[0031] Like SARS-CoV and MERS-CoV, the novel SARS-CoV-2 belongs to the genus Betacoronavirus. The SARS-CoV-2 genome has approximately 30,000 base pairs and encodes many structural and non-structural proteins. Structural proteins include the spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. As reported by Zhou et al. (Cell Discovery (2020) 6:14), SARS-CoV-2 shows the greatest nucleotide sequence similarity with SARS-CoV (79.7%). Specifically, the envelope and nucleocapsid proteins of SARS-CoV-2 are two evolutionarily conserved regions, with sequence similarity of 96% and 89.6% respectively compared to SARS-CoV. While the spike protein of SARS-CoV-2 has been reported to exhibit the lowest sequence conservation (77% sequence agreement) between SARS-CoV-2 and SARS-CoV, the spike protein of SARS-CoV-2 has only 31.9% sequence agreement with the spike protein of MERS-CoV. Several non-structural proteins encoded by open reading frames ORF 1ab, ORF 3a, ORF 3b, ORF 6, ORF 7a, ORF 7b, ORF 8, ORF 9a, ORF 9b, and ORF 10 were predicted for SARS-CoV-2 (Srinivasan et al. Viruses (2020) 12:360). In the meantime, several variants of SARS-CoV-2 have been identified. For example, lineage B.1.1.7 of SARS-CoV-2 was first reported in the United Kingdom, lineage B.1.351 was first reported in South Africa, and subclade B.1.1.28 was first reported in Brazil and renamed P.1 (Galloway et al., MMWR Morb Mortal Wkly Rep. 2021 Jan 22; 70(3): 95-99). According to Galloway et al., these variants possess a group of genetic mutations, including the S protein receptor-binding domain, which is essential for binding to the host cell angiotensin-converting enzyme-2 (ACE-2) receptor, facilitating viral entry. These variants appear to spread more efficiently.

[0032] Various reports on SARS-CoV suggest protective roles in both humoral and cell-mediated immune responses. The S protein is the most exposed protein, and antibody responses against the SARS-CoV S protein have been shown to protect against SARS-CoV infection in mouse models. Antibody responses may be effective but short-lived. Conversely, T cell responses have been shown to provide long-term protection against SARS-CoV. In addition, numerous studies have shown the production of antibodies against the SARS-CoV N protein, and extending this to SARS-CoV-2, the N protein is considered to be a highly immunogenic protein that is abundantly expressed during infection. Furthermore, T cell responses to the S and N proteins among structural proteins have been reported to be the most dominant and longest-lasting (Ahmed et al. Viruses (2020) 12:254). Salmonella Typhi Ty21a, an attenuated strain of Salmonella, is one of the Salmonella enterica species. Attenuated derivatives of Salmonella enterica are an attractive medium for delivering heterologous antigens to the mammalian immune system. This is because S. enterica strains can potentially be delivered through mucosal immunization, i.e., orally or nasally. This offers the advantage of being simpler and safer compared to parenteral administration. Furthermore, Salmonella strains induce robust humoral and cellular immune responses at both systemic and mucosal compartmental levels. Batch preparation costs are low, and live bacterial vaccine formulations are highly stable. Attenuation can be achieved by deletion of various genes (including genes related to virulence, regulation, and metabolism).

[0033] Several strains of Salmonella typhi weakened by allomutation have been shown to be safe and effective delivery vehicles for heterologous antigens in animal models.

[0034] Attenuated Salmonella Typhi Ty21a strains have been shown to be safe and effective as a vaccine against typhoid fever and as a delivery medium for heterologous antigens for human vaccination (primarily against tumor antigens and / or stroma antigens).

[0035] The live, attenuated Salmonella Typhi Ty21a strain is the active ingredient in Typhoral L® (also known as Vivotif®), manufactured by Berna Biotech Ltd., Crucell Company, Switzerland. This is currently the only approved live oral vaccine against typhoid fever. The vaccine has been extensively studied for patient toxicity and transmission to third parties and has been proven safe (Wahdan et al., J. Infectious Diseases 1982, 145:292-295). The vaccine is approved in over 40 countries and has been used for prophylactic vaccination against typhoid fever in millions of individuals, including thousands of children. The marketing authorization number for Typhoral L® is PL 15747 / 0001, dated December 16, 1996. One dose of the vaccine contains at least 2 × 10⁶ doses. 9 A viable Salmonella Typhi Ty21a colony-forming unit and at least 5 × 10⁻⁶ 9 Contains non-surviving Salmonella Typhi Ty21a cells.

[0036] This well-tolerated oral vaccine for typhoid fever is derived from the chemical mutagenesis of the wild-type highly virulent bacterial isolate Salmonella Typhi Ty2, which has a loss-of-function mutation in the galE gene and is therefore unable to metabolize galactose. This attenuated bacterial strain is also unable to reduce sulfates to sulfides. These sulfides distinguish the attenuated strain from the wild-type Salmonella Typhi Ty2 strain. Regarding its serological characteristics, Salmonella Typhi Ty21a strain contains the O9-antigen, a polysaccharide in the outer membrane of the bacterium, but lacks the O5-antigen, which in turn is a characteristic component of Salmonella Typhi. This serological characteristic supports the rationale for including each test in a group identity test for batch shipments.

[0037] The SARS-CoV-2 S protein is a glycoprotein with 66 N-linked glycosylation sites per trimer. This protein also contains O-linked glycans at residues S673, T678, and S686. Furthermore, the S protein contains two functional domains: a receptor-binding domain and a second domain containing a sequence that mediates the fusion of the viral membrane with the cell membrane. The S glycoprotein is required for cell entry because it must be cleaved by a cellular protease to allow exposure of the fusion sequence. From the SARS-CoV-2 protein sequence, it is revealed that a furin cleavage sequence (PRRARS|V) exists at residues 681-687 due to the insertion of the sequence PRRA. Since furin proteases are abundant in the respiratory tract, the SARS-CoV-2 S glycoprotein can be cleaved when it leaves epithelial cells, and as a result, it can efficiently infect other cells.

[0038] The expression cassette used in relation to the DNA vaccine of the present invention is a eukaryotic expression cassette. In the context of the present invention, the term “eukaryotic expression cassette” means an expression cassette that enables the expression of open reading frames in eukaryotic cells. It has been shown that the amount of heterologous antigen required to induce a sufficient immune response may be toxic to bacteria, potentially leading to cell death, excessive attenuation, or loss of expression of the heterologous antigen. Using a eukaryotic expression cassette that does not express in the bacterial plasmid vector but expresses only in target cells may overcome this toxicity problem, and the expressed protein typically exhibits a eukaryotic glycosylation pattern.

[0039] The eukaryotic expression cassette contains regulatory sequences (preferably a promoter and a polyadenylation signal) capable of controlling the expression of open reading frames in eukaryotic cells. The promoter and polyadenylation signal contained in the recombinant DNA molecule contained in the attenuated Salmonella strain of the present invention are preferably selected to function in the target cells to be immunized. Non-limiting examples of promoters particularly suitable for the manufacture of human DNA vaccines include promoters from cytomegalovirus (CMV) (such as the potent CMV early promoter), promoters from Simian virus 40 (SV40), promoters from mouse mammary tumor virus (MMTV), promoters from human immunodeficiency virus (HIV) (such as the HIV long terminal repeat (LTR) promoter), promoters from Moloney virus, promoters from Epstein-Barr virus (EBV), and promoters from Roussarcoma virus (RSV), synthetic CAG promoters consisting of CMV early enhancer elements, promoters of the chicken beta-actin gene, the first exon and first intron, and splice acceptors of the rabbit beta-globin gene, as well as promoters from human genes (such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein). In a particular embodiment, the eukaryotic expression cassette contains the CMV promoter. In the context of this invention, the term "CMV promoter" means a potent, early cytomegalovirus promoter.

[0040] Non-limiting examples of polyadenylation signals particularly suitable for the production of human DNA vaccines include bovine growth hormone (BGH) polyadenylation sites, SV40 polyadenylation signals, and LTR polyadenylation signals. In a particular embodiment, the eukaryotic expression cassette contained in a recombinant DNA molecule consisting of a Salmonella attenuated strain of the present invention includes a BGH polyadenylation site.

[0041] In addition to regulatory elements necessary for the expression of heterologous SARS-CoV-2 S protein or parts thereof (such as promoters and polyadenylation signals), other elements can also be included in recombinant DNA molecules. Such additional elements are enhancers. Possible enhancers include, for example, human actin, human myosin, human hemoglobin, and human muscle creatine enhancers, as well as viral enhancers (such as enhancers from CMV, RSV, and EBV).

[0042] In the context of the present invention, it is generally advantageous to use a gene (or open reading frame) encoding the SARS-CoV-2 S protein or a portion thereof (as well as optional further SARS-CoV-2 proteins or portions thereof (such as the SARS-CoV-2 N protein or a portion thereof)) whose codons are optimized for expression in mammals, particularly in humans. Therefore, in one embodiment, the eukaryotic expression cassette comprises at least a codon-optimized sequence encoding the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof.

[0043] Non-limiting examples of the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof encoded by the DNA vaccine of the present invention include (a) the SARS-CoV-2 full-length S protein; (b) the SARS-CoV-2 S protein ectodomain; (c) the SARS-CoV-2 protein subunit S1; (d) the SARS-CoV-2 receptor-binding domain (RBD); or (e) at least three immunodominant epitopes of the SARS-CoV-2 S protein.

[0044] In one embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein is the SARS-CoV-2 full-length S protein. The SARS-CoV-2 full-length S protein may include an amino acid sequence having the amino acid sequence of SEQ ID NO: 1, or a sequence that is at least 95% identical to SEQ ID NO: 1. In one preferred embodiment, the SARS-CoV-2 full-length S protein has an amino acid sequence having a sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In one embodiment, the SARS-CoV-2 full-length S protein has an amino acid sequence having a sequence that is at least 98% to 100% identical to SEQ ID NO: 1. In one particular embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein is the SARS-CoV-2 full-length S protein consisting of an amino acid sequence having the amino acid sequence of SEQ ID NO: 1, or a sequence that is at least 95% identical to SEQ ID NO: 1. The amino acid sequence of Sequence ID No. 1 has GenBank accession number MN_908947 and has been published by Wu et al. (Nature 2020, 579: 265-269). In a particular embodiment, the full-length S protein of a SARS-CoV-2 variant (such as lineage B.1.1.7, B.1.351, or P.1) can also be used as the full-length S protein of SARS-CoV-2.

[0045] We have identified various SARS-CoV-2 S protein sequences available in GenBank as follows: MN_908947(QHD434616.1), MN_988668(QHQ62107.1), NC_045512(YP_009724390.1), MN_938384.1(QHN73795.1). Alignment and comparison with MN_975262.1(QHN73810.1), MN_985325.1(QHQ60594.1), MN_988713.1(QHQ62877.1), MN_994467.1(QHQ71963.1), MN_994468.1(QHQ71973.1), and MN997409.1(QHQ82464.1) revealed no significant differences. However, slight differences have been previously reported in SARS-CoV-2 S proteins. For example, the following substitutions have been described in clinical isolates by Wrapp et al. (Science, 2020, 367: 1260-1263): F32I, H49Y, S247R, N354D, D364Y, V367F, D614G, V1129L, and E1262G. Furthermore, the substitutions H49Y and V860Q have been reported by Wang et al. (J. Med. Virol. March 13, 2020: 1-8). Further homology analysis of SARS-CoV-2 sequences published by the same authors revealed that the nucleotide homology of the S protein is 99.82%–100% and the amino acid homology of the S protein is 99.53%–100%. The identified variants B.1.1.7, B.1.351, and P.1 have several mutations. The S protein of variant B.1.1.7 has the deletions 69-70HV and 144Y, and the following mutations: N501Y, A570D, D614G, P681H, T761I, S982A, and D1118H. Variant B.1.351 has the following mutations in its S protein: K417N, E484K, N501Y, D614G, and A701V.The P.1 variant contains mutations in the S protein such as L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T1027I (Galloway et al., MMWR Morb Mortal Wkly Rep. 2021 Jan 22; 70(3): 95-99). However, further substitutions or variants may arise or be identified over time.

