Vaccine compositions and methods of use thereof

Abstract

The present disclosure provides compositions and methods for use in vaccines comprising a polynucleotide encoding one or more viral antigenic proteins and an enhancer protein, where the enhancer protein is a picornavirus leader (L) or a functional variant thereof. The compositions and methods provided herein may improve the production of functional virus-like particles (VLPs).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 162,496, filed March 17, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] Incorporating sequence tables The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Copy of Sequence Listing in Computer Readable Format (Filename: EXCI_003_03WO_SeqList_ST25, Recorded Date: March 17, 2022, File Size: 266 KB). [Background technology]

[0003] Viral infections, such as influenza, are highly prevalent and often contagious. Viral diseases produce a wide variety of symptoms that vary in character and severity, depending on the type of viral infection and other factors, including the person's age and overall health. Treatment of viral infections can be with varying degrees of success, depending on the type of virus and other factors. Sometimes treatment may only involve managing symptoms.

[0004] The most effective way to combat viral infections is through vaccination, which can induce a global cellular and / or humoral immune response that is protective against future infections. Vaccination provides tremendous public health and economic benefits by preventing the occurrence of viral infections or minimizing their severity. Although vaccines are available to prevent over 20 life-threatening diseases, including viral infections, the emergence of new infectious viruses, e.g., SARS CoV2, may necessitate rapid research and development of vaccines against new viral targets. However, vaccine development remains time-consuming and expensive, even with the latest genome sequencing and other technological advances.

[0005] Thus, there is a need for methods and compositions that are versatile enough to be rapidly adapted for the development of vaccines targeting different viruses. [Brief description of the drawings]

[0006] [Figure 1A] Plasmid maps of CoVEG2 (FIG. 1A) and CoVEG1 (FIG. 1B) are shown, along with the relative positions of the cytomegalovirus (CMV) enhancer and promoter, and Simian virus 40 Poly A (SV40PA). [Figure 1B] Plasmid maps of CoVEG2 (FIG. 1A) and CoVEG1 (FIG. 1B) are shown, along with the relative positions of the cytomegalovirus (CMV) enhancer and promoter, and Simian virus 40 Poly A (SV40PA).

[0007] [Diagram 2] 1 shows expression of SARS-CoV-2 S protein, SARS-CoV-2 M protein, SARS-CoV-2 E protein and SARS-CoV-2 N protein from CoVEG2 in HEK293 cells.

[0008] [Figure 3A]We show that SARS-CoV-2 S, N, M, and E proteins expressed from CoVEG2 can assemble into VLPs and are secreted from cells. See Example 3. Figure 3A shows results from an SDS PAGE experiment showing S, N, M, and E protein bands in the size-exclusion chromatography void sample. Figure 3B shows a chromatogram of a size-exclusion VLP isolation run, while the void volume peak containing VLPs is indicated by an arrow. Figure 3C shows results from a Western blotting experiment showing S, N, M, and E protein bands in the size-exclusion chromatography void sample. E: envelope protein, M: membrane protein, N: nucleocapsid protein, S: spike protein, NT: non-transfected. [Figure 3B] We show that SARS-CoV-2 S, N, M, and E proteins expressed from CoVEG2 can assemble into VLPs and are secreted from cells. See Example 3. Figure 3A shows results from an SDS PAGE experiment showing S, N, M, and E protein bands in the size-exclusion chromatography void sample. Figure 3B shows a chromatogram of a size-exclusion VLP isolation run, while the void volume peak containing VLPs is indicated by an arrow. Figure 3C shows results from a Western blotting experiment showing S, N, M, and E protein bands in the size-exclusion chromatography void sample. E: envelope protein, M: membrane protein, N: nucleocapsid protein, S: spike protein, NT: non-transfected. [Figure 3C]We show that SARS-CoV-2 S, N, M, and E proteins expressed from CoVEG2 can assemble into VLPs and are secreted from cells. See Example 3. Figure 3A shows results from an SDS PAGE experiment showing S, N, M, and E protein bands in the size-exclusion chromatography void sample. Figure 3B shows a chromatogram of a size-exclusion VLP isolation run, while the void volume peak containing VLPs is indicated by an arrow. Figure 3C shows results from a Western blotting experiment showing S, N, M, and E protein bands in the size-exclusion chromatography void sample. E: envelope protein, M: membrane protein, N: nucleocapsid protein, S: spike protein, NT: non-transfected.

[0009] [Figure 4] 1 shows the study design for testing the immunogenicity of CoVEG1 and CoVEG2 plasmids in mice.

[0010] [Figure 5A] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5B] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5C] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5D] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5E] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5F] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5G] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5H] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5I] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5J]The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5K] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5L] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5M] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5N] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5O] The plasmid maps of CoVEG3 to 17 are shown respectively. [Figure 5P] The plasmid map of the control S-only plasmid is shown. CMV: cytomegalovirus, SV40PA: simian virus 40 polyA.

[0011] [Figure 6-1] Schematic diagrams of the expression cassettes in each of CoVEG3-17 are shown. Each of the plasmids CoVEG3-17 differs in the genes encoded, the order of genetic elements, and the presence or absence of regulatory elements, such as viral packaging signals. The boxes marked "M", "S", "N", and "E" indicate genes encoding membrane proteins, spike proteins, nucleocapsid proteins, and envelope proteins, respectively. The box marked "L" refers to the gene encoding the L enhancer protein. "S(Mut)" indicates a prefusion conformation-stabilized spike protein mutant in which the internal endogenous furin cleavage site has been mutated to contain the following amino acid substitutions: R682G, R683S, R685S, and further has two amino acid substitutions, K986P and V987P. [Figure 6-2]Schematic diagrams of the expression cassettes in each of CoVEG3-17 are shown. Each of the plasmids CoVEG3-17 differs in the genes encoded, the order of genetic elements, and the presence or absence of regulatory elements, such as viral packaging signals. The boxes marked "M", "S", "N", and "E" indicate genes encoding membrane proteins, spike proteins, nucleocapsid proteins, and envelope proteins, respectively. The box marked "L" refers to the gene encoding the L enhancer protein. "S(Mut)" indicates a prefusion conformation-stabilized spike protein mutant in which the internal endogenous furin cleavage site has been mutated to contain the following amino acid substitutions: R682G, R683S, R685S, and further has two amino acid substitutions, K986P and V987P.

[0012] [Figure 7] Shown are images from anti-spike (S) protein antibody immunostaining of HEK293T cells transfected with each of the indicated plasmids or a control plasmid expressing only the S protein without other viral proteins (M, N, or E). The images confirm expression of the S protein in these cells.

[0013] [Figure 8] Results from Western blot analysis of preparations of viral-like particles (VLPs) isolated from cell culture supernatants when transiently transfected with the indicated CoVEG constructs are shown. "S" indicates transient transfection with a control plasmid expressing only the S protein without other viral proteins (M, N, or E). Staining with anti-RBD (left) and anti-N protein (right) antibodies indicates that the tested plasmids are capable of expressing and secreting viral structural proteins into the cell culture supernatant as VLPs. Red arrows indicate full-length proteins.

[0014] [Figure 9A]Shown are total signal values ​​obtained from ELISA analysis using 1:500 diluted serum samples from BALB / c mice injected with each of the indicated CoVEG plasmids after 56 days. [Figure 9B] Shown are endpoint titer values ​​obtained from ELISA analysis using serum samples from BALB / c mice injected with each of the indicated CoVEG plasmids after 56 days. Endpoint titer refers to the reciprocal of the highest antibody dilution at which the ELISA signal (absorbance at 450 nm) exceeds 3 standard deviations above the background signal.

[0015] [Figure 10] Figure 1 shows the percent (%) inhibition of in vitro binding of the spike protein receptor binding domain (RBD) to the ACE2 receptor by serum samples obtained from mice injected with each of the indicated plasmids using the commercially available cPass™ Neutralization Assay (GenScript). The results show that serum samples obtained from mice injected with CoVEG5 and CoVEG8 have neutralizing antibodies. The positive and negative controls are assay controls for the cPass Neutralization Assay Kit and contain known amounts of SARS-CoV-2 neutralizing antibodies or seronegative samples, respectively, and are used according to the manufacturer's protocol.

[0016] [Figure 11] Shown are results from Western blot analysis of preparations of virus-like particles (VLPs) isolated from cell culture supernatants when transiently transfected with the indicated CoVEG constructs. Staining with anti-RBD (left) and anti-N protein (right) antibodies indicates that the tested plasmids are capable of expressing and secreting viral structural proteins as VLPs into the cell culture supernatant with varying efficiency.

[0017] [Figure 12]Shown are results from Western blot analysis of preparations of virus-like particles (VLPs) isolated from cell culture supernatants when transiently transfected with the indicated CoVEG constructs. Staining with anti-RBD (left) and anti-N protein (right) antibodies indicates that the tested plasmids are capable of expressing and secreting viral structural proteins as VLPs into the cell culture supernatant with varying efficiency.

[0018] [Figure 13] Shown are the total signal values ​​obtained from ELISA analysis from BALB / c mice injected either intradermally (ID) or intramuscularly (IM) with each of the indicated CoVEG plasmids 15 days later. "S only" indicates administration of spike protein alone.

[0019] [Figure 14A] 1 shows maps of plasmids encoding West Nile Virus proteins, preM protein, and envelope protein, along with an enhancer protein (EMCV L1 protein). [Figure 14B] Maps of a control plasmid encoding only West Nile virus proteins, the preM protein, and the envelope protein are shown. CMV: cytomegalovirus, SV40PA: simian virus 40 polyA.

[0020] [Figure 15A] The total signal values ​​obtained from the ELISA analysis of VLP secretion in HEK293 cells are shown. Figure 15A shows the ELISA analysis of VLP secretion performed using anti-spike (S protein) antibody. Figure 15B shows the ELISA analysis of VLP secretion performed using anti-nucleocapsid (N protein) antibody. [Figure 15B]The total signal values ​​obtained from the ELISA analysis of VLP secretion in HEK293 cells are shown. Figure 15A shows the ELISA analysis of VLP secretion performed using anti-spike (S protein) antibody. Figure 15B shows the ELISA analysis of VLP secretion performed using anti-nucleocapsid (N protein) antibody.

[0021] [Figure 16] Transmission electron micrographs (TEM) of CoVEG10 and CoVEG20 protein expression are shown. Figure 16, left, shows a TEM of CoVEG10 containing the L regulatory protein. Figure 16, right, shows a TEM of CoVEG20 lacking the L regulatory protein.

[0022] [Figure 17] Shown are images from anti-nucleocapsid (N) protein antibody immunostaining of HEK293T cells transfected with each of the indicated plasmids.

[0023] [Figure 18] Shown are results from Western blot analysis of preparations of virus-like particles (VLPs) isolated from cell culture supernatants when transiently transfected with the indicated CoVEG constructs. Staining with anti-RBD (left) and anti-N protein (right) antibodies indicates that the tested plasmids are capable of expressing and secreting viral structural proteins as VLPs into the cell culture supernatant with varying efficiency.

[0024] [Figure 19] Western blot results of co-immunoprecipitation experiments of CoVEG10 plasmids are shown. (Left) Receptor binding domain (RBD) pull-down signal of N protein during elution indicates that N protein was retained within the particles. (Right) Co-IP was performed without anti-RBD antibody, indicating that N protein did not bind nonspecifically to the precipitating resin.

[0025] [Figure 20]1 shows antibody binding titers from CoVEG5 and 9-14 plasmids, and spike (S) protein containing plasmid alone, following intramuscular (IM) or intradermal (ID) injection in C57BL / 6 mice.

[0026] [Figure 21] ELISA analysis of neutralizing antibodies from the samples shown in FIG. 20 is shown.

[0027] [Figure 22] 1 shows antibody binding titers from CoVEG 9, 10, and 18-20 plasmids, and spike (S) protein containing plasmid alone, following intramuscular (IM) or intradermal (ID) injection in C57BL / 6 mice.

[0028] [Diagram 23] ELISA analysis of neutralizing antibodies produced in response to intramuscular (IM) or intradermal (ID) injection of CoVEG 9, 10 and 20 plasmids in C57BL / 6 mice.

[0029] [Figure 24A] Figure 24 shows the results of T cell analysis of CoVEG10 and CoVEG20. Figure 24A shows the results of T cell analysis of CoVEG10. Figure 24B shows the results of T cell analysis of CoVEG20. [Figure 24B] Figure 24 shows the results of T cell analysis of CoVEG10 and CoVEG20. Figure 24A shows the results of T cell analysis of CoVEG10. Figure 24B shows the results of T cell analysis of CoVEG20.

[0030] [Diagram 25]Figure 1 shows ELISA analysis of VLPs purified from cell culture supernatants of HEK293T cells transfected with isolated and resuspended West-Nile Virus (WNV) constructs with (WNV+EG, circles) and without (WNC, squares) enhancer protein. The specificity of the ELISA was tested against a plasmid containing GFP that gave no signal in the specific ELISA assay. The ELISA analysis revealed that isolation of VLPs by ultracentrifugation with the addition of enhancer protein contained more active VLPs than in the absence of enhancer protein. This was particularly surprising since the total amount of protein produced was higher in the absence of enhancer protein, further indicating that the addition of enhancer protein increased the quality of the expressed target protein.

[0031] [Figure 26A] Figure 26 shows a time course analysis of cell culture supernatants obtained from HEK293T cells overexpressing West Nile Virus (WNV) constructs with (WNV+L) and without (WNV) enhancer protein and supernatants containing the constructs. Figure 26A shows an ELISA analysis showing that VLP concentration in the WNV+L (left) construct peaked at 72 hours post-transfection and gradually decreased thereafter. This was evidence of VLP secretion from healthy cells, as the expression profile followed the expected production of VLPs from transient transfection and the associated VLP particle half-life. In contrast, the WNV construct in the absence of enhancer protein showed a constant increase in VLPs in the supernatant (right), which is more consistent with non-specific release of proteins from cells during cell death rather than active secretion. Figure 26B confirms the ELISA analysis with cell images. The WNV+L cell image (left) showed little to no cell death, whereas the WNV cell image showed visible signs of cell death starting 72 hours after transfection (right, cell death indicated by a black asterisk). [Figure 26B]Figure 26 shows a time course analysis of cell culture supernatants obtained from HEK293T cells overexpressing West Nile Virus (WNV) constructs with (WNV+L) and without (WNV) enhancer protein and supernatants containing the constructs. Figure 26A shows an ELISA analysis showing that VLP concentration in the WNV+L (left) construct peaked at 72 hours post-transfection and gradually decreased thereafter. This was evidence of VLP secretion from healthy cells, as the expression profile followed the expected production of VLPs from transient transfection and the associated VLP particle half-life. In contrast, the WNV construct in the absence of enhancer protein showed a constant increase in VLPs in the supernatant (right), which is more consistent with non-specific release of proteins from cells during cell death rather than active secretion. Figure 26B confirms the ELISA analysis with cell images. The WNV+L cell image (left) showed little to no cell death, whereas the WNV cell image showed visible signs of cell death starting 72 hours after transfection (right, cell death indicated by a black asterisk).

[0032] [Figure 27A] Analysis of neutralizing antibodies for CoVEG9, spike constructs with enhancer protein (Spike+L), and spike constructs without enhancer protein (Spike) at 42 and 70 days after immunization of mice. The presence of neutralizing antibodies was detected using a commercially available cPass™ SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) kit (GenScript). Analysis showed that all constructs lost some neutralizing capacity over time, but the addition of enhancer protein prolonged the presence of desired neutralizing antibodies in the serum of test animals (CoVEG9 and Spike+L). Figure 27A shows individual animals per group at 42 and 70 days. Figure 27B shows the median neutralizing antibody levels in each treatment group for the same data. [Figure 27B]Analysis of neutralizing antibodies for CoVEG9, spike constructs with enhancer protein (Spike+L), and spike constructs without enhancer protein (Spike) at 42 and 70 days after immunization of mice. The presence of neutralizing antibodies was detected using a commercially available cPass™ SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) kit (GenScript). Analysis showed that all constructs lost some neutralizing capacity over time, but the addition of enhancer protein prolonged the presence of desired neutralizing antibodies in the serum of test animals (CoVEG9 and Spike+L). Figure 27A shows individual animals per group at 42 and 70 days. Figure 27B shows the median neutralizing antibody levels in each treatment group for the same data.

