Compositions and vaccines for treating and / or preventing viral infections, including coronavirus infection, and methods of using the same.

Abstract

This invention provides novel compositions and methods for effectively stimulating antiviral immunity. [Solution] A composition comprising (a) a vector comprising a plasmid encoding at least one viral antigen; and (b) a vector comprising a CD1d recognition antigen; and (c) at least one pharmaceutically acceptable carrier, wherein at least one of vectors (a) and (b) is an intact bacterial minicell or a dead bacterial cell.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Application 62 / 994,057 filed on 24 March 2020, the entirety of which is incorporated herein by reference.

[0002] Severe acute respiratory syndrome (SARS, 2002-2004 [Ksiazek et al., 2003; Drosten et al., 2003]) and Middle East respiratory syndrome (MERS, 2012-present [Zaki et al., 2012]), which have occurred over the past 20 years, pose a significant threat to global public health. [Background technology]

[0003] Respiratory syndromes caused by coronaviruses (CoVs) are known to be transmitted from person to person through close contact, leading to high morbidity and mortality rates among infected individuals. SARS and MERS initially present with mild, flu-like symptoms such as fever, shortness of breath, and cough, but as they worsen, they become characterized by atypical interstitial pneumonia and diffuse alveolar damage. Both SARS-CoV and MERS-CoV can cause alveolar inflammation, pneumonia, and hypoxic lungs, leading to respiratory failure and multi-organ disease, and can cause acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, in which 50% of ARDS patients die [Lew et al., 2003].

[0004] Over the decades, the development of antiviral agents has been ongoing. Many of them are non-structural proteins involved in viral replication and assembly. Many of these proteins are highly conserved and are known to have broad antiviral activity. Structural proteins and accessory proteins are poorly conserved and have a high mutation rate, so mutated viruses can escape the effects of antiviral agents. Examples of successful antiviral agents include oseltamivir (Tamiflu) and zanamivir (Relenza), neuraminidase inhibitors used for the treatment and prevention of influenza A and B, and ribavirin, a guanosine analog that shows activity in vitro against a number of highly lethal emerging viruses.

[0005] Monoclonal antibodies (mAbs) are thought to be useful for the prevention and treatment of highly pathogenic viral diseases by neutralizing viral structural proteins. Unfortunately, these mAbs need to be directed against surface-exposed structural proteins, which tend to mutate at a high frequency. Therefore, although mAbs effective against CoV infection in animal models target a very diverse range of spike glycoproteins, these mAbs have been found to lack cross-protection against other related CoVs [Agnihothram et al, 2014]. It is advisable for preclinical and clinical mAb formulations to include cocktails of multiple mAbs targeting different epitopes so that the virus cannot escape neutralization.

[0006] Vaccines have long been considered the gold standard for preventing and eradicating infectious diseases in human populations and have also provided the benefit of long-term immune protection to individuals. Unfortunately, in human infections with highly pathogenic coronaviruses such as SARS-CoV and MERS-CoV, the most vulnerable populations are patients aged 65 and older or those with comorbidities, and it is said that designing effective vaccines for these groups of patients is difficult. The vaccine formulations developed against SARS-CoV not only failed to protect the animal models of the elderly population but also resulted in the enhancement of SARS disease in the vaccinated groups challenged with SARS-CoV later, causing immunopathology in the young population [Bolles et al., 2011; Sheahan et al., 2011].

Summary of the Invention

Problems to be Solved by the Invention

[0007] Due to the diversity of bat - CoV, current treatment strategies targeting specific SARS-CoV or MERS-CoV antigens are likely to be ineffective against coronaviruses that may emerge in the human population in the future. Vaccines prescribed against the epidemic antigen of SARS-CoV do not provide effective protection against SARS-like bat - CoV currently circulating in bat populations [Menachery et al., 2015].

[0008] Therefore, new compositions and methods for effectively stimulating antiviral immunity are needed. The present invention meets such needs.

Means for Solving the Problems

[0009] The present disclosure is directed to a composition comprising (a) a vector comprising a plasmid encoding at least one viral antigen; and (b) a vector comprising a CD1d - recognizing antigen; and (c) at least one pharmaceutically acceptable carrier, wherein at least one of vector (a) and vector (b) is an intact, bacterially derived minicell or a killed bacterial cell.

[0010] In another embodiment, vector (a) is a first intact bacterial minicell or dead bacterial cell, and vector (b) is a second intact bacterial minicell or dead bacterial cell. In yet another embodiment, vectors (a) and (b) are identical intact bacterial minicells or dead cells containing plasmids encoding a CD1d recognition antigen and at least one viral antigen.

[0011] In one embodiment of the compositions described herein, one of vector (a) and vector (b) is not an intact bacterial minicell or dead bacterial cell, while the other of vector (a) and vector (b) is an intact bacterial minicell or dead bacterial cell.

[0012] In all compositions described herein, the viral antigen is: alpha coronavirus; bat coronavirus CDPHE15 and other coracoviruses; bat coronavirus HKU10 or rhinolophas fermecinum alpha coronavirus HuB-2013 and other decacoviruses; human coronavirus 229E and other dubinacoviruses; rat coronavirus Rn rat coronavirus; minakoviruses such as ferret coronavirus or mink coronavirus 1; minunacoviruses such as miniopters bat coronavirus 1 or miniopters bat coronavirus HKU8; myoctacoviruses such as myotis rickettii alpha coronavirus Sax-2011; nyctacocviruses such as nyctalas bertinus alpha coronavirus SC-2013; pedacocviruses such as swine epidemic diarea virus or scotophilus bat coronavirus 512; rhinacoviruses such as rhinolophas bat coronavirus HKU2; human coronavirus NL63 or NL63-related coronavirus Cetracoviruses such as Mori coronavirus strain BtKYNL63-9b; Tegacoviruses such as Alpha coronavirus 1; Embecoviruses such as Beta coronavirus 1, Human coronavirus OC43, China rat coronavirus HKU24, Human coronavirus HKU1 or Muscle coronavirus; Hibecoviruses such as Bat Hp-Beta coronavirus Cho-Chiang 2013; Marbecoviruses such as Hedgehog coronavirus 1, Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV), Pipistrelle bat coronavirus HKU5 or Tyronycteris bat coronavirus HKU4; Novecoviruses such as Russet bat coronavirus GCCDC1, Russet bat coronavirus HKU9, Survecoviruses such as Severe Acute Respiratory Syndrome-associated coronavirus, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2, COVID-19); Delta coronavirus; Andecoviruses such as Wijon coronavirus HKU20;The group includes, or has characteristics of, viruses selected from the group consisting of: buldecoviruses such as buldecovirus HKU11, swine coronavirus HKU15, munia coronavirus HKU13, or white-eye coronavirus HKU16; herdecoviruses such as night heron coronavirus HKU19; mudecoviruses such as van coronavirus HKU21; gamma coronaviruses; segacoviruses such as beluga whale coronavirus SW1; and avian coronaviruses such as gacoviruses.

[0013] In another embodiment, the viral antigen may be encoded by a polynucleotide containing the sequence of SARS-CoV-2, or by a polynucleotide having at least 80% sequence identity with a polynucleotide containing the sequence of SARS-CoV-2. In yet another embodiment, the viral antigen may contain or be characterized by human coronavirus 229E, human coronavirus OC43, SARS-CoV, HCoV NL63, HKU1, MERS-CoV, or SARS-CoV-2. Furthermore, the viral antigen may contain or be characterized by SARS-CoV-2.

[0014] In another embodiment, the plasmid encodes at least one of the following SARS-CoV-2 proteins: the spike (S) protein, the nucleocapsid (N) protein, the membrane (M) protein, and the envelope (E) protein. Furthermore, the plasmid may encode the spike (S) protein, the nucleocapsid (N) protein, the membrane (M) protein, or the envelope (E) protein.

[0015] In one embodiment, the CD1d recognition antigen includes sphingoglycolipids. For example, the CD1d recognition antigen includes α-galactosylceramide (α-GalCer), C-glycoside of α-galactosylceramide (α-C-GalCer), 12-carbon acyl of galactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), l,2-diacyl-3-O-galactosyl-sn-glycerol (BbGL-II), diacylglycerol-containing glycolipid (Glc-DAG-s2), and ganglioside The following can be selected from the group consisting of (GD3), gangliotriosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), alpha-glucuronide ceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide (iGb3), lipophosphoglycan (LPG), lysophosphatidylcholine (LPC), alpha-galactosylceramide analog (OCH), slate ceramide, and any derivative thereof.

[0016] In another embodiment, the CD1d recognition antigen includes α-GalCer. Furthermore, the CD1d recognition antigen may include synthetic α-GalCer analogs selected from 6′-deoxy-6′-acetamide α-GalCer (PBS57), naptylurea α-GalCer (NU-α-GC), NC-α-GalCer, 4ClPhC-α-GalCer, PyrC-α-GalCer, α-carba-GalCer, carba-α-D-galactose α-GalCer analog (RCAI-56), 1-deoxy-neo-inositol α-GalCer analog (RCAI-59), 1-O-methylated α-GalCer analog (RCAI-92), and HS44 aminocyclitol ceramide.

[0017] In one embodiment, the CD1d-recognizing antigen is an IFNγ agonist.

[0018] The compositions described herein may be formulated for any pharmaceutically acceptable use. Examples of pharmaceutically acceptable formulations include, but are not limited to, oral administration, injection, intranasal administration, intrapulmonary administration, or topical administration.

[0019] This disclosure also includes methods for treating and / or vaccinating against viral infections, which include administering the compositions described herein to subjects requiring them.

[0020] In one embodiment, subjects are suffering from or at risk of lymphopenia. In another embodiment, subjects are considered to be at risk of severe illness and / or serious complications due to viral infection. For example, “elderly” subjects at high risk of severe illness and / or serious complications due to viral infection are those approximately 50 years of age or older, approximately 55 years of age or older, approximately 60 years of age or older, or approximately 65 years of age or older.

[0021] In another aspect of the method described herein, the subject has one or more pre-existing conditions selected from the group consisting of diabetes, asthma, respiratory disease, hypertension, and heart disease. In yet another aspect, the subject is immunocompromised. For example, the subject may become immunocompromised due to AIDS, cancer, cancer treatment, hepatitis, autoimmune disease, steroid administration, immunosenescence, or any combination thereof.

[0022] In one embodiment, administration of the composition described herein enhances the likelihood of survival after exposure to coronavirus. For example, the likelihood of survival can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as measured using any clinically recognized technique.

[0023] In yet another embodiment, administration of the compositions described herein reduces the risk of coronavirus infection. For example, the reduction in infection risk may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as measured using any clinically recognized technique.

[0024] In all methods described herein, the administration step may be carried out by any pharmaceutically acceptable method.

[0025] In another embodiment, the subject may or is expected to be exposed to an individual infectious with coronavirus. Furthermore, an individual infected with coronavirus may have one or more symptoms selected from the group consisting of fever, cough, shortness of breath, diarrhea, sneezing, runny nose, and sore throat.

[0026] In one embodiment, the subjects of the method described herein are healthcare workers, persons aged 60 years or older, frequent travelers, military personnel, caregivers, or persons with pre-existing conditions that increase the risk of death associated with infection.

[0027] In another embodiment, the method further comprises administering one or more antiviral agents. For example, one or more antiviral agents may be selected from the group consisting of chloroquine, darunavir, galidesivir, interferon-beta, lopinavir, ritonavir, remdesivir, and triazavirin.

[0028] In the method of this disclosure, the CD1d-recognizing antigen induces a Th1 cytokine response in the subject. For example, the cytokine may include IFNγ.

[0029] In another embodiment, a first minicell containing a CD1d-recognizing antigen and a second minicell containing a plasmid encoding at least one viral antigen are administered simultaneously to a subject. In yet another embodiment, a first minicell containing a CD1d-recognizing antigen and a second minicell containing a plasmid encoding at least one viral antigen are administered sequentially to a subject. Alternatively, the disclosure includes a method in which a first minicell containing a CD1d-recognizing antigen and a second minicell containing a plasmid encoding at least one viral antigen are repeatedly administered to a subject.

[0030] In the method described herein, a first minicell containing a CD1d recognition antigen and a second minicell containing a plasmid encoding at least one viral antigen may be administered to a subject at least once, twice, three times, or four times per week.

[0031] Both the above summary and the following drawings and detailed description are illustrative and descriptive. They are intended to provide further details of the invention and are not to be construed as limiting. Other purposes, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention. [Brief explanation of the drawing]