[0046] As a SARS-CoV-2 full-length S protein, pre-fusion stabilized forms of the SARS-CoV-2 full-length S protein (such as those containing two or more stabilizing mutations) are also possible. In one embodiment, the pre-fusion stabilized form of the SARS-CoV-2 full-length S protein contains two stabilizing mutations to proline corresponding to amino acid positions K986 and V987 of the amino acid sequence of SEQ ID NO: 1.

[0047] Wrapp et al. described a pre-fusion stabilization form of the SARS-CoV-2 S protein by adding two stabilizing proline mutations to residues 986 and 987 in the C-terminal S2 fusion mechanism, utilizing a previous stabilization strategy proven effective in other betacoronavirus S proteins (Science, 2020, 367: 1260-1263). Furthermore, Wrapp et al. (Science, 2020, 367: 1260-1263) described a "GSAS" mutation at the position of residues 682-685 in the furin cleavage site, replacing the RRAR sequence at this position. Both of these mutations stabilize the protein and thus prevent fusion. This may not only improve the stability and expression of the S protein but also improve safety by preventing cell fusion. In one embodiment, the pre-fusion stabilized form of the SARS-CoV-2 full-length S protein includes two stabilizing mutations to proline corresponding to amino acid positions K986 and V987 of the amino acid sequence of SEQ ID NO: 1, and / or mutations in the furin cleavage sequence (PRRARS|V) corresponding to residues 681-687 of SEQ ID NO: 1 (e.g., R682G, R683S, and R685S mutations). The SARS-CoV-2 full-length S protein has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with SEQ ID NO: 1, and further comprises two stabilizing mutations K986P and V987P; or furin cleavage sequence mutations R682G, R683S, and R685S, or preferably two stabilizing mutations K986P and V987P and furin cleavage sequence mutations R682G, R683S, and R685S. Alternatively, there may be a deletion in the furin cleavage sequence (e.g., amino acids 680-683). Therefore, in one embodiment, the SARS-CoV-2 full-length S protein has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and further includes a deletion in the furin cleavage sequence (e.g., a deletion containing amino acids S680-R683 or consisting of amino acids S680-R683). Other amino acid substitutions or deletions that constitute a pre-fusion stabilization form of the S protein can also be used.

[0048] In some embodiments, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof includes the SARS-CoV-2 S protein ectodomain. The term "ectodomain" means the extracellular portion of the transmembrane protein SARS-CoV-2 S protein, i.e., the absence of the transmembrane domain and cytoplasmic domain. The ectodomain includes a distal membrane subunit S1 containing the receptor-binding domain and a proximal membrane subunit S2. The SARS-CoV-2 S protein ectodomain includes the amino acid sequence of amino acid residues 1-1208 of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with amino acid residues 1-1208 of SEQ ID NO: 1. However, as used herein, the SARS-CoV-2 S protein ectodomain may include a sequence corresponding to at least amino acid residues 1-1208 of SEQ ID NO: 1, or a slightly longer sequence, for example, up to the N-terminal 1213 amino acid residue of SEQ ID NO: 1, or a sequence having at least 95% agreement with amino acid residues 1-1213 of SEQ ID NO: 1. In one preferred embodiment, the SARS-CoV-2 S protein or a portion thereof includes a SARS-CoV-2 S protein ectodomain having an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of amino acid residues 1-1208 of SEQ ID NO: 1. In one embodiment, the SARS-CoV-2 S protein ectodomain has an amino acid sequence that is at least 98% to 100% identical to the sequence of amino acid residues 1-1208 of SEQ ID NO: 1. In a particular embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof is a SARS-CoV-2 S protein ectodomain having an amino acid sequence that is at least 95% identical to the sequence of amino acid residues 1-1208 of SEQ ID NO: 1, or a sequence that is at least 95% identical to the sequence of amino acid residues 1-1208 of SEQ ID NO: 1. In a further specific embodiment, the SARS-CoV-2 S protein ectodomain may also be the S protein ectodomain of a SARS-CoV-2 variant (such as lineage B.1.1.7, B.1.351, or P.1).

[0049] The SARS-CoV-2 S protein or a portion thereof may also include a pre-fusion stabilized form of the SARS-CoV-2 S protein ectodomain containing two or more stabilizing mutations. In one embodiment, the pre-fusion stabilized form of the SARS-CoV-2 S protein ectodomain contains two stabilizing mutations to proline corresponding to amino acid positions K986 and V987 in the amino acid sequence of amino acid residues 1-1208 of SEQ ID NO: 1.

[0050] In one embodiment, the SARS-CoV-2 S protein or a portion thereof comprises the amino acid sequence of amino acid residues 1-1208 of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with amino acid residues 1-1208 of SEQ ID NO: 1, and further comprises two stabilizing mutants K986P and V987P.

[0051] In one embodiment, the pre-fusion stabilization form of the SARS-CoV-2 S protein ectodomain includes two stabilizing mutations to proline corresponding to amino acid positions K986 and V987 in the amino acid sequence of amino acid residues 1-1208 of SEQ ID NO: 1, and / or mutations in the furin cleavage sequence (PRRARS|V) corresponding to residues 681-687 in the amino acid sequence of amino acid residues 1-1208 of SEQ ID NO: 1 (e.g., R682G, R683S, and R685S mutations). The SARS-CoV-2 S protein ectodomain has an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acid residues 1-1208 of SEQ ID NO: 1, and preferably includes two stabilizing mutations K986P and V987P; or Furin cleavage sequence mutations R682G, R683S, and R685S, or two stabilizing mutations K986P and V987P and Furin cleavage sequence mutations R682G, R683S, and R685S. Alternatively, the amino acids in the Furin cleavage sequence may be deleted, such as amino acids 680-683. Therefore, in one embodiment, the SARS-CoV-2 full-length S protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, and includes a deletion in the Furin cleavage sequence (such as including amino acids S680-R683 or a deletion consisting of amino acids S680-R683). Other amino acid substitutions or deletions can be used to obtain a pre-fusion stabilization form of the S protein ectodomain.

[0052] The SARS-CoV-2 ectodomain may further contain a fusion domain for stabilization and / or improved expression and / or improved secretion. Possible fusion domains include trimerization domains (such as the C-terminal T4 fibrintin trimerization motif). The trimerization domain of bacteriophage T4 fibrintin (referred to as "foldon") has the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 10), which corresponds to amino acid residues 457-483 of the fibrintin protein.

[0053] The sequence encoding the SARS-CoV-2 S protein or a portion thereof preferably includes a signaling sequence encoding a signaling peptide. The signaling peptide of the SARS-CoV-2 S protein may have, for example, the amino acid sequence corresponding to amino acid residues 1-15 of SEQ ID NO: MFVFLVLLPLVSSQC (SEQ ID NO: 3), or an equivalent functional signaling peptide having a sequence that is at least 80%, preferably at least 90%, identical to the amino acid sequence of SEQ ID NO: 3. In one embodiment, the signal peptide of the SARS-CoV-2 S protein signaling peptide invariant chain, preferably in one embodiment, has amino acid residues 1-12 of SEQ ID NO: 1 substituted with amino acid residues 1-29 of SEQ ID NO: 15.

[0054] In one embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof includes a SARS-CoV-2 S protein subunit S1. The SARS-CoV-2 S protein subunit S1 includes an amino acid sequence of amino acid residues 1-681 of SEQ ID NO: 1, or an amino acid sequence having a sequence that is at least 95% identical to amino acid residues 1-681 of SEQ ID NO: 1. In a preferred embodiment, the SARS-CoV-2 S protein or a portion thereof includes a SARS-CoV-2 S protein subunit S1 having an amino acid sequence having a sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of amino acid residues 1-681 of SEQ ID NO: 1. In one embodiment, the SARS-CoV-2 S protein subunit S1 has an amino acid sequence having a sequence that is at least 98% to 100% identical to amino acid residues 1-681 of SEQ ID NO: 1. In one particular embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof is a SARS-CoV-2 S protein subunit S1 having or consisting of an amino acid sequence of amino acid residues 1-681 of SEQ ID NO: 1, or an amino acid sequence that is at least 95% identical to amino acid residues 1-681 of SEQ ID NO: 1. In a further particular embodiment, the SARS-CoV-2 S protein subunit S1 may also be an S protein subunit S1 of a variant of SARS-CoV-2 (e.g., lineage B.1.1.7, B.1.351, or P.1).

[0055] In one embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof includes a SARS-CoV-2 S protein receptor-binding domain (RBD). The SARS-CoV-2 S protein RBD includes an amino acid sequence of amino acid residues 319-541 of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with amino acid residues 319-541 of SEQ ID NO: 1. In a preferred embodiment, the SARS-CoV-2 S protein or a portion thereof includes a SARS-CoV-2 S protein RBD having a sequence having at least 96%, at least 97%, at least 98%, or at least 99% agreement with amino acid residues 319-541 of SEQ ID NO: 1. In one embodiment, the SARS-CoV-2 S protein RBD has an amino acid sequence having at least 98%-100% agreement with amino acid residues 319-541 of SEQ ID NO: 1. In one particular embodiment, the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof is a SARS-CoV-2 S protein RBD having or consisting of an amino acid sequence that is at least 95% identical to amino acid residues 319-541 of SEQ ID NO: 1, or an amino acid sequence that is at least 95% identical to amino acid residues 319-541 of SEQ ID NO: 1. In one particular embodiment, the SARS-CoV-2 S protein RBD may also be an S protein RBD of a SARS-CoV-2 variant (such as lineage B.1.1.7, B.1.351, or P.1).

[0056] One advantage of using SARS-CoV-2 full-length S protein, SARS-CoV-2 S protein ectodomain, SARS-CoV-2 protein subunit S1, or SARS-CoV-2 RBD is that it provides a polyclonal humoral immune response (including a neutralizing antibody response) while maintaining efficacy against mutated SARS-CoV-2, and that the cellular immune response, in addition to the humoral immune response, is not MHC-limited and therefore not limited to patients with a particular type of HLA.

[0057] In the context of this invention, the expression "sequence that is at least 95% identical to" refers to proteins whose amino acid sequence (such as the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of amino acid residues 1-1208, 1-681, or 319-541 of SEQ ID NO: 1 (including the corresponding portion thereof)) and / or the nucleic acid sequence encoding that amino acid sequence may differ from the reference sequence. The S protein or any part thereof can be of natural origin (e.g., a mutant version or variation of the SARS-CoV-2 S protein having the amino acid sequence of SEQ ID NO: 1), or it can be an engineered protein (e.g., an engineered glycoprotein derivative) modified by introducing site-directed mutation, cloning, or a combination thereof. Codon utilization is known to differ between species. Therefore, when expressing heterologous S proteins in target cells, it may be necessary, or at least helpful, to adapt the nucleic acid sequence to the codon utilization of the target cells. Methods for designing and constructing derivatives of a given protein are well known to those skilled in the art. Adapting a nucleic acid sequence to the codon utilization of a target cell is also known as codon optimization.

[0058] An S protein or a portion thereof that shares a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 or its corresponding portion may contain one or more mutations, including the addition, deletion, and / or substitution of one or more amino acids. According to the teachings of the present invention, the deleted, added, and / or substituted amino acids can be consecutive amino acids or scattered over the length of the amino acid sequence of the S protein or portion thereof that shares a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 or its corresponding portion. According to the teachings of the present invention, any number of amino acid additions, deletions, and / or substitutions are possible, as long as the amino acid sequence match with the amino acid sequence of SEQ ID NO: 1 or its corresponding portion is at least about 95%. In a particular embodiment, the sequence match between the amino acid sequence of the S protein or portion thereof and the amino acid sequence of SEQ ID NO: 1 or its corresponding portion is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and preferably at least 99%. All percentage values ​​relate to the amino acid sequence of Sequence ID No. 1 or its corresponding portion (e.g., amino acid residues 1-1208, 1-681, or 329-541). Methods and algorithms for determining sequence matching (including comparisons of the parent protein with its derivatives that have deletions, additions, and / or substitutions compared to the parent sequence) are well known to those skilled in the art. At the DNA level, nucleic acid sequences encoding S proteins or parts thereof that share a sequence that is at least about 95% identical to the amino acid sequence of Sequence ID No. 1 may differ due to genetic code degeneracy and possibly codon optimization.