[0033] [Figure 28] Immunofluorescence images from cells overexpressing West Nile Virus (WNV) constructs with (WNV+L, left) or without (WNV, right) enhancer proteins are shown. As observed in other examples, the total amount of protein was reduced when enhancer proteins were added (WNV+L, left), as indicated by a weaker Alexa Fluor 488 Fluor signal of the secondary antibody used in immunostaining, compared to WNV (right). However, the absence of enhancer proteins led to the formation of nuclear foci consistent with aggregation or misfolding of the expressed protein (right, arrows), indicating a lower quality of expressed protein compared to constructs with enhancer proteins (WNV+L). Summary of the Invention [Means for solving the problem]

[0034] The present disclosure provides a composition for use as a vaccine, comprising an expression cassette comprising a polynucleotide encoding a viral protein and a polynucleotide encoding an enhancer protein. In some embodiments, the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof. In some embodiments, the amino acid sequence of the enhancer protein has at least 95% identity to SEQ ID NO:1 or at least 95% identity to SEQ ID NO:2. In some embodiments, the amino acid sequence of the enhancer protein is SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the polynucleotide encoding the enhancer protein is operably linked to a polynucleotide encoding an internal ribosome entry site (IRES). In some embodiments, the polynucleotide encoding the IRES is SEQ ID NO:24.

[0035] In some embodiments, the viral protein is a viral antigen. In some embodiments, the viral protein is from a virus selected from the group consisting of coronavirus, influenza virus, Hepatitis B virus, Human Papilloma virus (HPV), West Nile virus, and Human Immunodeficiency Virus (HIV). In some embodiments, the viral protein is from a coronavirus. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the betacoronavirus is a severe acute respiratory syndrome (SARS) virus. In some embodiments, the SARS virus is a SARS-CoV-2 virus. In some embodiments, the betacoronavirus is a Middle East respiratory syndrome (MERS) virus.

[0036] In some embodiments, the coronavirus protein is a coronavirus spike protein. In some embodiments, the spike protein shares at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 13. In some embodiments, the spike protein is SEQ ID NO: 13. In some embodiments, the coronavirus protein is a coronavirus membrane (M) protein. In some embodiments, the M protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 33. In some embodiments, the M protein is SEQ ID NO: 33. In some embodiments, the coronavirus protein is a coronavirus envelope (E) protein. In some embodiments, the E protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:22. In some embodiments, the E protein is SEQ ID NO:22. In some embodiments, the coronavirus protein is a coronavirus nucleocapsid (N) protein. In some embodiments, the N protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:20. In some embodiments, the N protein is SEQ ID NO:20. In some embodiments, the coronavirus proteins form virus-like particles (VLPs).

[0037] In some embodiments, the viral protein is derived from West Nile Virus, hi some embodiments, the viral protein is a precursor membrane protein (preM), an envelope glycoprotein (E), or a combination thereof.

[0038] The present disclosure provides a vector for use as a vaccine, comprising an expression cassette comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:30.

[0039] The present disclosure provides a vector for use as a vaccine, comprising an expression cassette comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:31.

[0040] The present disclosure provides a vector for use as a vaccine comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35-49, 55, and 62.

[0041] In some embodiments, the vector is a naked polynucleotide. In some embodiments, the vector is a deoxyribonucleic acid (DNA) polynucleotide. In some embodiments, the vector is a ribonucleic acid (RNA) polynucleotide. In some embodiments, the vector comprises a plasmid. In some embodiments, the vector comprises linear DNA. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, and the like, and in some cases may replicate autonomously or integrate into a host cell chromosome. Vectors can also be naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, polylysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, and the like, that do not replicate autonomously.

[0042] In some embodiments, the vector is an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector, a replication-competent adenoviral vector, a replication-deficient adenoviral vector, a herpesvirus vector, a baculovirus vector, a non-viral plasmid, a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, an artificial chromosome, or an adenoviral vector. In some embodiments, the vector is a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).

[0043] In some embodiments, the expression cassette comprises a promoter operably linked to each of the polynucleotide sequences of the expression cassette. In some embodiments, the vector comprises a DNA polynucleotide, the DNA polynucleotide encoding a viral packaging signal. In some embodiments, the viral packaging signal is an RNA polynucleotide. In some embodiments, the viral packaging signal is derived from a coronavirus.

[0044] The present disclosure provides a vaccine composition comprising any one of the vectors disclosed herein and a pharma- ceutically acceptable carrier. In some embodiments, the vaccine composition comprises an adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is monophosphoryl lipid A (MPL).

[0045] The present disclosure provides a method of expressing a viral antigen in a eukaryotic cell, comprising contacting the cell with any one of the vectors disclosed herein. In some embodiments, contacting the cell with the vector results in (i) expression of the antigen at a higher expression level, and / or (ii) expression of the antigen for a longer period of time, and / or (iii) expression of the antigen with better protein quality than a vector lacking an enhancer protein. In some embodiments, contacting the cell with the vector results in (i) expression of a virus-like particle (VLP) comprising the antigen at a higher expression level, and / or (ii) expression of the VLP comprising the antigen for a longer period of time, and / or (iii) expression of the VLP comprising the antigen with better protein quality than a vector lacking an enhancer protein.

[0046] In some embodiments, the vector comprises a polynucleotide encoding a viral packaging signal, and contacting a cell with the vector results in expression of the viral packaging signal and VLPs encapsidating the viral packaging signal. In some embodiments, the vector results in the formation of a greater number of VLPs compared to a control vector lacking a polynucleotide encoding a viral packaging signal.

[0047] The present disclosure provides a method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of any one of the vaccine compositions disclosed herein. In some embodiments, the subject's tissue at the administration site expresses the antigen and / or VLPs comprising the antigen. In some embodiments, the subject's tissue at the administration site (i) expresses the antigen and / or VLPs comprising the antigen at a higher expression level, and / or (ii) expresses the antigen and / or VLPs comprising the antigen for a longer period of time, and / or (iii) expresses the antigen and / or VLPs comprising the antigen with better protein quality than when a vector lacking an enhancer protein is administered. In some embodiments, the vector comprises a polynucleotide encoding a viral packaging signal, the subject's tissue at the administration site expresses the viral packaging signal, and the VLPs encapsidate the viral packaging signal. In some embodiments, the vector results in the expression of a greater number of VLPs compared to a control vector lacking a polynucleotide encoding a viral packaging signal. In some embodiments, VLPs that encapsidate a viral packaging signal are more immunogenic than control VLPs that contain an antigen but lack the viral packaging signal.

[0048] In some embodiments, the method induces an antibody response in the subject. In some embodiments, the antibody response is a neutralizing antibody response. In some embodiments, the method induces a cellular immune response. In some embodiments, the method induces a prophylactic, protective, and / or therapeutic immune response in the subject. In some embodiments, administration is intradermal, intramuscular, subcutaneous, or intranasal.

[0049] The present disclosure provides a polynucleotide comprising an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof. In some embodiments, the amino acid sequence of the enhancer protein has at least 95% identity to SEQ ID NO:1 or at least 95% identity to SEQ ID NO:2. In some embodiments, the amino acid sequence of the enhancer protein is SEQ ID NO:1 or SEQ ID NO:2.

[0050] In some embodiments, the polynucleotide encoding the enhancer protein is operably linked to a polynucleotide encoding an internal ribosome entry site (IRES). In some embodiments, the polynucleotide encoding the IRES is SEQ ID NO: 24. In some embodiments, the coronavirus proteins form virus-like particles (VLPs).

[0051] The present disclosure provides a polynucleotide comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 30. The present disclosure provides a polynucleotide comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 31.

[0052] In some embodiments, the polynucleotide is a naked polynucleotide. In some embodiments, the polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide. In some embodiments, the polynucleotide is a ribonucleic acid (RNA) polynucleotide. In some embodiments, the expression cassette comprises a promoter operably linked to each of the polynucleotide sequences of the expression cassette.

[0053] The disclosure provides a kit comprising a vector, the vector comprising an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof.

[0054] The present disclosure provides a vector comprising an expression cassette, the expression cassette comprising a promoter linked to a target gene, and the vector comprising a nucleic acid sequence encoding a viral packaging element. In some embodiments, the viral packaging element is an RNA polynucleotide. In some embodiments, the viral packaging element is derived from a coronavirus. In some embodiments, the viral packaging element is derived from SARS-CoV2. In some embodiments, the nucleic acid sequence encoding the viral packaging element has at least about 70% identity to the nucleic acid sequence of SEQ ID NO:34.

[0055] The present disclosure provides a method for expressing a target protein in a eukaryotic cell, comprising contacting the cell with any one of the vectors disclosed herein. In some embodiments, contacting the cell with the vector results in the formation of a virus-like particle (VLP) comprising the target protein. In some embodiments, contacting the cell with the vector results in the formation of a greater number of virus-like particles (VLPs) comprising the target protein, compared to a control vector comprising an expression cassette but lacking a nucleic acid sequence encoding a viral packaging element. In some embodiments, the nucleic acid sequence encoding a viral packaging element has at least about 70% identity to the nucleic acid sequence of SEQ ID NO: 34.

[0056] The disclosure provides a vector for use as a vaccine comprising an expression cassette comprising, in 5' to 3' order, the following elements: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises the amino acid sequence of SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a first proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises the amino acid sequence of SEQ ID NO:20 or at least 95% identical thereto, a polynucleotide encoding a second proteolytic cleavage site, a polynucleotide encoding an S protein, wherein the S protein comprises the amino acid sequence of SEQ ID NO:13 or at least 95% identical thereto, a polynucleotide encoding a third proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises the amino acid sequence of SEQ ID NO:22 or at least 95% identical thereto, a polynucleotide encoding an IRES sequence, wherein the IRES sequence comprises a polynucleotide sequence of SEQ ID NO:24 or at least 95% identical thereto, and a polynucleotide encoding an enhancer L protein, wherein the L protein comprises the amino acid sequence of SEQ ID NO:2 or at least 95% identical thereto.

[0057] The disclosure also provides a polypeptide comprising the following elements, in 5' to 3' order: a promoter, a polynucleotide encoding an M protein or an amino acid sequence at least 95% identical thereto, wherein the M protein comprises SEQ ID NO:33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a first proteolytic cleavage site, a polynucleotide encoding an S protein, wherein the S protein comprises SEQ ID NO:13 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a second proteolytic cleavage site, a polynucleotide encoding an E protein or a polynucleotide sequence at least 95% identical thereto, wherein the protein comprises SEQ ID NO:22 or an amino acid sequence at least 95% identical thereto, an IRES sequence, and a polynucleotide encoding an IRES sequence, wherein the IRES sequence comprises SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises SEQ ID NO:2 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO:20 or an amino acid sequence at least 95% identical thereto, and a polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 95% identical thereto.

[0058] The disclosure also provides a vector for use as a vaccine comprising an expression cassette comprising, in 5' to 3' order, the following elements: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises the amino acid sequence of SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises the amino acid sequence of SEQ ID NO:20 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, wherein the mutated S protein comprises the amino acid sequence of SEQ ID NO:51 or SEQ ID NO:52, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises the amino acid sequence of SEQ ID NO:22 or at least 95% identical thereto, a polynucleotide encoding an IRES sequence, wherein the IRES sequence comprises the polynucleotide sequence of SEQ ID NO:24 or at least 95% identical thereto, and a polynucleotide encoding an enhancer L protein, wherein the L protein comprises the amino acid sequence of SEQ ID NO:2 or at least 95% identical thereto.

[0059] The disclosure provides a nucleic acid sequence encoding a nucleic acid vector comprising, in 5' to 3' order, the following elements: a promoter, a first polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an M protein, wherein the M protein comprises SEQ ID NO:33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO:20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, wherein the S protein comprises SEQ ID NO:51 or SEQ ID NO:52 or an amino acid sequence at least 95% identical thereto. and a second polynucleotide encoding a viral packaging signal, the viral packaging signal comprising a polynucleotide having a sequence similar to that of SEQ ID NO:34 or a polynucleotide sequence at least 95% identical thereto. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] Virus-like particles (VLPs) are composed of viral structural proteins. Although VLPs are immunogenic, they are non-infectious. Therefore, VLPs have great potential for use in vaccine development. VLPs can be produced in vitro and then administered to subjects that require immunization. Alternatively, VLPs can be produced in vivo in subjects.

[0061] The production of VLPs is difficult, mainly because it often requires the expression of two or more structural proteins from two or more plasmids. In some cases, several plasmids carrying different structural proteins may need to be introduced into host cells in a defined ratio to support the formation of VLPs. This process can be unreliable and often fails to produce sufficient levels of VLPs of the required quality. If the multiple structural proteins required for the formation of VLPs could be expressed from a single plasmid or a single RNA transcript, the process of making VLPs would be greatly simplified, thus providing a much-needed boost to the development of vaccines that contain VLPs.

[0062] The compositions and methods disclosed herein allow for reliable formation of high levels of VLPs in vivo, and therefore robust induction of immune responses against viral antigens on the VLPs. Furthermore, these compositions and methods can be used to induce immune responses against different viruses, such as coronaviruses (e.g., SARS CoV-2), influenza viruses, and West Nile viruses. definition

[0063] As used herein and in the appended claims, the singular forms "a," "an," and "the" include their plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "antigen" can refer to one or more antigens, and reference to a "method" includes reference to equivalent steps and / or methods known to those of skill in the art and / or others.

[0064] As used herein, the term "about" or "approximately" preceding a numerical value refers to the value plus or minus a range of 10%. For example, "about 100" includes 90 and 110.

[0065] As used herein, unless otherwise indicated, nucleotide sequences are listed in a 5' to 3' orientation and amino acid sequences are listed in an N-terminal to C-terminal orientation.

[0066] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0067] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, a teenager between 13 and 19 years of age, a child (2 to 14 years of age), an infant (1 month to 24 months), or a newborn (up to 1 month). In some embodiments, the adult is about 65 years of age or older, or an elderly person about 60 years of age or older. In some embodiments, the subject is a pregnant woman or a woman who is about to become pregnant. In other embodiments, the subject is not a human, for example, a non-human primate, such as a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject may be a pet, such as a dog or cat.

[0068] As used herein, the terms "immunogen," "antigen," and "epitope" refer to a substance that is capable of eliciting an immune response, such as proteins, including glycoproteins, and peptides.

[0069] As used herein, an "immunogenic response" in a subject results in the development in the subject of a humoral and / or cellular immune response to an antigen.

[0070] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve a result, e.g., to affect a beneficial or desired outcome. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, etc., which can be easily determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system through which it is delivered.

[0071] As used herein, the term "virus-like particle" (VLP) refers to a structure that resembles a virus in at least one attribute, but has not been shown to be infectious. Virus-like particles according to the present disclosure do not carry genetic information that encodes the proteins of the virus-like particle. In general, virus-like particles lack a viral genome and are therefore non-infectious. Furthermore, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified.

[0072] As used herein, an amino acid substitution at a particular position on a protein sequence, synonymously referred to as an amino acid replacement, is indicated herein in the following format: "one letter code of the WT amino acid residue-amino acid position-one letter code of the amino acid residue replacing the WT residue." For example, spike (S) protein, which is an R682G mutant, refers to an S protein in which the wild type residue at amino acid position 682 (R, arginine) is replaced with G, glycine. vector

[0073] The present disclosure provides a vector comprising an expression cassette comprising a polynucleotide encoding an antigen and a polynucleotide encoding an enhancer protein, in some embodiments, the vector is used as a vaccine or as part of a vaccine composition.