[0032] [Figure 1] This is an illustration of the EnGeneIC Dream Vehicle (EDV™), a composition comprising a combination of intact bacterial minicells carrying α-galactosylceramide (α-GalCer), a CD1d-restricted iNKT cell antigen that stimulates IFNγ, and bacterial minicells carrying plasmids encoding viral antigens. [Figure 2]This figure shows peripheral blood mononuclear cells (PBMCs) from patient 1-CB04-1 (a 72-year-old male) with advanced hepatocellular carcinoma, treated for 2 and 3 cycles with intact bacterial minicells containing EGFR-targeted, PNU-containing cells and intact bacterial minicells containing α-galactose-treated ceramide, showing elevated CD8+ cytotoxic T cells (Figure 2A), NK cells (Figure 2B), NKT cells (Figure 2C), and iNKT cells (Figure 2D). It should be noted that this patient is elderly and severely immunocompromised. PNU is PNU-159682, a morpholinyl anthracycline derivative. [Figure 3] This figure shows the activation of major immune cells in PBMCs of a 45-year-old woman with advanced colorectal cancer. The patient's CD8+ effector cytotoxic T cells (CD45RA+ CCR7-) significantly increased by the second and third cycles (Figure 3A). Similarly, an increase in NK cells was observed in the control PBMCs in the second and third cycles (Figure 3B). Interestingly, ELISA analysis of the patient's serum 3 hours after administration of each intact bacterial-derived minicell showed a spike in IFNγ, which occurs when α-galactosylceramide is effectively presented from antigen-presenting cells (APCs) to iNKT cells, triggering the release of IFNγ, a key mediator in fighting viral infection (Figure 3C). [Figure 4] This figure shows the white blood cell count (average value for 9 patients) before administration and 3 hours after administration. Eight of the nine patients were elderly, all had stage IV pancreatic cancer, and were severely immunocompromised patients for whom all conventional treatments had been ineffective. Interestingly, there was a significant increase in white blood cells (WBCs) 3 hours after administration, which occurred with each subsequent administration. This suggests that the initial administration of intact bacterial-derived minicells mobilized fresh monocytes from the bone marrow, following activation signals from macrophages, dendritic cells, and NK cells, and that these cells were fully activated, matured, and proliferated by the third administration. [Figure 5A]This figure shows the activation of major immune cells in PBMCs of a 45-year-old woman with advanced colorectal cancer. The patient's CD8+ effector cytotoxic T cells (CD45RA+ CCR7-) increased significantly by the second and third cycles (Fig. 5A). [Figure 5B] This figure shows the activation of major immune cells in PBMCs of a 45-year-old woman with advanced colorectal cancer. Similarly, an increase in NK cells was observed in the patient's PBMCs during the second and third cycles (Figure 5B). [Figure 5C] This figure shows the activation of major immune cells in PBMCs of a 45-year-old woman with advanced colorectal cancer. Interestingly, ELISA analysis of the patient's serum 3 hours after administration of intact bacterial-derived minicells (EDV™) showed a spike in IFNγ (Figure 5C). This likely occurs when α-galactosylceramide is effectively presented from antigen-presenting cells to iNKT cells, inducing the release of IFNγ, a key mediator in combating viral infection. [Figure 6A] This diagram shows the structure of the expression cassette. [Figure 6B] This figure shows that bacterial minicells carrying Covid-αGC (EDVCovid-αGC) successfully delivered αGC to JAWSII cells. [Figure 6C] This figure shows a Western blot analysis demonstrating the successful integration of the spike protein into the EDV membrane. [Figure 7A] This figure shows the results of measuring serum IgG titers one week after administration of various bacterial minicell (EDV) formulations to mice. Here, it was found that intramuscular (IM) injection of bacterial minicells carrying Covid-αGC (EDVCovid-αGC) produced the highest S protein-specific IgG titer compared to subcutaneous (SC) injection. [Figure 7B] This bar graph shows the total AUC of IgG one week after administration of various bacterial minicell (EDV) formulations to mice. AUG analysis revealed that mice injected with IM showed the highest AUG. [Figure 7C]This figure shows that mice injected with EDUCovid-αGC via IM had the highest serum IFNα levels 8 hours after injection. [Figure 7D] This figure shows that mice injected with EDUCovid-αGC via IM had the highest serum IFNγ levels 8 hours after injection. [Figure 7E] This figure shows that mice injected with EDUCovid-αGC via IM had the highest serum IL12 levels (Figure 7E) 8 hours after injection. [Figure 7F] This figure shows that mice injected with EDUCovid-αGC via IM had the highest serum IL-6 levels 8 hours after injection. [Figure 7G] This figure shows that mice injected with EDUCovid-αGC via IM had the highest serum TNFα levels 8 hours after injection. [Figure 8A] This figure shows that mice injected with bacterial minicells carrying Covid-αGC (EDVCovid-αGC) had the highest serum IgM levels four weeks after the initial injection (boost on day 21). [Figure 8B] This figure shows that mice injected with bacterial minicells carrying Covid-αGC (EDVCovid-αGC) had the highest serum IgG levels four weeks after the initial injection (boost on day 21). [Figure 8C] This figure shows an ELISA analysis demonstrating that bone marrow-derived B cells, when incubated with spike protein, were able to produce spike protein-specific IgG ex vivo. [Figure 8D] This figure shows the analysis of neutralizing antibodies 4 weeks after the initial dose. [Figure 8E] This figure shows the IgG subtype analysis of EDVCovid and EDVCovid-αGC. [Figure 9A]This is a FACS analysis of mouse splenocytes showing that mice injected with EDVCovid-αGC had the highest amount of antigen-specific memory CD137+CD69+ cytotoxic T cells 4 weeks after the initial injection (1 boost on day 21). For example, it shows that the number of CD137+CD69+ populations was significantly higher in the cytotoxic T cell population in EDVCovid-αGC-treated mice compared to all other treatment groups. [Figure 9B] This figure shows an AIMS assay demonstrating that Covid-αGC (EDVCovid-αGC)-treated cytotoxic T cells from the spleen express virus antigen-specific CD69 monopositive cytotoxic T cells after spike protein stimulation in a similar manner to when stimulated with PHA (e.g., when ex vivo exposed to spike protein). Splenocytes from mice treated with EDVCovid showed similar characteristics, but to a lesser degree. This was not observed in other treatment groups. [Figure 10A] This figure shows the pUC57-Kan construct, with insertion sites of 5'Kpnl and 3'Sall. [Figure 10B] This figure shows the in vitro synthesis of a synthetically modified lactamase promoter. The nucleotide sequences of the P-lactamase promoter (A1) and the synthetically modified version (B2) are shown. The -35 and -10 regions, the +1 transcription start site, the ribosome binding site (RBS), and the ATG translation start codon are shown. The newly introduced restriction enzyme sites for EcoRI, XhoI, NdeI, and BamHI are also shown. [Figure 11] Figures A and B show that JAWSII cells are expressed on the cell surface via the CD1d ligand after being treated with EDTCovid-αGC. The expression level was better than that of JAWSII cells treated with free αGC alone (A). Western blot analysis of EDTCovid-αGC confirmed that the spike protein was incorporated into the EDV structure (B). [Figure 12A]This figure shows a detailed ELISA analysis of the initial interferon response in mouse serum after IM injection of EDV, EDVαGC, EDVControl, EDVControl-αGC, EDUCovid, and EDUCovid-αGC (serum IFNα concentration 8 hours after IM injection). The results showed that the initial interferon response in mice was predominantly induced by administration of αGC carried by EDV with or without antigen-specific plasmids (e.g., administration of EDVαGC with or without EDVPlasmid), and IM injection dramatically increased IFNα, IFNγ, TNFα, IL12, and IL6 8 hours after the first dose. [Figure 12B] This figure shows a detailed ELISA analysis of the initial interferon response in mouse serum after IM injection of EDV, EDVαGC, EDVControl, EDVControl-αGC, EDUCovid, and EDUCovid-αGC (serum IFNγ concentration 8 hours after IM injection). The results showed that the initial interferon response in mice was predominantly induced by administration of αGC carried by EDV with or without antigen-specific plasmids (e.g., administration of EDVαGC with or without EDVPlasmid), and IM injection dramatically increased IFNα, IFNγ, TNFα, IL12, and IL6 within 8 hours after the first dose. [Figure 12C] This figure shows a detailed ELISA analysis of the initial interferon response in mouse serum after IM injection of EDV, EDVαGC, EDVControl, EDVControl-αGC, EDUCovid, and EDUCovid-αGC (IL6 serum concentration 8 hours after IM injection). The results showed that the initial interferon response in mice was predominantly induced by administration of αGC carried by EDV with or without antigen-specific plasmids (e.g., administration of EDVαGC with or without EDVPlasmid), and IM injection dramatically increased IFNα, IFNγ, TNFα, IL12, and IL6 8 hours after the first dose. [Figure 12D]This figure shows a detailed ELISA analysis of the initial interferon response in mouse serum after IM injection of EDV, EDVαGC, EDVControl, EDVControl-αGC, EDUCovid, and EDUCovid-αGC (serum TNFα concentration 8 hours after IM injection). The results showed that the initial interferon response in mice was predominantly induced by administration of αGC carried by EDV with or without antigen-specific plasmids (e.g., administration of EDVαGC with or without EDVPlasmid), and IM injection dramatically increased IFNα, IFNγ, TNFα, IL12, and IL6 8 hours after the first dose. [Figure 12E] This figure shows a detailed ELISA analysis of the initial interferon response in mouse serum after IM injection of EDV, EDVαGC, EDVControl, EDVControl-αGC, EDUCovid, and EDUCovid-αGC (IL12p40 serum concentration 8 hours after IM injection). The results showed that the initial interferon response in mice was predominantly induced by administration of αGC carried by EDV with or without antigen-specific plasmids (e.g., administration of EDVαGC with or without EDVPlasmid), and IM injection dramatically increased IFNα, IFNγ, TNFα, IL12, and IL6 within 8 hours after the first dose. [Figure 13A] Figures 13A and 13B show that FACS analysis of extracted mouse spleens revealed a high proportion of CD3+CD8+ cytotoxic T cells in EDUCovid-αGC-treated mice (Figure 13A). Stimulation of spleen cells with the Covid-19 spike protein induced a greater increase in the number of CD69+ CD137+ cells within the cytotoxic T cell population compared to stimulation with PHA (+ve control) (Figure 13B). [Figure 13B]Figures 13A and 13B show that FACS analysis of extracted mouse spleens revealed a high proportion of CD3+CD8+ cytotoxic T cells in EDUCovid-αGC-treated mice (Figure 13A). Stimulation of spleen cells with the Covid-19 spike protein induced a greater increase in the number of CD69+ CD137+ cells within the cytotoxic T cell population compared to stimulation with PHA (+ve control) (Figure 13B). [Figure 14A] Figures 14A, B, and C show that high levels of spike protein-specific IgG were found in the serum of mice treated with EDUCovid-αGC four weeks after the initial injection (A). [Figure 14B] This has also been observed with spike protein-specific IgM (B). [Figure 14C] Interestingly, while serum from mice treated with EDPCovid-αGC showed the highest degree of inhibition of spike protein binding to the hACE receptor protein, treatment with EDVαGC also demonstrated the ability to prevent spike protein binding (C). [Figure 15] This figure shows the results of an experiment in which B cells extracted from the bone marrow of EDPCovid-αGC-treated mice secreted the highest levels of spike protein-specific IgG (Figure 15A) and IgM (Figure 15B) detected by an improved ELISA at 4 weeks. [Modes for carrying out the invention]

[0033] I. Overview This disclosure is directed toward novel compositions useful for the treatment and / or vaccination of a target viral infection, including but not limited to coronavirus infection. The compositions comprise a combination of (a) a vector comprising a plasmid encoding at least one viral antigen from a virus to be treated / vaccinated; and (b) a combination of vectors comprising a CD1d-recognizing antigen, wherein at least one of the two vectors is an intact, bacterial-derived minicell or dead bacterial cell, wherein the two vectors are present in at least one pharmaceutically acceptable carrier. As an antigen recognized by CD1d, alpha-galactosylceramide (α-GalCer) is exemplified and is known to stimulate IFNγ, which is important for viral immunity. In another embodiment, both of the two vectors are intact, bacterial-derived minicells or dead bacterial cells, either contained in either two separate bacterial-derived minicells or dead bacterial cells, or contained together in a single bacterial-derived minicell or dead bacterial cell. In another embodiment, the vector or a minicell derived from intact bacteria may consist of all four major structural proteins of the coronavirus, or their antigenic fragments, such as the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein.

[0034] In another embodiment, one or the other (but not both) of the plasmid payload and the CD1d recognition payload described above can be administered via a vector that is not a intact bacterial minicell or a dead bacterial cell. Examples of such non-minicell vectors include liposomes, macromolecular vectors, reconstituted viral envelopes (virosomes), and immunostimulatory complexes (ISCOMs). For example, Bunger et al. (2002), Kersten et al. (2003), Daemen et al. (2005), Chen et al. (2012), Yue et al. (2013). https: / / www.ncbi.nlm.nih.gov / pubmed / 12428908 (Bungener); https: / / www.meta.org / papers / liposomes-and-iscoms / 12547602 (Kersten); https: / / www.ncbi.nlm.nih.gov / pubmed / 15560951 (Daemen); https: / / journals.plos.org / plosone / article?id=10.1371 / journal.pone.0039039 (Chen); See https: / / pubs.rsc.org / en / content / articlelanding / 2013 / bm / c2bm00030j#!divAbstract(Yue).

[0035] Mature SARS-CoV-2 viruses possess four structural proteins: the envelope, membrane, nucleocapsid, and spike. All of these proteins are thought to function as antigens that stimulate neutralizing antibodies and increase the CD4+ / CD8+ T cell response.

[0036] This composition may be administered by any pharmaceutically acceptable method, including, but is not limited to, injection (parenteral, intramuscular, intravenous, intrahepatic, peritoneal, subcutaneous, intratumoral, or intradermal administration), oral administration, application of the formulation to body cavities, inhalation, pneumoperitoneum, nasal administration, pulmonary administration, or any combination of these routes.

[0037] This composition can be administered as a vaccine to subjects at risk of viral infection, or as a therapeutic agent to subjects suffering from viral infection.

[0038] In terminally ill cancer patients with severely weakened immune systems, intact bacterial mini-cell therapy (EnGeneIC Dream Vector) TM EDV TM(1) Activation and proliferation of CD8+ T cells, macrophages, NK cells, dendritic cells, and iNKT cells have been confirmed. This result is exactly what is needed for CoV-2 therapeutic drugs / vaccines.

[0039] In one embodiment, the present disclosure provides the use of recombinant intact bacterial minicells in the preparation of a composition, the minicells containing plasmids encoding viral proteins for use in a method of treating and / or preventing a disease by administering the composition to a person infected with a virus or at risk of viral infection. The disease addressed herein is a viral infection.

[0040] This disclosure is directed toward compositions useful for the treatment and / or vaccination of viral infections. Exemplary viral infections to be treated or vaccinated include, but are not limited to, coronaviruses that cause SARS-CoV-2 coronavirus and coronavirus disease 2019 (COVID-19). Accordingly, this specification describes, as an example, the development of treatments and / or vaccines based on intact bacterial minicells against SARS-CoV-2 coronavirus infection in humans.

[0041] In yet another embodiment, the composition includes a combination of (a) an intact bacterial minicell containing at least one viral antigen derived from SARS-CoV-2 and (b) an intact bacterial minicell containing the CD1d recognition antigen α-GalCer. Furthermore, the intact bacterial minicell containing at least one viral antigen derived from SARS-CoV-2 may contain all four constituent proteins of SARS-CoV-2.

[0042] Currently, the main areas of research regarding treatments / vaccines against SARS-CoV-2 are as follows: (1) antiviral agents (e.g., Gilead Sciences: nucleotide analog Remdesivir), (2) cocktail monoclonal antibodies (e.g., Regeneron), and (3) sterilized viruses as vaccines that stimulate a strong antibody response against viral proteins. Each of these strategies faces its own challenges, but most importantly, they cannot address the problem of lymphopenia in elderly and immunocompromised patients, which can hinder viral infection. Without a robust immune system, this patient group remains the most vulnerable and likely to succumb to the disease.

[0043] In previous disclosures, EnGeneIC demonstrated the use of plasmid-packaged minicells for the treatment of neoplasms, with the primary function of the plasmids being to encode siRNA or miRNAs that silence genes in cancer cells that cause cell proliferation or drug resistance.

[0044] In this disclosure, the function of the complete plasmid-packaged minicell component (including CD1d-recognizing antigens such as α-GC-packaged minicells) is novel and has not been previously shown or described. Specifically, viral proteins are encoded using the plasmid in parental bacterial cells, and the proteins segregate into minicells during asymmetric cell division. These viral proteins are transported to the lysosomes of antigen-presenting cells (APCs) such as macrophages and dendritic cells. After antigen treatment, the epitopes of the viral proteins are expected to be displayed on the APC surface via MHC class I and class II molecules, triggering a strong antibody response against the viral proteins. Furthermore, because the plasmid itself is a double-stranded nucleic acid, it is recognized by the nucleic acid-sensing proteins of APCs, triggering the secretion of type I interferons (IFNα, IFNβ).