[0059] According to the present invention, in one embodiment, the DNA vaccine may include Salmonella Typhi Ty21a strain comprising a DNA molecule containing a eukaryotic expression cassette that encodes at least the SARS-CoV-2 S protein or a portion thereof and an enhancer sequence (such as a complement peptide sequence, more preferably three copies of complement protein C3d (SEQ ID NO: 4), each of the three C3ds preferably separated by a GS linker (3C3d; SEQ ID NO: 5)) from the N-terminus to the C-terminus. Such sequences have been described as enhancing the humoral immune response, particularly inducing a stronger antibody response. If the SARS-CoV-2 S protein or a portion thereof comprises the SARS-CoV-2 S protein ectodomain, the SARS-CoV-2 S protein subunit S1, or the SARS-CoV-2 S protein RBD, the eukaryotic expression cassette may further encode a trimerizing domain (such as the C-terminal T4 fibrintin trimerizing motif (SEQ ID NO: 10)) preferably fused to the SARS-CoV-2 S protein portion. Therefore, in one embodiment, the DNA vaccine may also include a Salmonella Typhi Ty21a strain comprising a DNA molecule containing a eukaryotic expression cassette that encodes at least a SARS-CoV-2 protein or a portion thereof, a trimer domain, and optionally an enhancer sequence (such as a complement peptide sequence), from the N-terminus to the C-terminus, including the SARS-CoV-2 S protein ectodomain, SARS-CoV-2 S protein subunit S1, or SARS-CoV-2 S protein RBD (preferably the SARS-CoV-2 S protein ectodomain), a trimer domain, and optionally an enhancer sequence (such as a complement peptide sequence).

[0060] Representative enhancer sequences (such as ubiquitin peptide sequences or complement peptide sequences that promote antigen presentation in MHC class I or II molecules, respectively) are known in this field. Plasmid vectors encoding MHC class I antigens and ubiquitin peptides, delivered to mice by Salmonella typhi, have been demonstrated to enhance antigen-specific T cell responses and tumor control in a B16 tumor challenge model (Xiang et al, PNAS, 2000). Antibody responses to B cell epitopes encoded by DNA vectors have been shown to be enhanced by introducing three copies of the complement protein C3d peptide, which binds to the CR2 (CD21) receptor found on the surface of B cells and follicular dendritic cells and enhances antigen-specific B cell activation (Moveseyan, J Neuroimmunol, 2008; Yang, Virus Res, 2010; Hou, Virology J, 2019). Therefore, to enhance the B cell response, a complement peptide sequence (such as three copies of complement protein C3d (KFLTTAKDKNRWEDPGKQLYNVEATSYA; SEQ ID NO: 4)) can be added to the C-terminus of a sequence encoding the SARS-CoV-2 S protein or a portion thereof. These three 28-amino acid peptides are preferably separated by a GS linker (such as GS(G4S)2GS of SEQ ID NO: 5) (3C3d). Furthermore, to improve the nuclear translocation of a DNA molecule (such as a plasmid) containing a eukaryotic expression cassette encoding the SARS-CoV-2 S protein or a portion thereof from at least the cytoplasm, the DNA molecule may further contain a DNA nuclear target sequence (such as one or more copies of the SV40 DNA nuclear target sequence (DTS; SEQ ID NO: 16) (preferably two or more copies of DTS)).

[0061] The DNA vaccine of the present invention may further encode another SARS-CoV-2 protein or a portion thereof (preferably a SARS-CoV-2 N protein or a portion thereof). In a preferred embodiment, the SARS-CoV-2 N protein or a portion thereof includes the sequence of SEQ ID NO: 8 or a portion thereof, or a sequence that is at least 95% identical to SEQ ID NO: 8 or a corresponding portion thereof. Preferably, the SARS-CoV-2 N protein or a portion thereof has an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 8. In one embodiment, the SARS-CoV-2 N protein or a portion thereof has an amino acid sequence that is at least 98% to 100% identical to the sequence of SEQ ID NO: 8 or a corresponding portion thereof. In a further embodiment, the SARS-CoV-2 N protein or a portion thereof may also have the amino acid sequence of a variant of SARS-CoV-2 (e.g., lineage B.1.1.7, B.1.351, or P.1).

[0062] Another SARS-CoV-2 protein or a portion thereof can be expressed by a further DNA vaccine comprising Salmonella Typhi Ty21a strain, which comprises a DNA molecule containing a eukaryotic expression cassette encoding at least a COVID-19 coronavirus (SARS-CoV-2) protein different from the spike (S) protein or a portion thereof. These two DNA vaccines can be administered simultaneously to induce an immune response to the SARS-CoV-2 S protein and the other SARS-CoV-2 protein. Alternatively, another SARS-CoV-2 protein or a portion thereof can be expressed by the DNA vaccine of the present invention, which further comprises a second DNA molecule encoding that other SARS-CoV-2 protein. Thus, the DNA vaccine comprises Salmonella Typhi Ty21a strain, which comprises a first DNA molecule containing a eukaryotic expression cassette encoding at least a COVID-19 coronavirus (SARS-CoV-2) protein spike (S) protein or a portion thereof, and a second DNA molecule containing a eukaryotic expression cassette encoding at least a COVID-19 coronavirus (SARS-CoV-2) protein different from the spike (S) protein or a portion thereof. The first and second DNA molecules are preferably plasmids, and more preferably expression plasmids. It is more preferable that the plasmids have the same plasmid vector backbone (e.g., the pVAX10 backbone). It is also conceivable that another SARS-CoV-2 protein or a portion thereof be expressed by the same DNA molecule comprising a first expression cassette encoding a SARS-CoV-2 S protein or a portion thereof, and a second expression cassette encoding another SARS-CoV-2 protein or a portion thereof. All these embodiments can be freely combined with the embodiments mentioned above, and further, expression cassettes encoding at least a SARS-CoV-2 S protein or a portion thereof and optionally including an enhancer sequence and / or trimer domain are further defined.

[0063] The DNA molecule may further comprise a eukaryotic expression cassette encoding the SARS-CoV-2 S protein or a portion thereof, and another SARS-CoV-2 protein or a portion thereof. Therefore, in one embodiment, the DNA vaccine comprises a Salmonella Typhi Ty21a strain comprising a DNA molecule containing a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof, and another COVID-19 coronavirus (SARS-CoV-2) protein (structural or non-structural). Preferably, the SARS-CoV-2 S protein or a portion thereof is expressed at the N-terminus, and the other SARS-CoV-2 protein or a portion thereof is expressed at the C-terminus. The following embodiments can be freely combined with the embodiments mentioned above, and in particular, further define expression cassettes encoding at least the SARS-CoV-2 S protein or a portion thereof and optionally comprising an enhancer sequence and / or trimerizing domain. In one preferred embodiment, the DNA vaccine comprises Salmonella Typhi Ty21a strain containing a DNA molecule comprising a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof and the COVID-19 coronavirus (SARS-CoV-2) N protein or a portion thereof. The SARS-CoV-2 N protein or a portion thereof may include a sequence having sequence number 8 or a portion thereof, or a sequence having at least 95% agreement with sequence number 8 or a corresponding portion thereof. Preferably, the SARS-CoV-2 N protein or a portion thereof has an amino acid sequence having a sequence having at least 96%, at least 97%, at least 98%, or at least 99% agreement with sequence number 8. In one embodiment, the SARS-CoV-2 N protein or a portion thereof has an amino acid sequence having a sequence having at least 98% to 100% agreement with sequence number 8 or a corresponding portion thereof. In one embodiment, the SARS-CoV-2 N protein or a portion thereof may also have the amino acid sequence of a SARS-CoV-2 variant (such as lineage B.1.1.7, B.1.351, or P.1).The SARS-CoV-2 S protein or a portion thereof can be ligated to another SARS-CoV-2 protein via a 2A self-cleaving peptide (2A peptide) or an internal ribosome entry site (IRES), preferably via the 2A peptide. Examples of 2A peptides are P2a, which has the amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 6), or T2a, which has the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 7).

[0064] According to the present invention, the DNA vaccine may include Salmonella Typhi Ty21a strain comprising a DNA molecule containing a eukaryotic expression cassette encoding at least one SARS-CoV-2 protein or a portion thereof, a 2A peptide or IRES sequence, and another SARS-CoV-2 protein or a portion thereof (preferably a SARS-CoV-2 N protein or a portion thereof) from the N-terminus to the C-terminus. Following the other SARS-CoV-2 protein or a portion thereof, a SARS-CoV-2 protein subunit S2 may be further added, particularly if the SARS-CoV-2 S protein or a portion thereof is a SARS-CoV-2 protein subunit S1. In some embodiments, the SARS-CoV-2 protein subunit S2 comprises amino acid residues 686-1208 of SEQ ID NO: 1, or a sequence that is at least 95% identical to amino acid residues 686-1208 of SEQ ID NO: 1. In one embodiment, subunit S2 includes amino acid residues 686-1273 of SEQ ID NO: 1, or a sequence that is at least 95% identical to amino acid residues 686-1273 of SEQ ID NO: 1.

[0065] Further enhancer sequences (such as ubiquitin sequences) may precede another SARS-CoV-2 protein or a portion thereof. Ubiquitin is conserved between mouse and human and has the amino acid sequence MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDY NIQKESTLHLVLRLRG (SEQ ID NO: 9). If not constrained by theory, the N-terminal ubiquitin sequence may enhance the T cell response to the antigen. Therefore, a DNA vaccine containing Salmonella typhi Ty21a strain is also conceivable, which includes a DNA molecule containing a eukaryotic expression cassette comprising a DNA molecule that, from the N-terminus to the C-terminus, encodes at least a SARS-CoV-2 S protein or a portion thereof, a 2A peptide or IRES sequence, a ubiquitin sequence, and another SARS-CoV-2 protein or a portion thereof (preferably a SARS-CoV-2 N protein or a portion thereof), optionally followed by a SARS-CoV-2 protein subunit S2.

[0066] The N protein is thought to primarily induce a T cell response. Plasmid vectors encoding MHC class I antigens and ubiquitin peptides, delivered to mice by Salmonella typhi, have been demonstrated to enhance antigen-specific T cell responses and tumor control in a B16 tumor challenge model (Xiang et al, PNAS, 2000). Therefore, T cell-enhancing sequences can preferably be fused at the N-terminus to another SARS-CoV-2 protein or a portion thereof (such as the SARS-CoV-2 N protein or a portion thereof).

[0067] The terms “2A self-cleaving peptide,” “2A cleavage site,” or “2A peptide” are used herein as synonyms and refer to a single class of peptides, 18 to 22 amino acids in length, capable of inducing the cleavage of recombinant proteins within a cell. 2A peptides were originally found in the 2A region of viral genomes and were adopted as a tool for expressing polypeptides within a single expression cassette. 2A peptide-mediated cleavage occurs after translation and cleavage are initiated by the cleavage of the peptide bond between proline (P) and glycine (G) at the C-terminus of the 2A peptide. Sequences encoding the 2A peptide linker are known in this field and are given, for example, in SEQ ID NOs. 6 or 7.

[0068] The term "internal ribosome entry site" is abbreviated as IRES, and in this specification, it is an RNA element that enables translation initiation in a manner independent of the cap, and therefore translation in mRNA containing an IRES sequence also begins at the location of the IRES sequence.

[0069] In another embodiment, a DNA vaccine comprising Salmonella Typhi Ty21a strain comprising a DNA molecule comprising a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof (where the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof comprises at least three immunodominant epitopes of the SARS-CoV-2 S protein). In one embodiment, the expression cassette encodes at least three immunodominant epitopes of the SARS-CoV-2 S protein and an enhancer sequence (such as a complement peptide sequence as described above).