[0074] The term "vector" refers to a means by which a nucleic acid may be propagated and / or transported between organisms, cells, or cellular components. Vectors for use according to the present disclosure may include any vector known in the art. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, and the like, and may replicate autonomously or integrate into a host cell's chromosome. Vectors may also be non-autonomously replicating, naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, polylysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, and the like.

[0075] In some embodiments, the vector is an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication-competent adenovirus vector, a replication-deficient adenovirus vector, a herpesvirus vector, a baculovirus vector, a non-viral plasmid, a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, an artificial chromosome, or an adenovirus vector. In some embodiments, the vector is a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC). In some embodiments, the vector is a naked polynucleotide. In some embodiments, the vector is a deoxyribonucleic acid (DNA) polynucleotide. In some embodiments, the vector is a ribonucleic acid (RNA) polynucleotide.

[0076] In some embodiments, the vector comprises a first polynucleotide encoding an antigen and a second polynucleotide encoding an enhancer protein. In some embodiments, the vector has a design as shown in Figure 1A or Figure 1B. In some embodiments, the vector is CoVEG1. In some embodiments, the vector is CoVEG2.

[0077] Table 1 shows the nucleic acid sequences of important regions of CoVEG1 and CoVEG2, and the amino acid sequences that these regions encode. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] i) Expression Cassette

[0078] The vectors disclosed herein may contain one or more expression cassettes. The phrase "expression cassette" as used herein refers to a defined segment of a nucleic acid molecule that contains the minimum elements required for the production of another nucleic acid or protein encoded by that nucleic acid molecule. In some embodiments, the expression cassette contains a promoter. In some embodiments, the promoter is operably linked to each of the polynucleotide sequences of the expression cassette.

[0079] In some embodiments, the vector may comprise an expression cassette, the expression cassette comprising a first polynucleotide encoding an antigen and a second polynucleotide encoding an enhancer protein. In some embodiments, the expression cassette comprises a first promoter operably linked to the first polynucleotide and a second promoter operably linked to the second polynucleotide. In some embodiments, the expression cassette comprises a shared promoter operably linked to both the first polynucleotide and the second polynucleotide.

[0080] In some embodiments, the expression cassette comprises a coding polynucleotide comprising a first polynucleotide and a second polynucleotide linked by a polynucleotide encoding a separation element (e.g., a ribosomal skipping site or a 2A element), wherein the coding polynucleotides are operably linked to a shared promoter.

[0081] In some embodiments, the expression cassette comprises a coding polynucleotide that encodes an enhancer protein and an antigen, linked by a separation element (e.g., a ribosomal skipping site or a 2A element), and the coding polynucleotide is operably linked to a shared promoter.

[0082] In some embodiments, the expression cassette is configured for transcription of a single messenger RNA encoding both the antigen and the enhancer protein, linked by a separation element (e.g., a ribosomal skipping site or a 2A element), and translation of the messenger RNA results in expression of the antigen and the enhancer protein (e.g., the L protein) as separate polypeptides. In some embodiments, the expression cassette disclosed herein comprises one or more proteolytic cleavage sites, for example, 1, 2, 3, 4, or 5 proteolytic cleavage sites. In some embodiments, the proteolytic cleavage site is located between a polynucleotide encoding a first antigen and another polynucleotide encoding a second antigen. In some embodiments, the proteolytic cleavage site is located between a polynucleotide encoding an antigen and a polynucleotide encoding an enhancer protein. In some embodiments, the proteolytic cleavage site comprises the nucleic acid sequence of SEQ ID NO: 50. In some embodiments, the proteolytic cleavage site is a furin cleavage site.

[0083] In some embodiments, the expression cassette disclosed herein comprises a nucleic acid sequence encoding a viral accessory protein, such as an ORF3a protein. In some embodiments, the polynucleotide encoding the ORF3 protein has a nucleic acid sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the nucleic acid sequence of SEQ ID NO:54. In some embodiments, the polynucleotide encoding the ORF3 protein has a nucleic acid sequence of SEQ ID NO:54. In some embodiments, the ORF3 protein has an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the amino acid sequence of SEQ ID NO:53. In some embodiments, the ORF3 protein has the amino acid sequence of SEQ ID NO:53.

[0084] In some embodiments, the vector is selected from the group consisting of CoVEG3-17. In some embodiments, the vector comprises a nucleic acid sequence having at least about 70% identity, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35-49. In some embodiments, the vector comprises a nucleic acid sequence having at least 70% (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100%) identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35-49.

[0085] The nucleic acid sequence of the expression cassette for CoVEG3-17 and the genetic elements therein are listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16]

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

Table 2-44

Table 2-45

Table 2-46

Table 2-47

[0086] In some embodiments, a vector disclosed herein comprises a polynucleotide derived from any one of CoVEG1-17 that encodes a SARS CoV2 spike protein. In some embodiments, a vector disclosed herein comprises a polynucleotide derived from any one of CoVEG1-17 that encodes a SARS CoV2 membrane protein. In some embodiments, a vector disclosed herein comprises a polynucleotide derived from any one of CoVEG1-17 that encodes a SARS CoV2 envelope protein. In some embodiments, a vector disclosed herein comprises a polynucleotide derived from any one of CoVEG1-17 that encodes a SARS CoV2 nucleocapsid protein. In some embodiments, a vector disclosed herein comprises a polynucleotide derived from any one of CoVEG1-17 that encodes an EMCV L protein. In some embodiments, a vector disclosed herein comprises a polynucleotide derived from any one of CoVEG1-17 that encodes an internal ribosome entry site (IRES). In some embodiments, a vector disclosed herein comprises a polynucleotide derived from any one of CoVEG1-17 that encodes a viral packaging signal. ii) Polynucleotides

[0087] Polynucleotides of the present disclosure may include DNA, RNA, and DNA-RNA hybrid molecules. In some embodiments, polynucleotides are isolated from natural sources, prepared in vitro using techniques such as PCR amplification or chemical synthesis, prepared in vivo, for example, via recombinant DNA technology, or prepared or obtained by any suitable method. In some embodiments, polynucleotides are of any shape (linear, circular, etc.) or topology (single-stranded, double-stranded, linear, circular, supercoiled, staggered, nicked, etc.). Polynucleotides may also include nucleic acid derivatives, such as peptide nucleic acids (PNAS) and polypeptide-nucleic acid complexes, nucleic acids having at least one chemically modified sugar residue, backbone, internucleotide linkage, base, nucleotide, nucleoside, or nucleotide analog or derivative, as well as nucleic acids having chemically modified 5' or 3' termini, and nucleic acids having two or more such modifications. Not all linkages in a polynucleotide need be identical.

[0088] A polynucleotide is said to "encode" a protein if it comprises a nucleic acid sequence that can be transcribed and translated (e.g., DNA → RNA → protein) or translated (RNA → protein) to produce an amino acid sequence that corresponds to the amino acid sequence of the protein. In vivo (e.g., within a eukaryotic cell) transcription and / or translation is performed by endogenous or exogenous enzymes. In some embodiments, transcription of the polynucleotides of the disclosure is performed by the endogenous polymerase II (pol II) of the eukaryotic cell. In some embodiments, an exogenous RNA polymerase is provided on the same or a different vector. In some embodiments, a viral polymerase can be used alternatively or additionally. In some embodiments, a vector promoter is used in combination with one or more viral polymerases. In some embodiments, the RNA polymerase is selected from T3 RNA polymerase, T5 RNA polymerase, T7 RNA polymerase, H8 RNA polymerase, EMCV RNA polymerase, HIV RNA polymerase, influenza RNA polymerase, SP6 RNA polymerase, CMV RNA polymerase, T3 RNA polymerase, T1 RNA polymerase, SP01 RNA polymerase, SP2 RNA polymerase, Phi15 RNA polymerase, etc. The viral polymerase is an RNA priming or capping polymerase. In some embodiments, an IRES element is used in conjunction with the viral polymerase.

[0089] The polynucleotides disclosed herein may encode one or more antigens and / or one or more enhancer proteins. In some embodiments, the polynucleotide encodes one antigen. In some embodiments, the polynucleotide encodes one enhancer protein. In some embodiments, the polynucleotide encodes two or more antigens, two or more enhancer proteins, and / or one or more separate elements.

[0090] In some embodiments, the polynucleotide may encode a non-antigenic polypeptide. In some embodiments, the non-antigenic polypeptide may form part of a VLP. Thus, the present disclosure provides a vector comprising a polynucleotide encoding one or more antigens, and / or a polynucleotide encoding one or more non-antigenic polypeptides, and / or a polynucleotide encoding one or more enhancer proteins. In some embodiments, the one or more antigens and the one or more non-antigenic polypeptides can form a virus-like particle (VLP). In some embodiments, the one or more antigens may be derived from one or more proteins of a first virus, and the one or more non-antigenic polypeptides may be derived from one or more proteins of a second virus. iii) Separation element

[0091] In some embodiments, the antigen and enhancer protein according to the present disclosure are encoded on the same vector. In some embodiments, the antigen and enhancer protein according to the present disclosure are encoded on separate vectors. In some embodiments, when the nucleic acid sequences encoding one or more antigens and one or more enhancer proteins are present in the same vector, the vector may contain a separate element for separate expression of the proteins. In some embodiments, the vector is a bicistronic vector or a polycistronic vector. The separate element may be an internal ribosome entry site (IRES) or a 2A element. In some embodiments, the vector may contain a nucleic acid encoding a 2A element or a nucleic acid encoding an IRES.

[0092] In some embodiments, the first polynucleotide or the second polynucleotide, or both, are operably linked to a polynucleotide encoding a 2A element. In some embodiments, a polynucleotide encoding an enhancer protein and / or a polynucleotide encoding an antigen is operably linked to a polynucleotide encoding a 2A element. Non-limiting examples of 2A elements include P2A, E2A, F2A, and T2A. In some embodiments, the amino acid sequence of the 2A peptide has at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween)) to SEQ ID NO: 17. In some embodiments, the amino acid sequence of the 2A peptide is SEQ ID NO: 17.

[0093] In some embodiments, the nucleic acid sequence encoding the 2A peptide has at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween)) to SEQ ID NO: 18 or 69. In some embodiments, the nucleic acid sequence encoding the 2A peptide is SEQ ID NO: 18 or 69.

[0094] In some embodiments, the first polynucleotide or the second polynucleotide, or both, are operably linked to a polynucleotide encoding an internal ribosome entry site (IRES). In some embodiments, the polynucleotide encoding an enhancer protein and / or the polynucleotide encoding an antigen are operably linked to a polynucleotide encoding an IRES. In some embodiments, the polynucleotide encoding an IRES has a nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values ​​and subranges therebetween) to the nucleic acid sequence of SEQ ID NO: 24 or 67. In some embodiments, the polynucleotide encoding an IRES has a nucleic acid sequence of SEQ ID NO: 24 or 67.

[0095] In some embodiments, the antigen and enhancer protein are included in a single fusion protein. In some embodiments, the fusion protein may include a linking element. In some embodiments, the linking element may include a cleavage site for enzymatic cleavage in cis or trans (e.g., a furin, cathepsin, or intein cleavage site). In other embodiments, the fusion protein or linking element does not include a cleavage site, and the expressed fusion protein includes both the target protein and the enhancer protein. In some embodiments, the linking element is a 2A element. iv) Promoter

[0096] A vector according to the present disclosure may include one or more promoters. The term "promoter" refers to a region or sequence located upstream or downstream of the transcription start site that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A polynucleotide or vector according to the present disclosure may include one or more promoters. The promoter may be any promoter known in the art. The promoter may be a forward promoter or a reverse promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the one or more promoters are native promoters. In some embodiments, the one or more promoters are non-native promoters. In some embodiments, the one or more promoters are non-mammalian promoters. Non-limiting examples of RNA promoters for use in the disclosed compositions and methods include U1, human elongation factor-1 alpha (EF-1 alpha), cytomegalovirus (CMV), human ubiquitin, spleen focus-forming virus (SFFV), U6, H1, tRNA Lys , tRNA Ser , and tRNA Arg , CAG, PGK, TRE, UAS, UbC, SV40, T7, Sp6, lac, araBad, trp, and Ptac promoters.

[0097] As used herein, the term "operably linked" refers to elements or structures in a nucleic acid sequence that are linked by their ability to operate and not by their physical location. The elements or structures are characterized by being capable of or achieving a desired operation. It will be recognized by those skilled in the art that elements or structures in a nucleic acid sequence need not be in tandem or contiguous order to be operably linked.

[0098] In some embodiments, the promoter contained by a vector according to the present disclosure is an inducible promoter.

[0099] In some embodiments, the vector according to the present disclosure may further comprise a polynucleotide sequence encoding a polymerase. In some embodiments, the polymerase is a viral polymerase. In some embodiments, the vector disclosed herein comprises a polynucleotide sequence encoding a T7 RNA polymerase. In some embodiments, for example, the vector may comprise a T7 promoter configured for transcription by T7 RNA polymerase of either or both of the polynucleotide encoding the antigen and the second polynucleotide encoding an enhancer protein. antigen

[0100] In some embodiments, the expression or quality of the antigen is significantly improved by expression by the disclosed methods, for example in conjunction with one or more enhancer proteins. In some embodiments, the antigen is derived from a single protein. In some embodiments, the antigen is derived from multiple proteins. In some embodiments, the antigen is a chimeric antigen that includes amino acid sequences from one or more proteins. In some embodiments, the antigen is a viral antigen. A viral antigen may include, but is not limited to, all or part of an amino acid sequence derived from any viral protein. In some embodiments, the viral antigen is a viral protein. In some embodiments, the amino acid sequence of the viral protein is all or part of a structural protein or structural proteins of the virus. In some embodiments, the antigen(s) are assembled into VLPs and released from the expressing cell.

[0101] In some embodiments, viral antigens include, but are not limited to, any whole or antigenic fragment of a coronavirus protein. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the betacoronavirus is a severe acute respiratory syndrome (SARS) virus. In some embodiments, the betacoronavirus is a Middle East Respiratory Syndrome (MERS) virus, OC43, or HKU1. In some embodiments, the SARS virus is SARS-CoV-1. In some embodiments, the SARS virus is SARS-CoV-2.

[0102] In some embodiments, the viral antigen comprises a whole or antigenic fragment of any one or more of the following proteins: coronavirus spike protein, coronavirus M protein, coronavirus N protein, and coronavirus E protein.

[0103] In some embodiments, the coronavirus spike protein is selected from the group consisting of SARS-Cov-2 spike protein, Middle East Respiratory Syndrome (MERS) spike protein, and SARS-CoV spike protein. In some embodiments, the coronavirus M protein is selected from SARS-Cov-2 M protein, MERS M protein, and SARS-CoV M protein. In some embodiments, the coronavirus N protein is selected from SARS-Cov-2 N protein, MERS N protein, and SARS-CoV N protein. In some embodiments, the coronavirus E protein is selected from SARS-Cov-2 E protein, MERS E protein, and SARS-CoV E protein.

[0104] In some embodiments, a polynucleotide encoding a coronavirus spike protein has a nucleic acid sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween) to the nucleic acid sequence of SEQ ID NO: 14 or 70. In some embodiments, a polynucleotide encoding a coronavirus spike protein has the nucleic acid sequence of SEQ ID NO: 14 or 70.

[0105] In some embodiments, the amino acid sequence of the coronavirus spike protein has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the amino acid sequence of the coronavirus spike protein is SEQ ID NO: 13.