[0045] Thus, a unique dual trigger—the antibody response against viral proteins and the type I interferon response—not only clears viral particles released from infected cells, but also allows immune system cells to recognize and destroy virus-infected cells. In particular, the fact that type I interferon triggers a previously unknown mechanism for recognizing and destroying virus-infected cells was previously unknown.

[0046] This disclosure demonstrates that IFNγ triggering after presentation of α-GC / CD1d to iNKT cell receptors is key to enhancing antiviral immunity. Although the exact mechanism of action is unknown, IFNγ is important for identifying and destroying virus-infected cells.

[0047] In the United States, several clinical trials are underway in which bacterial minicells carrying anticancer drugs or microRNA mimics are administered to humans as a treatment for cancer. See, for example, ClinicalTrials.gov Identifier Nos. NCT02766699, NCT02687386, and NCT02369198. In Australia, bacterial minicells carrying α-GC were administered to terminally ill cancer patients in a Phase IIa clinical trial. The results showed that intact bacterial minicells carrying α-GC were potent IFN-γ stimulants. See Trial ID No. ACTRN12619000385145. Thus, the in vivo efficacy of intact bacterial minicells carrying CD1d recognition antigens in humans has been demonstrated, and further, the efficacy of intact bacterial minicells carrying target compounds (e.g., anticancer compounds instead of viral antigens) in humans has also been shown.

[0048] Furthermore, the disclosed composition has another important function that enables elderly and immunocompromised patients to recover from lymphopenia (rapid depletion of lymphocytes, including macrophages, dendritic cells, NK cells, and CD8+ T cells), which is a major cause of infection in these patients when viruses such as SARS-CoV-2 take over, ultimately leading to death from respiratory distress syndrome. Specifically, the minicells of the composition themselves recognize pathogen-associated molecular patterns (PAMPs) such as LPS, thereby activating macrophages. This signals dormant monocytes in the bone marrow to activate, mature, and proliferate, significantly increasing the number of activated macrophages and dendritic cells. In addition, PAMPs involving the minicells also activate NK cells, promoting their proliferation. Furthermore, the activated macrophages and dendritic cells home in the infection site and engulf apoptotic virus-infected cells. They then migrate to the excretory lymph nodes, activating and proliferating naive CD8+ T cells.

[0049] Therefore, the minicell components of the composition can overcome lymphopenia in these elderly and immunocompromised patients through PAMP signaling, and the activation of these lymphocytes helps overcome viral infections and prevent patients from turning to respiratory distress and death.

[0050] A. Background regarding coronavirus infection Coronaviruses are a family of hundreds of viruses that cause fever, respiratory problems, and sometimes gastrointestinal symptoms. SARS-CoV-2 is one of seven members of this family known to infect humans and is the third case of animal-to-human transmission in the last 30 years. Since its emergence in China in December 2019, this novel coronavirus has caused a global health emergency, infecting more than 350,000 people worldwide, and as of March 22, 2020, approximately 15,000 people worldwide have recorded deaths attributable to COVID-19. For now, coronaviruses appear to be more deadly than seasonal influenza. However, there are still many uncertainties regarding the mortality rate of COVID-19. The annual influenza mortality rate is typically around 0.1% in the United States, and the CDC (Centers for Disease Control and Prevention) has reported that the mortality rate in the US for the 2019-20 influenza season to date is 0.05%. In contrast, recent data, including a study published in the China CDC Weekly on February 18, suggests that the COVID-19 mortality rate is more than 20 times higher, at approximately 2.3%. Mortality rates vary depending on factors such as region and age.

[0051] Patients infected with SARS-CoV or MERS-CoV present with mild flu-like symptoms such as fever, shortness of breath, and cough. Most patients recover from the illness. However, the most vulnerable groups are patients over 65 years of age and those with immunosuppressive comorbidities such as cancer and HIV. As the disease progresses, it becomes characterized by atypical interstitial pneumonia and diffuse alveolar damage. Both SARS-CoV and MERS-CoV can cause acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, in which alveolar inflammation, pneumonia, and hypoxic lungs lead to respiratory failure and multi-organ disease, with 50% of ARDS patients dying. Lymphopenia is commonly observed as the disease progresses. Many deaths from CoV-2 infection are a result of immunocompromised patients developing severe lymphopenia, leading to the progression of the disease and the development of ARDS.

[0052] Coronavirus (SARS-CoV-2; COVID-19) causes atypical pneumonia in infected individuals, presenting with symptoms such as fever, dry cough, and fatigue. Most patients experience lymphopenia (a decrease in white blood cell count, particularly T cells, B cells, and NK cells). Currently, it is known that those most likely to die from this disease are immunocompromised patients (the elderly and those with immunosuppressive diseases such as cancer), and those with underlying conditions such as diabetes, hypertension, heart disease, and respiratory diseases. In the former group, lymphopenia makes it difficult to control viral replication and infection in both lungs, often leading to the development of acute respiratory distress syndrome (ARDS).

[0053] When key immune cells such as T cells, B cells, macrophages, and NK cells are depleted, viral replication begins. In older patients, immune function is not as robust as in younger individuals. Studies have shown that many people have normal immune function even in their 60s or 70s. After the age of 75 or 80, immune function declines further.

[0054] COVID-19 spreads rapidly through human-to-human transmission, with a median incubation period of 3.0 days (range, 0–24.0) and a time from symptom onset to pneumonia development of 4.0 days (range, 2.0–7.0) (Guan et al., 2020). Common symptoms of COVID-19 include fever, dry cough, and fatigue (Huang et al., 2020). Most patients show lymphopenia and bilateral subsurface glass turbidity changes on chest CT scans (Huang et al., 2020; Duan and Qin, 2020). There are no specific antiviral treatments or vaccines. The development of a SARS-CoV-2-based vaccine is urgently needed.

[0055] The preparation of vaccines using viral particles, such as inactivated vaccines and attenuated viral vaccines, is considered desirable because it is based on previous research on the prevention and control of seasonal influenza (Grohskopf et al., 2018). The genome sequence of the first SARS-CoV-2 (Wuhan-Hu-1) has been completed (Genbank Accession no. MN908947.3; Wu et al., 2020). Large-scale culture of SARS-CoV-2 has been performed, and inactivated viral vaccines have been prepared using established physical and chemical methods such as ultraviolet light, formaldehyde, and β-propiolactone (Jiang et al., 2005). Furthermore, the development of attenuated viral vaccines is possible by screening serially replicating SARS-CoV-2, which exhibits reduced pathogenicity compared to wild-type viruses, such as minimal induction of lung damage, limited reduction in neutrophil influx, and increased expression of anti-inflammatory cytokines (Regla-Nava et al., 2015). Inactivated virus vaccines and attenuated virus vaccines each have their own drawbacks and side effects (Table 1; reprinted from Shang et al., 2020).

[0056] [Table 1]

[0057] All new treatments currently under development are either (i) antiviral agents to stop the virus from multiplying throughout the body, or (ii) attenuated viruses to stimulate a strong antibody response against viral proteins as vaccines.

[0058] None of these treatments appear to be able to prevent deaths in immunocompromised patients, as is currently seen in influenza virus infections. Every year, the majority of deaths from influenza infections occur among immunocompromised patients and the elderly.

[0059] Effective immunotherapy strategies for diseases such as cancer rely on the activation of both innate and adaptive immune responses. Cells of the innate immune system interact with pathogens via conserved pattern recognition receptors. Cells of the adaptive immune system, on the other hand, recognize pathogens through a variety of antigen-specific receptors generated by rearrangement of somatic DNA. Invariant natural killer T (iNKT) cells are a subset of lymphocytes (type I NKTs) that bridge the gap between the innate and adaptive immune systems. iNKT cells express an invariant α-chain T cell receptor (Vα24-Jα18 in humans, Vα14-Jα18 in mice) and are specifically activated by certain glycolipids presented in the context of the non-polymorphic MHC class I-like protein CD1d. CD1d binds to various dialkyl lipids and glycolipids, such as the sphingoglycolipid α-galactosylceramide (α-GalCer). When the TCR of iNKT cells recognizes the CD1d-lipid complex, inflammatory cytokines, including the Th1 cytokine interferon-gamma (IFNγ), and regulatory cytokines are released. The release of cytokines activates adaptive cells such as T cells and B cells, as well as innate cells such as dendritic cells and NK cells.

[0060] α-GalCer (also known as KRN7000, chemical formula C50H99NO9) is a synthetic glycolipid obtained from structure-activity relationship studies of galactosylceramide isolated from the sponge Agelas mauritianus. α-GalCer is a potent immunostimulant and exhibits strong antitumor activity in many in vivo models. A major challenge in using α-GalCer in immunotherapy is that it is presented by other CD1d-expressing cells, such as B cells in peripheral blood, leading to anergy induction in iNKT cells. Furthermore, α-GalCer delivery has been shown to cause hepatotoxicity.

[0061] B. Background on Coronavirus and SARS-CoV-2 Severe Acute Respiratory Syndrome (SARS), which occurred in 2003, and more recently, Middle East Respiratory Syndrome (MERS), demonstrate the lethality of COVID-19 when it crosses species barriers and infects humans.

[0062] Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome. Their genome is 26-32 kilobases (kb) long, making them the largest genomes among RNA viruses. Based on genetic and antigenic criteria, CoVs are classified into three groups: α-CoV, β-CoV, and γ-CoV. While coronaviruses primarily cause outbreaks in birds and mammals, in recent decades they have become known to infect humans, causing illnesses ranging from upper respiratory tract infections (URTIs) similar to the common cold to lower respiratory tract infections (LRTIs) such as bronchitis and pneumonia, and even severe acute respiratory syndrome (SARS).

[0063] Because there is no effective treatment for CoV infection, a more detailed and comprehensive understanding of the molecular biology of coronaviruses is necessary, with particular focus on structural proteins and their associated proteins.

[0064] The coronavirus genome encodes four major structural proteins: the spike (S) protein, the nucleocapsid (N) protein, the membrane (M) protein, and the envelope (E) protein, all of which are necessary for the formation of a structurally complete viral particle. Some CoVs do not require a complete ensemble of structural proteins to form a complete infectious virion. This suggests that some structural proteins are unnecessary, or that these CoVs encode additional proteins with redundant compensatory functions. Each protein is individually primarily involved in the structure of the viral particle, but also in other aspects of the replication cycle. The S protein attaches the virus to receptors on the host cell surface and then mediates the fusion of the virus with the host cell membrane, facilitating viral entry into the host cell. In some CoVs, expressing S on the cell membrane can mediate intercellular fusion between infected cells and adjacent uninfected cells. The formation of this large multinucleated cell (synthia) has been proposed as a strategy to directly spread the virus between cells and disrupt antiviral antibodies.

[0065] It has been shown that the SARS-CoV-2 spike (S) glycoprotein enters human cells by binding to angiotensin-converting enzyme 2 (ACE2), a cell membrane protein. COVID-19 has been shown to bind to ACE2 via the S protein on its surface. Upon infection, the S protein is cleaved into two subunits, S1 and S2. S1 contains a receptor-binding domain (RBD) that allows the coronavirus to directly bind to the peptidase domain (PD) of ACE2. S2 is thought to play a role in membrane fusion.

[0066] Unlike other major structural proteins, N is the only protein whose primary function is to bind to the CoV RNA genome that constitutes the nucleocapsid. While N is heavily involved in processes related to the viral genome, it is also involved in other aspects of the CoV replication cycle and the host cell's response to viral infection. Transient expression of N has been shown to significantly increase the production of virus-like particles (VLPs) in some CoVs, suggesting that it is not required for envelope formation but rather for complete virion formation.

[0067] The M protein is the most abundant structural protein and determines the shape of the viral envelope. It also interacts with all other major coronavirus structural proteins and is considered the central organizer of CoV assembly. Homotype interactions of the M protein are the main driving force for virion envelope formation, but not alone. The binding of M and N stabilizes the nucleocapsid (N protein-RNA complex) and the virion's internal core, ultimately completing the viral assembly. M and E together constitute the viral envelope, and their interactions are sufficient for VLP generation and release.

[0068] The CoV envelope (E) protein is the smallest of the major structural proteins. This membrane protein is involved in several aspects of the viral life cycle, including aggregation, budding, envelope formation, and pathogenicity. During the replication cycle, E is abundantly expressed in infected cells, but only a small portion is incorporated into the virion's envelope. The majority of the protein is localized to intracellular transport sites, where it is involved in CoV assembly and budding. Recombinant CoVs lacking E exhibit significantly reduced viral titers, inhibited viral maturation, or produce non-proliferative offspring, demonstrating the importance of E for viral production and maturation.

[0069] Coronaviruses are viruses whose genome is single-stranded mRNA with a 3'-UTR and a poly-A tail. In a subset of coronaviruses, including 2019-nCoV, SARS, and MERS, the 3'-UTR has a highly conserved sequence (other messages vary considerably) that folds into a unique structure called s2m (stem two motif). The s2m sequence appears to be extremely conserved and necessary for viral survival, but its exact function is unknown. The 2019 Wuhan Novel Coronavirus (COVID-19, formerly 2019-nCoV) has almost the same s2m sequence (and therefore structure) as SARS.

[0070] The genome sequence of SARS-CoV-2 has been made public, and multiple complete nucleotide sequences from viruses around the world, as well as sequences of specific viral genes such as the S gene, N gene, and M gene, are available at https: / / www.ncbi.nlm.nih.gov / genbank / sars-cov-2-seqs / (downloaded March 24, 2020). Examples include GenBank accession numbers MN908947.3, MN975262.1, NC_045512.2, MN997409.1, MN985325.1, MN988669.1, MN988668.1, MN994468.1, MN994467.1, MN988713.1, and MN938384.1. SARS-CoV-2 is a periphery-type single-stranded, forward-chain RNA virus with a genome consisting of 29,891 nucleotides, encoding 12 putative open reading frames involved in the synthesis of viral structural and non-structural proteins (Wu et al., 2020; Chen et al., 2020). Mature SARS-CoV-2 has four structural proteins: envelope, membrane, nucleocapsid, and spike (Chen et al., 2020). All of these proteins may function as antigens that stimulate neutralizing antibodies and increase the CD4+ / CD8+ T cell response (Jiang et al.). However, subunit vaccines require multiple booster shots and appropriate adjuvants to be effective, and certain subunit vaccines, such as hepatitis B surface antigen, PreS1, and PreS2, may not show a protective response in clinical trials. DNA and mRNA vaccines remain in the experimental stage because they are easy to design and can move very quickly into clinical trials. Furthermore, viral vector-based vaccines can be constructed rapidly and may even be used without adjuvants. However, the development of such vaccines may not begin until antigens containing neutralizing epitopes are identified. The E and M proteins play crucial roles as coronavirus aggregates, while the N protein is necessary for viral RNA synthesis.Since the pathogenicity of CoV disappears when the E protein is deleted, several studies have explored the potential of attenuated live vaccines of recombinant SARS-CoV or MERS-CoV with mutated E protein. While the M protein can enhance the immune response induced by the N protein DNA vaccine against SARS-CoV, the conserved N protein across the CoV family means it is not a suitable candidate for vaccine development, and antibodies against the SARS-CoV-2 N protein do not provide immunity against infection. The important glycoprotein S of SARS-CoV-2 is involved in viral binding and entry. The SARS-CoV-2 S precursor protein is cleaved into S1 (685 aa) and S2 (588 aa) subunits by proteolysis. The S2 protein is well conserved across SARS-CoV-2 viruses and has 99% identity with the bat SARS-CoV protein. Vaccine designs based on the S2 protein may enhance broad antiviral efficacy and are worth testing in animal models. Antibodies against the conserved stem region of influenza hemagglutinin exhibit broad cross-reactivity immunity but are not very effective at neutralizing influenza A virus. The S1 subunit, on the other hand, consists of a receptor-binding domain (RBD) that mediates the entry of the virus into susceptible cells via the host's angiotensin-converting enzyme 2 (ACE2) receptor. The 2019-nCoV S1 protein shares approximately 70% identity with the human SARS-CoV S1 protein. The external subdomain, where the virus and host receptors directly interact, contains the most amino acid variations within the RBD.