[0070] The expression "at least three immunodominant epitopes of the SARS-CoV-2 S protein" means, as used herein, one polypeptide or two or more polypeptides that together contain three or more immunodominant epitopes of the SARS-CoV-2 S protein. It is not important whether the three or more immunodominant epitopes of the SARS-CoV-2 S protein are part of the same polypeptide or part of different polypeptides. Thus, three or more immunodominant epitopes of the SARS-CoV-2 S protein can be expressed as one polypeptide or as two or more polypeptides. In one embodiment, a eukaryotic expression cassette encodes at least one polypeptide that contains at least three immunodominant epitopes of the SARS-CoV-2 S protein. The immunodominant epitopes contained in the at least one polypeptide are three or more, five or more, ten or more, twenty or more, thirty or more, fifty or more, or more than fifty immunodominant epitopes. In the context of the Salmonella Typhi Ty21a strain used herein, a eukaryotic expression cassette encoding at least three immunodominant epitopes of the SARS-CoV-2 S protein can encode a single polypeptide containing up to 50 immunodominant epitopes, or more, for example, up to 300. Antigens presented as peptides on the surface of MHC class I or II (in human HLA) are typically 11–30 amino acids in length (CD4 antigen) for MHC II and 8–10 amino acids (CD8 antigen) for MHC I. Therefore, preferred ranges of immunodominant epitopes contained in at least one polypeptide are 3–300, 5–300, 10–300, 20–300, or 50–300 immunodominant epitopes. Thus, the polypeptide may further contain immunodominant epitopes from other structural proteins of SARS-CoV-2 (such as the E protein, M protein, or N protein, preferably the N protein). The preferred range of immunodominant epitopes for the SARS-CoV-2 S protein, expressed by a eukaryotic expression cassette or contained within at least one polypeptide, is 3–25, 3–20, or 5–15.Each polypeptide containing the fused immunodominant epitope is cleaved into the epitope by proteolysis within antigen-presenting cells, presented via HLA, and induces a T cell response.

[0071] Because the SARS-CoV-2 S protein and SARS-CoV are genetically very similar (76%), it may be possible to predict the T and B epitopes of SARS-CoV-2 by utilizing existing immunological studies of SARS-CoV (Ahmed et al, Viruses, 2020). T and B cell epitopes may also be predictable using bioinformatics approaches and validated algorithms for recognizing amino acid motifs that bind to MHC class I and class II proteins of various HLA molecules (Grifoni et al, Cell, 2020). Proposed T and B cell epitopes can be generated using publicly available resources (such as the Immune Epitope Database and Analysis Resource (IEDB), NetMHCPan, and NetMHCIIPan). These approaches may allow for the design of multi-epitope vaccines that encompass epitope-rich S protein compartments. One particularly interesting region is the receptor-binding motif (RBM) of the S protein, which interacts with the angiotensin-converting enzyme 2 (ACE2) receptor on human target cells to facilitate viral entry. Antibodies against the SARS-CoV RBM are neutralizing antibodies, but the RBMs of SARS-CoV and SARS-CoC-2 only match 50%, and these antibodies do not cross-neutralize (Ju et al, BioRxiv, 2020 - submitted; Walls et al, Cell, 2020).

[0072] According to the present invention, at least three immunodominant epitopes of the SARS-CoV-2 S protein may include CD8 T cell antigen and / or CD4 T cell antigen. Preferably, at least three immunodominant epitopes of SARS-CoV-2 S include CD8 T cell antigen and CD4 T cell antigen.

[0073] A single immunodominant epitope is typically a peptide having 8 to 30 amino acids, preferably 8 to 20, and more preferably 8 to 12 amino acids.

[0074] For a vaccine containing an immunodominant epitope of the SARS-CoV-2 S protein, it is beneficial if the vaccine also targets multiple immunodominant epitopes of the S protein, and preferably additional structural proteins (such as the N protein), because this reduces the risk of immune evasion due to mutations in the S protein.

[0075] Alternatively, in some embodiments, the DNA vaccine comprises a Salmonella Typhi Ty21a strain containing a DNA molecule comprising a eukaryotic expression cassette encoding at least three immunodominant epitopes of the SARS-CoV-2 S protein from the N-terminus to the C-terminus, and optionally an enhancer sequence, a 2A peptide or IRES sequence, optionally a ubiquitin sequence, and another SARS-CoV-2 protein or a portion thereof (preferably the SARS-CoV-2 N protein or a portion thereof). As a portion of the SARS-CoV-2 N protein, at least three immunodominant epitopes of the SARS-CoV-2 N protein are possible.

[0076] The advantages of the DNA vaccine of the present invention, which includes Salmonella Typhi Ty21a as a carrier for at least the SARS-CoV-2 S protein or a portion thereof (such as the SARS-CoV-2 S protein, the full-length S protein, the S protein ectodomain, the S protein subunit S1, or three immunodominant epitopes of the S protein RBD), are established quality control assays, individual plasmid differences only within the antigen-coding insert, no need for proliferation, and no need for sterility testing due to oral administration. Furthermore, in addition to an expression plasmid suitable for transformation, the Salmonella Typhi Ty21a strain as a carrier enables large inserts (such as the full-length S protein or multiple immunodominant epitopes). Moreover, it enables the introduction of another SARS-CoV-2 protein or a portion thereof (such as the SARS-CoV-2 N protein or a portion thereof) linked to the SARS-CoV-2 S protein or a portion thereof via a 2A peptide or IRES sequence.

[0077] The immunodominant epitope of the SARS-CoV-2 S protein (or possibly the N protein) can be inserted into a plasmid as a string-like bead, possibly separated by a linker (expressed as one or more polypeptides). Possible linkers include, but are not limited to, GS linkers, 2A cleavage sites, or IRES sequences. Due to the rapid generation and limited need for quality control, the time required to generate the Salmonella typhi Ty21a strain containing a DNA molecule with at least one eukaryotic expression cassette encoding the SARS-CoV-2 S protein or a portion thereof is short, and can be achieved, for example, within 15 days, preferably 14 days or less, after identifying the antigen (including the immunodominant epitope or a novel clinical isolate or variant). An overnight culture is sufficient, and the bacterial yield is high, with a net yield of 10 in 1 L of culture. 11Because it is in the range of colony-forming units (CFUs), upscaling is not necessary. This allows for short manufacturing times and low manufacturing costs. Furthermore, this drug product has been shown to be stable for at least three years. Therefore, this DNA vaccine is suitable for the rapid development and manufacture of an effective SARS-CoV-2 preventive and / or therapeutic vaccine for use in a large number of target populations that need it. In addition, it is easy to store and does not require trained medical staff to administer.

[0078] At least one DNA sequence encoding the SARS-CoV-2 S protein or a portion thereof can be separated from another SARS-CoV-2 protein or a portion thereof using a linker (possibly a GS linker, a 2A cleavage site, or an IRES sequence).

[0079] Methods for detecting immunodominant epitopes in proteins and reliably predicting and identifying peptides that bind with high affinity to autologous human leukocyte antigen (HLA) molecules are known in this field. Subsequently, peptides predicted to have a high probability of binding to the patient's autologous HLA-A or HLA-B protein, or peptides that are dominant within the population, are selected. This can be confirmed, for example, by in vitro interferon-gamma enzyme-coupled immunospot (ELISPOT).

[0080] In one embodiment, the DNA molecule, or the DNA molecule comprising at least one eukaryotic expression cassette, comprises an antibiotic resistance gene (such as a kanamycin antibiotic resistance gene), an ori (such as a pMB1 ori or pUC), and a potent promoter (such as a CMV promoter). In a particular embodiment, the DNA molecule, or the DNA molecule comprising at least one eukaryotic expression cassette, is a plasmid (such as a plasmid based on or derived from a commercially available pVAX1® expression plasmid (Invitrogen, San Diego, California)).

[0081] This expression plasmid vector can be modified by replacing the high-copy pUC replication origin with the low-copy pMB1 replication origin of pBR322. The low-copy modification was made to reduce the metabolic burden and make the construct more stable. The resulting expression plasmid vector backbone was named pVAX10.

[0082] Expression vectors can also be designed to include enhancers (such as ubiquitin or complement) to facilitate antigen presentation in MHC class I or II molecules. Plasmid vectors encoding MHC class I antigens and ubiquitin peptides, delivered to mice by Salmonella typhi, have been demonstrated to enhance antigen-specific T cell responses and tumor control in a B16 tumor challenge model (Xiang et al, PNAS, 2000). Antibody responses to B cell epitopes encoded by DNA vectors have been shown to be enhanced by including three copies of complement protein C3d (SEQ ID NO: 4), which binds to the CR2 (CD21) receptor found on the surface of B cells and follicular dendritic cells to enhance antigen-specific B cell activation (Moveseyan, J Neuroimmunol, 2008; Yang, Virus Res, 2010; Hou, Virology J, 2019).

[0083] Several methods have been used to facilitate the translation of multiple genes using a single plasmid vector, including inserting internal ribosome entry sites (IRESs) (Ma et al, Hum Vaccin Immunother, 2013) or 2A peptides (Liu et al, Scientific Reports, 2017) between peptide gene sequences.

[0084] In a particular embodiment, the expression plasmid contains the DNA molecule of SEQ ID NO: 2 (vector backbone pVAX10), which correlates with the sequence of the expression vector pVAX10, which lacks the multiple cloning region located between restriction sites NheI and XhoI. In one embodiment, the expression plasmid contains the nucleic acid sequence of SEQ ID NO: 2 and a sequence encoding the amino acid sequence of SEQ ID NO: 1 or a portion thereof, or an amino acid sequence having at least 95% agreement with SEQ ID NO: 1 or a portion thereof.

[0085] An expression plasmid was generated when the SARS-CoV-2 S protein encoding an ORF containing the nucleic acid sequence encoding Sequence ID No. 1 was inserted into this expression vector backbone via NheI / XhoI. A schematic diagram of this expression plasmid, pVAX10.SCV-1, is shown in Figure 2.

[0086] The DNA vaccine of the present invention can be in the form of a pharmaceutical composition. Therefore, in one embodiment, a DNA vaccine comprising Salmonella Typhi Ty21a strain containing a DNA molecule comprising a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof, further comprises one or more pharmaceutically acceptable excipients. In one embodiment, the DNA vaccine is an oral dosage form. The DNA vaccine of the present invention can be in the form of a solution, a suspension, or any other form suitable for intended oral use. Alternative dosage forms are enteric-coated capsules or lyophilized powders. Typically, the DNA vaccine of the present invention is provided as a drinking solution, preferably as a suspension, more preferably as an aqueous suspension. This embodiment offers the advantage of improved patient compliance and enables rapid, feasible, and affordable large-scale vaccination programs, particularly in impoverished areas.

[0087] The present invention also provides a pharmaceutical composition containing the DNA vaccine of the present invention.

[0088] In the context of this invention, the term “excipient” means a natural or synthetic substance formulated together with the active ingredient of a pharmaceutical. Suitable excipients include solvents, antifouling agents, binders, coatings, disintegrants, fragrances, colorants, lubricants, flow enhancers, adsorbents, preservatives, and sweeteners.

[0089] In the context of this invention, the expression “pharmaceutically acceptable” means a molecular compound and other components of a pharmaceutical composition that are physiologically tolerable and typically do not produce undesirable reactions when administered to mammals (e.g., humans). The expression “pharmaceutically acceptable” may also mean that it is approved by a federal or state regulatory authority, or that it is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in mammals, or more precisely, in humans.

[0090] In some embodiments, the DNA vaccine or pharmaceutical composition of the present invention is in the form of an enteric-coated capsule, a lyophilized powder, or a suspension. A suitable suspension includes means to neutralize stomach acid to at least some extent, i.e., means to bring the pH of the stomach acid closer to pH 7. Therefore, in some embodiments, the suspension is a buffered suspension obtained by suspending the attenuated strain of Salmonella of the present invention in a suitable buffer, preferably a buffer that neutralizes stomach acid to at least some extent, preferably a buffer containing 2.6 g of sodium bicarbonate, 1.7 g of L-ascorbic acid, 0.2 g of lactose monohydrate, and 100 ml of drinking water.