[0106] In some embodiments, the SARS-Cov-2 spike protein is a mutant S protein (also designated "S(Mut)") that comprises one or more amino acid mutations compared to SEQ ID NO: 13. In some embodiments, the mutant S protein is expressed at a higher level compared to the wild-type S protein. In some embodiments, the mutant S protein is a pre-fusion conformation-stabilizing spike protein. In some embodiments, the mutations in the S protein stabilize the trimeric state of the S protein. In some embodiments, the mutant S protein comprises one or more mutations in an internal endogenous proteolytic cleavage site of the S protein. In some embodiments, the mutant S protein comprises a deletion of an internal endogenous proteolytic cleavage site of the S protein. In some embodiments, the one or more mutations in the proteolytic cleavage site of the S protein inhibit cleavage of the S protein during the assembly process. In some embodiments, a VLP comprising any one or more of the mutant S proteins disclosed herein is more immunogenic than a VLP comprising a wild-type S protein, e.g., an S protein comprising the amino acid sequence of SEQ ID NO: 13.

[0107] In some embodiments, the mutant S protein comprises a modification (e.g., a substitution) of at least one amino acid residue selected from the group consisting of R682, R683, A684, R685, K986, and V987 in SEQ ID NO: 13. In some embodiments, the mutant S protein comprises at least one amino acid substitution selected from the group consisting of R682G, R683S, R685S, K986P, and V987P in SEQ ID NO: 13. In some embodiments, the mutant S protein comprises the amino acid substitutions R682G, R683S, R685S, K986P, and V987P in SEQ ID NO: 13. In some embodiments, the mutant S protein comprises the following amino acid substitutions in an internal endogenous furin cleavage site: R682G, R683S, R685S. That is, in some embodiments, the mutant S protein contains the following amino acids at the internal endogenous furin cleavage site: G at amino acid residue 682, S at amino acid residue 683, A at amino acid residue 684, and S at amino acid residue 685.

[0108] In some embodiments, the mutant S protein has at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween) to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the amino acid sequence of the mutant S protein is SEQ ID NO:51.

[0109] In some embodiments, a polynucleotide encoding a mutant S protein has a nucleic acid sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, a polynucleotide encoding a coronavirus spike protein has the nucleic acid sequence of SEQ ID NO:52.

[0110] In some embodiments, a polynucleotide encoding a coronavirus M protein has a nucleic acid sequence having at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the nucleic acid sequence of SEQ ID NO: 19 or 66. In some embodiments, a polynucleotide encoding a coronavirus M protein has the nucleic acid sequence of SEQ ID NO: 19 or 66.

[0111] In some embodiments, the amino acid sequence of the coronavirus M protein has at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the amino acid sequence of the coronavirus M protein is SEQ ID NO:33.

[0112] In some embodiments, a polynucleotide encoding a coronavirus N protein has a nucleic acid sequence having at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any value and subrange therebetween), to the nucleic acid sequence of SEQ ID NO:21 or 71. In some embodiments, a polynucleotide encoding a coronavirus N protein has the nucleic acid sequence of SEQ ID NO:21 or 71.

[0113] In some embodiments, the amino acid sequence of the coronavirus N protein has at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the amino acid sequence of the coronavirus N protein is SEQ ID NO: 20.

[0114] In some embodiments, a polynucleotide encoding a coronavirus E protein has a nucleic acid sequence having at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any value and subrange therebetween), to the nucleic acid sequence of SEQ ID NO: 23 or 72. In some embodiments, a polynucleotide encoding a coronavirus E protein has the nucleic acid sequence of SEQ ID NO: 23 or 72.

[0115] In some embodiments, the amino acid sequence of the coronavirus E protein has at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the amino acid sequence of the coronavirus E protein is SEQ ID NO: 22.

[0116] In some embodiments, the viral protein is from any one of virus groups I, II, III, IV, V, VI, or VII according to the Baltimore classification. In some embodiments, the viral protein is from an enveloped, negative-stranded, single-stranded, segmented RNA virus (e.g., influenza virus). In some embodiments, the viral protein is from an enveloped DNA virus (e.g., hepatitis B virus). In some embodiments, the viral protein is from a non-enveloped DNA virus (e.g., human papilloma virus). In some embodiments, the viral protein is from a positive-stranded enveloped RNA virus (e.g., coronaviruses, e.g., SARS CoV2, and flaviviruses, e.g., West Nile virus). In some embodiments, the viral antigen comprises any whole or antigenic fragment of a protein from a virus selected from the group consisting of SARS-CoV-1, MERS-CoV, Chikungunya virus, African swine fever virus, Dengue virus, Zika virus, influenza virus (e.g., A, B, C), human immunodeficiency virus (HIV), Ebola virus, hepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), West Nile virus, and human papillomavirus.

[0117] In some embodiments, the viral antigen comprises any whole or antigenic fragment of a protein derived from West Nile Virus. In some embodiments, the West Nile Virus protein is precursor membrane (prM), envelope glycoprotein (E), or a combination thereof. In some embodiments, the vector encoding one or more West Nile Virus proteins, e.g., prM and / or E protein, is a West Nile Virus minimal plasmid (WNV minimal plasmid) as shown in FIG. 14A or a West Nile Virus standard plasmid (WNV standard plasmid) as shown in FIG. 14B. In some embodiments, the vector encoding one or more West Nile Virus proteins, e.g., prM and / or E protein, comprises a nucleic acid sequence having at least 70% (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100%) identity to SEQ ID NO:55. In some embodiments, a vector encoding one or more West Nile Virus proteins, e.g., prM and / or E protein, includes an expression cassette having a nucleic acid sequence having at least 70% (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100%) identity to SEQ ID NO:64.

[0118] In some embodiments, a polynucleotide encoding a West Nile virus E protein has a nucleic acid sequence having at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any value and subrange therebetween), to the nucleic acid sequence of SEQ ID NO: 60. In some embodiments, a polynucleotide encoding a West Nile virus E protein has the nucleic acid sequence of SEQ ID NO: 60.

[0119] In some embodiments, a polynucleotide encoding a West Nile virus prM protein has a nucleic acid sequence having at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% (including any values ​​and subranges therebetween), to the nucleic acid sequence of SEQ ID NO: 59. In some embodiments, a polynucleotide encoding a West Nile virus prM protein has the nucleic acid sequence of SEQ ID NO: 59.

[0120] The nucleic acid sequences of vectors encoding the West Nile virus antigens disclosed herein, and the genetic elements therein, are listed in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0121] In some embodiments, the viral antigen comprises a whole or antigenic fragment of any protein derived from an influenza virus. The strain of influenza virus can be any strain currently known or later discovered, for example, H1N1, H3N2, or influenza B strain, without limitation. In some embodiments, the influenza virus protein is an HA protein, an NA protein, an M1 protein, an M2 protein, or any combination thereof. In some embodiments, the viral antigen comprises a whole or antigenic fragment of any protein derived from a hepatitis B virus. In some embodiments, the hepatitis B virus protein is an sAg (S protein), an sAg (M protein), an sAg (L protein), preS1, preS2, cAg (core antigen), or any combination thereof. In some embodiments, the viral antigen comprises a whole or antigenic fragment of any protein derived from a human papilloma virus. In some embodiments, the human papilloma virus protein is an HPV6 L1 protein, an HPV11 L1 protein, an HPV16 L1 protein, an HPV18 L1 protein, or any combination thereof.

[0122] In some embodiments, the viral antigen comprises any one or more whole or antigenic fragments of the proteins from each of the viruses listed below in Table 4. For example, in some embodiments, the viral antigen may comprise any whole or antigenic fragment of any protein from avian influenza virus (H5N3). [Table 4] Enhancer Proteins

[0123] Without wishing to be bound by any theory, it is believed that co-expression of an enhancer protein with an antigen may improve one or more aspects of antigen expression, including but not limited to, yield, quality, folding, post-translational modifications, activity, localization, and downstream activity, or may reduce one or more of misfolding, altered activity, incorrect post-translational modifications, and / or toxicity.

[0124] In some embodiments, the enhancer protein is a picornavirus leader (L) protein, or a functional variant thereof. In some embodiments, the picornavirus leader (L) protein can block nuclear pores, thereby inhibiting nucleocytoplasmic transport (NCT). As used herein, the term "functional variant" refers to a protein that is homologous to a picornavirus leader (L) protein and / or shares substantial sequence similarity (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity) with a picornavirus leader (L) protein. In some embodiments, the functional variant shares one or more functional characteristics of a picornavirus leader (L) protein. For example, in some embodiments, a functional variant of a picornavirus leader (L) protein retains the ability to inhibit NCT.

[0125] In some embodiments, the picornavirus leader (L) protein is an L protein from the Cardiovirus, Hepatovirus, or Aphthovirus genera. For example, the enhancer protein can be from bovine rhinitis A virus, bovine rhinitis B virus, equine rhinitis A virus, foot and mouth disease virus, hepatovirus A, hepatovirus B, Himalayan marmot hepatovirus, Phopivirus, encephalomyocarditis virus, Cardiovirus B, Theiler's Murine encephalomyelitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), Theiler-like rat virus (TRV), or Saffold virus (SAF-V).

[0126] In some embodiments, the picornavirus leader (L) protein is a Theiler's virus L protein or a functional variant thereof. In some embodiments, the L protein shares at least 90% identity to SEQ ID NO: 1. In some embodiments, the enhancer protein may comprise or consist of SEQ ID NO: 1. In some embodiments, the enhancer protein may share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to a SEQ ID NO: 1.

[0127] In some embodiments, the picornavirus leader (L) protein is the L protein of Encephalomyocarditis virus (EMCV) or a functional variant thereof. In some embodiments, the L protein may share at least 90% identity to SEQ ID NO:2. In some embodiments, the enhancer protein may comprise or consist of SEQ ID NO:2. In some embodiments, the enhancer protein may share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:2.

[0128] In some embodiments, the nucleic acid sequence encoding the enhancer protein may comprise or consist of SEQ ID NO: 68. In some embodiments, the nucleic acid sequence encoding the enhancer protein may share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:68.

[0129] In some embodiments, the picornavirus leader (L) protein is selected from the group consisting of a poliovirus L protein, an HRV16 L protein, a Mengovirus L protein, and a Saffold virus 2 L protein, or a functional variant thereof.

[0130] In some embodiments, the picornavirus leader (L) protein is selected from the proteins listed in Table 5 or a functional variant thereof. A polynucleotide encoding a picornavirus leader (L) protein may encode an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence listed in Table 2. The amino acid sequence of the picornavirus leader (L) protein may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence listed in Table 2. The amino acid sequence of a picornavirus leader (L) protein may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 12. In some embodiments, the enhancer protein may have an amino acid sequence that comprises or consists of one of the amino acid sequences listed in Table 2. In some embodiments, the enhancer protein may have an amino acid sequence that comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 12. [Table 5]

[0131] The antigens, enhancer proteins, and / or fusion proteins, or the polynucleotides encoding them, may be modified to include one or more markers, labels, or tags. For example, in some embodiments, the proteins of the present disclosure may be labeled with any label that allows for its detection, such as a radioactive label, a fluorescent agent, biotin, a peptide tag, an enzyme fragment, etc. The proteins may include affinity tags, such as His tags, GST tags, Strep tags, biotin tags, immunoglobulin binding domains, such as IgG binding domains, calmodulin binding peptides, etc. In some embodiments, the polynucleotides of the present disclosure include a selection marker, such as an antibiotic resistance marker. Viral packaging signals

[0132] In some embodiments, the vectors disclosed herein comprise a polynucleotide sequence encoding a viral packaging signal (interchangeably referred to herein as a "viral packaging sequence" or packaging signal" or "psi sequence"). In some embodiments, the polynucleotide sequence encoding the viral packaging signal is a DNA polynucleotide, an RNA polynucleotide, or a combination thereof. In some embodiments, the viral packaging signal is an RNA polynucleotide. In some embodiments, the vector comprises more than one copy of the polynucleotide sequence encoding the viral packaging signal, e.g., 2, 3, 4, or 5 copies of the polynucleotide sequence.

[0133] The viral packaging signal may be derived from any virus. In some embodiments, the viral packaging signal is derived from the same virus as the antigen expressed from the vector. In some embodiments, the viral packaging signal is derived from a different virus than the antigen expressed from the vector. In some embodiments, the viral packaging signal is derived from a virus selected from the group consisting of SARS-CoV-2, SARS-CoV-1, MERS-CoV, Chikungunya virus, African swine fever virus, Dengue virus, Zika virus, influenza virus (e.g., A, B, C), human immunodeficiency virus (HIV), Ebola virus, hepatitis virus (e.g., Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, and Hepatitis E), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), West Nile virus, and human papillomavirus.

[0134] In some embodiments, a polynucleotide encoding a viral packaging element has at least about 70% (e.g., about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100%) identity to a polynucleotide of SEQ ID NO:34 (including all values ​​and subranges therebetween).

[0135] The position of the polynucleotide encoding the viral packaging signal on the vector is not limited. In some embodiments, the position of the polynucleotide encoding the viral packaging signal on the vector can be 5' to all the nucleic acid sequences encoding viral antigens. In some embodiments, the position of the polynucleotide encoding the viral packaging signal on the vector can be 3' to all the nucleic acid sequences encoding viral antigens. In some embodiments, the position of one copy of the polynucleotide encoding the viral packaging signal on the vector is 5' to all the nucleic acid sequences encoding viral antigens, and the position of the other copy of the polynucleotide encoding the viral antigen is 3' to all the nucleic acid sequences encoding viral antigens. Furthermore, the size of the viral packaging signal is not limited and may be in the range of about 50 bps to about 3 kb, for example, about 100 bps, about 200 bps, about 300 bps, about 400 bps, about 500 bps, about 550 bps, about 600 bps, about 650 bps, about 700 bps, about 800 bps, about 900 bps, about 1 kb, about 2 kb, or about 3 kb, including all values ​​and subranges therebetween. In some embodiments, the size of the viral packaging signal is about 600 to about 700 bps, for example, about 650 bps. In some embodiments, the size of the viral packaging signal is about 661 bps.

[0136] The present disclosure further provides a vector comprising an expression cassette, the expression cassette comprising a promoter linked to a target gene, the vector comprising a polynucleotide encoding any one of the viral packaging elements disclosed herein. Order of genetic elements in expression cassettes

[0137] In the vectors disclosed herein, the polynucleotide sequences encoding one or more viral antigens, and the polynucleotide sequences encoding enhancers, and / or one or more regulatory elements (e.g., polynucleotides encoding an IRES sequence, CMV proteins, polynucleotides encoding viral packaging signals, and polynucleotides encoding proteolytic cleavage sites) may be ordered in any possible combination. For example, the order of elements in the expression cassette may be as shown for any one of plasmids CoVEG3-17 in FIG. 6. Without being bound by theory, it is believed that the order of elements in the expression cassette may relate to the expression of antigens encoded by the vector and / or the formation of VLPs. Furthermore, it is believed that higher protein expression and more stable VLP formation may result when the expression cassette contains genes M, N, S, and E in the following 5' to 3' order.

[0138] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG3. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a polynucleotide encoding an M protein, where the M protein comprises an amino acid sequence set forth in SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding an IRES, where the IRES sequence comprises SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto, a first polynucleotide encoding an enhancer L protein, where the L protein comprises an amino acid sequence set forth in SEQ ID NO:2 or at least 95% identical thereto, a polynucleotide encoding an S protein, where the S protein comprises an amino acid sequence set forth in SEQ ID NO:13 or at least 95% identical thereto. a second polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO:24 or an amino acid sequence at least 95% identical thereto; a second polynucleotide encoding an enhancer L protein, wherein the L protein comprises SEQ ID NO:2 or an amino acid sequence at least 95% identical thereto; a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO:20 or an amino acid sequence at least 95% identical thereto; and a polynucleotide encoding an E protein, wherein the E protein comprises SEQ ID NO:22 or an amino acid sequence at least 95% identical thereto.