[0071] C. Overview of how the disclosed compositions work for the treatment and vaccination of viral infections This invention aims to intervene before infection with viruses such as coronaviruses like SARS-CoV and MERS-CoV, or at an early stage after infection. This composition and method addresses issues such as (i) overcoming lymphopenia to prevent viral infection / disease from overcoming the patient's own immune defenses, and (ii) stimulating high-titer systemic antibodies against proteins exposed on the viral surface to rapidly capture viral particles released from infected cells, thereby limiting infection to other healthy cells. This interferon is known to rapidly address a variety of different viral infections through many effects, including specific stimulation of antiviral immunity and removal of virus-infected cells.

[0072] To address these and other needs, the present invention provides, in one embodiment, a composition comprising a vector which may be optionally recombinant intact bacterial minicells and is packaged with a plasmid encoding a viral protein which functions to stimulate an antibody response to a viral protein and stimulate type I interferon; (ii) an optionally recombinant intact bacterial minicell packaged with a CD1d-recognizing antigen; and (iii) at least one pharmaceutically acceptable carrier. The vector packaged with a CD1d-recognized antigen such as α-GalCer has the function of stimulating type II interferon. The minicell vector itself has the function of promoting the activation, maturation, and proliferation of cells in the immune system. In another embodiment, intact bacterial minicells may also be used to replace dead bacterial cells.

[0073] Accordingly, the present invention relates to compositions comprising, in certain embodiments, an immunogenically effective amount of (a) a vector or intact bacterial minicell or dead bacterial cell that encapsulates one or more viral antigens and plasmids, and (b) a combination of a vector or intact bacterial minicell or dead bacterial cell that encapsulates a CD1d-recognizing antigen such as α-galactosylceramide (α-GalCer). In some embodiments, the encapsulated CD1d-recognizing antigen can be taken up by phagocytic cells such as dendritic cells or macrophages. The cell antigen recognized by CD1d forms a complex with CD1d in the lysosome of the phagocytic cell, is then transported to the surface of the phagocytic cell, and the CD1d-bound CD1d-recognizing antigen is presented to and recognized by iNKT cells. In some embodiments, the CD1d-recognizing cell antigen induces a Th1 cytokine response, particularly IFNγ, by iNKT cells that recognize the CD1d-bound CD1d-recognizing cell antigen on the surface of the phagocytic cell. IFNγ is known to elicit a potent antiviral immune response. CD1d-restricted NKT cells activate innate and adaptive immune responses, suggesting that these cells can modulate immunity against infectious diseases. Furthermore, CD1d-restricted iNKT cells express various effector molecules that can exert antibacterial effects, potentially contributing directly to host resistance. The CD1 protein is an antigen-presenting molecule that presents lipid antigens to T cells.

[0074] In one embodiment, the intention to administer the compositions described herein to subjects in need is to rapidly relieve them from lymphopenia and, at the same time, activate key cells of the immune system to fight viral infection, particularly in elderly and immunocompromised patients. This can prevent the worsening of viral infections and the resulting death of the patient. As a result, infected individuals experience milder flu-like symptoms and recover more quickly as their immune system begins to recover.

[0075] In one aspect of this disclosure, all genes encoding the four SARS-CoV-2 structural proteins (envelope, membrane, nucleocapsid, and spike) are cloned into a plasmid having a bacterial origin of replication, and the proteins are EDV TM Expressed only in production bacterial cells and EDV TM The gene is transcribed using a bacterial gene expression promoter so that it is shifted to the cytoplasm. In this way, all four types of SARS-CoV-2 proteins can be expressed from a single bacterial expression promoter. Alternatively, the gene can be transcribed under a mammalian gene expression promoter so that the protein is expressed only by mammalian cells. The recombinant plasmid can be transformed into minicell-producing strains of Salmonella typhimurium. Such recombinant intact bacterial-derived minicell therapeutics are expected to elicit a potent antibody response against all four CoV-2 proteins.

[0076] Furthermore, when recombinant intact bacterial minicells are systemically administered to patients infected with the CoV-2 virus, they are rapidly taken up by phagocytic cells such as macrophages and dendritic cells, and then degraded in lysosomes, releasing plasmid DNA. This DNA is recognized by intracellular DNA sensors such as cGAS, AIM2, and IFI16, which triggers the type I interferon (IFNα, IFNβ) response. These interferons are known to be potent inducers of antiviral defense.

[0077] It is well known that in the early stages of infection, IFN stimulation alters the cell's transcriptional program, leading to an antiviral state characterized by the activation of numerous host genes with partially defined antiviral functions [Schoggins et al., 2011].

[0078] In some embodiments, the CD1d recognition antigen is α-galactosylceramide (α-GalCer), C-glycoside of α-galactosylceramide (α-C-GalCer), 12-carbon acyl of galactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), l,2-diacyl-3-O-galactosyl-sn-glycerol (BbGL-II), diacylglycerol-containing glycolipid (Glc-DAG-s2), The sphingoglycolipid is selected from the group consisting of ganglioside (GD3), gangliotriosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), α-glucuronide ceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide (iGb3), lipophosphoglycan (LPG), lysophosphatidylcholine (LPC), α-galactosylceramide analog (OCH), slate ceramide, and any derivative thereof. In some embodiments, the sphingoglycolipid is α-GalCer. In some embodiments, the sphingoglycolipid is a synthetic α-GalCer analog. In some embodiments, the synthetic α-GalCer analog is selected from 6′-deoxy-6′-acetamide α-GalCer (PBS57), naptylurea α-GalCer (NU-α-GC), NC-α-GalCer, 4ClPhC-α-GalCer, PyrC-α-GalCer, α-carba-GalCer, carba-α-D-galactose α-GalCer analog (RCAI-56), 1-deoxy-neo-inositol α-GalCer analog (RCAI-59), 1-O-methylated α-GalCer analog (RCAI-92), and HS44 aminocyclitol ceramide. In some embodiments, the CD1d recognition antigen is derived from a bacterial antigen, a fungal antigen, or a protozoan antigen.

[0079] In some embodiments, the immune response produced in target cells includes the production of type I interferons, including interferon-α and / or interferon-β.

[0080] Treatment using these bacterial minicells should reduce the severity of the illness, shorten the duration of the disease, and make it feel like a common cold in almost all patients.

[0081] Alternatively, the proteins encoded by recombinant plasmids mounted on minicells can be administered to healthy individuals as a vaccine to protect them from viral infections carried by the virus.

[0082] In one embodiment, the adjuvant composition comprises (a) an immunogenically effective amount of encapsulated CD1d-recognizing antigen and (b) a minicell having a recombinant plasmid encoding one or more viral antigens.

[0083] In one embodiment, the CD1d recognition antigen and recombinant plasmid are packaged within two intact bacterial minicells or dead bacterial cells.

[0084] The CD1d-recognizing antigen is present in a first intact bacterial minicell or dead cell, while the recombinant plasmid encoding the viral antigen is present in a second intact bacterial minicell or dead cell.

[0085] In some embodiments, minicells containing an encapsulated CD1d-recognizing antigen (e.g., α-GalCer) and a recombinant plasmid encoding at least one viral antigen are administered simultaneously. In some embodiments, minicells containing an encapsulated CD1d-recognizing antigen (e.g., α-GalCer) and a recombinant plasmid encoding a viral antigen are administered sequentially. In some embodiments, minicells containing an encapsulated CD1d-recognizing antigen (e.g., α-GalCer) and a recombinant plasmid encoding a viral antigen are administered multiple times. In some embodiments, minicells containing an encapsulated CD1d-recognizing antigen (e.g., α-GalCer) and a recombinant plasmid encoding a viral antigen are administered at least once, twice, three, or four times a week until the disease is resolved.

[0086] The objectives of this treatment after SARS-CoV-2 infection are as follows: (1) To activate innate and adaptive immunity by recruiting fresh monocytes and dendritic cells from the bone marrow and activating NK cells. This will maintain a high level of immunity in the patient even as the disease progresses and prevent the development of lymphopenia. (2) To secrete physiologically resistant type I (IFNα, IFNβ) and type II (IFNγ) interferons. It is well known that in the early stages of viral infection, IFN stimulation alters the cell's transcriptional program, resulting in an antiviral state characterized by the activation of numerous host genes with partially defined antiviral functions. This activation allows for the rapid elimination of virus-infected cells and suppression of viral replication. (3) To secrete antibodies against the four structural proteins of the virus (envelope, membrane, spike, and nucleocapsid) and sweep away the many viral particles released from infected cells. Based on the above, it is thought that this treatment has little toxicity.

[0087] II. Intact Bacterial Minicells A "minicell" is a derivative of a chromosome-free bacterial cell that arises from a disruption in the coordination of cell division and DNA segregation during binary fission. Minicells are distinct from other vesicles, such as so-called "membrane bleeps" (size approximately 0.2 μm or less), which are spontaneously generated and released under specific conditions but are not the result of specific gene rearrangements or episomal gene expression. The bacterial-derived minicells employed in this disclosure are completely intact and distinguish them from other chromosome-free forms of bacterial cell derivatives characterized by the destruction, degradation, or even removal of the outer membrane or defining membrane. The intact membrane characterizing the minicells of this disclosure allows for the retention of a therapeutic payload within the minicell until the payload is released.

[0088] EDVs TMThese are nucleated, non-living nanoparticles produced by inactivating genes that control bacterial cell division and suppressing cell polarity. Furthermore, while current stealth liposome drug carriers, such as DOXIL (liposomal doxorubicin), can only encapsulate up to 14,000 molecules per particle, and "armed antibodies" can carry fewer than 5 molecules, bacterial minicells can easily carry up to 1 million drug molecules.

[0089] The minicells used in this disclosure can be prepared from bacterial cells, such as E. coli and S. typhymurium. Chromosome replication in prokaryotes is associated with normal double division accompanied by the formation of cell septa. In E. coli and other bacteria, mutations in min genes such as minCD can remove the inhibition of septum formation at the cell pole during cell division, resulting in the generation of normal daughter cells and chromosome-less minicells.

[0090] In addition to mutations in the min operon, chromosome-less minicells can occur following various other gene rearrangements and mutations that affect septum formation, such as divIVB1 in B. subtilis. Furthermore, altering the gene expression levels of proteins involved in cell division and chromosome segregation can also lead to minicell formation. For example, overexpression of minE can induce polar division and generate minicells. Similarly, chromosome-less minicells can result from defects in chromosome segregation, such as the smc mutation in Bacillus subtilis, the spoOJ deletion in B. subtilis, the mukB mutation in E. coli, and the parC mutation in E. coli. Additionally, CafA can increase the rate of cell division and / or inhibit post-replication chromosome segregation, potentially leading to the formation of chain cells or chromosome-less minicells.

[0091] Therefore, minicells can be prepared for this disclosure from any bacterial cell, which may be of Gram-positive or Gram-negative origin, due to the conserved nature of bacterial cell division in these bacteria. Furthermore, the minicells used in this disclosure should have intact cell walls (i.e., be "intact minicells") as described above, and should be isolated from other vesicles, such as membrane breeches, which are not due to specific gene rearrangements or episomal gene expression.

[0092] In a given embodiment, the parent (source) bacteria of the minicell may be Gram-positive or Gram-negative. In one embodiment, the parent bacteria are one or more selected from BV4, which includes Terra- / Glidobacteria (BV1), Proteobacteria (BV2), Spirochaetes, Sphingobacteria, and Plantobacteria. In another embodiment, the bacteria are one or more selected from Firmicutes (BV3), such as Bacilli, Clostridia, or Tenericutes / Mollicutes, or Actinobacteria (BV5), such as Actinomycetales or Bifidobacteriales.

[0093] In accordance with this disclosure, dead bacterial cells are abiogenous cells of bacteria, cyanobatelia, eubacteria, and archaebacteria, as defined in Bergey's Manual of Systematic Biology, 2nd edition. Such cells are considered “intact” if they have an intact cell wall and / or cell membrane and contain the genetic material (nucleic acid) inherent to the bacterial species. Methods for preparing dead bacterial cells are described, for example, in U.S. 2008 / 0038296.

[0094] In yet another aspect, the bacterium is one or more selected from Eubacteria (Chloroflexi, Deinococcus-Thermus), Cyanobacteria, Thermodesulfobacteria, thermophiles (Aquificae, Thermotogae), Alpha, Beta, Gamma (Enterobacteriaceae), Delta or Epsilon Proteobacteria, Spirochaetes, Fibrobacteres, Chlorobi / Bacteroidetes, Chlamydiae / Verrucomicrobia, Planctomycetes, Acidobacteria, Chrysiogenetes, Deferribacteres, Fusobacteria, Gemmatimonadetees, Nitrospirae, Synergistetes, Dictyoglomi, Lentisphaerae Bacillales, Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillales, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Streptococcaceae, Clostridiales, Halanaerobiales, Thermoanaerobacterales, Mycoplasmatales, Entomoplasmatales, Anaeroplasmatales, Acholeplasmatales, Haloplasmatales, Actinomycineae, Actinomycetaceae, Corynebacterineae, Nocardiaceae, Corynebacteriaceae, Frankineae, Frankiaceae, Micrococcineae, Brevibacteriaceae, and Bifidobacteriaceae.

[0095] For pharmaceutical applications, the compositions of this disclosure preferably contain minicells or dead bacterial cells that have been as thoroughly isolated as possible from immunogenic components and other harmful contaminants. A method for purifying bacterial minicells to remove free endotoxins and parent cells is described, for example, in WO 2004 / 113507. The purification process can remove (a) vesicles such as membrane brives, generally smaller than 0.2 μm, (b) endotoxins released from the cell membrane, and (c) parent bacteria and their remnants, whether alive or dead, which are also sources of free endotoxins. Such removal can be carried out, in particular, with a 0.2 μm filter to remove smaller vesicles and cell debris, a 0.45 μm filter to remove parent cells after inducing them to form filaments, antibiotics to kill living bacterial cells, antibodies against free endotoxins, etc.