[0091] In one embodiment, the DNA vaccine of the pharmaceutical composition of the present invention further comprises one or more adjuvants.

[0092] In the context of this invention, the term "adjuvant" means an activator, i.e., a drug that alters the effect of the attenuated Salmonella strain of the present invention. Adjuvants can enhance the immune response to an antigen, thereby making it possible to minimize the amount of antigen administered.

[0093] In the context of the present invention, the term “vaccine” means a drug that, when administered, can induce an immune response in a target. Preferably, the vaccine can prevent, improve, or treat a disease. The vaccine of the present invention contains Salmonella Typhi Ty21a, a live attenuated strain of Salmonella typhi. The vaccine of the present invention is a DNA vaccine and therefore further comprises at least one copy of a DNA molecule containing a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof.

[0094] The terms “DNA vaccine” or “DNA vaccination” as used herein mean a vaccine for protecting against or treating a disease or infection by delivering genetically engineered linear DNA, preferably a plasmid containing the DNA sequence, encoding an antigen (such as the SARS-CoV-2 S protein or a portion thereof) that is to be elicited to induce an immune response, to target cells in a patient who needs it. Thus, the antigen is produced by the target cells to induce an immune response. DNA vaccines have potential advantages over conventional vaccines, including the ability to induce a broader type of immune response (such as humoral immune responses and / or cell-mediated immune responses). Plasmids can be delivered to tissues in several ways (including by injection in saline, gene guns, liposomes, or by carriers (such as bacterial and viral vectors)). The DNA vaccine of the present invention comprises Salmonella Typhi Ty21a strain as a carrier for delivering a DNA molecule containing at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof. The DNA molecule delivered by the attenuated Salmonella Typhi Ty21a strain is preferably a plasmid.

[0095] The bio-attenuated Salmonella strain of the present invention stably supports a DNA molecule encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof. This can be used as a medium for oral delivery of this DNA molecule. Such a delivery plasmid vector containing a DNA molecule encoding a heterologous antigen (such as the SARS-CoV-2 S protein or a portion thereof) is referred to as a DNA vaccine in the context of the present invention.

[0096] Genetic immunization may offer advantages over conventional vaccination. Target DNA can be detected over a considerable period, acting as an antigen depot. Sequence motifs within some plasmids (such as GpC islands) are immunostimulant and can act as adjuvants, facilitated by immune stimulation from LPS and other bacterial components.

[0097] Live attenuated Salmonella plasmid vectors (such as Salmonella typhi Ty21a) produce their own immunomodulatory factors (such as lipopolysaccharide (LPS)) in situ, which may constitute an advantage compared to other administration methods (such as microencapsulation). Furthermore, the mucosal DNA vaccine of this invention utilizes the natural entry site of the coronavirus, which may prove beneficial. Mucosal vaccination has an intralymphatic mode of action. After the attenuated vaccine of this invention is taken up, the modified bacteria invade macrophages and other cells in Peyer's patches in the intestine. The bacteria are taken up by these phagocytic cells. Due to its attenuated mutation, the Salmonella typhi Ty21 strain bacteria cannot survive in these phagocytic cells and die at this point. DNA molecules are released from the bacteria and endosomes and then transferred to the cytosol of phagocytic immune cells via a special transport system or by endosomal leakage. Finally, the recombinant DNA molecules enter the nucleus, where they are transcribed, leading to the massive expression of the SARS-CoV-2 S protein in the phagocytic cells. Infected cells undergo apoptosis, loading S protein antigens that are then taken up and processed by the intestinal immune system. The danger signal of bacterial infection acts as a potent adjuvant in this process, leading to a strong antigen-specific CD8+ T cell response and antibody response at both the systemic and mucosal compartmental levels. The intralymphatic mucosal vaccination route is particularly useful for large-scale vaccination and for pathogens that utilize mucosal entry (such as coronaviruses).

[0098] Salmonella vaccines containing eukaryotic plasmids can induce a B-cell response to the antigen encoded by the plasmid. In mice orally immunized with Salmonella typhi containing a pCMVb eukaryotic expression vector encoding the antigens listeriolisin or ActA, antigen-specific antibodies could be detected in the serum up to 4 weeks after immunization (Darji et al, Cell, 1997; Darji et al, FEMS Immunol Med Microbiol, 2000).

[0099] The Salmonella Typhi Ty21a vaccine strain has an unparalleled safety record. There is no available data indicating that Salmonella Typhi Ty21a can enter the systemic bloodstream. Therefore, a live attenuated Salmonella Typhi Ty21a vaccine strain allows for safe, well-tolerated, and specific targeting of the intestinal immune system. Conversely, adenovirus-based DNA vaccines may carry the inherent risk of unexpected viral replication. In addition, existing immunity to adenoviruses has been shown to limit vaccine efficacy in humans.

[0100] This specification also provides the DNA vaccine of the present invention for use in the treatment and / or prevention of coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection. This specification also provides a method comprising administering the DNA vaccine of the present invention to a patient in need as a method for treating and / or preventing coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection.

[0101] If adverse events similar to hypersensitivity reactions mediated by histamine, leukotrienes, or cytokines occur, treatment options are available for fever, anaphylaxis, blood pressure instability, bronchospasm, and dyspnea. Treatment options for unwanted T-cell-derived autoimmunity are derived from the standard treatment scheme applied in acute and chronic graft-versus-host disease after stem cell transplantation. Cyclosporine and glucocorticoids are proposed as treatment options.

[0102] In the unlikely case of a systemic typhi bacillus Ty21a infection, appropriate antibiotic therapy using fluoroquinolones (including ciprofloxacin or ofloxacin) is recommended. Bacterial infections of the gastrointestinal tract should be treated with their respective medications (such as rifaximin).

[0103] In a preferred embodiment, the DNA vaccine of the present invention, comprising Salmonella Typhi Ty21a strain, is administered orally. This includes a DNA molecule containing a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof. Oral administration is simpler, safer, and more comfortable than parenteral administration. The DNA vaccine of the present invention may also be administered by any other suitable route, but oral administration is preferred. It is preferable that a therapeutically effective dose is administered to the subject, but this dose may depend on the specific use (especially whether the DNA vaccine is therapeutic or prophylactic, the subject's weight, age, sex, and health status, mode of administration, and formulation, etc.). Administration may be a single or multiple dose as needed.

[0104] The DNA vaccine of the present invention can be provided in the form of a solution, suspension, lyophilized product, enteric-coated capsule, or any other suitable form. Typically, the attenuated Salmonella strain of the present invention is formulated as a drinking solution. This embodiment provides improved patient compliance. The drinking solution preferably includes means to neutralize gastric acid to at least some extent, i.e., means to bring the pH of the gastric acid closer to pH 7. The drinking solution is preferably a buffered suspension containing the attenuated Salmonella strain of the present invention. In one particular embodiment, the buffered suspension is obtained by suspending the attenuated Salmonella strain of the present invention in a suitable buffer preferably containing 2.6 g of sodium bicarbonate, 1.7 g of L-ascorbic acid, 0.2 g of lactose monohydrate, and 100 ml of drinking water.

[0105] In a particular embodiment, the treatment and / or prevention of COVID-19 or SARS-CoV-2 infection may further include the administration of a further SARS-CoV-2 vaccine or anti-SARS-CoV-2 treatment. The treatment and / or prevention of COVID-19 and / or SARS-CoV-2 infection may further include a DNA vaccine comprising a strain of Salmonella Typhi Ty21a containing a DNA molecule comprising a eukaryotic expression cassette encoding at least another SARS-CoV-2 protein or a portion thereof (such as the COVID-19 coronavirus (SARS-CoV-2) envelope (E) protein, membrane (M) protein, or nucleocapsid (N) protein, or a portion thereof, preferably the SARS-CoV-2 N protein or a portion thereof). These two DNA vaccines may be administered simultaneously or sequentially, but it is preferable to administer the two DNA vaccines simultaneously.

[0106] In some embodiments, the treatment and / or prevention of COVID-19 and / or SARS-CoV-2 infection includes prime / boost vaccination against SARS-CoV-2. In the context of the present invention, the term “prime / boost vaccination” means an immunization program that includes immunizing a subject with a prime vaccination and at least one subsequent boost vaccination. In preferred embodiments, the prime vaccine and the boost vaccine are the same; i.e., prime / boost vaccination is homogeneous prime / boost vaccination. In particular, the DNA vaccine of the present invention is administered as both prime and boost vaccine. In other embodiments, the prime vaccine and the boost vaccine are different types of vaccines against the same pathogen; i.e., prime / boost vaccination is heterogeneous prime / boost vaccination. In some embodiments, the DNA vaccine of the present invention may be administered as a prime vaccine, and a further SARS-CoV-2 vaccine may be administered as a boost vaccine. In other specific embodiments, a further beta-coronavirus vaccine may be administered as a prime vaccine, and an attenuated Salmonella strain of the present invention may be administered as a boost vaccine. Prime / boost vaccination may induce a better immune response than vaccination using only a single prime dose. Improved initial T-cell response, antibody response, and / or long-lasting immune response may be achievable through prime / boost vaccination.

[0107] In one embodiment, the administration of the prime and boost DNA vaccines of the present invention is carried out within 8 consecutive weeks, more specifically within 3 to 6 consecutive weeks. The prime vaccine and the boost vaccine can be administered by the same route or different routes. The prime and boost DNA vaccines of the present invention are preferably administered by the same route, and more preferably the prime and boost DNA vaccines are administered orally. Also, the DNA vaccines of the present invention can be administered once or several times at the same dose or different doses. Optimizing the prime / boost vaccination schedule (including optimizing the timing and dose of vaccine administration) is within the ability of those skilled in the art.

[0108] In a particular embodiment, a single-dose DNA vaccine contains the Ty2 strain of Salmonella typhi of the present invention at about 10 5 ~ about 10 11 or about 1×10 6 ~ about 1×10 10 more preferably about 1×10 6 ~ about 1×10 9 about 1×10 6 ~ about 1×10 8 or about 1×10 6 ~ about 1×10 7 colony forming units (CFU). In one embodiment, a single-dose DNA vaccine contains the Ty2 strain of Salmonella typhi at about 1×10 [[ID=We]] 6 ~ about 1×10 9 colony forming units (CFU). Administration of this live-attenuated bacterial DNA vaccine at a low dose minimizes the risk of excretion and thus the risk of transmission to third parties. It has previously been shown that excretion cannot be detected at all at less than 1×10 9 CFU.

[0109] In this context, the term "about" or "roughly" means within 3 times, or within 2 times (including within 1.5 times) of a given value or range. [[ID=We]]

[0110] In one embodiment, the treatment and / or prevention of COVID-19 or SARS-CoV-2 infection comprises multiple doses of the DNA vaccine of the present invention. The single doses of the DNA vaccine may be the same or different, but the single doses may be the same and contain approximately 1 × 10⁻⁶ doses of Salmonella Typhi Ty21a strain. 6 ~Approx. 1×10 9 Preferably, the vaccine is contained in colony-forming units (CFUs). In particular, treatment and / or therapy for COVID-19 or SARS-CoV-2 infection involves 1, 2, 3, 4, 5, or 6 doses of the DNA vaccine of the present invention. Preferably, treatment and / or therapy for COVID-19 or SARS-CoV-2 infection involves administering the DNA vaccine 2 to 4 times per week for priming (as a prime vaccination), and optionally following with one or more single-dose boosting doses. In some embodiments, the DNA vaccine is administered 2 to 4 times within the first week (as a prime vaccination), followed by one or more single-dose boosting doses (as booster vaccinations) at least 2 weeks later each. That is, a prime vaccination in the first week and single-dose booster vaccinations from the third week onward, optionally followed by one (or more) further single-dose booster vaccinations at least 2 weeks later. In an alternative embodiment, the DNA vaccine is administered 2 to 4 times within the first week (as a prime vaccine), followed by one or more single-dose boostings at least 4 weeks later (as booster vaccines). That is, the prime vaccine is administered in the first week, followed by single-dose booster vaccines from week 5 onwards, and possibly one (or more) further single-dose booster vaccines at least 4 weeks later. [Examples]

[0111] Example 1: Preparation of recombinant plasmid pVAX10.SCV-1 The DNA encoding the SARS-CoV-2 S protein (1273 amino acids, SEQ ID NO: 1) is cloned into the pVAX10 backbone derived from pVAX10.VR2-1 (WO 2013 / 091898). The S protein DNA fragment is generated by double-strand gene synthesis, during which oligonucleotides are linked together using a heat-stable ligase. The resulting linked product is amplified by PCR. After amplification, the in vitro synthesized S protein DNA fragment is cloned into the pVAX10 backbone via NheI / XhoI (the VEGFR-2 coding region of the recombinant plasmid pVAX10.VR2-1 is replaced with the S protein coding region). For quality control, the entire plasmid is sequenced after transformation in E. coli and aligned with each reference sequence to confirm that there are no errors. The resulting plasmid is named pVAX10.SCV-1 (Figure 2). Other suitable constructs are shown in Figure 3.