[0139] In some embodiments, the vector comprises an expression cassette that comprises elements in the same 5' to 3' order as CoVEG4. In some embodiments, the vector comprises an expression cassette that comprises the following elements in 5' to 3' order: a promoter, a polynucleotide encoding an M protein, where the M protein comprises the amino acid sequence of SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein, where the S protein comprises the amino acid sequence of SEQ ID NO:13 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, where the E protein comprises the amino acid sequence of SEQ ID NO:22 or at least 95% identical thereto, a polynucleotide encoding an IRES, where the IRES sequence comprises the polynucleotide sequence of SEQ ID NO:24 or at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, where the L protein comprises the amino acid sequence of SEQ ID NO:2 or at least 95% identical thereto.

[0140] In some embodiments, the vector comprises an expression cassette that contains elements in the same 5' to 3' order as CoVEG5. In some embodiments, the vector comprises an expression cassette comprising, in 5' to 3' order, the following elements: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises the amino acid sequence of SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises the amino acid sequence of SEQ ID NO:20 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein, wherein the S protein comprises the amino acid sequence of SEQ ID NO:13 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises the amino acid sequence of SEQ ID NO:22 or at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises a polynucleotide sequence of SEQ ID NO:24 or at least 95% identical thereto, and a polynucleotide encoding an enhancer L protein, wherein the L protein comprises the amino acid sequence of SEQ ID NO:2 or at least 95% identical thereto.

[0141] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG6. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a polynucleotide encoding an S protein, where the S protein comprises an amino acid sequence of SEQ ID NO: 13 or at least 95% identical thereto, a polynucleotide encoding an IRES, where the IRES sequence comprises SEQ ID NO: 24 or a polynucleotide sequence at least 95% identical thereto, an enhancer, a polynucleotide encoding an L protein, where the L protein comprises an amino acid sequence of SEQ ID NO: 2 or at least 95% identical thereto, a polynucleotide encoding an M protein, where the M protein comprises an amino acid sequence of SEQ ID NO: 33 or at least 95% identical thereto. The expression cassettes include a polynucleotide comprising an amino acid sequence of SEQ ID NO:22 or at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO:24 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises SEQ ID NO:2 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO:20 or an amino acid sequence at least 95% identical thereto, and a polynucleotide encoding an E protein, wherein the E protein comprises SEQ ID NO:22 or an amino acid sequence at least 95% identical thereto.

[0142] In some embodiments, the vector comprises an expression cassette that contains elements in the same 5' to 3' order as CoVEG7. In some embodiments, the vector comprises an expression cassette comprising, in 5' to 3' order, the following elements: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises the amino acid sequence of SEQ ID NO:33, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein, wherein the S protein comprises the amino acid sequence of SEQ ID NO:13, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises the amino acid sequence of SEQ ID NO:22, or at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises a polynucleotide sequence of SEQ ID NO:24, or at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises the amino acid sequence of SEQ ID NO:2, or at least 95% identical thereto, a polynucleotide encoding an N protein, wherein the N protein comprises the amino acid sequence of SEQ ID NO:20, or at least 95% identical thereto.

[0143] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG8. In some embodiments, the expression cassette includes the following elements in 5' to 3' order: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises an amino acid sequence of SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein, wherein the S protein comprises an amino acid sequence of SEQ ID NO:13 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises an amino acid sequence of SEQ ID NO:22 or at least 95% identical thereto, a polynucleotide encoding an IRES sequence. The expression cassette comprises a polynucleotide having an IRES sequence comprising SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises an amino acid sequence of SEQ ID NO:2 or at least 95% identical thereto, a polynucleotide encoding an N protein, wherein the N protein comprises an amino acid sequence of SEQ ID NO:20 or at least 95% identical thereto, and a polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 9% identical thereto.

[0144] In some embodiments, the vector comprises an expression cassette that contains elements in the same 5' to 3' order as CoVEG9. In some embodiments, the vector comprises an expression cassette comprising, in 5' to 3' order, the following elements: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises the amino acid sequence of SEQ ID NO:33, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises the amino acid sequence of SEQ ID NO:20, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, wherein the S protein comprises the amino acid sequence of SEQ ID NO:51 or SEQ ID NO:52, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises the amino acid sequence of SEQ ID NO:22, or at least 95% identical thereto, a polynucleotide encoding an IRES sequence, wherein the IRES sequence comprises SEQ ID NO:24, or a polynucleotide sequence at least 95% identical thereto, and a polynucleotide encoding an enhancer L protein, wherein the L protein comprises the amino acid sequence of SEQ ID NO:2, or at least 95% identical thereto.

[0145] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG10. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises SEQ ID NO:33, or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO:20, or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, wherein the S protein comprises SEQ ID NO:51 or SEQ ID NO:52, or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, The expression cassettes include a polynucleotide encoding an enhancer L protein, a polynucleotide encoding an E protein, where the E protein comprises SEQ ID NO:22 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an IRES, where the IRES sequence comprises SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, where the L protein comprises SEQ ID NO:2 or an amino acid sequence at least 95% identical thereto, and a polynucleotide encoding a viral packaging signal, where the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 9% identical thereto.

[0146] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG11. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging signal, where the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 9% identical thereto, a polynucleotide encoding an M protein, where the M protein comprises an amino acid sequence of SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, where the N protein comprises an amino acid sequence of SEQ ID NO:20 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, where the S protein comprises SEQ ID NO:51 or SEQ ID NO:52, or at least one identical thereto. The expression cassette comprises a polynucleotide comprising an amino acid sequence at least 95% identical to that of SEQ ID NO: 34, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises SEQ ID NO: 22 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO: 24 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises SEQ ID NO: 2 or an amino acid sequence at least 95% identical thereto, and a second polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises SEQ ID NO: 34 or a polynucleotide sequence at least 9% identical thereto.

[0147] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG12. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises an amino acid sequence set forth in SEQ ID NO:33, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises an amino acid sequence set forth in SEQ ID NO:20, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein, wherein the S protein comprises an amino acid sequence set forth in SEQ ID NO:13, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, The expression cassette comprises a polynucleotide encoding an E protein, wherein the E protein comprises SEQ ID NO:22 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises SEQ ID NO:2 or a polynucleotide sequence at least 95% identical thereto, and a polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 9% identical thereto.

[0148] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG13. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging signal, where the viral packaging signal comprises a polynucleotide sequence set forth in SEQ ID NO:34 or at least 9% identical thereto, a polynucleotide encoding an M protein, where the M protein comprises an amino acid sequence set forth in SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, where the N protein comprises an amino acid sequence set forth in SEQ ID NO:20 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein, where the S protein comprises an amino acid sequence set forth in SEQ ID NO:13 or at least 95% identical thereto. The expression cassette comprises a polynucleotide comprising an amino acid sequence 5% identical to that of SEQ ID NO: 34, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises an amino acid sequence of SEQ ID NO: 22 or at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO: 24 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises an amino acid sequence of SEQ ID NO: 2 or at least 95% identical thereto, and a second polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises a polynucleotide sequence of SEQ ID NO: 34 or a polynucleotide sequence at least 9% identical thereto.

[0149] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG14. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging signal, where the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 9% identical thereto, a polynucleotide encoding an M protein, where the M protein comprises SEQ ID NO:33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, where the N protein comprises SEQ ID NO:20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein, where the S protein comprises SEQ ID NO:13 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, the polynucleotide encoding an enhancer L protein, wherein the L protein comprises an amino acid sequence set forth in SEQ ID NO:2 or at least 95% identical thereto; a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto; a polynucleotide encoding an enhancer L protein, wherein the L protein comprises an amino acid sequence set forth in SEQ ID NO:2 or at least 95% identical thereto; a polynucleotide encoding ORF3a, wherein ORF3a encodes an amino acid sequence set forth in SEQ ID NO:53 or at least 95% identical thereto; and a second polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises a polynucleotide sequence set forth in SEQ ID NO:34 or a polynucleotide sequence at least 9% identical thereto.

[0150] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG15. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging signal, where the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an M protein, where the M protein comprises an amino acid sequence of SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, where the N protein comprises an amino acid sequence of SEQ ID NO:20 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, The expression cassette comprises a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises an amino acid sequence of SEQ ID NO:22 or at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto, and a second polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises a polynucleotide sequence of SEQ ID NO:34 or a polynucleotide sequence at least 9% identical thereto.

[0151] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG16. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises an amino acid sequence set forth in SEQ ID NO:33, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises an amino acid sequence set forth in SEQ ID NO:20, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein, wherein the S protein comprises an amino acid sequence set forth in SEQ ID NO:13, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E ... The expression cassette comprises a polynucleotide comprising an amino acid sequence of SEQ ID NO:22 or at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises an amino acid sequence of SEQ ID NO:2 or at least 95% identical thereto, a polynucleotide encoding ORF3a, which encodes SEQ ID NO:53 or an amino acid sequence having at least 95% identity thereto, and a polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 95% identical thereto.

[0152] In some embodiments, the vector comprises an expression cassette that includes elements in the same 5' to 3' order as CoVEG17. In some embodiments, the vector includes the following elements in 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging signal, where the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence at least 9% identical thereto, a polynucleotide encoding an M protein, where the M protein comprises SEQ ID NO:33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, where the N protein comprises SEQ ID NO:20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S, where the S protein comprises SEQ ID NO:51 or SEQ ID NO:52 or an amino acid sequence at least 95% identical thereto ... The expression cassette comprises a polynucleotide encoding a proteinolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises SEQ ID NO:22 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO:24 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an enhancer L protein, wherein the L protein comprises SEQ ID NO:2 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding ORF3a, wherein ORF3a comprises SEQ ID NO:53, and a second polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises SEQ ID NO:34 or a polynucleotide sequence having at least 95% identity thereto. Expression of antigens and VLPs in cells

[0153] The present disclosure provides a method of expressing an antigen in a eukaryotic cell, comprising contacting the cell with any one of the vectors disclosed herein. In some embodiments, the vector is contacted with the cell in vitro, ex vivo, or in vivo. In some embodiments, the vector is contacted with the cell in a subject (in vivo).

[0154] In some embodiments, expression of one or more antigens results in the formation of a virus-like particle (VLP). In some embodiments, the VLP is immunogenic. In some embodiments, the VLP is capable of eliciting an immune response in a subject. In some embodiments, the VLP has an envelope. In some embodiments, the VLP does not have an envelope. The number of antigens present in the VLP is not limited. In some embodiments, the VLP comprises one antigen, two antigens, three antigens, four antigens, five antigens, six antigens, seven antigens, eight antigens, nine antigens, ten antigens, or more. In some embodiments, the VLP comprises three antigens. In some embodiments, the VLP comprises four antigens. In some embodiments, the structural proteins forming the VLP and the immunogenic viral antigens that are part of the VLP are derived from the same virus (i.e., a native VLP). In some embodiments, the structural viral proteins forming the VLP are derived from one virus and the immunogenic viral antigens incorporated into the VLP are derived from another virus (i.e., a chimeric VLP). In some embodiments, viral proteins are mutated to enhance VLP assembly, VLP secretion and / or loading of immunogenic antigen(s) into the VLP.

[0155] In some embodiments, the vector comprises a DNA polynucleotide encoding a viral packaging signal such that contacting a cell with the vector results in expression of the viral packaging signal. In some embodiments, the VLP encapsidates the viral packaging signal. In some embodiments, expression of the viral packaging signal increases or promotes the formation of VLPs. In some embodiments, more VLPs are formed in the presence of the viral packaging signal compared to the absence of the viral packaging signal. In some embodiments, contacting a cell with any one of the disclosed vectors encoding a viral packaging signal results in the expression of a greater number of VLPs compared to a control vector lacking a DNA polynucleotide encoding a viral packaging signal. In some embodiments, contacting a cell with any one of the disclosed vectors encoding a viral packaging signal results in packaging of the viral packaging signal in a VLP, which in turn results in an enhanced immune response due to improved adjuvanting properties or other mechanisms. In some embodiments, the packaging signal and protein are from the same virus from which the VLP is formed (i.e., native packaging). In some embodiments, the packaging signal and protein are from another virus with a known packaging mechanism (i.e., chimeric packaging).

[0156] In some embodiments, the expression cassette comprises a polynucleotide sequence encoding a first antigen, a second antigen, a third antigen, a fourth antigen, or a combination thereof. In some embodiments, the expression cassette comprises a polynucleotide sequence encoding a first antigen, a second antigen, and a third antigen. In some embodiments, the expression cassette comprises a polynucleotide sequence encoding a first antigen, a second antigen, a third antigen, and a fourth antigen.

[0157] In some embodiments, the first antigen is a coronavirus spike protein, the second antigen is a coronavirus membrane (M) protein, and the third antigen is a coronavirus envelope (E) protein. In some embodiments, the first antigen is a coronavirus spike protein, the second antigen is a coronavirus membrane (M) protein, the third antigen is a coronavirus envelope (E) protein, and the fourth antigen is a coronavirus nucleocapsid (N) protein.

[0158] In some embodiments, the vector causes (i) expression of the antigen at a higher expression level, and / or (ii) expression of the antigen for a longer period, and / or (iii) expression of the antigen with better protein quality, compared to a vector lacking the enhancer protein. In some embodiments, the vector causes (i) expression of a virus-like particle (VLP) comprising the antigen at a higher expression level, and / or (ii) expression of a VLP comprising the antigen for a longer period, and / or (iii) expression of a VLP comprising the antigen with better protein quality, compared to a vector lacking the enhancer protein. As used herein, "protein quality" refers to, but is not limited to, protein folding, post-translational modification, functional activity, localization, and downstream activity. Thus, in some embodiments, an antigen co-expressed with an enhancer protein using any of the methods or vectors or compositions disclosed herein may have improved protein folding, improved post-translational modification, improved functional activity, improved localization, and improved downstream activity, compared to an antigen not co-expressed with the enhancer protein.

[0159] As used herein, the terms "transfection," "transduction," and "transformation" refer to the process of introducing a nucleic acid into a cell (e.g., a eukaryotic cell). The vectors disclosed herein can be introduced into a cell (e.g., a eukaryotic cell) using any method known in the art. For example, the vector can be introduced into a cell using chemical, physical, biological, or viral means. Methods of introducing a vector into a cell include, but are not limited to, the use of calcium phosphate, dendrimers, cationic polymers, lipofection, fugene, cell-penetrating peptides, peptide dendrimers, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, gene guns, magnetofection, microprojectile guns, nucleofection, viral transduction, injection, transformation, transfection, direct uptake, projectile bombardment, and liposomes. Other non-limiting examples of methods include viral transfection, direct uptake, projectile bombardment, direct injection with or without electroporation / sonoporation, with or without cationic polymers, lipids, lipid formulations, and jet genetic devices. Antigens and enhancer proteins can be stably or transiently expressed in cells using expression vectors. Expression techniques in eukaryotic cells are well known to those skilled in the art. (See Current Protocols in Human Genetics: Chapter 12 "Vector Therapy" & Chapter 13 "Delivery Systems for Gene Therapy").

[0160] In some embodiments, the vector can be introduced into the host cell by insertion into the genome, optionally through lentiviral transfection, baculovirus gene transfer into mammalian cell (BacMam), retroviral transfection, CRISPR / Cas9, and / or the use of transposons, using standard methods for generating stable cell lines. In some embodiments, the polynucleotide or vector can be introduced into the host cell for transient transfection. In some embodiments, transient transfection can be performed through the use of viral vectors, helper lipids, e.g., PEI, Lipofectamine, and / or Fectamine 293. The genetic elements can be encoded, e.g., as DNA on a vector, or as RNA, e.g., from PCR. The genetic elements can be separated on different vectors or combined on the same vector.