[0096] This purification method is based on a discovery made by the inventors. Despite differences in bacterial sources, all intact minicells are approximately 400 nm in size, meaning they are larger than membrane bullets and other small vesicles, but smaller than the parent bacteria. Minicell size determination can be achieved using solid-state techniques such as electron microscopy, or liquid-based techniques such as dynamic light scattering. There is an error in the size values ​​obtained by such techniques, and the values ​​may vary slightly depending on the technique. Thus, the size of a dry minicell, when measured with an electron microscope, is approximately 400 nm ± 50 nm. Using dynamic light scattering, the same minicell can be measured as approximately 500 nm ± 50 nm. Furthermore, minicells of ligand targets containing drugs can also be measured as approximately 400 nm to 600 nm ± 50 nm using dynamic light scattering.

[0097] Furthermore, in dead bacterial cells and minicells derived from Gram-negative bacteria, the O-polysaccharide component of lipopolysaccharide (LPS) is embedded in the outer membrane via a lipid A anchor. The component is a chain consisting of repeating units of sugar residues, with 70 to 100 repeating units of 4 to 5 sugars per repeating unit. Because this chain is not rigid, in a liquid environment such as within a living organism, it can adopt a flexible, undulating structure similar to seaweed in coral reefs; that is, the chain remains fixed to the minicell membrane while moving with the liquid.

[0098] Influenced by the O-polysaccharide component, dynamic light scattering can provide minicell size values ​​ranging from approximately 500 nm to approximately 600 nm, as described above. However, minicells of Gram-negative and Gram-positive bacteria easily pass through a 0.45 μm filter, demonstrating that the effective minicell size is 400 nm ± 50 nm. Such size variations are inherent in the present invention and are indicated by the modifier "approximately" in expressions such as "size of approximately 400 nm".

[0099] With respect to hazardous contaminants, the compositions of this disclosure preferably contain less than about 350 EU of free endotoxins. Examples in this regard include free endotoxin levels of approximately 250 EU or less, approximately 200 EU or less, approximately 150 EU or less, approximately 100 EU or less, approximately 90 EU or less, approximately 80 EU or less, approximately 70 EU or less, approximately 60 EU or less, approximately 50 EU or less, approximately 40 EU or less, approximately 30 EU or less, approximately 20 EU or less, approximately 15 EU or less, approximately 10 EU or less, approximately 9 EU or less, approximately 8 EU or less, approximately 7 EU or less, approximately 6 EU or less, approximately 5 EU or less, approximately 4 EU or less, approximately 3 EU or less, approximately 2 EU or less, approximately 1 EU or less, approximately 0.9 EU or less, approximately 0.8 EU or less, approximately 0.7 EU or less, approximately 0.6 EU or less, approximately 0.5 EU or less, approximately 0.4 EU or less, approximately 0.3 EU or less, approximately 0.2 EU or less, approximately 0.1 EU or less, approximately 0.05 EU or less, and approximately 0.01 EU or less.

[0100] The compositions disclosed herein also contain at least about 10 9 A number of minicells or dead bacterial cells, e.g., at least about 1 × 10⁶ 9, at least about 2×10 9 , at least about 5×10 9 , or at least 8×10 9 It can contain. In some embodiments, the composition has about 10 11 or fewer minicells or dead bacterial cells, e.g., about 1×10 11 or fewer, or about 9×10 10 or fewer, or about 8×10 10 or fewer.

[0101] III. CD1d-Recognized Antigen The compositions and methods of the present invention include vectors that can be intact bacteria-derived minicells containing CD1d-recognized antigens. Such antigens will increase the level (e.g., activity or expression level) of type II interferon, e.g., IFN-γ (γ). IFN-γ is involved in the regulation of immune and inflammatory responses. In humans, there is only one type of interferon-γ. It is produced by activated T cells and natural killer cells. IFN-γ enhances the effects of type I IFN. IFN-γ released from Th1 cells attracts leukocytes to the site of infection, resulting in increased inflammation. It also stimulates macrophages to kill ingested bacteria. IFN-γ released from Th1 cells is also important for controlling the Th2 response.

[0102] The IFNγ cytokine is released by natural killer (NK) cells, which are part of the innate immune system, upon binding to an antigen, while sphingolipid compounds are known to function as potent activators of both the innate and adaptive immune systems. Sphingolipids induce a strong cytokine response, including type II interferon, IFN-γ, and many interleukins (Th1-, Th2-, Th17-), by natural killer T cells (iNKT) of the innate immune system. iNKT cells induce the maturation of DCs, exert T cell helper-like functions, and lead to the development of cytotoxic T cell responses.

[0103] Examples of glycosphingolips useful for inducing IFN type II responses are described herein and include C-glycosides of α-galactosylceramide (α-C-GalCer), α-galactosylceramide (α-GalCer), 12-carbon acyl form of galactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), l,2-diacyl-3-0-galactosyl-SN-glycerol (BbGL-II), diacylglycerol-containing glycolipids (Glc-DAG-s2), gangliosides (GD3), gangliotriosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), and α-glucuronide ceramide (GSL-1 or GSL-4). This includes isoglobotrihexosylceramide (iGb3), lipophosphoglycan (LPG), lysophosphatidylcholine (LPC), α-galactosylceramide analog (OCH), slate ceramide, and the like. In certain embodiments, the minicells disclosed herein comprise α-galactosylceramide (α-GalCer) as a type II IFN agonist.

[0104] α-GC is a type II agonist of INF and is known to stimulate the immune system by activating a type of white blood cell called natural killer T cells (NKT cells).

[0105] This minicell can directly deliver a type II IFN agonist to immune system cells with the aim of enhancing iNKT cell activation and type II interferon IFN-γ production in vivo. The non-targeted, intact bacterial minicell is taken up by immune system phagocytic cells, degraded in endosomes, and αGC is presented to iNKT cells for immunostimulation. Therefore, in some embodiments, the minicell provides targeted delivery of a type II interferon agonist. In other embodiments, the composition disclosed herein comprises a non-targeted minicell containing a type II interferon agonist.

[0106] IFN-γ production is regulated by cytokines secreted by antigen-presenting cells (APCs), particularly interleukin (IL)-12 and IL-18. These cytokines play a bridging role in the innate immune response, linking infection and IFN-γ production. When macrophages recognize many pathogens, the secretion of IL-12 and chemokines is induced. These chemokines attract NK cells to the site of inflammation, and IL-12 promotes IFN-γ synthesis in these cells. In macrophages, natural killer cells, and T cells, IFN-γ production is further increased by a combination of IL-12 and IL-18 stimulation. Therefore, any one or a combination of these proteins would be suitable agents for the purposes of this disclosure.

[0107] Negative regulators of IFN-γ production include IL-4, IL-10, transforming growth factor β, and glucocorticoids. Proteins and nucleic acids that inhibit these factors can promote IFN-γ production.

[0108] Furthermore, suitable for use in this context are polynucleotides encoding IFN-γ, or genes that activate the production and / or secretion of IFN-γ.

[0109] Drugs that increase IFN-γ may also be viral vaccines. Many viral vaccines exist that can induce IFN-γ production without causing side effects such as infection. Influenza vaccines are one example of this type of viral vaccine.

[0110] The serum concentration of IFN-γ required to effectively activate the host immune response is lower when drug-conjugated bispecific antibody-targeted minicells or dead bacterial cells are administered. Therefore, in one embodiment, the methodology of the present invention results in an increase in serum IFN-γ concentration not higher than about 30,000 pg / mL. In another embodiment, the serum IFN-γ concentration is increased so as not to exceed about 5,000 pg / mL, 1,000 pg / mL, 900 pg / mL, 800 pg / mL, 700 pg / mL, 600 pg / mL, 500 pg / mL, 400 pg / mL, 300 pg / mL, 200 pg / mL, or 100 pg / mL. In a further embodiment, the resulting serum IFN-γ concentration is at least about 10 pg / mL, or at least about 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, or 500 pg / mL.

[0111] In some embodiments, the drug is an IFN-γ protein, or an artificial protein or analog. In some embodiments, the administration achieves approximately 0.02 ng to 1 microgram of IFN-γ per ml of host blood. In one embodiment, the achieved IFN-γ concentration in host blood is approximately 0.1 ng to approximately 500 ng / ml, approximately 0.2 ng to approximately 200 ng / ml, approximately 0.5 ng to approximately 100 ng / ml, approximately 1 ng to approximately 50 ng / ml, or approximately 2 ng to approximately 20 ng / ml.

[0112] IV. Loading of viral antigens and CD1d-recognizing antigens into minicells or dead bacterial cells Similar to CD1d-recognizing antigens, viral antigens can also be directly packaged into minicells or dead bacterial cells by co-incubating them with the antigen in a buffer. The buffer composition can be varied to optimize antigen loading in the minicells as a function of conditions well known in this field. The buffer can also be varied depending on the antigen (e.g., depending on the nucleotide sequence or length of the nucleic acid loaded into the minicell in the case of nucleic acid payloads). Exemplary buffers suitable for loading include, but are not limited to, phosphate-buffered saline (PBS). Once packaged, the antigen remains within the minicell and is protected from degradation. Long-term incubation studies using minicells with siRNA packages incubated in sterile saline have shown, for example, no siRNA leakage.

[0113] Antigens, such as proteins encoded by nucleic acids, can be introduced into minicells by transforming parent bacterial cells with a vector, such as a plasmid, that encodes the antigen. When minicells are formed from parent cells, they hold the plasmid and / or expression product, e.g., a specific copy of the antigen. Details of packaging into minicells and expression products are described in WO 03 / 033519.

[0114] The data presented in WO 03 / 033519 demonstrates, for example, that recombinant minicells containing mammalian gene expression plasmids can be delivered to both phagocytic and non-phagocytic cells. WO 03 / 033519 also describes the genetic transformation of minicell-producing parent bacterial strains using heterologous nucleic acids supported on episomal replicated plasmid DNA. Upon separation of the parent bacteria from the minicells, a portion of the episomal DNA was sequestered in the minicells. The resulting recombinant minicells were readily taken up by mammalian phagocytic cells and degraded within the phagolysosome. Furthermore, a portion of the recombinant DNA escaped the phagolysosome membrane and was transported to the mammalian cell nucleus, where the recombinant gene was expressed. In other embodiments, multiple antigens can be packaged in the same minicell.

[0115] By creating an antigen concentration gradient between the extracellular medium, which consists of minicells, and the cytoplasm of the minicells, the antigen can be packaged within the minicells. If the extracellular medium has a higher antigen concentration than the minicell cytoplasm, the antigen will naturally move down this concentration gradient into the minicell cytoplasm. However, if the concentration gradient is reversed, the antigen will not move out of the minicells. Details of the activator loading process and its remarkable properties are described, for example, in U.S. Patent Application Publication No. 2008 / 0051469.

[0116] V. Formulations The disclosure includes, to the extent of its scope, compositions comprising (a) a vector, intact bacterial minicell, or dead bacterial cell containing at least one viral antigen as a payload; and (b) a combination of a vector, intact bacterial minicell, or dead bacterial cell containing at least one CD1d-recognizing antigen as a payload, both of which are present on at least one pharmaceutically acceptable carrier. The at least one viral antigen and the at least one CD1d-recognizing antigen may be present in the same or different vector, intact bacterial minicell, or dead bacterial cell. At least one of the viral antigen and the CD1d-recognizing antigen is present in the intact bacterial minicell.

[0117] In another embodiment, the viral antigen and one of at least one CD1d-recognizing antigens are present in a non-bacterial cell carrier, such as a liposome carrier.

[0118] In some aspects, the CD1d-recognized antigen is α-galactosylceramide, an interferon type II agonist.

[0119] The compositions of this disclosure may be presented in unit dose forms, e.g., ampoules or vials, or in multi-dose containers, with or without preservatives. The compositions may be solutions, suspensions, or emulsions in oily or aqueous vehicles and may include formulations such as suspending agents, stabilizers, and / or dispersants. Suitable solutions are isotonic with the recipient's blood, and examples include saline, Ringer's solution, and glucose solution. Alternatively, the formulation may be in the form of a lyophilized powder and reconstituted with a suitable vehicle, e.g., sterile pyrogen-free water or saline. The formulation may also be in the form of a depot formulation. Such long-acting formulations may be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. In some embodiments, administration includes enteral or parenteral administration. In some embodiments, administration may include administration selected from oral, buccal, sublingual, intranasal, rectal, vaginal, intravenous, intramuscular, and subcutaneous injections.

[0120] In some embodiments, minicell-containing compositions are provided that include a therapeutically effective amount of viral antigen, as well as a therapeutically effective amount of CD1d-recognizing antigen. A “therapeutably effective” amount of antigen is, in accordance with this disclosure, an amount that elicits a pharmacological response when administered to a subject.

[0121] Therefore, in the context of this disclosure, a therapeutically effective dose may be measured by reference to the prevention or improvement of a viral infection in either an animal model or a human subject when a minicell with a therapeutic payload is administered, as further described below. In a particular case, a dose that proves to be a “therapeutally effective dose” for a particular subject may not be effective for 100% of subjects treated similarly for a viral infection, even if such a dose is considered a “therapeutally effective dose” by those skilled in the art. In this respect as well, the appropriate dose will vary as a function of, for example, the stage and severity of the viral infection, whether the subject has an underlying disease, is over 60 years of age, or is immunocompromised.

[0122] a. Route of administration The formulations of the present invention can be administered to various sites within the mammalian body via various routes to achieve desired therapeutic effects locally or systemically. Delivery can be achieved by any pharmaceutically acceptable route, such as oral administration, application of the formulation to body cavities, inhalation, nasal administration, pulmonary administration, pneumoperitoneum, or injection (e.g., parenteral, intramuscular, intravenous, intrahepatic, peritoneal, subcutaneous, intratumoral, or intradermal administration). Multiple routes can also be used in combination.

[0123] b. Purity Bacterial minicells are substantially free from contamination by the parent bacteria. Therefore, the minicell-containing formulation is preferably 10 7 Less than 1 contaminated parent bacterial cell per minicell, 10 8 Less than 1 contaminated parent bacterial cell per minicell, 10 9 Less than 1 contaminated parent bacterial cell per minicell, 10 10 Approximately less than 1 contaminated parent bacterial cell per minicell, or 10 11 Each minicell consists of less than approximately one contaminated parent bacterial cell.

[0124] Methods for purifying minicells are known in the art and are described in PCT / IB02 / 04632. One such method is a combination of cross-flow filtration (feed flow parallel to the membrane surface; Forbes, 1987) and dead-end filtration (feed flow perpendicular to the membrane surface). Optionally, differential centrifugation at low centrifugal force can be performed before the filtration combination to remove some of the bacterial cells, thereby concentrating the supernatant for use in minicells.