[0112] Example 2: Transformation of attenuated Salmonella strains using recombinant plasmid pVAX10.SCV-1 Salmonella Typhi Ty21a is transformed with plasmid pVAX10.SCV-1. Transformation is performed by electroporation.

[0113] Preparation of competent Salmonella cells: Glycerol cultures of Salmonella Typhi Ty21a were inoculated into LB plates ([ACF] soy peptone without animal components). The plates were incubated overnight at 37°C. One colony was used for overnight liquid-preparation culture. 3 ml of LB medium (ACF soy peptone) inoculated with one colony was incubated overnight at 180 rpm at 37°C. To prepare competent cells, 3 ml of the overnight culture was inoculated into 2 × 300 ml of LB medium (ACF soy peptone) and incubated at 180 rpm at 37°C. 600 The cultures were incubated until the concentration was approximately 0.5. These cultures were then placed on ice for 10 minutes. Subsequently, the bacteria were centrifuged at 3000 × g for 10 minutes at 4°C, and each pellet was placed in 500 mL of ice-cold H2O. destThe pellets were resuspended in [the solution]. After a new centrifugation step, the bacterial pellets were washed twice with 10% ice-cold glycerol. Both pellets were placed together in 2 ml of 10% glycerol and finally frozen on dry ice to produce multiple 50 μL aliquots. The glycerol used was completely free of animal components (Sigma Aldrich, G5150).

[0114] Transformation of competent Salmonella cells: For each transformation reaction, thaw one 50 μl aliquot of competent typhibacterium Ty21a cells on ice for 10 minutes. Add 3–5 μl of plasmid DNA pVAX10.SCV-1, then incubate the mixture on ice for 5 minutes. Transfer the mixture to a pre-chilled cuvette (1 mm thick). Apply an electrical pulse at 12.5 kV / cm. Immediately afterward, add 1 ml of LB medium (ACF soy peptone) to the cells, transfer the cells to a 2 ml Eppendorf tube, and shake at 37°C for 1 hour. After a short centrifugation step on a bench centrifuge (16600 rcf, 20 seconds), resuspend the bacterial pellet in 200 μl of LB (ACF soy peptone) antibiotic-free medium. Using a Drigalski spatula, spread this mixture onto an LB plate (ACF soy peptone) containing kanamycin (concentration = 25 μg / ml or 50 μg / ml). Incubate the plate overnight at 37°C.

[0115] Plasmid preparation of recombinant Salmonella clones: Three clones of recombinant Salmonella Typhi Ty21a strain were incubated overnight at 37°C in 3 ml of LB medium (ACF soy peptone) containing kanamycin (50 μg / ml). The bacterial culture was then pelleted by centrifugation (16600 rcf, 30 seconds). Plasmid isolation was performed using the NucleoSpin plasmid kit from Macherey-Nagel. Plasmid DNA was eluted from the silica gel column with 50 μl of water. 5 μl of the eluent was used as a control in agarose gel electrophoresis.

[0116] To prepare a 1 ml glycerol culture of a positive clone for long-term storage, add 172 μl of glycerol (without animal components) in a 1 low ml screw microtube to 828 μl of medium for a 3 ml logarithmic growth stage culture. Store the sample at -70°C until use.

[0117] Complete sequencing of recombinant plasmid DNA isolated from Salmonella: One colony of recombinant Salmonella (pVAX10.SCV-1 with Salmonella typhi Ty21a) is inoculated into 3 ml of liquid LB-Kan medium (ACF soy peptone) and incubated overnight at 37°C at 180 rpm. This overnight culture is pelletized by centrifugation at 1300 rpm for 30 seconds on a bench centrifuge (Biofuge pico, Heraeus). Plasmid isolation is performed using the NucleoSpin plasmid kit from Macherey-Nagel. After alkaline lysis and sedimentation of high molecular weight genomic DNA and cellular components, the plasmid DNA is loaded onto a silica-membrane column. After washing, the plasmid is eluted from the column with 50 μl of sterile water and sequenced. The sequences are then compared with their respective reference sequences by clone-specific alignment. That is, each plasmid sequence of Salmonella clone is aligned with a reference sequence, and it is checked that all sequences match their respective reference sequences. This recombinant Salmonella strain is named VXM-SCV-1 (plasmid pVAX10.SCV-1 containing Salmonella typhi Ty21a).

[0118] Example 3: Large-scale production of VXM-SCV-1 Bacterial fermentation is carried out as described in WO2013 / 091898. Downstream processing consists of dialysis, dilution, and filling. 1-10 × 10 fermentation runs of 100 L each. 10Approximately 5 liters of CFU / ml vaccine are produced. The vaccine is further diluted in appropriate aliquots and stored at -70°C. The aliquots can be transported on dry ice. Dilute the aliquots in application buffer at the site to make a ready-to-use vaccine (100 ml of drinking solution, prepared in bulk).

[0119] Example 4: Design of a preclinical study - Evaluation of the immune response induced by VXM-SCV-1 in healthy mice The immune response to SARS-CoV-2 in healthy C57Bl / 6 mice, BALBc mice, or CD1 mice will be evaluated by antibody ELISA. The mice will be given Salmonella typhi containing plasmid pVAX10.SCV-1 (10 8 -10 9 Vaccinate with CFU / dose. Salmonella typhi containing plasmid pVAX10.SCV-1 is prepared as described above for Salmonella typhi Ty21a. A plasmid vector control group (10 per dose of Salmonella typhi without the expression plasmid) is used as a negative control. 8 ~10 10 Include CFUs in the study setup and identify the desired immune response from any nonspecific background stimulation caused by the Salmonella-free plasmid vector. Perform immune monitoring at one or more time points after vaccination.

[0120] 1. Animal husbandry and management Healthy female mice, 6 weeks old at the time of arrival, are observed for 7 days in a specific pathogen-free (SPF) animal housing unit before commencing the procedure. Animals are maintained in a room under controlled conditions of temperature (23±2°C), humidity (45±10%), photoperiod (12 hours light / 12 hours dark), and ventilation. Animals are maintained under SPF conditions. Room temperature and humidity are continuously monitored. The air treatment system is programmed for 14 air changes per hour, with no recirculation. Fresh outside air is passed through a series of filters and then evenly distributed to each room. Positive pressure (20±4 Pa) is maintained in the laboratory to prevent contamination or the spread of pathogens within the rodent colony. Animals are housed in polycarbonate cages (Techniplast, Limonest, France) equipped with food and water dispensers. The standard cage size used was 800 cm². 2 The maximum number of mice per cage is 10 (from the same group). The animals' bedding is sterile corn cob bedding (ref: LAB COB 12, SERLAB, Cergy-Pontoise, France), which is changed twice a week. The animals' food is purchased from DIETEX (Saint-Grathien, France). Irradiated RM1 is used as sterilization-controlled granules. Food is freely provided from water bottles equipped with rubber stoppers and sipper tubes. The water bottles are sterilized by sterile filtration and changed twice a week. On day 0, the mice are divided into two groups according to their individual body weight using Vivo Manager® software (Biosystemes, Couternon, France). The average body weights of the two groups (then divided into groups 1-5 and 6-10 respectively) are statistically insignificant (analysis of variance).

[0121] 2. Detection of antibody response in mice BALBc mice and CD1 mice were divided into six groups of eight mice each. Mice in groups 1-3 received a plasmid vector control, while mice in groups 4-6 received Salmonella typhi containing plasmid pVAX10.SCV-1. Both Salmonella typhi strains were thawed and administered within 30 minutes, and the working solution was discarded after use. The treatment dose was 100 μl per administration. 8The CFU is administered by forced oral administration (PO) through a 0.1 ml cannula. Regardless of the animal group, each animal receives a pre-administration buffer to neutralize stomach acid before administration (100 μl / animal / application). This buffer is prepared by dissolving 2.6 g of sodium bicarbonate, 1.7 g of L-ascorbic acid, and 0.2 g of lactose monohydrate in 100 ml of drinking water and is applied within 30 minutes before administering the Salmonella typhi strain. The treatment schedule is as follows:

[0122] The mice in Group 1 (n=8) and Group 4 (n=8) were each 10 8 CFU receives three doses of typhoid bacilli administered every two weeks (Q2WK×3).

[0123] The mice in group 2 (n=8) and group 5 (n=8) were each 10 8 The patient receives daily PO administration of Salmonella typhi (CFU) every two days for four consecutive weeks (Q2D x 4).

[0124] The mice in group 3 (n=8) and group 6 (n=8) were each 10 8 The patient received daily PO administration of Salmonella typhi (CFU) every two days for four consecutive weeks (Q2D x 4), followed by two booster doses every two weeks (Q2WK x 2).

[0125] The animals' lifespan and behavior will be recorded daily, and their weight will be measured twice a week. Serum will be collected at weeks 3, 4, 8, 12, 16, 20, 24, and 28 of the study and stored at -20°C until analysis. At the end of the study, all treated animals will undergo necropsy (macroscopic examination of the heart, lungs, liver, spleen, kidneys, and gastrointestinal tract).

[0126] In short, a 96-well EIA plate is coated overnight at 4°C with an N or S protein epitope or recombinant total N or S protein in 1 microgram per 1 ml of sodium carbonate buffer (pH 9.5). The next day, the plate is washed with 100 mmol of Tris-buffered saline / Tween® (TBST) and blocked with 3% gelatin at 37°C for 1 hour. After thoroughly washing the plate with TBST, serum is added to the top row of each plate, and 1:1 dilutions are prepared downwards for each column using TBST. Each plate contains a negative control column without serum. The plate is incubated overnight at 4°C. For development, the plate is washed with TBST and incubated with a 1:1000 dilution of alkaline phosphatase-bound protein G (Calbiochem, USA) at 37°C for 1 hour. OD405 is measured using an ELISA plate reader. The antibody endpoint titer is determined as the reciprocal of the dilution required to obtain an OD405 that is one standard deviation greater than the mean OD405 of the negative control.

[0127] 3. Detection of T cell response in C57BL6 mice or BALBc mice BALBc mice and C57BL6 mice were divided into six groups of 12 mice each. Mice in groups 1-3 received a plasmid vector control, while mice in groups 4-6 received Salmonella typhi containing plasmid pVAX10.SCV-1. Both Salmonella typhi strains were thawed and administered within 30 minutes, and the working solution was discarded after use. The treatment dose was 100 μl per administration. 8 The CFU is administered by forced oral administration (PO) through a 0.1 ml cannula. Regardless of the animal group, each animal receives a pre-administration buffer to neutralize stomach acid before administration (100 μl / animal / application). This buffer is prepared by dissolving 2.6 g of sodium bicarbonate, 1.7 g of L-ascorbic acid, and 0.2 g of lactose monohydrate in 100 ml of drinking water and is applied within 30 minutes before administering the Salmonella typhi strain. The treatment schedule is as follows:

[0128] The mice in Group 1 (n=12) and Group 4 (n=12) were each 10 8 CFU receives three doses of typhoid bacilli administered every two weeks (Q2WK×3).

[0129] The mice in group 2 (n=12) and group 5 (n=12) were each 10 8 The patient receives daily PO administration of Salmonella typhi (CFU) every two days for four consecutive weeks (Q2D x 4).