[0161] Host cells used to express antigens and enhancer proteins can include, but are not limited to, prokaryotic hosts (e.g., E. coli) or eukaryotic hosts (e.g., Saccharomyces cerevisiae, insect cells such as Sf21 cells, or mammalian cell lines and primary cells such as NIH 3T3, HeLa, COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; see also, e.g., Ausubel et al., supra). Non-limiting examples of insect cells are Spodoptera frugiperda (S1) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells. Examples of fungal (including yeast) host cells are S. cerevisiae, Kluyveromyces lactis (K. lactis), Candida species including C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, HeLa cells, MDCK cells, Vero, and Hep-2 cells. Xenopus laevis oocytes or other cells of amphibian origin may also be used. Prokaryotic host cells include bacterial cells, such as E. coli, B. subtilis, and mycobacteria. Vaccine Compositions

[0162] The present disclosure provides a vaccine composition comprising any one of the vectors disclosed herein and at least one pharma- ceutically acceptable carrier, excipient, and / or vehicle, such as a solvent, buffer, solution, dispersion medium, coating, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents. As used herein, the term "pharma- ceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the US Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopeia, for use in mammals, more particularly in humans. In some embodiments, the pharma- ceutically acceptable carrier, excipient, and / or vehicle may include saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. In some embodiments, the pharma- ceutically acceptable carrier, excipient, and / or vehicle comprises phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, polyol, (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), or suitable mixtures thereof. In some embodiments, the compositions disclosed herein further comprise minor amounts of emulsifying or wetting agents, or pH buffering agents.

[0163] In some embodiments, the compositions are in a solid form, such as a lyophilized powder, liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder suitable for reconstitution. In some embodiments, delivery vehicles, such as liposomes, nanocapsules, nanoparticles, microparticles, microspheres, lipid particles, vesicles, polymers, peptides, and the like, may be used for the introduction of the vectors and vaccine compositions disclosed herein into suitable host cells. In some embodiments, the vectors and vaccine compositions disclosed herein may be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, and the like.

[0164] In some embodiments, the compositions disclosed herein contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, such as chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, or albumin. In some embodiments, the compositions disclosed herein contain antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, or thimerosal, isotonic agents, such as sugars or sodium chloride, and / or agents that delay absorption, such as aluminum monostearate and gelatin.

[0165] In some embodiments, the vaccine composition comprises an adjuvant. As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances or otherwise alters or modifies the immune response elicited against the immunogen. Modification of the immune response can include enhancing or broadening the specificity of either or both the antibody and cellular immune responses.

[0166] In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is monophosphoryl lipid A (MPL). In some embodiments, other adjuvants may be used in addition or instead. Inclusion of any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)" (herein incorporated by reference in its entirety for all purposes) is contemplated within the scope of this disclosure. Other adjuvants include complete Freund's adjuvant (a non-specific stimulator of immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant and aluminum hydroxide adjuvant, GMCSP, BCG, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL), MF-59, three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and RIBI containing cell wall skeleton (CWS) in a 2% squalene / Tween® 80 emulsion. In some embodiments, the adjuvant can be paucilamellar lipid vesicles, such as Novasomes®. Novasomes® are paucilamellar nonphospholipid vesicles ranging from about 100 nm to about 500 nm. They include Brij® 72, cholesterol, oleic acid, and squalene. Novasomes have been shown to be effective adjuvants (see U.S. Patent Nos. 5,629,021, 6,387,373, and 4,911,928). In some embodiments, the composition may not include added adjuvants. Alum-free compositions that induce a strong immune response are particularly useful in adults over about 60 years of age. Methods for inducing an immune response in a subject

[0167] The present disclosure further provides a method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of any one of the vaccine compositions disclosed herein. In some embodiments, the subject's tissue at the administration site expresses the antigen and / or VLPs comprising the antigen. In some embodiments, the subject's tissue at the administration site (i) expresses the antigen and / or VLPs comprising the antigen at a higher expression level, and / or (ii) expresses the antigen and / or VLPs comprising the antigen for a longer period of time, and / or (iii) expresses the antigen and / or VLPs comprising the antigen with better protein quality, compared to when a vector lacking an enhancer protein is administered.

[0168] In some embodiments, the method induces an antibody response in the subject. In some embodiments, the antibody response is a neutralizing antibody response. In some embodiments, the method induces a cellular immune response. In some embodiments, the method induces a prophylactic, protective, and / or therapeutic immune response in the subject.

[0169] In some embodiments, the vector comprises a DNA polynucleotide encoding a viral packaging signal, such that the tissue at the site of administration of the subject expresses the viral packaging signal. In some embodiments, the VLP encapsidates the viral packaging signal. In some embodiments, the VLP encapsidates a polynucleotide comprising a viral packaging signal. In some embodiments, the VLP encapsidates a polynucleotide consisting of a viral packaging signal. In some embodiments, the VLP encapsidates a VLP that encapsidates a viral packaging signal is more immunogenic than a control VLP that comprises an antigen but lacks a viral packaging signal. Without being bound by theory, it is believed that more VLPs may be formed in the presence of a viral packaging signal compared to the absence of a viral packaging signal. Thus, in some embodiments, the disclosed vectors that encode a viral packaging signal promote the formation of a greater number of VLPs compared to a control vector that does not encode a viral packaging signal. Without being bound by theory, it is also believed that the RNA viral packaging signal may act as an adjuvant by acting as an agonist of Toll-like receptors (TLRs).

[0170] Methods of administering any one of the compositions or vectors disclosed herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral or pulmonary routes or by suppository), subcutaneous, and intraperitoneal. In some embodiments, the compositions of the present invention are administered intramuscularly, intravenously, subcutaneously, transdermally, or intradermally. The compositions or vectors may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, bladder, and intestinal mucosa, etc.), and may be administered together with other biologically active agents. In some embodiments, the administration is intradermal. In some embodiments, the administration is intramuscular. In some embodiments, the administration is subcutaneous. In some embodiments, the administration is intranasal. In some embodiments, the composition or vector disclosed herein is administered by injection. In some embodiments, the injection is performed using a needle, syringe, microneedle, or needle-free injection device. In some embodiments, the compositions or vectors disclosed herein are administered intranasally, either by droplets, large particle aerosol (greater than about 10 microns), or spray to the upper respiratory tract, or by small particle aerosol (less than 10 microns) or spray to the lower respiratory tract. In some embodiments, injection is followed by electroporation.

[0171] The vector or vaccine compositions disclosed herein may be administered in a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule or a booster immunization schedule. In a multiple dose schedule, various doses may be administered by the same or different routes, e.g., parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. In some embodiments, subsequent boost doses are administered within a period of about 1 hour to about years after the previous dose (e.g., about 12 hours, about 1 day, about 2 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 1 month, about 6 months, about 1 year, about 2 years, including all values ​​and subranges therebetween). Immunogenic effects

[0172] In some embodiments, inclusion of an enhancer protein in a polynucleotide encoding one or more viral antigen proteins increases functional virus-like particle (VLP) production compared to a polynucleotide that does not contain the enhancer protein, in some embodiments, inclusion of the enhancer protein increases functional VLP production by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, or about 1000% compared to a vector that does not contain the enhancer protein. As used herein, functional VLP production may be measured by methods known in the art, including but not limited to, the level of protein aggregation, the titer of neutralizing antibodies in vivo, the induced Th1 response, the amount of VLPs over time relative to the VLP half-life, and / or cell death associated with misfolded VLPs.

[0173] In some embodiments, the inclusion of an enhancer protein in a polynucleotide encoding one or more viral antigen proteins increases the duration or amount of neutralizing antibodies in a subject compared to a vaccine composition that does not include the enhancer protein, hi some embodiments, the inclusion of an enhancer protein increases the duration or amount of neutralizing antibodies in a subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold compared to a vaccine composition that does not include the enhancer protein.

[0174] In some embodiments, the inclusion of an enhancer protein increases a Th1 cellular response compared to a vaccine composition that does not include the enhancer protein, hi some embodiments, the inclusion of an enhancer protein increases the Th1 response by about 25%, about 50%, about 100%, about 200%, about 300%, about 400%, about 500%, or about 1000% compared to a vaccine composition that does not include the enhancer protein. Kits and manufactured products

[0175] The present disclosure provides kits comprising any one or more of the vectors disclosed herein. The present disclosure further provides kits comprising any one or more of the polynucleotides disclosed herein. The present disclosure also provides kits comprising any one or more of the vaccine compositions disclosed herein. The kits disclosed herein are useful for carrying out or aiding in carrying out the disclosed methods. In some embodiments, the kits comprise a pharma- ceutically acceptable carrier. In some embodiments, the kits comprise instructions for proper use of the product or formulation and safety information. In some embodiments, the kits comprise dosage information based on the application and method of administration as determined by a physician.

[0176] The present application also provides an article of manufacture comprising any one of the vaccine compositions or kits described herein. An example of an article of manufacture is a vial (e.g., a sealed sterile vial).

[0177] In some embodiments, the kit comprises one or more containers or vials filled with one or more of the components of the vaccine compositions disclosed herein. In some embodiments, the kit comprises two containers, one containing a vector, or polynucleotide, or vaccine composition disclosed herein, and the other containing an adjuvant. In some embodiments, the kit further comprises a notice reflecting approval by a governmental agency for manufacture, use, or sale for human administration.

[0178] The present invention is further illustrated by the following additional examples which should not be construed as limiting: Those of skill in the art will, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLES

[0179] Example 1: Construction of polynucleotides encoding SARS-CoV-2 and L proteins The CoVEG1 and CoVEG2 plasmids encode the SARS-CoV-2 and L enhancer proteins.

[0180] Plasmid CoVEG1 contains polynucleotides encoding the full-length S protein (SEQ ID NO: 14), M protein (SEQ ID NO: 19), and E protein (SEQ ID NO: 23) viral proteins of SARS-CoV-2. Plasmid CoVEG2 contains polynucleotides encoding the full-length S protein, M protein, N protein (SEQ ID NO: 21), and E protein viral proteins of SARS-CoV-2. The backbone of the CoVEG1 and CoVEG2 plasmids is shown in Figure 1. Additionally, the CoVEG1 and CoVEG2 plasmids also contain a polynucleotide encoding the L protein (SEQ ID NO: 16) from EMCV.

[0181] The nucleic acid sequence of the complete insert in CoVEG2 is represented by SEQ ID NO: 30. See Table 1. Expression of this construct results in three polypeptides: a SARS-CoV-2 spike protein having the amino acid sequence of SEQ ID NO: 13, a CoVEG2 polypeptide 1 having the amino acid sequence of SEQ ID NO: 25, and a CoVEG2 polypeptide 2 having the amino acid sequence of SEQ ID NO: 26. The nucleic acid sequence of the insert in CoVEG1 is represented by SEQ ID NO: 31. Expression of this construct results in two polypeptides: a SARS-CoV-2 spike protein having the amino acid sequence of SEQ ID NO: 13, and a CoVEG1 polypeptide having the amino acid sequence of SEQ ID NO: 32. See Table 1.

[0182] The plasmid backbone (based on the design principles of the pVax1 plasmid) and the inserts of both plasmids were generated using gene synthesis and contain no animal or human origin material. The plasmid backbone consists of a kanamycin resistance gene, a ColE1 origin of replication, a human cytomegalovirus immediate early promoter and a simian virus (SV40) polyA signal. Polynucleotides encoding viral proteins were cloned between the CMV promoter and the SV40 polyA signal. After gene synthesis and plasmid preparation, the plasmids were transformed into E. coli for cloning and then screened using kanamycin. A representative colony was selected, its plasmid sequence verified and used as the source plasmid for further development. After transcription, the viral proteins were expressed from a single polycistronic mRNA.

[0183] Without wishing to be bound by any particular theory, it is believed that when co-expressed, the S, E, and M proteins assemble into VLPs that are secreted by the expressing cells, and when the N protein is also expressed together with the S, E, and M proteins, VLP secretion is significantly increased. Example 2: Expression of SARS-CoV-2 S, E, M, and N proteins in eukaryotic cells as observed by immunofluorescence

[0184] HEK 293 (eukaryotic) cells were transfected with the pCoVEG2 plasmid. After 24 hours, the cells were fixed, permeabilized, and analyzed by immunocytochemistry using a commercially available Alexa Fluor 568 fluorescently labeled secondary antibody for detection. Figure 2 shows the expression of S, M, N, and E proteins in HEK 293 cells, demonstrating that pCoVEG2 as disclosed herein expresses viral antigens in cells. Example 3: Expression of SARS-CoV-2 S, E, M, and N proteins in eukaryotic cells as observed by SDS-PAGE and Western blot

[0185] HEK 293 cells were transfected with pCoVEG2 plasmid and incubated for 96 hours. The cell culture supernatant was then harvested and concentrated. The concentrate was loaded onto Superose 6GL resin packed in a Tricorn 10 / 300 column using PBS as eluent. The void fraction containing the secreted VLPs was analyzed by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and / or Western blotting using monoclonal antibodies against S, N, M, or E to show the presence of S, N, M, and E proteins. See Figure 3.

[0186] These data show that the DNA vector CoVEG2 disclosed herein expresses all SARS-CoV-2 viral structural proteins (S, M, E, and N proteins) in HEK293 cells, and secreted VLP components can be detected in the cell culture supernatant. These results suggest that the CoVEG1 and CoVEG2 plasmids can potentially be used as highly effective DNA vaccines against SARS-CoV-2. Example 4: Immunogenicity of Polynucleotide Vaccines

[0187] To determine the immunogenicity of CoVEG1 and CoVEG2, these plasmids are injected intradermally into 6-week-old BALB / c mice at 2-week intervals, for a total of three injections on days 1, 15, and 29. The induced humoral immune response [anti-S antibody titers using respective enzyme-linked immunosorbent assays (ELISA)] as well as the cellular immune response [presence of antigen-reactive T cells using respective IFN-γ and IL-4 enzyme-linked immunosorbent spot (Dual color ELISpot) assays] are measured. To measure neutralization against total antibodies, an in vitro virus neutralization assay is performed. For this, serum isolated from day 43 is diluted and incubated with SARS-CoV-2 live virus before being added to VERO cells. Virus isolation is determined by the lack of successful infection of cells compared to the native virus.

[0188] Anti-SARS-CoV-2 antibody analysis, including anti-S protein antibody ELISA assays, are performed based on commercially available materials. Alternatively, in-house developed cell-based and VLP-based ELISA assays are used. For ELISpot analysis, spleens are collected and T cells are isolated. ELISpot evaluation is performed by priming T cells with Miltenyi Biotec Peptivator SARS-CoV-2 peptide pools to activate SARS-CoV-2 reactive T cells. Additionally, the toxicokinetic and pharmacodynamic properties of the plasmids are determined. See Figure 4.

[0189] Female BALB / c mice (6-8 weeks old) weighing 15-25 grams are randomly assigned to 4 groups, each group containing 10 animals. Mice are administered intradermally either vehicle-PBS, reference product EG-BB encoding an enhancer protein under the control of the CMV promoter, and two doses of CoVEG1 and CoVEG2 at 1 and 25 μg. Mice are assessed twice daily for mortality and moribundity. Clinical observations and body weights are collected weekly starting at week -1 and at least every 2 weeks thereafter for the duration of the study.

[0190] Dosed mice are bled at pre-dosing time points and serum is separated by centrifugation. The resulting serum samples are then analyzed for antibodies against full-length recombinant S protein (S1+S2) using a quantitative ELISA, as shown in Table 3 below. [Table 3-6]

[0191] At day 43, the resulting serum is split into two approximately equal aliquots, one for anti-vaccine antibody (AVA) analysis and the second reserved for testing for neutralizing antibodies. The aliquots are immediately frozen on dry ice or in a freezer set to maintain -80°C.

[0192] At the end of the study, all animals are euthanized. Spleens are harvested using a cell culture lavage procedure for IFN-γ and IL-4 assessment by ELISpot.