[0125] In particular, purification methods utilizing bacterial filamentation are effective in increasing the purity of minicells. Therefore, a method for purifying minicells may include (a) subjecting a sample containing minicells to conditions that induce the parent bacterial cells to take on a filamentous morphology, and then (b) filtering the sample to obtain a purified minicell preparation.

[0126] Furthermore, it is possible to combine known mini-cell purification methods. The following are some highly effective combinations: Step A: Differential centrifugation of the minicell-producing bacterial culture. This step can be performed at 2,000 g for approximately 20 minutes, removing most of the parent bacteria and leaving the minicells in the supernatant. Step B: Density gradient centrifugation using an isotonic and non-toxic density gradient medium. This step separates the minicells from many contaminants, including parent bacteria, and minimizes minicell loss. Preferably, this step is repeated within the purification method. Step C: Cross-flow filtration is performed using a 0.45 μm filter to further reduce contamination by parent bacteria. Step D. Filamentation of residual parent fungi due to stress. This can be achieved by exposing the minicell suspension to one of several stress-inducing environmental conditions. Step E: Antibiotic treatment to kill the parent bacteria cells. Step F: Cross-flow filtration removes small contaminants such as membrane bleed, membrane fragments, bacterial debris, nucleic acids, and culture medium components, and concentrates the minicells. A 0.2 μm filter can separate the minicells from small contaminants, and a 0.1 μm filter can concentrate the minicells. Step G: Remove dead filamentous fungi by dead-end filtration. A 0.45 μm filter can be used in this step; and Step H: Remove endotoxins from the mini-cell preparation solution. Magnetic beads coated with anti-lipid A can be used in this step.

[0127] c. Operating Schedule In general, the formulations disclosed herein can be used in appropriate doses defined in routine trials to obtain optimal physiological effects while minimizing potential toxicity. The dosage can be selected according to various factors such as the patient's age, weight, sex, medical condition, severity of the symptoms to be treated, route of administration, and the patient's renal and hepatic function.

[0128] To optimize the concentration of minicells and drugs within the range that provides maximum efficacy while minimizing side effects, regimens based on the target site and rate of arrival of minicells and antigens to target cells may be optimal. When determining the optimal concentration for a treatment regimen, the distribution, equilibrium, and excretion of minicells and antigens may be considered. When using minicells and antigens in combination, the dosage can be adjusted to achieve the desired effect.

[0129] Furthermore, the dosage of the formulation can be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more dosing regimens may be selected, and the pharmacokinetic / pharmacodynamic profiles of one or more dosing regimens may be determined using a pharmacokinetic / pharmacodynamic model. Then, one of the dosing regimens can be selected for administration that achieves the desired pharmacokinetic / pharmacodynamic response based on the specific pharmacokinetic / pharmacodynamic profile. See, for example, WO 00 / 67776.

[0130] Specifically, the formulation can be administered at least once a day for several days (3-4 days), or until the symptoms of the viral infection subside. In one embodiment, the formulation is administered at least once a day until the viral disease subsides.

[0131] More specifically, the formulation may be administered at least once a day for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. Alternatively, the formulation may be administered once daily, or once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days or more.

[0132] The composition may be administered once a day, or the total daily dose may be divided and administered in two, three, or four doses per day.

[0133] VI. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the relevant field.

[0134] For convenience, the meanings of certain terms and phrases used in this specification, the examples, and the appended claims are given below. Other terms are defined herein.

[0135] The singular forms "a," "an," and "the" imply a reference to a plural form unless the context clearly indicates otherwise.

[0136] As used herein, the term "about" is expected to be understood by those skilled in the art and to vary to some extent depending on the context in which it is used. If there is a usage that is not clear to those skilled in the art from the context in which the term is used, "about" will mean a range of plus or minus 10% of a particular term.

[0137] As used herein, unless otherwise required by context, variations of the terms “comprise,” “comprises,” and “comprised” are not intended to exclude other additives, ingredients, integers, or steps.

[0138] The terms "biologically active" and "biological activity" are sometimes used to modify or indicate the effect of a compound or composition on living organisms. Thus, a substance is considered to be biologically active if it interacts with or affects any cell tissue in the body of a human or animal by reacting with intracellular proteins, nucleic acids, or other molecules.

[0139] The terms “individual,” “subject,” “host,” and “patient,” as used interchangeably herein, refer to any mammalian subject for which diagnosis, treatment, or therapy is desired. The individual, subject, host, or patient may be human or a non-human animal. Therefore, preferred subjects may include, but are not limited to, non-human primates, cattle, horses, dogs, cats, guinea pigs, rabbits, rats, and mice.

[0140] Terms such as "treatment," "procedure," and "therapy" mean obtaining a desired pharmacological and / or physiological effect in a patient. This effect may be either a preventative effect, completely or partially preventing a viral infection or its symptoms, or a therapeutic effect against a viral infection. Alternatively or additionally, the desired therapeutic effect may be an increase in the patient's overall survival, progression-free survival, or reduction in side effects.

[0141] The term "pharmaceutical grade" means that the product is free from parent cell contamination, cell debris, free endotoxins, and other pyrogens, and fully meets regulatory requirements for intravenous administration to humans. See, for example, "Guidance for Industry - Pyrogen and Endotoxins Testing," US Food and Drug Administration (June 2012).

[0142] In this specification, “payload” refers to the identification or modification of a biologically active substance that is intended to be loaded into a minicell for delivery to a target host cell, or that has been loaded into a minicell.

[0143] "Substantially" generally means a similarity of 90% or more. In some embodiments, "substantially" refers to ±0.2° in the context that the first powder X-ray diffraction pattern is substantially as shown in the second powder X-ray diffraction pattern. In some embodiments, "substantially" refers to ±0.4°C in the context that the first differential scanning calorimetry thermogram is substantially as shown in the second differential scanning calorimetry thermogram. In some embodiments, "substantially" refers to ±0.4% by weight in the context that the first thermogravimetric analysis is substantially as shown in the second thermogravimetric analysis. In some embodiments, "substantially purified" refers to a purity of at least 95%. This includes a purity of at least 96%, 97%, 98%, or 99%. In further embodiments, "substantially purified" refers to a purity of about 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, including its increments.

[0144] As used herein, "therapeutic activity" or "activity" may refer to activity whose effect matches a desired therapeutic outcome in humans, or a desired effect in non-human mammals or other species or organisms. Therapeutic activity can be measured in vivo or in vitro. For example, the desired effect can be assayed in cell culture.

[0145] As used herein, the phrase “therapeutic dose” means the dose of an agent that elicits a specific pharmacological response in a significant number of subjects requiring such treatment. It is emphasized that a therapeutic dose of an antigen administered to a particular subject in a particular instance is not necessarily effective in treating the viral infections described herein, even if such a dose is considered therapeutically effective by those skilled in the art.

[0146] The technology of the present invention, as generally described above, will be more readily understood by referring to the following examples. These examples are provided for illustrative purposes and are not intended to limit the technology of the present invention. [Examples]

[0147] Example 1 Figure 1 depicts an exemplary composition containing a first intact bacterial minicell containing a plasmid encoding a viral protein, which functions to stimulate an antibody response to the viral protein. The double-stranded DNA of the plasmid is recognized by intracellular nucleic acid sensors, triggering IFNα and IFNβ responses. Also shown is a second intact bacterial minicell containing α-galactosylceramide, an IFNγ-stimulating compound.

[0148] Since the genome sequence of the SARS-CoV-2 virus is known, it is possible to create plasmids that express all four types of SARS-CoV-2 proteins expressed from a single bacterial expression promoter. Then, these plasmids can be used to create minicells derived from intact bacteria (i.e., EnGeneIC Nanocell Dream Vector (EDV) TM It can be encapsulated in )). Another method involves packaging bacterial cell-derived minicells with glycolipids (α-galactosylceramide: EDVα-GC).

[0149] This product is freeze-dryable. Based on intact bacterial minicells, the product is extremely stable, and freeze-dried vials containing anticancer compounds and α-GC have already shown stability for over 3 years when stored at 4°C in a standard hospital pharmacy refrigerator. It can be shipped worldwide via courier and has been proven effective in US cancer trials using EDV.

[0150] Administration to patients: When administering to patients, the vial can be reconstituted with 1 ml of sterile saline solution and injected intravenously as a bolus injection.

[0151] If this plasmid is directly used to transform a bacterial minicell-producing strain, the viral protein will be expressed in the bacterial cytoplasm. When minicells are produced from intact bacterial cells through asymmetric cell division, many proteins are sequestered in the cytoplasm of these intact minicells. This has been demonstrated in several studies in which heterologous proteins were expressed in intact bacterial minicell-producing bacterial cells, and the proteins were isolated in the cytoplasm of the intact bacterial minicells.

[0152] The expected results from intact bacterial minicells encapsulated in plasmids are antibody responses to all four viral proteins and type I interferon responses.

[0153] The injected, intact bacterial minicells are rapidly engulfed by immune system cells (macrophages, NK cells, and dendritic cells) in the lymph nodes, liver, and spleen. Normally, intact bacterial minicells enter endosomes, are broken down in lysosomes, and release plasmids that escape into the cytoplasm.

[0154] Cytoplasmic DNA sensors that recognize plasmid DNA are a type of pattern recognition receptor (PRR) that induce the production of type I interferons (IFNα, IFNβ), triggering a rapid and efficient innate immune response. Type I interferons are well known to have potent antiviral effects.

[0155] Viral proteins are released from degraded, intact bacterial minicells via lysosomes and undergo antigen processing and presentation on the cell surface via MHC class II. This triggers a potent antibody response against the viral antigenic epitope. This further induces a CD4+ / CD8+ T cell response to virus-infected cells, which should enhance the antiviral response.

[0156] The activation, maturation, and proliferation of fresh bone marrow-derived monocytes, as well as the activation and proliferation of macrophages, dendritic cells, NK cells, B cells, and T cells, are expected to overcome the lymphopenia observed in elderly, immunocompromised SARS-CoV-2 patients.

[0157] Expected effects from intact bacterial minicells encapsulating α-galactosylceramide - Induction of IFN-γ response: EDV TM α-GC is also engulfed by immune system cells (macrophages, NK cells, and dendritic cells) in lymph nodes, the liver, and the spleen. Undamaged bacterial minicells are degraded by intracellular lysosomes, and the released α-GC is picked up by CD1d (an MHC class I-like molecule involved in the presentation of foreign glycolipids) bound to the lysosome and transported to the cell surface. This α-GC / CD1d complex is recognized by the invariant T cell receptor on invariant NKT cells, which rapidly releases IFN-γ. IFN-γ is known to strongly stimulate specific antiviral immune responses, and as a result, it is expected to enhance the rejection response to viral infection.

[0158] This bacterial-derived, intact mini-cell therapy has already been administered to more than 140 human cancer patients over 1,500 times, and its safety has been confirmed with virtually no side effects even with repeated administration.

[0159] Example 2 Figure 2 shows peripheral blood mononuclear cells (PBMCs) from patient 1-CB04-1 (72-year-old male) with advanced hepatocellular carcinoma. This shows an increase in CD8+ cytotoxic T cells (Figure 2A), NK cells (Figure 2B), NKT cells (Figure 2C), and iNKT cells (Figure 2D) after treatment with epidermal growth factor receptor (EGFR) antibody-targeted therapy, up to the second and third cycles. (PNU packaging intact bacterial-derived minicells (i.e., EDV)) TM ) + α-galactosylceramide packaging intact bacterial-derived minicells (EDV TM ) was administered.

[0160] It should be noted that this patient is elderly and suffers from severe immunodeficiency. PNU refers to PNU-159682, a morpholinyl anthracycline derivative.

[0161] [Table 2]

[0162] Preparation of epidermal growth factor receptor (EGFR)-antibody targets in PNU-packaged, intact bacterial minicells is described, for example, in WO2020 / 021437.

[0163] Detailed results are shown in the figures. Figures 2A-D show the positive effects on the immune system after administration of combination compositions of intact bacterial minicells packaged with an anticancer compound (PNU-159682) and bacterial minicells packaged with a CD1d-recognizing antigen (α-galactosylceramide). In particular, Figure 2A is a graph of the percentage of CD8+ T cells (Y axis) and T cell subsets of naive (first 4 columns) and effector (last 4 columns). The T cell subsets are C1D1, C1D9, C2D7, and C3D7.

[0164] Populations of specifically differentiated T cells play a crucial role in regulating and shaping immune responses by providing various immune-related functions. One such function is immune-mediated cell death, which T cells perform in several ways. CD8+ T cells, also known as "killer cells," are cytotoxic; that is, they can directly kill virus-infected cells and cancer cells. Furthermore, CD8+ T cells can use small signaling proteins called cytokines to attract other cells when initiating an immune response.

[0165] Figure 2B shows the proportion of white blood cells and subsets of NK cells (C1D1, C1D9, C2D7, C3D7). Figure 2C shows the proportion of T cells and subsets of NKT cells (C1D1, C1D9, C2D7, C3D7). Finally, Figure 2D shows the proportion of NKT cells and subsets of iNKT cells (C1D1, C1D9, C2D7, and C3D7).

[0166] Example 3 Figure 3 shows the activation of major immune cells in PBMCs from a 45-year-old woman with advanced colorectal cancer. The patient's CD8+ effector cytotoxic T cells (CD45RA+ CCR7-) significantly increased by the second and third cycles (Figure 3A). Similarly, an increase in NK cells was observed in the control PBMCs in the second and third cycles (Figure 3B). Interestingly, ELISA analysis of the patient's serum 3 hours after administration of each intact bacterial-derived minicell showed a spike in IFNγ (Figure 3C), which occurs when α-galactosylceramide is effectively presented from antigen-presenting cells (APCs) to iNKT cells, triggering the release of IFNγ, a key mediator in fighting viral infection.

[0167] [Table 3]

[0168] Similar to Example 2, the intact bacilli-derived minicells administered to the subjects contained a combined bacterial minicell composition consisting of an anticancer agent (PNU-159682) in an intact bacilli-derived minicell package and an intact bacilli-derived minicell package plus a CD1d-recognizing antigen (α-galactosylceramide).

[0169] Figure 3A is a graph of percentage CD8+ T cells versus CD8+ memory T cell subsets, with the first four columns corresponding to naive trial results and the second four columns corresponding to effector trial results. Patient CD8+ effector cytotoxic T cells (CD45RA+ CCR7-) significantly increased by cycles 2 and 3.

[0170] Figure 3B is a graph comparing the percentage of white blood cells and NK cell subsets (C1D1, C1D9, C2D7, C3D7). As a result, an increase in NK cells was observed in the target PBMCs by the second and third cycles.

[0171] Finally, Figure 3C shows a comparison of IFNγ (pg / mL) and IFNγ measured for each dose. This occurs when α-galactosylceramide is effectively presented from antigen-presenting cells (APCs) to iNKT cells, inducing the release of IFNγ.