[0130] The mice in group 3 (n=12) and group 6 (n=12) were each 10 8 The patient received daily PO administration of Salmonella typhi (CFU) every two days for four consecutive weeks (Q2D x 4), followed by two booster doses every two weeks (Q2WK x 2).

[0131] The animals' survival and behavior were recorded daily, and their weight was measured twice a week. One-third of the mice (n=4) in each group were euthanized on day 14, one-third (n=4) on day 28, and the remaining one-third (n=4) on day 56. Spleen and blood samples were collected at the end of the study. Blood was processed to obtain serum, which was stored at -20°C until analysis. The spleens were processed to form single-cell suspensions. The immunogenicity of the vaccine in the spleen cell preparations was evaluated using IFN-gamma ELISPOT. Briefly, spleen cells were loaded into wells of an ELISPOT plate pre-coated with anti-IFN-gamma (500,000 cells in 0.1 ml). Peptide epitopes from N or S proteins were added to double wells at 10 micrograms per milliliter. The plates were incubated at 37°C for 18 hours. The following day, plates were developed using an AEC kit (Sigma, USA), and individual IFN-gamma secreting cells were counted using an Immunospot plate reader (Cellular Technologies Ltd, USA). Antibodies in serum samples were detected by ELISA. Briefly, 96-well EIA plates were coated overnight at 4°C with either an N or S protein epitope or recombinant total N or S protein at a concentration of 1 microgram per milliliter of sodium carbonate buffer (pH 9.5). The following day, plates were washed with 100 mmol Tris-buffered saline / Tween® (TBST) and blocked with 3% gelatin at 37°C for 1 hour. After thoroughly washing the plates with TBST, serum was added to the top row of each plate, and 1:1 dilutions were prepared downwards for each column using TBST. Each plate contained a negative control column without serum. Plates were incubated overnight at 4°C. For development, plates were washed with TBST and incubated at 37°C for 1 hour with a 1:1000 dilution of alkaline phosphatase-bound protein G (Calbiochem, USA). OD405 was measured using an ELISA plate reader. Antibody endpoint titer was determined as the reciprocal of the dilution required to yield an OD405 that is one standard deviation greater than the mean OD405 of the negative control.

[0132] 4. Antigen expression analysis Antigen expression analysis will be performed by transfecting mouse 3T3 cells and human 293T cells with the plasmid pVAX10.SCV-1. Cells will be collected and lysed 24 and 48 hours after infection. The resulting whole cell lysates will be analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by analysis by Western blotting on a PVDF membrane. RNA expression will also be confirmed by RT / PCR.

[0133] Example 5: Preclinical study - Evaluation of the immune response induced by VXM-SCV-3 in healthy mice The pVAX10-SCV-3 plasmid (insert SCV-3; SEQ ID NO: 11) encodes the SARS-CoV-2 spike protein (SEQ ID NO: 1) with the furin domain (amino acid residues 680-683) removed, and the SARS-CoV-2 N protein (SEQ ID NO: 8) (accession number YP_009724397). These antigens are separated by a 2A self-cleaving peptide sequence (SEQ ID NO: 7) derived from the capsid protein precursor of the Thosea asigna virus (see Figure 3).

[0134] Salmonella typhi SL7207 vaccine containing pVAX10-SCV-3 was prepared by electroporation. Competent bacteria were incubated on ice with 100-500 ng of plasmid DNA, and then electroporated in GenePulsar II at 2.5 KB. The bacteria were incubated in SOC medium on a shaker plate at 37°C for 1 hour, and then 100 μL was seeded onto a TSB agar plate with 50 μg / mL of kanamycin at 37°C overnight. Individual colonies were grown and frozen in 25% glycerol at -80°C.

[0135] We purchased pathogen-free female BALBc mice, aged 4-6 weeks, from Charles River Laboratories (Saint Constant, Quebec, Canada) and housed them in a facility where they had free access to food and water, in accordance with facility guidelines.

[0136] A group of 10 mice was treated with the SL-SCV-3 vaccine. For each treatment, the mice were pre-treated by forced oral administration of 100 microliters of administration buffer (310 mmol sodium bicarbonate, 100 mmol L-ascorbic acid, 5 mmol lactose monohydrate), followed by 1.5–2 × 10⁶ per milliliter. 9 Mice were administered 100 microliters of a dosing buffer containing a dose of CFU vaccine. Mice were treated on days 0, 2, 5, 7, 21, and 35. Mice were induced to bleed before the study (pre-immunization) and afterward at weeks 2, 4, 6, and 8.

[0137] The efficacy of the vaccine was evaluated by enzyme-linked immunosorbent assay (ELISA), a method that allows for the detection of antigen-specific antibody levels in the serum of immunized animals. Briefly, a 96-well EIA plate was coated overnight with the antigen, SARS-CoV-2 spike protein (ACROBiosystems), at 4°C, blocked at 37°C for 1 hour with 2% bovine serum albumin, and incubated overnight at 4°C with serial dilutions of serum, typically starting at a dilution of 1 / 200. The secondary reagent (goat anti-mouse IgG(H+L) peroxidase, Jackson ImmunoResearch) was then added to each well at a dilution of 1 / 5000 and incubated at 37°C for 1 hour. The plate was thoroughly washed, and the reaction was stopped by adding 3,3′,5,5′-tetramethylbenzidine substrate (Life Technologies) to the wells for 5–10 minutes and then adding 0.16N H2SO4. The absorbance at 450 nanometers in each well was measured using a microtitration plate reader (Cytation 5, Biotek). The endpoint titer was calculated as described by Frey A. et al. (Journal of Immunological Methods, 1998, 221:35-41). The calculated titer represents the maximum dilution, at which a statistically significant increase in absorbance is observed in serum samples from immunized mice compared to serum samples from naive, unimmunized control mice.

[0138] Of the 10 mice vaccinated with the SL-SCV-3 vaccine, two mice produced an antibody response greater than the assay background of 1 / 400. One mouse achieved a peak antibody titer of 1 / 800 by week 4, and another mouse achieved and maintained a peak antibody titer of 1 / 3200 by week 6 (see Figure 4). This demonstrates that a Salmonella-based SARS-CoV-2 vaccine construct targeting the spike protein can induce an antigen-specific immune response against the spike protein, i.e., a humoral immune response.

[0139] Example 6: Preclinical study - Evaluation of immune response induced by VXM-SCV-30 in healthy mice The pVAX10-SCV-30 plasmid (insert SCV-30; SEQ ID NO: 12) encodes the SARS-CoV-2 RBD domain of the spike protein (amino acids 319-541 of SEQ ID NO: 1), followed by three repeats of mouse C3d (3C3d; SEQ ID NO: 17; KFLNTAKDRNRWEEPDQQLYNVEATSYA), then a 2A self-cleaving peptide sequence derived from the capsid protein precursor of the Thosea asigna virus (SEQ ID NO: 7), followed by ubiquitin fused to the SARS-CoV-2 N protein (SEQ ID NO: 8) (SEQ ID NO: 9) (accession number YP_009724397) (see Figure 3).

[0140] Salmonella typhi SL7207 vaccine was prepared using pVAX10-SCV-30 as described in Example 5.

[0141] We purchased pathogen-free female BALBc mice, aged 4-6 weeks, from Charles River Laboratories (Saint Constant, Quebec, Canada) and housed them in a facility where they had free access to food and water, in accordance with facility guidelines.

[0142] A group of 10 mice was treated with the SL-SCV-30 vaccine. For each treatment, the mice were pre-treated by forced oral administration of 100 microliters of administration buffer (310 mmol sodium bicarbonate, 100 mmol L-ascorbic acid, 5 mmol lactose monohydrate), followed by 1.5–2 × 10⁶ per milliliter. 9 Mice were administered 100 microliters of administration buffer containing a dose of CFU vaccine. Mice were treated on days 0, 2, 5, 7, 21, and 35. Mice were induced to bleed before the study (pre-immunization) and thereafter at weeks 3, 4, 6, and 12.

[0143] The serum was analyzed as described in Example 5 to search for antibodies against the SARS-CoV-2 spike protein.

[0144] One out of ten mice vaccinated with the SL-SCV-30 vaccine developed an antibody response greater than the assay background of 1 / 400, reaching 1 / 3200 by week 3 (see Figure 5). This demonstrates that a Salmonella-based SARS-CoV-2 vaccine construct targeting the RBD domain of the spike protein induces an antigen-specific immune response against the spike protein.

[0145] Example 7: Preclinical study - Evaluation of the immune response induced by VXM-SCV-42 in healthy mice The pVAX10-SCV-42 plasmid (insert SCV-42; SEQ ID NO: 13) encodes the SARS-CoV-2 S1 domain of the spike protein (amino acids 1-681 of SEQ ID NO: 1), followed by three repeats of mouse C3d (SEQ ID NO: 17; 3C3d, SEQ ID NO: 18), then a 2A self-cleaving peptide sequence derived from the capsid protein precursor of the Thosea asigna virus (SEQ ID NO: 7), followed by a ubiquitin-mediated 2A self-cleaving peptide sequence fused to the SARS-CoV-2 N protein (SEQ ID NO: 8) (SEQ ID NO: 9), and the SARS-CoV-2 S2 domain of the spike protein (Ser686-Thr1273 of SEQ ID NO: 1) (see Figure 3).

[0146] Salmonella typhi SL7207 vaccine was prepared using pVAX10-SCV-42 as described in Example 5.

[0147] We purchased pathogen-free female BALBc mice, aged 4-6 weeks, from Charles River Laboratories (Saint Constant, Quebec, Canada) and housed them in a facility where they had free access to food and water, in accordance with facility guidelines.

[0148] A group of 10 mice was treated with the SL-SCV-42 vaccine. For each treatment, the mice were pre-treated by forced oral administration of 100 microliters of administration buffer (310 mmol sodium bicarbonate, 100 mmol L-ascorbic acid, 5 mmol lactose monohydrate), followed by 1.5–2 × 10⁶ per milliliter. 9 Mice were administered 100 microliters of administration buffer containing a dose of CFU vaccine. Mice were treated on days 0, 2, 5, 7, 21, and 35. Mice were induced to bleed before the study (pre-immunization) and afterward at weeks 2, 4, 6, and 8.

[0149] The serum was analyzed as described in Example 5 to search for antibodies against the SARS-CoV-2 spike protein.

[0150] Two out of ten mice vaccinated with the SL-SCV-42 vaccine developed an antibody response greater than the assay background of 1 / 400, reaching 1 / 1600 by week 3 (see Figure 6). This demonstrates that a Salmonella-based SARS-CoV-2 vaccine construct targeting the S1 and / or S2 subunits of the spike protein elicits an antigen-specific immune response against the spike protein.

[0151] Example 8: Preclinical study - Evaluation of the immune response induced by VXM-SCV-53 in healthy mice The pVAX10-SCV-53 plasmid (insert SCV-53; SEQ ID NO: 14; full plasmid sequence is SEQ ID NO: 19) encodes a SARS-CoV-2 spike protein (SEQ ID NO: 1) with the furin domain removed (deletion of amino acid residues 680-683) (however, the signal domain (Met1-Ser12 in SEQ ID NO: 1) is replaced by the signal domain of the immutable chain (Met1-Arg29 in SEQ ID NO: 15)), followed by a 2A self-cleaving peptide sequence (SEQ ID NO: 7) derived from the capsid protein precursor of the Thosea asigna virus, and then ubiquitin (SEQ ID NO: 9) fused to the SARS-CoV-2 N protein (SEQ ID NO: 8). This plasmid also contains a 72-nucleotide SV40 DNA nuclear target sequence (DTS) (SEQ ID NO: 16) within a larger SV40 ori enhancer sequence (SEQ ID NO: 20) upstream of the kanamycin resistance gene (see Figure 3).

[0152] Salmonella typhi SL7207 vaccine was prepared using pVAX10-SCV-53 as described in Example 5.

[0153] We purchased pathogen-free female BALBc mice, aged 4-6 weeks, from Charles River Laboratories (Saint Constant, Quebec, Canada) and housed them in a facility where they had free access to food and water, in accordance with facility guidelines.