[0193] For the evaluation of T cell-mediated toxicity, a quantitative evaluation is performed using an ELISpot assay. Splenocytes from harvested spleens are stimulated with Miltenyi Biotec's SARS-CoV-2 PepTivator Peptide Pools, covering the sequence of S, M, and N SARS-CoV-2 proteins. Splenocytes are tested with two concentrations of three different SARS-CoV-2 peptide pools in addition to a negative control (medium) and a positive control (phorbol myristate acetate / ionomycin). Example 5: Immunogenicity of Polynucleotide Vaccines in Humans

[0194] To evaluate the safety, reactogenicity, and immunogenicity of CoVEG1 and CoVEG2, an open-label, multicenter, dose-ranging study will be conducted in males and non-pregnant females starting at age 18 (inclusive) who are healthy and meet all eligibility criteria. Approximately 45 subjects will be enrolled in one of three cohorts (1, 25, and 200 μg). Subjects will receive intradermal injections (100 μl) of CoVEG1 and CoVEG2 on days 1 and 29 and will be followed up until 12 months after the second vaccination (day 394). Follow-up visits will be conducted 1, 2, and 4 weeks after each vaccination (days 8, 15, 29, 36, and 57), and 3, 6, and 12 months after the second vaccination (days 119, 209, and 394).

[0195] The safety and reactogenicity of a two-dose vaccination schedule of CoVEG1 and CoVEG2 administered as an intradermal injection in healthy adults will be evaluated based on the percentage of participants with Adverse Events (AEs), the percentage of participants with administration (injection) site reactions, and the percentage of participants with Adverse Events of Special Interest (AESIs) over two doses, 28 days apart.

[0196] To assess immunogenicity, the following parameters will be assessed on days 15, 29 (before the second dose), and 57 according to the two-dose vaccination schedule for CoVEG1 and CoVEG2: (a) IgG, IgM, and / or IgA ELISA for S antibodies using validated ELISA methods; (b) Immune memory indications are assessed by immunophenotyping PBMCs by flow cytometry and validated ELISpot assays focusing on CD49b+T-bet+ resting memory Th cell progenitors and CXCR4+S1P1+ memory plasma cell progenitors on days 15, 29, 57, 180, 270, and / or 394. Measurements include geometric mean fold rise (GMFR) of IgG, IgM, and / or IgA titers from baseline (days 1-394), antibody geometric mean titers (GMT) (days 15, 29, 57, 180, 270, and 394), and percentage of subjects who seroconverted (days 1-15, 29, 57, 180, 270, and 394). Seroconversion is defined as a four-fold change in antibody titer from baseline. (c) IFN-γ responses as a measure of CD8 T cell responses and memory immune cell phenotypes are measured in PBMCs isolated from subjects by a validated ELISpot assay. PBMCs are isolated on days 15, 29, 57, 180, 270, and 394. Example 6: Design of further polynucleotides for expression of SARS-CoV-2 S, E, M, and N proteins, and variants with and without enhancer L protein

[0197] Plasmids CoVEG3-17 contain expression cassettes encoding different viral proteins in the order shown in Figure 6. The plasmid backbone (based on the design principle of pVax1 plasmid) and the inserts of the plasmids were generated using gene synthesis. The plasmid backbone consists of a kanamycin resistance gene, a ColE1 origin of replication, a human cytomegalovirus immediate early promoter and a simian virus (SV40) polyA signal. Polynucleotides encoding viral proteins were cloned between the CMV promoter and the SV40 polyA signal. After gene synthesis and plasmid preparation, the plasmids were transformed into E. coli for cloning and then screened using kanamycin. A representative colony was selected, its plasmid sequence verified and used as the source plasmid for further development. After transcription, the viral proteins were expressed from a single polycistronic mRNA. Example 7: Expression of SARS-CoV-2 S and N proteins observed by immunofluorescence

[0198] 24 hours prior to transfection, HEK293T cells were seeded at 40,000 cells / well in 24-well plates. Cells were transfected with pCoVEG3~20 plasmid using PEI complexes according to the manufacturer's instructions. 12 hours after transfection, the medium was changed and cells were incubated for 48 hours at 37°C and 5% CO2. Cell medium was removed and cells were permeabilized with 0.2% Triton® X-100 in PBS for 10 minutes and then fixed with 10% neutral buffered formalin for 10 minutes. Before immunostaining, nonspecific binding was blocked by adding EZ-Block (SCYTEK). Staining with anti-spike (S) protein antibody (also referred to herein as "anti-RBD"), which binds to the receptor binding domain (RBD), was added at a dilution of 1:500 and incubated for 1 hour at room temperature. The stain was removed, cells were washed, and secondary antibody (Alexa Fluor 568 fluorescently labeled secondary anti-rabbit, 1:1000 dilution) was added. The stain was incubated for 1 hour at room temperature, after which the stain was removed and washed. Cells were imaged using the EVOS Cell Imaging System. Figure 7 shows the expression of S protein in HEK293 cells, demonstrating that all CoVEG plasmids tested are capable of expressing the spike protein.

[0199] Furthermore, to check the expression of VLPs, cells were analyzed for the expression of nucleocapsid (N) protein using immunofluorescence staining. For this experiment, HEK293T cells were seeded at 40,000 cells / well in 24-well plates 24 h before transfection. Cells were transfected with pCoVEG5, 9-12, and 14-20 plasmids using PEI complexes according to the manufacturer's instructions. 12 h after transfection, the medium was changed and cells were incubated for 48 h at 37 °C and 5% CO2. Cell medium was removed and cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and then fixed with 10% neutral buffered formalin for 10 min. Nonspecific binding was blocked by adding EZ block (SCYTEK) before immunostaining. Staining with anti-nucleocapsid (N) protein antibody was added at a dilution of 1:1000 and incubated for 1 h at room temperature. The stain was removed, cells were washed, and secondary antibody (Alexa Fluor 488 fluorescently labeled secondary anti-mouse, 1:1000 dilution) was added. The secondary antibody was incubated at room temperature for 1 hour, after which the stain was removed and washed. Cells were imaged using the EVOS Cell Imaging System. Figure 17 shows the expression of N protein in HEK293 cells, demonstrating that all CoVEG plasmids tested are capable of expressing nucleocapsid protein. Example 8: L protein required for detectable SARS-CoV-2 VLP formation

[0200] To isolate intact virus-like particles (VLPs), culture 4 × 10 cells in a 150 mm dish using PEI complexes according to the manufacturer's instructions. 6HEK293 cells were transfected with pCoVEG3-20 plasmid. 12 hours after transfection, the medium was changed and the cells were incubated at 37 °C, 5% CO2 for 72 hours. The VLP-containing supernatant was collected, spun down (1,500 × g, 15 min), and concentrated using Amicon centrifugal filter units (100 kDa cutoff). The concentrate was spun down (4,500 × g, 15 min) to remove the precipitate, and the VLPs were pelleted through a 20% sucrose cushion (100,000 × g, 1.5 hours). The VLPs were resuspended in PBS and analyzed by Western blot, as shown in Figure 8. Figure 8 shows that the CoVEG3-8 plasmid can express the S protein, and the CoVEG3 and 5-8 plasmids can express the N protein. These data indicate that the DNA vectors CoVEG3 and 5-8 disclosed herein express the SARS-CoV-2 viral structural proteins required for VLP formation in HEK293 cells, and that secreted VLP components can be detected in the cell culture supernatant.

[0201] Figure 11 shows the expression of S and N proteins from CoVEG5, 9, 11, 16, 10, and 15 plasmids. The results showed that expression of mutant S proteins (from CoVEG9, 11, 10, and 15) increased the amount of spike protein expressed and displayed on the VLPs. Expression of ORF3 protein (from CoVEG16) appeared to decrease the amount of S and N proteins in the VLPs. In the absence of Enhancer L protein upon expression of CoVEG15 plasmid, a similar amount of S protein was obtained, but much more N protein was obtained. Without being bound by theory, it is believed that in the absence of Enhancer L protein, more N protein is expressed, resulting in unbalanced VLP formation.

[0202] Figure 12 shows the expression of S and N proteins from the CoVEG5, 12, 14, 13, 10, 9, and 11 plasmids. These data show that expressing mutant S proteins (from CoVEG9, 10, and 11 plasmids) increased the amount of spike protein expressed and displayed on the VLPs compared to the comparison plasmid that expressed the wild-type S protein, while the amount of N protein appeared to be constant across the plasmids.

[0203] Figure 18 shows expression of the S and N proteins from the CoVEG5, 8, 9, 10, 15, 16, 17, and 20 plasmids. Notably, the presence of the enhancer L protein resulted in different S and N protein expression ratios, as shown, for example, by the CoVEG10 (L protein) vs. CoVEG20 (no L protein) S and N Western blots in Figure 18.

[0204] The presence of secreted VLPs was also confirmed by ELISA. For this experiment, 24,000 HEK293 cells were transfected with pCoVEG5 and 9-14 plasmids or with plasmids containing either spike protein or nucleocapsid protein as controls. The experiment was performed in 24-well plates using PEI complexes according to the manufacturer's instructions. 12 hours after transfection, the medium was changed and the cells were incubated for 72 hours at 37°C and 5% CO2. The VLP-containing supernatant was harvested, spun down (1,500 x g, 15 min), and 75 μl of the clarified supernatant was used to coat an ELISA plate overnight at 4°C. After incubation, the plate was washed twice with 0.05% Tween-20 in PBS and the wells were blocked using EZ-block (2 hours at 37°C). The plate was washed twice with 0.05% Tween-20 in PBS. To detect VLPs in the coating material, anti-RBD (Sino Biological, Mouse anti-RBD SARS-CoV-2 (2019-nCoV) spike neutralizing antibody, Mouse Mab, 40592-MM57 SARS-CoV-2, diluted 1:500 in EZ Block) or anti-N (Novus Biologicals, Mouse anti-SARS-CoV-2 Nucleocapsid, Clone:B3449M, N2787B09, diluted 1:1000 in EZ Block) were added to the wells. The antibodies were incubated for 1 hour at room temperature, followed by washing three times with 0.05% Tween-20 in PBS and adding 75 μl of secondary antibody (goat anti-mouse, HRP conjugate, 1:2,000 dilution, Southern Biotech, goat anti-mouse IgG(H+L), horseradish peroxidase, HRP, polyclonal, OB103405) and incubating for 1 hour at room temperature. Wells were washed extensively (five times with 0.05% Tween-20 in PBS) and binding was developed using 75 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Surmodisc Inc TMB One Component HRP Microwell Substrate).The reaction was carried out with 75 μl of Stop Solution (Surmodisc Inc 450 NM LIQ STOP REAGENT) for 30 minutes, and the absorbance was measured at 450 nm.

[0205] Figure 15 shows the ELISA results from VLP secretion of CoVEG5 and 9-14 plasmids compared to single protein S and N expression vectors. Both spike and nucleocapsid proteins were secreted from HEK293 cells. However, while the S protein showed a high ELISA VLP signal compared to single protein expression, the N protein showed a significantly lower VLP signal compared to single protein expression. The N signal in VLPs may be lower than the S signal in VLPs because the N protein is inside the VLP and cannot be accessed by antibodies.

[0206] To further ensure that the expressed structural proteins from SARS-CoV2 formed intact VLPs, 20 × 10 6HEK293 cells were transfected with the pCoVEG10 plasmid in 5-150 mm dishes using PEI complexes according to the manufacturer's instructions. 12 h after transfection, the medium was changed and the cells were incubated at 37 °C, 5% CO2 for 72 h. The VLP-containing supernatant was collected, spun down (1,500 × g, 15 min), and concentrated using Amicon centrifugal filter units (100 kDa cutoff). The concentrate was spun down (4,500 × g, 15 min) to remove the precipitate, and the VLPs were pelleted through a 20% sucrose cushion (100,000 × g, 1.5 h). The VLPs were resuspended in PBS and used for co-immunoprecipitation (co-IP). For co-IP, resuspended VLPs were incubated with anti-S RBD antibody (Sino Biological, Mouse anti-RBD SARS-CoV-2 (2019-nCoV) spike neutralizing antibody, Mouse Mab, 40592-MM57 SARS-CoV-2) for 60 min, followed by addition of 100 μl of washed protein A / G agarose resin (Thermo Fisher scientific). The resin was incubated for 120 min and then eluted with 0.1 M glycine pH 2. The eluate was immediately neutralized by adding 5 volumes of 1 M Tris pH 8.0. Fractions were analyzed for the presence of N protein by Western blot using anti-N antibody (Novus Biologicals, Mouse anti-SARS-CoV-2 Nucleocapsid, Clone:B3449M, N2787B09).

[0207] Figure 19 shows the Western blot results of a co-IP experiment of the CoVEG10 plasmid. The N protein is packed into intact VLPs, as shown by the presence of the N signal in the elution fraction after incubation with RBD (left, arrow). This signal, compared to the absence of signal in the wash fraction, indicates that the N protein is retained within the particle. As a control for possible nonspecific N protein binding to the outside of the particle, co-IP was performed in parallel without anti-RBD antibody (right). The N protein signal was not detectable in the elution fraction, indicating that the N protein did not bind nonspecifically to the resin.

[0208] To visualize secreted VLPs, 20 x 10 6 HEK293 cells were transfected with pCoVEG10 and 20 plasmids in 5×150 mm dishes using PEI complexes and according to the manufacturer's instructions. 12 hours after transfection, the medium was changed and the cells were incubated at 37° C., 5% CO2 for 72 hours. The VLP-containing supernatant was collected, spun down (1,500×g, 15 min), and concentrated using an Amicon 100 kDa centrifugal filter unit. The concentrate was spun down (4,500×g, 15 min) to remove precipitate, and the VLPs were pelleted through a 20% sucrose cushion (100,000×g, 1.5 h). The VLPs were resuspended in PBS, flash frozen, and stored at −80° C. until used for transmission electron microscopy (TEM).

[0209] For TEM experiments, VLPs were ultracentrifuged for 2 h at 25000 g on a 20% sucrose cushion using a TLS-55 (Optima TLX Ultracentrifuge, Beckman). 10 μl was placed on a microscope copper grid (Sigma Aldrich) and fixed for 5 min with 2% (v / v) paraformaldehyde. Samples were then negatively stained with 5 mL of phosphotungstic acid (Sigma Aldrich). Grids were examined using a Hitachi HT7700 TEM operating at 100 keV.

[0210] Figure 16 shows the presence of VLPs in isolated material for CoVEG10. The presence of large particles with a distinct spike trimerized surface could be observed for CoVEG10 (see magnified inset). CoVEG20, which is identical to CoVEG10 except for the presence of the L regulatory protein, failed to produce recognizable VLPs.

[0211] Intact and immunogenic VLPs are highly dependent on the ratio of all VLP-forming proteins. It has been shown herein that the L protein controls the expression of all VLP-forming proteins and the correct formation of VLPs. Example 9: L protein increased neutralizing antibodies against SARS-CoV-2 proteins in vivo and was required for Th1 responses

[0212] To determine the immunogenicity of plasmids CoVEG3-8, the plasmids were diluted to 1 mg / ml in PBS and 50 μl was injected intramuscularly into 6-week-old BALB / c mice at 2-week intervals for a total of two injections on days 1 and 15. Blood was collected on days 14, 28, 42, and 56, and serum was isolated and flash frozen in the presence of anticoagulant.

[0213] To determine the immunogenicity of plasmids CoVEG5, 8, and 9-14, as well as the S-only plasmid, plasmids were diluted to 2 mg / ml or 0.5 mg / ml in PBS and 50 μl was injected intramuscularly (2 mg / ml) or intradermally (0.5 mg / ml) into 6-week-old BALB / c mice at 2-week intervals for a total of two injections on days 1 and 15. Blood was collected on days 14, 28, 42, and 56, and serum was isolated and flash frozen in the presence of anticoagulant.