[0172] Example 4 Figure 4 shows the white blood cell counts (mean values ​​for 9 patients) before and 3 hours after administration. Eight of the nine patients were elderly, all had stage IV pancreatic cancer, and were severely immunocompromised patients for whom all conventional treatments had been ineffective. This suggests that, in accordance with activation signals from macrophages, dendritic cells, and NK cells, initial administration of bacterial-derived minicells mobilized fresh monocytes from the bone marrow, and by the third administration, they were fully activated, matured, and proliferated.

[0173] [Table 4]

[0174] This result is significant, as detailed in Figure 4, because the proliferation of macrophages, dendritic cells, and NK cells is essential for successful immune defense against viral infection.

[0175] Example 5 Figure 5 shows the activation of major immune cells in PBMCs of a 45-year-old woman with advanced colorectal cancer. The patient's CD8+ effector cytotoxic T cells (CD45RA+ CCR7-) increased significantly by the second and third cycles (Figure 5A). Similarly, an increase in NK cells was observed in the patient's PBMCs in the second and third cycles (Figure 5B). Interestingly, intact bacterial-derived minicells (EDV) were also observed. TM ELISA analysis of patient serum 3 hours after administration revealed spikes in IFNγ (Figure 5C). This likely occurs when α-galactosylceramide is effectively presented from antigen-presenting cells to iNKT cells, inducing the release of IFNγ, a key mediator in combating viral infection.

[0176] [Table 5]

[0177] Example 6 This example uses EDV as a vaccine against SARS-CoV-2. Covid-αGC (EDVC ovid-αGC This research aims to evaluate the feasibility of using bacterial minicells equipped with ( ).

[0178] Plasmids encoding the DNA sequences of α-GC and spike protein are one EDV (EDV Covid-αGC It can be successfully incorporated into the IV molecule. Subsequently, EDV was administered by subcutaneous (SC), intravenous (IV), and intramuscular (IM) injection. The results showed that intramuscular injection yielded the strongest initial interferon response at 8 hours post-injection and the highest spike protein-specific IgG titer at 1 week post-injection compared to all other methods.

[0179] After that, EDVCovid-αGC When the corresponding control was administered by intramuscular injection, αGC was incorporated into EDV, resulting in a dramatic increase in the production of IFNα, TNFα, IFNγ, IL12, and IL6 8 hours after administration. Covid-αGC The treatment was accompanied by an increase in the amount of cytotoxic T cells in the spleen of the treated mice. These T cells responded to stimulation with spike proteins ex vivo and expressed CD69+ and CD137+.

[0180] EDV Covid-αGC Mice administered with EDV had the highest levels of spike protein-specific IgG and IgM four weeks after administration compared to all controls tested. B cells extracted from these mice were able to produce IgG and IgM in vitro in response to spike protein stimulation. Covid-αGC Splenocytes from administered mice contained the highest number of antiviral CD69+ CD137+ cytotoxic T cells. Ex vivo stimulation of these spleen cells with spike protein increased the number of virus antigen-specific CD69+ cytotoxic T cells. Furthermore, EDV Covid-αGC The serum of mice injected with showed the strongest inhibition of spike protein binding to the hACE receptor in vitro, indicating that the produced antibodies were neutralizing. Interestingly, the serum of mice administered with αGC also showed a measurable, non-antigen-specific antiviral effect.

[0181] Based on the above, EDV Covid It was found that incorporating αGC is important to maximize the effectiveness of the anti-SARS-CoV-2 spike protein. The results of this study are relevant to EDV Covid-αGC This indicates that IM administration is an effective strategy against the current Covid-19 pandemic.

[0182] Materials and methods Design of a SARS-CoV-2 spike protein bacterial expression plasmid: The expression cassette was generated by placing the coding nucleotide sequence of the SARS-CoV-2 (Covid-19) spike protein (Genebank MN908947.3) at the 3' end of a modified β-lactamase promoter previously tested for expression in Salmonella tiphimurium strains (Su, Brahmbhatt et al., Infection and Immunity, 60(8):3345-3359 (1992)). Next, this expression cassette was inserted between the Kpn 5' site and the Sal I 3' site at the M13 multicloning site of the PUC57-Kan backbone plasmid to create P-Blac-Cov2S. The Cov2S sequence was removed from P-Blac-Cov2S to create the control plasmid P-Blac (Figure 10A, Figure 10B).

[0183] Cloning of P-Blac-Cov2S and P-Blac-Cov2S into Salmonella Typhimurium EDV-producing strains, and subsequent incorporation of P-Blac-Cov2S and spike protein into EDV: P-Blac-Cov2S and P-Blac-Cov2S were used in a chemically competent Salmonella typhimurium intermediate strain (4004) that lacks plasmid restriction mechanisms, and then used in Gene Pulser Xcell. TMElectroporation was performed using Bio-Rad (Hercules CA) at a setting of 200Ω, 25Hz, and 2.5 mV. Transformants were collected in TSB medium at 37°C for 1.5 hours and then plated onto TSB agar plates containing 75 μg / ml kanamycin (#K4000, Sigma-Aldrich, St. Louis, Missouri). Isolates were collected in TSB broth with 75 μg / ml kanamycin, and plasmid DNA was extracted using a Qiagen miniprep kit according to the manufacturer's instructions (#27104, Qiagen, Hilden, Germany). Subsequently, the plasmid DNA extracted from strain 4004 was electroporated with the EDV-producing bacterium Salmonella typhimurium strain (ENSm001) (EnGeneIC Pty., Inc.) in the same manner as above. Bacteria containing P-Blac-Cov2S produced the encoded Covid2 spike protein, which was incorporated into EDV alone along with the plasmid DNA. COVID This will generate the following. The EDV (EDVCONT) containing P-Blac will be used as the control.

[0184] EDV COVID and EDV CONT To measure the plasmid content, we used the Qiaprep Spin miniprep kit (Qiagen) to measure 2x10 9 Plasmids were extracted from each EDV according to the manufacturer's instructions. Empty EDVs were processed similarly and used as controls. Subsequently, the amount of DNA plasmid was measured by absorbance at 260 nm using a biophotometer (Eppendorf). The plasmid copy number was calculated using the following formula.

[0185]

number

[0186] Western blot: 2 × 10 10EDVCOVID protein was collected in 100 μL B-PER samples supplemented with 10% (v / v) lysozyme (Sigma-Aldrich) and 1% (v / v) DNase I (Qiagen). TM Extraction was performed using (Thermo Fisher) bacterial protein extraction reagent. The extracted samples were then centrifuged at 12,000 g for 10 minutes, and the supernatant was collected. The remaining pellet was also collected and resuspended in 100 μl of PBS. 23 μl of the supernatant and pellet protein samples were incubated with 5 μl of loading buffer and 2 μl of DTT (Sigma-Aldrich) at 80°C for 20 minutes. The entire volume of each sample was then loaded onto a NuPAGE 4-12% Bis-Tris mini gel (Life Technologies) and measured at 190 V for approximately 80 minutes. The samples were then transferred using an iBlot 2 machine, the membranes were blocked with Superblock blocking buffer (Thermo Fisher), and subsequently stained with 1:2000 rabbit polyclonal SARS-CoV2 spike antibody (cross-reacts with S1 subunit, Sino Biological, Beijing, China), and incubated overnight at 4°C. Subsequently, the membrane was washed with PBST and incubated with HRP-conjugated anti-rabbit secondary antibody (1:5000) (Abcam) at room temperature for 1 hour. The blot was developed using Lumi-Light Western Blot substrate (Roche) and visualized using Chemidoc MP (Biorad).

[0187] Immobilization of α-galactosylceramide onto EDVCOVID and cell culture: EDVCOVID-αGC was prepared by immobilizing α-galactosylceramide glycolipid adjuvant (α-GC) onto EDVCOVID using Engenic's proprietary method.

[0188] JAWSII cells (ATCC) were treated with EDVCOVID-αGC at a rate of 1 x 10⁴ cells per cell in a 96-well Perfecta3D hanging drop plate (Sigma-A). JAWSII cells treated with 4 μg / mL α-GC were used as a positive control. The cultures were then incubated in 5% CO₂ at 37°C for 24 hours, and the cells were harvested, stained with CD1d-αGC antibody (ThermoFisher), and analyzed using a Gallios flow cytometer (Beckman). The results were analyzed using Kaluza Analysis software (Beckman).

[0189] For the animal experiments, female Balb / c mice aged 6-7 weeks were obtained from Animal Resources Company in Western Australia. The mice were acclimatized for one week before the start of the experiments. The mice were injected with appropriate therapeutic agents via SC and IM, and serum was collected from the tail vein 8 hours, 1 week, and 4 weeks after injection. The spleen and bone marrow were also collected.

[0190] Enzyme-linked immunosorbent assay: The concentrations of IL-12p40, IFN-γ, TNFα, IL-6, IL2, IFNα, and IFNβ in mouse serum were measured using a standard sandwich enzyme-linked immunosorbent assay (ELISA) from R&D Systems, following the manufacturer's instructions. The concentrations of present proteins were determined by calculating the absorbance of the samples against a standard curve constructed using the same assay with purified proteins.

[0191] For the analysis of anti-RBD-specific IgG and IgM antibodies, 96-well plates (Immulon 4 HBX; Thermo Fisher Scientific) were coated with 50 μl per well of a 2 μg / ml solution of anti-mold spike RBD protein (Genetex) suspended in PBS (GIBCO) at 4°C. The following day, the coating protein solution was removed, and 100 μl of 3% nonfat milk prepared in PBS containing 0.1% Tween 20 (PBST) was added to each well, and the plates were blocked at room temperature for 1 hour. During this time, serial dilutions of mouse serum were prepared in 1% nonfat milk prepared in PBST. Subsequently, the blocking solution was removed, and 100 μl of each serially diluted serum sample was added to the plates and incubated at room temperature for 2 hours. After the incubation period, the plate was washed three times with 250 μl of water per well before adding 100 μl of a 1:3000 dilution of goat anti-mouse IgG / IgM-horseradish peroxidase (HRP) conjugated secondary antibody (ThermoFisher) prepared in 0.1% PBST. After incubation at room temperature for 1 hour, the plate was washed three more times with 0.1% PBST. After complete drying, the sample was visualized by incubation with TMD. The reaction was then terminated, and the sample was read at 490 nm using a KC Junior plate reader (BioTek Instruments).

[0192] Antibody titers were measured by ELISA after serially diluting treated mouse serum samples 1:3, and expressed as the reciprocal of the highest dilution at which a positive result was obtained.

[0193] Statistical analysis: Student's T-tests and one-way ANOVA were performed using Prism 8 (GraphPad). A p-value < 0.05 is considered statistically significant.

[0194] result To effectively and efficiently administer the vaccine with a single injection, EDV Covid αGC is co-encapsulated in EDV Covid-αGC I made it. EDV Covid-αGCThe function of the encapsulated αGC was verified by examining its presentation on JAWSII cells via CD1d ligand after treatment. The results showed that a high percentage of treated JAWSII cells expressed CD1d-αGC, at levels equivalent to or higher than those observed after treatment with 3 μg / mL of free αGC (Figure 11A). EDV Covid-αGC Western blot analysis was performed to confirm that the incorporated spike protein was not affected by secondary incorporation by αGC (Figure 11B).

[0195] Next, EDV Covid-αGC The effects of different delivery methods were evaluated in vivo. Serum samples were collected from mice treated with subcutaneous (SC), intravenous (IV), and intramuscular (IM) injections, and the concentrations of IFNα (Figure 7C; serum IFNα concentration 8 hours after injection), IFNγ (Figure 7D; serum IFNγ concentration 8 hours after injection), IL12 (Figure 7E; IL12p40 serum concentration), IL6 (Figure 7F; IL6 serum concentration 8 hours after injection), and TNFα (Figure 7G; serum TNFα concentration 8 hours after injection) were analyzed by ELISA. As a result, EDV Covid-αGC When administered via IM, it was found to be overwhelmingly superior in inducing the production of all tested cytokines in mice 8 hours after administration.

[0196] EDV Covid-αGC The differences in administration methods were further demonstrated by analyzing spike protein-specific antibodies one week after the initial infusion. EDV Covid-αGC In mice administered IM, higher spike protein-specific IgG titers were detected in the serum compared to those administered SC (Figure 7A). EDV Covid-αGC Based on the initial interferon response and subsequent high IgG titers, it was concluded that administration via IM injection is preferable.

[0197] EDV, EDV αGC EDV Control EDV Control-αGC EDV Covid EDV Covid-αGCDetailed analysis of the initial interferon response after IM administration revealed that the initial interferon response in mice is primarily induced by the administration of αGC delivered by EDV, with or without antigen-specific plasmids. See Fig. 12A (serum IFNα concentration 8 hours after IM injection); Fig. 12B (serum IFNγ concentration 8 hours after IM infusion); Fig. 12C (IL6 serum concentration 8 hours after IM injection); Fig. 12D (serum TNFα concentration 8 hours after IM infusion), and Fig. 12E (IL12p40 serum concentration 8 hours after IM administration).

[0198] FACS analysis of mouse spleen cells one week after injection showed that EDV was superior to the saline group. Covid-αGC Injected mice showed an increase in the number of CD3+ CD8+ cytotoxic T cells (Figure 13A). When AIMS assays were performed on ex vivo spleen cells and stimulated with spike proteins, the virus antigen-specific CD69+ CD137+ population within the cytotoxic T cell population was found to be increased to a higher level compared to PHA-stimulated positive controls (Figure 13B).

[0199] Four weeks after the first injection, EDV Covid-αGC In mice administered via IM injection, the highest levels of spike protein-specific IgG (Figure 14A) and IgM (Figure 14B) were observed in their serum. Interestingly, EDV Control-αGC The serum of mice administered with the drug was found to contain "specific" antibodies against the spike protein. This finding was confirmed by neutralizing antibody analysis. EDV Covid-αGC The serum of mice administered with EDV contained the most neutralizing antibodies, but EDV Control-αGC EDV Covid and EDV αGC Even in the serum of mice administered αGC, the inhibition of spike protein binding to the hACE receptor was measurable (Figure 14C). αGC alone possesses antiviral activity, suggesting that its administration can suppress the binding of the virus to cells in the body. On the other hand, EDV without αGC... Covid On its own, EDV Covid-αGCCompared to when other substances were administered, neutralizing antibodies were produced in the serum, but at a much lower level. This indicates that incorporating αGC as an immune adjuvant into this system is important as a key component of a functional vaccine.

[0200] Furthermore, to demonstrate the specificity of the antibody response, B cells were extracted from the bone marrow of treated mice 4 weeks after the initial injection and stimulated with spike protein in vitro for 48 hours. EDV Covid-αGC B cells from mice treated with this method produced the highest levels of spike protein-specific IgG (Figure 15A) and IgM (Figure 15B) compared to all other treatment groups.