[0154] A group of 10 mice was treated with the SL-SCV-53 vaccine. For each treatment, the mice were pre-treated by forced oral administration of 100 microliters of administration buffer (310 mmol sodium bicarbonate, 100 mmol L-ascorbic acid, 5 mmol lactose monohydrate), followed by 1.5–2 × 10⁶ per milliliter. 9 Mice were administered 100 microliters of a dosing buffer containing a dose of CFU vaccine. Mice were treated on days 0, 2, 5, 7, 21, and 35. Mice were induced to bleed before the study (pre-immunization) and afterward at weeks 2, 4, 6, and 8.

[0155] The serum was analyzed as described in Example 5 to search for antibodies against the SARS-CoV-2 spike protein.

[0156] Three out of ten mice vaccinated with the SL-SCV-53 vaccine exhibited an antibody response greater than the assay background of 1 / 400, reaching 1 / 800 (see Figure 7). This demonstrates that a Salmonella-based SARS-CoV-2 vaccine construct targeting a signal domain-modified spike protein induces an antigen-specific immune response against the spike protein.

[0157] Example 9: VXM-SCV-X Phase I clinical trial; study design The objective of this Phase I trial is to investigate the safety, tolerability, and immune response to VXM-SCV-X. This randomized, placebo-controlled, double-blind, dose-escalation study will include 45 participants. Participants will receive four doses of either VXM-SCV-X or placebo on days 1, 3, 5, and 7. 6 10 from CFU 9 This trial will evaluate VXM-SCV-X doses up to CFU. An independent Data Safety Monitoring Committee (DSMB) will be involved in dose escalation decisions. In addition to safety as the primary endpoint, VXM-SCV-1-specific immune responses will be evaluated.

[0158] The objective is to investigate the safety, tolerability, and immune response to the anti-SARS-CoV-2 virus vaccine VXM-SCV-X, as well as to identify the maximum tolerance dose (MTD) of VXM-SCV-1. The MTD is defined as the highest dose level at which fewer than two out of six patients treated with VXM-SCV-X experience dose-limiting toxicity (DLT).

[0159] The primary endpoints for safety and tolerability are adverse events and serious adverse events according to the CTCAE criteria.

[0160] Secondary endpoints include evaluating the effectiveness of the experimental vaccine in inducing a specific immune response against the SARS-CoV-2 S protein, including the number of immunopositive patients.

[0161] VXM-SCV-X was manufactured according to Good Manufacturing Practices (GMP) and provided in a buffered solution. The placebo control consisted of an isotonic sodium chloride solution.

[0162] The starting dose is 10 6 The solution consists of colony-forming units (CFUs) of VXM-SCV-X. This VXM-SCV-X dose was selected for safety reasons. For comparison, the single dose of Typhoral®, an approved vaccine for typhoid fever, is approximately 1000 times the VXM-SCV-1 starting dose, equivalent to 2 × 10⁻¹⁰. 9 ~6×10 9 It contains CFU of Salmonella Typhi Ty21a. This dose is increased in logarithmic steps, which is considered justified with respect to live bacterial vaccines.

[0163] In accordance with guidelines for human first-dose trials, patients in the single-dose group were treated in multiple cohorts. The initial dose of VXM-SCV-X in the arbitrary-dose group was administered to one patient. The second cohort in each dose group consisted of two patients receiving VXM-SCV-X. This staggered administration in one preceding patient, i.e., only one patient receiving VXM-SCV-X initially, helps reduce risk.

[0164] The third cohort of patients (3 people receiving VXM-SCV-X) are included in all treatment groups.

[0165] Environmental risks inherent in oral vaccines include the potential for release into the environment and subsequent vaccination of individuals outside the target population. All study patients are confined to the study site for the day of vaccination and for three days thereafter. All fecal samples from study patients are collected and incinerated. Bodily fluid and fecal samples are examined for VXM-SCV-X shedding.

[0166] Hygienic precautions are applied to protect researchers from accidental ingestion. Researchers are specially trained for this aspect of the research.

[0167] In addition, specific T-cell activation and antibody formation will be measured in this patient setting. A placebo control will be included to gain further knowledge regarding specific safety issues related to the active vaccine and background treatments. Furthermore, a pooled group of placebo patients will serve as a valid comparison factor for evaluating specific immune activation.

[0168] Example 10: VXM-SCV-1 specific T cell response and B cell response The response to VXM19 is evaluated by monitoring the frequency of SARS-CoV-2 virus S protein-specific T cells, as detected by IFNγ ELISpot, in the peripheral blood of patients treated with VXM-SCV-X and placebo at different time points before, during, and after vaccination.

[0169] First, T cells and peptide-pulsed DCs are added to wells coated with anti-INFγ antibody. After the incubation period, the cells are removed with the secreted INFγ bound to the coated antibody. Then, a detection antibody is added to detect the bound INFγ, and a single amplification is performed. The final yield can then be seen as a "color spot" representing a single activated specific T cell.

[0170] B-cell response is measured by ELISA. Briefly, a 96-well EIA plate is coated overnight at 4°C with an N or S protein epitope, or recombinant total N or S protein, at a concentration of 1 microgram per milliliter of sodium carbonate buffer (pH 9.5). The following day, the plate is washed with 100 mmol of Tris-buffered saline / Tween® (TBST) and blocked with 3% gelatin at 37°C for 1 hour. After thoroughly washing the plate with TBST, serum is added to the top row of each plate, and 1:1 dilutions are prepared downwards for each column using TBST. Each plate contains a negative control column without serum. The plate is incubated overnight at 4°C. For development, the plate is washed with TBST and incubated at 37°C for 1 hour with a 1:1000 dilution of alkaline phosphatase-bound protein G (Calbiochem, USA). OD405 is measured using an ELISA plate reader. The antibody endpoint titer is determined as the reciprocal of the dilution required to obtain an OD405 that is one standard deviation greater than the mean OD405 of the negative control.

[0171] Example 11: Anti-carrier immunity To evaluate the immune response to the bacterial medium, anti-typhi immunoglobulins IgG and IgM are detected by ELISA using two commercially available assay kits (Typhi IgG ELISA, catalog no. ST0936G and Typhi IgM ELISA, catalog no. ST084M; Calbiotech, Inc., 10461 Austin Dr, Spring Valley, CA 91978, USA). These assays are qualitative assays. These assays are to be used as described in the respective package inserts (App. I / I), but may be modified as part of a research plan in accordance with the aforementioned validation study 580.132.2785.

[0172] Enzyme-linked immunosorbent assay technology is used in both assays. Calibrator, negative control, positive control, and sample are analyzed in a double-row system. Diluted patient serum (dilution 1:101) is added to wells coated with purified antigen. Specific antibodies against IgG or IgM, if present, bind to the antigen. All unbound material is washed away, and the enzyme complex is added to bind to the antibody-antigen complex (if present). Excess enzyme is washed away, and the substrate is added. The plate is incubated to allow enzymatic hydrolysis of the substrate. The intensity of the resulting color is proportional to the amount of IgG or IgM-specific antibody in the sample. Color intensity is measured at 450 nm using a spectrophotometric microtitration plate reader. The cutoff is calculated as follows: Calibrator OD × Calibrator Factor (CF).

[0173] The antibody index for each measurement is obtained by dividing the OD value of each sample by the cutoff value.

[0174] Interpretation of antibody indices: <0.9 Antibodies against Salmonella typhi IgG or IgM cannot be detected by ELISA. 0.9~1.1 borderline positive >1.1 Antibodies against Salmonella typhi IgG or IgM can be detected by ELISA.

[0175] Example 12: Vaccination Schedule Single dose, i.e., 10 6 ~10 8 The CFU VXM19 is administered orally as a 100 ml drinking solution. Single doses are given on days 1, 3, and 5, and possibly on day 7. The peak immune response is expected to occur approximately 10 days after the last vaccination. Boosting can be considered 2–4 weeks later, or even 3–6 months later. The recommended schedule is derived from the vaccine strain Ty21a.

Claims

1. A DNA vaccine containing Salmonella typhi Ty21a strain, which includes a DNA molecule containing a eukaryotic expression cassette encoding at least the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof.

2. The aforementioned COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof (a) SARS-CoV-2 full-length S protein; (b) SARS-CoV-2 S protein ectodomain; (c) SARS-CoV-2 S protein subunit S1; (d) SARS-CoV-2 S protein receptor-binding domain (RBD); or (e) The DNA vaccine according to claim 1, comprising at least three immunodominant epitopes of the SARS-CoV-2 S protein.

3. The DNA vaccine according to claim 2, wherein the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein is the SARS-CoV-2 full-length S protein, and optionally the SARS-CoV-2 full-length S protein comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with SEQ ID NO:

1.

4. The DNA vaccine according to claim 2, wherein the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof comprises the SARS-CoV-2 S protein ectodomain, and optionally the SARS-CoV-2 S protein ectodomain comprises the amino acid sequence of amino acid residues 1 to 1208 of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with amino acid residues 1 to 1208 of SEQ ID NO:

1.

5. The DNA vaccine according to claim 2, wherein the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof comprises the SARS-CoV-2 S protein subunit S1, and optionally the SARS-CoV-2 S protein subunit S1 comprises the amino acid sequence of amino acid residues 1 to 681 of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with amino acid residues 1 to 681 of SEQ ID NO:

1.

6. The DNA vaccine according to claim 2, wherein the COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof comprises the SARS-CoV-2 S protein receptor-binding domain (RBD), and optionally the SARS-CoV-2 RBD comprises the amino acid sequence of amino acid residues 319-541 of SEQ ID NO: 1, or an amino acid sequence having at least 95% agreement with amino acid residues 319-541 of SEQ ID NO:

1.

7. The aforementioned COVID-19 coronavirus (SARS-CoV-2) spike (S) protein or a portion thereof is a pre-fusion stabilized form of the SARS-CoV-2 full-length S protein or the SARS-CoV-2 S protein ectodomain, which includes two stabilizing mutations to proline corresponding to amino acid positions K986 and V987 of the amino acid sequence of SEQ ID NO: 1, and preferably the aforementioned SARS-CoV-2 S protein or a portion thereof is (a) The amino acid sequence of SEQ ID NO: 1, which includes two stabilizing mutants K986P and V987P, or an amino acid sequence having a sequence that is at least 95% identical to SEQ ID NO: 1; or (b) The DNA vaccine according to claim 2, comprising an amino acid sequence of amino acid residues 1 to 1208 of SEQ ID NO: 1, which includes two stabilizing mutations K986P and V987P, or an amino acid sequence having a sequence that is at least 95% identical to amino acid residues 1 to 1208 of SEQ ID NO:

1.

8. The DNA vaccine according to any one of claims 1 to 7, wherein the eukaryotic expression cassette further encodes another SARS-CoV-2 protein or a portion thereof.

9. The DNA vaccine according to claim 8, wherein the other SARS-CoV-2 protein is the SARS-CoV-2 N protein.

10. A DNA vaccine according to any one of claims 1 to 9, further comprising one or more pharmaceutically acceptable excipients.

11. A DNA vaccine according to any one of claims 1 to 10, which is in oral dosage form.

12. The DNA vaccine according to claim 11, wherein the oral dosage form is an enteric-coated capsule, a lyophilized powder, or a suspension.

13. A DNA vaccine according to any one of claims 1 to 12, further comprising one or more adjuvants.

14. A DNA vaccine according to any one of claims 1 to 13, for use in the treatment and / or prevention of coronavirus disease 2019 (COVID-19) or SARS-CoV-2 infection.

15. A DNA vaccine for use according to claim 14, administered orally.

16. (a) A single dose of DNA vaccine kills Salmonella typhi Ty21a strain in approximately 10 6 ~10 9 Includes in terms of colony-forming units (CFUs), and / or (b) The DNA vaccine for use according to claim 14 or 15, wherein the DNA vaccine is administered 2 to 4 times per week for priming, and optionally followed by one or more single-dose boosting doses.

17. A DNA vaccine for use according to claim 16, administered two to four times within the first week, followed by one or more single-dose boostings at least two weeks apart.

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