[0201] FACS analysis of ex vivo spleen cells from treated mice showed that EDV Covid-αGC The treatment was shown to increase CD69+ CD137+ cytotoxic T cells compared to all other treatment conditions (Figure 9A). Furthermore, when ex vivo spherocytes were stimulated with spike proteins, EDV Covid-αGC and EDV Covid Compared to PHA-stimulated controls, a similar increase in virus antigen-specific CD69+ CD137- cells was observed within the cytotoxic T cell population of treated mice (Figure 9B). This was not observed in any of the other treatment groups. This suggests that EDV Covid-αGC Unlike antiviral responses induced by treatment, the antiviral properties of αGC suggest that they are not antigen-specific but rather broad-spectrum.

[0202] While specific embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with the ordinary art in the art, without departing from the broader aspects of the art as defined in the following claims.

[0203] Embodiments described herein as exemplary may be suitably carried out even without elements, limitations, or restrictions not specifically disclosed herein. Therefore, expressions such as “comprising,” “including,” and “containing” should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are for illustrative purposes only, not limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or any part thereof, although it should be acknowledged that various modifications are possible within the scope of the claimed technology. Moreover, the expression “essentially consisting of” should be understood to include the elements specifically described and any additional elements that do not materially affect the fundamental and novel characteristics of the claimed technology.

[0204] This disclosure is not limited in terms of the specific embodiments described herein. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and compositions within the scope of this disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. This disclosure is limited only by the terms of the appended claims, and together with the entire scope of equivalents to which such claims are entitled. It should be understood that this disclosure is not limited to any particular methods, reagents, compounds, or compositions, and these may, of course, change. It should also be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit them.

[0205] Furthermore, if any feature or aspect of this disclosure is described in terms of the Markush group, a person skilled in the art will recognize that this disclosure is also described in terms of any individual member or subgroup of a member of the Markush group.

[0206] As will be understood by those skilled in the art, for all purposes, particularly in terms of providing written descriptions, all scopes disclosed herein encompass all possible subranges and combinations of subranges, including the endpoint. The scopes described can be adequately described and readily recognized as being able to be broken down into at least half, one-third, one-quarter, one-fifth, one-tenth, and so on. As a non-limiting example, each scope described herein can be readily broken down into the lower third, middle third, upper third, and so on. Furthermore, as will be understood by those skilled in the art, all language such as “up to,” “at least,” “greater than,” and “less than” refers to a scope that includes the number mentioned and can then be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, the scope includes individual components.

[0207] All publications, patent applications, issued patents, and other documents referenced herein are incorporated herein by reference in the same manner as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated to be incorporated by reference in whole. Definitions contained in the text incorporated by reference are excluded to the extent that they conflict with the definitions in this disclosure.

[0208] Other embodiments are described in the following claims.

[0209] References Duan, YN & Qin, J. Pre- and post-treatment chest CT findings: 2019 novel coronavirus (2019-nCoV) pneumonia. Radiology 2020. Grosskopf, LA et al., Prevention and Control of Seasonal Influenza by Vaccines. Recommendations of the Consultative Committee on Immunization Implementation - United States, 2018-19 Influenza Season. MMWR Recommends. Rep. 67, 1-20 (2018). Guan, W. et al. Clinical characteristics of COVID-19 in China in 2019. medRxiv. (2020). Huang, C. et al. Clinical characteristics of COVID-19 patients in Wuhan, China in 2019. Lancet. 395, 497-506 (2020). Jiang, S., He, Y. & Liu, S. SARS vaccine development. Emergency Infectious Diseases. 11, 1016-1020 (2005). Wu, F. et al. A novel coronavirus associated with human respiratory disease in China. Nature. (2020). Shang W, Yang Y, Rao Y, and Rao X. The SARS-CoV-2 outbreak calls for a viral vaccine. npj Vaccines (2020) 5:18. Regulanaba, JA et al., found that a severe acute respiratory syndrome coronavirus with a mutation in the E protein is attenuated and is a promising vaccine candidate. J. Virol. 89, 3870-3887 (2015). Genomic characterization of a novel human pathogenic coronavirus 2019 isolated from a patient who developed atypical pneumonia after visiting Wuhan. Emergency Microbes Infect. 9, 221-236 (2020). Ksiazek TG, Erdman D, Goldsmith CS, et al. Novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 2003;348(20):1953-1966. Drosten C, Gunther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med. 2003;348(20):1967-1976. Zaki AM, van Boheemen S, Bestebroer TM, et al., published a novel coronavirus isolated from pneumonia patients in Saudi Arabia. N Engl J Med. 2012;367(19):1814-1820. Lew TWK, Kwek TK, Tai D, et al. Acute respiratory distress syndrome in patients with severe acute respiratory syndrome. JAMA 2003;290(3):374-380. Source: http: / / jama.ama-assn.org / cgi / content / abstract / 290 / 3 / 374. Agnihothram S, Gopal R, Yount BL, et al. evaluated the serological and antigenic relationships between Middle East Respiratory Syndrome coronavirus and other coronaviruses to develop a vaccine platform for rapid response to emerging coronaviruses. J Infect Dis. 2014;209(7):995-1006. Source: http: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3952667 / Bolles M, Deming D, Long K, et al. found that a dual-inactivated vaccine for severe acute respiratory syndrome coronavirus provides incomplete protection in mice and increases the eosinophilic proinflorable lung response upon challenge. J Virol. 2011;85(23):12201-12215. Source: http: / / jvi.asm.org / content / 85 / 23 / 12201.abstract Sheahan T, Whitmore A, Long K, et al. Successful vaccination strategy to protect aged mice from lethal challenges by influenza virus and severe acute respiratory syndrome coronavirus. J Virol. 2011;85(1):217-230. Available from: http: / / jvi.asm.org / content / 85 / 1 / 217.abstract Su, Brahmbhatt et.al., Infection and Immunity, 60(8):3345-3359 (1992). Menachery VD, Yount BL Jr, and Debbink K et al. suggest that circulating SARS-like clusters of bat coronaviruses have the potential to appear in humans. Nat Med. 2015;21:1508-1513. Schoggins JW, Wilson SJ, Panis M, et al., found that diverse gene products act as effectors in the type I interferon antiviral response. *Nature* 2011;472(7344):481-485.

Claims

1. (a) A vector comprising a plasmid encoding at least one viral antigen; and (b) A vector containing the CD1d recognition antigen; and (c) at least one pharmaceutically acceptable carrier, A composition comprising, wherein at least one of vector (a) and vector (b) is an intact, bacterial-derived minicell or a dead bacterial cell.

2. The composition according to claim 1, wherein vector (a) is a first intact bacterial minicell or dead bacterial cell, and vector (b) is a second intact bacterial minicell or dead bacterial cell.

3. The composition according to claim 1, wherein vector (a) and vector (b) are identical, intact, bacterial-derived minicells or dead bacterial cells containing plasmids encoding a CD1d recognition antigen and at least one viral antigen.

4. The composition according to claim 1, wherein one of vector (a) and vector (b) is not an intact bacterial minicell or dead bacterial cell, and the other of vector (a) and vector (b) is an intact bacterial minicell or dead bacterial cell.

5. The viral antigens include: alpha coronavirus; bat coronavirus CDPHE15 and other coracoviruses; bat coronavirus HKU10 or rhinolophas fermecinum alpha coronavirus HuB-2013 and other decacoviruses; human coronavirus 229E and other dubinacoviruses; rat coronavirus Rn rat coronavirus and other lucacoviruses; ferret coronavirus or minkovirus 1 and other minunacoviruses; miniopters bat coronavirus 1 or miniopters bat coronavirus HKU8 and other minunacoviruses; myotitis rickettii alpha coronavirus Sax-2011 and other myoctacoviruses; nyctaras bertinus alpha coronavirus SC-2013 and other nyctacoviruses; porcine epidemic diarea virus or scotophilus bat coronavirus 512 and other pedacoviruses; rhinolophas bat coronavirus HKU2 and other rhinacoviruses; human coronavirus NL63 or NL63-related bat coronavirus strains. Cetracoviruses such as BtKYNL63-9b; tegacoviruses such as alphacoronavirus 1; envelopecoviruses such as betacoronavirus 1, human coronavirus OC43, China rat coronavirus HKU24, human coronavirus HKU1 or murine coronavirus; hibecoviruses such as bat Hp-betacoronavirus Cho-Chiang 2013; marvecoviruses such as hedgehog coronavirus 1, Middle East respiratory syndrome-associated coronavirus (MERS-CoV), pipistrelle bat coronavirus HKU5 or tyronycteris bat coronavirus HKU4; novecoviruses such as russet bat coronavirus GCCDC1, russet bat coronavirus HKU9, surveycoviruses such as severe acute respiratory syndrome-associated coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19); deltacoronavirus; andecoviruses such as wijon coronavirus HKU20;A composition according to any one of claims 1 to 4, comprising, or having characteristics thereof, a virus selected from the group consisting of: buldecoviruses such as buldecovirus HKU11, swine coronavirus HKU15, munia coronavirus HKU13, or white-eye coronavirus HKU16; herdecoviruses such as night heron coronavirus HKU19; mudecoviruses such as van coronavirus HKU21; gamma coronavirus; segacoviruses such as beluga whale coronavirus SW1; and avian coronaviruses.

6. The composition according to any one of claims 1 to 5, wherein the viral antigen is encoded by a polynucleotide containing the sequence of SARS-CoV-2, or a polynucleotide having at least 80% sequence identity with a polynucleotide containing the sequence of SARS-CoV-2.

7. The composition according to any one of claims 1 to 5, wherein the viral antigen comprises or is characterized by human coronavirus 229E, human coronavirus OC43, SARS-CoV, HCoV NL63, HKU1, MERS-CoV, or SARS-CoV-2.

8. The composition according to claim 7, wherein the viral antigen comprises or has the characteristics of SARS-CoV-2.

9. The composition according to claim 8, wherein the plasmid encodes at least one of the following: the spike (S) protein, the nucleocapsid (N) protein, the membrane (M) protein, and the envelope (E) protein of SARS-CoV-2.

10. The composition according to claim 9, wherein the plasmid encodes a spike (S) protein, a nucleocapsid (N) protein, a membrane (M) protein, and an envelope (E) protein.

11. The composition according to any one of claims 1 to 10, wherein the CD1d recognition antigen comprises a sphingoglycolipid.

12. CD1d-recognizing antigens include α-galactosylceramide (α-GalCer), C-glycoside of α-galactosylceramide (α-C-GalCer), 12-carbon acyl of galactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), l,2-diacyl-3-O-galactosyl-sn-glycerol (BbGL-II), diacylglycerol-containing glycolipid (Glc-DAG-s2), ganglioside (GD3), and gangly A composition according to any one of claims 1 to 11, selected from the group consisting of otriosylceramide (Gg3Cer), glycosylphosphatidylinositol (GPI), α-glucuronide ceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide (iGb3), lipophosphoglycan (LPG), lysophosphatidylcholine (LPC), α-galactosylceramide analog (OCH), slatelceramide, and any derivative thereof.

13. The composition according to any one of claims 1 to 12, wherein the CD1d recognition antigen comprises α-GalCer.

14. The composition according to any one of claims 1 to 13, wherein the CD1d recognition antigen comprises a synthetic α-GalCer analog.

15. The CD1d recognition antigen includes synthetic α-GalCer analogs selected from 6′-deoxy-6′-acetamide α-GalCer (PBS57), naptylurea α-GalCer (NU-α-GC), NC-α-GalCer, 4ClPhC-α-GalCer, PyrC-α-GalCer, α-carba-GalCer, carba-α-D-galactose α-GalCer analog (RCAI-56), 1-deoxy-neo-inositol α-GalCer analog (RCAI-59), 1-O-methylated α-GalCer analog (RCAI-92), and HS44 aminocyclitol ceramide. The composition according to claim 14.

16. The composition according to any one of claims 1 to 15, wherein the CD1d recognition antigen is an IFNγ agonist.

17. The composition according to any one of claims 1 to 16, wherein the composition is formulated for oral administration, injection, intranasal administration, intrapulmonary administration, or topical administration.

18. A method for treating and / or vaccinating against a viral infection, comprising administering the composition described in any one of claims 1 to 16 to a subject in need of such a subject.

19. The target is, (a) If you have or are at risk of developing lymphopenia; and / or (b) When there is a risk of severe illness and / or serious complications due to viral infection; and / or (c) is approximately 50 years of age or older, approximately 55 years of age or older, approximately 60 years of age or older, or approximately 65 years of age or older; and / or (d) having one or more pre-existing conditions selected from the group consisting of diabetes, asthma, respiratory disease, hypertension, and heart disease; and / or (e) immunodeficient; and / or (f) Immunity is weakened due to AIDS, cancer, cancer treatment, hepatitis, autoimmune disease, steroid administration, immunosenescence, or a combination thereof. The method according to claim 18.

20. The administration is (a) to increase the likelihood of survival after exposure to coronavirus; and / or, (b) Reduce the risk of coronavirus infection, The method according to any one of claims 18 to 19.

21. (a) The chance of survival increases by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when measured using any clinically recognized technique; and / or (b) The reduction in infection risk, when measured using any clinically recognized technique, is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The method according to claim 20.

22. The method according to any one of claims 18 to 21, wherein the administration is carried out by any pharmaceutically acceptable method.

23. The method according to any one of claims 18 to 22, wherein the subject is exposed to or is expected to be exposed to an individual infected with the coronavirus.

24. The method according to claim 23, wherein an individual infectious with coronavirus has one or more symptoms selected from the group consisting of fever, cough, shortness of breath, diarrhea, sneezing, runny nose, and sore throat.

25. The method according to any one of claims 18 to 24, wherein the subjects are healthcare workers, persons aged 60 or older, frequent travelers, military personnel, caregivers, or persons with pre-existing conditions that increase the risk of death associated with infection.

26. The method according to any one of claims 18 to 25, further comprising administering one or more antiviral agents.

27. The method according to claim 26, wherein one or more antiviral agents are selected from the group consisting of chloroquine, darunavir, galidesivir, interferon beta, lopinavir, ritonavir, remdesivir, and triazavirin.

28. The method according to any one of claims 18 to 27, wherein the CD1d-recognizing antigen induces a Th1 cytokine response in a subject, and the cytokine optionally includes IFNγ.

29. (a) A first minicell containing a CD1d recognition antigen and a second minicell containing a plasmid encoding at least one viral antigen are administered to the subject simultaneously; and / or, (b) A first minicell containing a CD1d recognition antigen and a second minicell containing a plasmid encoding at least one viral antigen are administered sequentially to the subject; and / or, (c) A first minicell containing a CD1d recognition antigen and a second minicell containing a plasmid encoding at least one viral antigen are repeatedly administered to the subject; and / or (d) Administer to the subject at least once, twice, three times, or four times per week a first minicell containing a CD1d recognition antigen and a second minicell containing a plasmid encoding at least one viral antigen. The method according to any one of claims 18 to 28.

30. Use of the composition according to any one of claims 1 to 17 for the manufacture of a pharmaceutical product, wherein the pharmaceutical product is useful for the treatment and / or vaccination of a viral infection.

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