[0214] To determine the immunogenicity of plasmids CoVEG9, 10, and 20, and S-only plasmids with and without enhancer proteins, plasmids were diluted to 2 mg / ml or 0.2 mg / ml in PBS and 50 μl was injected intramuscularly into 6-week-old C57BL / 6 mice at 2-week intervals for a total of 1 to 3 injections on days 1, 15, and 29. Blood was collected on days 0, 14, 28, 42, 56, and 70, and serum was isolated and snap frozen in the presence of anticoagulant. To measure the binding antibody concentration, i.e., the humoral immune response elicited, enzyme-linked immunosorbent assays (ELISAs) were performed using purified SARS-CoV-2 spike RBD protein (Creative Diagnostics® DAGC149 Recombinant SARS-CoV-2 Spike Protein Receptor Binding Domain [His]) as the coating material. For this experiment, high-binding 96-well plates were coated with 75 μl of 2 μg / ml SARS-CoV-2-spiked RBD solution and the plates were incubated overnight at 4 °C. After incubation, the plates were washed twice with 0.05% Tween-20 in PBS and the wells were blocked using EZ-block (2 h at 37 °C). The plates were washed twice with 0.05% Tween-20 in PBS. Serum was collected from mice after 56 days and added to the wells (1:500 dilution for bound antibody detection, 1:100 to 1:7812500 for end-point titration). The serum was incubated for 1 h at room temperature, followed by washing three times with 0.05% Tween-20 in PBS and adding 75 μl of secondary antibody (goat anti-rabbit, HRP conjugated, 1:4,000 dilution) and incubating for 1 h at room temperature. Wells were washed extensively (5 times with 0.05% Tween-20 in PBS) and binding was developed using 75 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Surmodisc Inc TMB One Component HRP Microwell Substrate).The reaction was carried out for 30 minutes and then stopped with 75 μl of Stop Solution (Surmodisc Inc 450 NM LIQ STOP REAGENT). The absorbance was measured at 450 nm.

[0215] Figure 9A shows the total binding antibodies measured using ELISA, and Figure 9B shows the endpoint titers measured. These results indicate that the CoVEG3-8 plasmids are capable of eliciting a strong immune response when injected into mice, thus producing anti-SARS CoV2 antibodies. CoVEG8 was identified as being particularly superior in being able to induce a strong immune response in these mice. These results indicate that the plasmids disclosed herein are highly effective DNA vaccines against SARS-CoV-2.

[0216] Figure 13 shows ELISA results from either intramuscular (IM) or intradermal (ID) injection of CoVEG5, 9, 11, 10, 12, 13, 8, and 14 plasmids. Notably, intramuscular injection of CoVEG10 induced a higher immune response compared to injection of spike protein alone. Intramuscular injection of CoVEG5, CoVEG9, and CoVEG11 also induced a higher immune response compared to injection of S protein alone. These results further demonstrate that the plasmids disclosed herein are highly effective DNA vaccines against SARS CoV2 that perform better than vaccines expressing spike protein alone.

[0217] Figures 20 and 22 show antibody binding titers from CoVEG5 and 9-14, 18, 19, and 20 plasmids, as well as S alone, 42 days after IM or ID injection. Interestingly, all of the CoVEGs tested were able to induce an immune response, with CoVEG9 being the most effective. Notably, VLP-forming CoVEG9 performed better than the spike protein alone.

[0218] Furthermore, cellular immune responses were measured using the presence of antigen-reactive T cells using IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISpot) assays. For ELISpot analysis, spleens were collected and T cells were isolated. ELISpot assessment was performed by priming T cells with Miltenyi Biotec Peptivator SARS-CoV-2 peptide pool to activate SARS-CoV-2-reactive T cells. For analysis, mouse IL-4 single-color ELISPOT and mouse INF-γ single-color ELISPOT (Immunospot, Cellular Technology Limited) were used according to the manufacturer's instructions. Briefly, 96-well PVDF membrane plates were coated with IL-4 or INF-γ capture antibodies and incubated overnight at 4°C. After washing, 150,000 splenocytes in 100 μl of CTL test medium were seeded onto the pre-coated plates and incubated for 15 min at 37°C and 4% CO2. Cells were activated with either PMA / ionophore (as a positive control) or 0.6 μl per well of reconstituted Miltenyi Biotec Peptivator SARS-CoV-2 peptide pools (S, N, or M). Reactions were incubated for 24 hours before being developed and counted using an ImmunoSpot analyzer (CTL).

[0219] Figure 24 shows the results of T cell analysis of CoVEG10 and CoVEG20. Importantly, to generate a successful vaccine against respiratory viruses, such as SARS-CoV-2, Th1 preference (INFγ) or at least a balanced Th1 / Th2 response is required. Surprisingly, the addition of the L regulatory protein in CoVEG10 influenced the T cell immune response in favor of an INFγ (Th1) response. Notably, the absence of the regulatory protein in CoVEG20 reversed the cellular response to IL4 (Th2). A Th1 response is necessary to generate a long-lasting immune response against the virus.

[0220] To test whether serum has neutralizing antibodies against SARS CoV2 that bind to the spike protein, an in vitro virus neutralization assay using the cPass™ Neutralization Assay (GenScript) was performed according to the manufacturer's instructions. cPass™ allows for the detection of total neutralizing antibodies in a sample by mimicking the interaction between the virus and the host cell in vitro. In the assay, if neutralizing antibodies are present in the sample being tested, the binding of the receptor binding domain (RBD) to the host cell membrane receptor ACE2 is inhibited. However, if neutralizing antibodies are not present in the sample, the RBD can bind to ACE2. Figure 10 shows the percent (%) inhibition of RBD binding to the ACE2 receptor. If the inhibition of RBD binding to ACE2 is greater than 30% (red dotted line), the sample is identified as having neutralizing antibodies. As shown in Figure 10, serum samples obtained from mice injected with COVEG5 and COVEG8 showed high inhibition rates and therefore generated neutralizing antibodies. These results further highlight that the disclosed plasmids are highly effective DNA vaccines against SARS-CoV-2.

[0221] Figure 21 shows the neutralizing antibodies of the samples shown in Figure 20. These data show that not all generated antibodies have neutralizing capacity. Lower signals in this assay confirmed neutralization as defined by a signal less than 70% of the negative control (dashed line). Signals less than 30% of the negative control were confirmed as strongly neutralizing. The CoVEGs tested performed differently depending on the design and injection site. Surprisingly, ID injection did not induce strong neutralizing antibodies, while IM injection did. Furthermore, CoVEG13 and 14 did not meet the criteria for neutralization. However, CoVEG9 showed the highest neutralization of all tested constructs, including the S spike protein only construct, again showing that the VLP approach provides a more potent vaccine than the subunit S spike only vaccine.

[0222] Figure 23 shows neutralizing antibodies for CoVEG9, 10, and 20 plasmids, with plasmid 20 being a control in the absence of enhancer L protein. CoVEG20 demonstrated neutralizing capacity, however, the Th2 overhang is not a viable vaccine option as shown by T cell analysis.

[0223] Furthermore, the neutralization capacity in the presence and absence of enhancer protein over time was analyzed by cPass™ SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) kit. For this, serum samples from immunized animals (immunized with CoVEG9, spike + enhancer protein L, and spike without enhancer) were taken on days 42 and 70 and cPass™ was performed as described above.

[0224] Figure 27A shows the individual values ​​of the analyzed serum samples, while Figure 27B shows the median values ​​of the same data as a summary. Interestingly, the addition of the enhancer protein clearly showed a benefit in neutralizing antibodies over time. First, both constructs with enhancer protein L (CoVEG9 and spike+L) showed higher median neutralization values ​​(Figure 27B B, CoVEG9 circles, spike+L squares). Second, the levels of neutralizing antibodies remained higher even 70 days after injection compared to the construct without enhancer protein (Figure 27B, spike triangles).

[0225] This further demonstrated the benefit of adding an enhancer protein to a vaccine candidate. Example 10: L protein increased functional West Nile Virus (WNV) VLP production when co-expressed with E and M proteins

[0226] Plasmids encoding the precursor membrane protein (prM), envelope glycoprotein (E) and enhancer protein of the NY99 strain of WNV were constructed as described in Example 6 (see FIG. 14A, SEQ ID NO: 55). A control plasmid encoding only the precursor membrane protein (prM) and envelope glycoprotein (E) of the NY99 strain of WNV was also constructed (FIG. 11B). HEK293T cells were cultured in DMEM supplemented with 10% FBS at 37° C. and 5% CO2. On day 1, cells were seeded in 24-well plates at 20,000 cells per well and grown overnight. On day 2, cells seeded in 24-well plates were transiently transfected with plasmids encoding the precursor membrane protein (prM), envelope glycoprotein (E) and enhancer protein of the NY99 strain of WNV. In all experiments, a control plasmid encoding only the precursor membrane protein (prM) and envelope glycoprotein (E) of the NY99 strain of WNV, but not the enhancer protein, was used.

[0227] Each well of a 24-well plate was transfected with a plasmid / PEI complex formed using 0.5ug of the corresponding plasmid and 1ug of PEI in 50μl of Opti-MEM. The complex was allowed to form by incubating the plasmid / PEI mixture at room temperature for 30 minutes. The cell medium in the 24-well plate was replaced with fresh Opti-MEM and the complex was added to the well. On the third day, the complex was removed from the transfected cells and replaced with fresh Opti-MEM.

[0228] On day 4, cell culture supernatants were collected, cleared from cell debris by centrifugation at 500×g for 5 min, and stored for downstream analysis by ELISA. Cells were fixed using 250 μl of 10% neutral buffered formalin (10 min at room temperature), permeabilized using 0.2% Triton-X100 (10 min at room temperature), and washed.

[0229] Protein expression in cells was visualized using a fluorescent microscope as follows: cells were stained using mouse anti-WNV_E and rabbit anti-WNV_M primary antibodies (1:500 dilution in PBS, 1 hour at room temperature), washed, developed with goat anti-mouse Alexa Fluor 488 secondary antibody (1:1000 dilution in PBS, 1 hour at room temperature), washed, and imaged using a fluorescent microscope.

[0230] The results of the immunostaining experiment are shown in Figure 28. As observed in other experiments described herein, the amount of WNV+enhancer protein (EG) was lower compared to that of WNV without it. However, the quality appeared to be higher when the enhancer protein was present, as observed by the formation of nuclei (Figure 28, right, as indicated by the arrow). These data indicate the higher quality of the protein expressed using the compositions and methods provided herein.

[0231] An ELISA assay was used to demonstrate the secretion of the expressed antigen. For this, the supernatants from the transfected cells were collected on day 4 (48 hours after transfection), day 5 (72 hours), day 6 (96 hours), day 7 (120 hours), and day 8 (144 hours). High-binding 96-well plates were coated with cell culture supernatant using 75 μl per well and incubated overnight at +4° C. The next day, the coated wells were washed using PBST buffer and blocked for 2 hours at +37° C. using 200 μl per well of EZ Block™ reagent (Scytek Laboratories). The wells were washed 3 times with PBST and incubated with the primary antibody (mouse anti-WNV_E, 1:1000 dilution in EZ Block, 75 μl per well) for 1 hour at room temperature. The wells were then washed three times with PBST and incubated with goat anti-mouse HRP secondary antibody, 75 μl per well, diluted 1:1000 in EZ Block reagent for 1 hour at room temperature. The wells were then washed five times using PBST and 75 μl of TMB substrate was added to each well and incubated for 30 minutes at room temperature, followed by the addition of 75 μl of Stop Solution and measuring absorbance at 450 nm using a plate reader. To further demonstrate that VLP secretion was not caused by cell death and non-specific release of intracellular proteins, cells were imaged daily and ELISA results were compared to the images.

[0232] FIG. 26A shows the secretion of VLPs from transfected cells over time, as measured by ELISA. WNV constructs with enhancer proteins showed the highest secretion 72 hours after transfection, as expected for intact VLPs and healthy cells, while WNV constructs without enhancer proteins showed a steady increase in secreted material over time. The latter was more consistent with increased cell death and non-specific release of protein material, most likely not fully formed VLPs. The results were confirmed by analysis of cell images taken upon harvesting from the supernatant. WNV with enhancer proteins showed little or no cell death over the experimental time (as indicated by the asterisks), whereas WNV without enhancer proteins showed visible cell death (as indicated by the asterisks), which became progressively more pronounced over the experimental time after 72 hours. The cell images are further evidence that the released material from WNV without enhancer proteins is most likely due to protein release from cell death, and not from controlled secretion of VLPs.

[0233] This example further demonstrates that the methods and compositions of the disclosure improve the quality of the antigens produced. Example 11: L protein increased total West Nile Virus (WNV) VLP production when co-expressed with WNV E and M proteins

[0234] To isolate intact VLPs, 4 x 10 cells in a 150 mm dish 6HEK293 cells were transfected with plasmids encoding the precursor membrane protein (prM) and envelope glycoprotein (E) of the NY99 strain of WNV, as well as the enhancer protein. In all experiments, a control plasmid was used that encoded only the precursor membrane protein (prM) and envelope glycoprotein (E) of the NY99 strain of WNV, but not the enhancer protein. Transfections were performed using PEI complexes according to the manufacturer's instructions, using 40 μg plasmid and 80 μg PEI per 150 mm dish. The medium was changed 12 hours after transfection, and the cells were incubated at 37°C, 5% CO2 for 72 hours. The VLP-containing supernatant was collected, spun down (1,500 × g, 15 min), and concentrated using Amicon Ultra centrifugal filter units (100 kDa cutoff). The concentrate was spun down (4,500×g, 15 min) to remove precipitate and the VLPs were pelleted through a 20% sucrose cushion at 100,000×g for 1.5 h. VLPs were resuspended in PBS and analyzed by ELISA.

[0235] ELISA was performed as described above and as known in the art, for example, in Cold Spring Harb Protoc;doi:10.1101 / pdb.prot093708. Briefly, high-binding 96-well plates were coated with VLPs resuspended in PBS at serial dilutions from 1:20 to 1:72,000 to visualize the difference in expression between constructs with and without enhancer protein and incubated overnight at 4°C. Plates were washed and blocked with EZ-block (Scytek Laboratories) for 2 hours at 37°C. Anti-West Nile virus antibody, clone E16, was diluted (1:5,000) in EZ-block and plates were incubated for 1 hour at room temperature. Wells were washed and goat anti-mouse HRP-labeled detection antibody (Southern Biotech) was added for detection, followed by washing and incubation with TMB substrate and stop solution. Signal was read as absorbance at 450 nm using a plate reader.

[0236] 25 shows the difference between total expression and secretion of West Nile virus constructs with and without enhancer protein. As shown, the addition of enhancer protein (circles) resulted in higher expression of West Nile virus particles compared to constructs lacking enhancer protein (squares). This indicates that the compositions and methods of the present disclosure are beneficial for WNV VLP vaccine production. Example 12: Immunogenicity of West Nile Virus Proteins Coexpressed with the L Enhancer Protein in Mice

[0237] The ability to induce an immune response in vivo upon vaccination with plasmids encoding precursor membrane (prM), envelope glycoprotein (E) and enhancer protein of WNV is assessed using BALB / c mice as follows: Six-week-old female BALB / c mice are randomized into groups based on body weight. Mice are dosed with plasmids using intradermal or intramuscular injections on days 1 and 21. Mouse serum samples are collected on days 1 (pre-vaccination), 21 (pre-boost) and 42. On day 42, mice are sacrificed and splenocytes are isolated.

[0238] The induced humoral immune response is measured by assessing the titers of anti-M and anti-E antibodies by enzyme-linked immunosorbent assay (ELISA), respectively, and the cellular immune response is measured by assessing the presence of antigen-reactive T cells using IFN-γ and IL-4 enzyme-linked immunosorbent spot (Dual color ELISpot) assays, respectively. ELISA is performed as described herein and as known in the art, for example, as described in Cold Spring Harb Protoc; doi:10.1101 / pdb.prot093708. Briefly, high-binding 96-well plates are coated and blocked using recombinant prM and E proteins (Abcam) at a concentration of 2 μg / ml. Serum samples are serially diluted with EZ Block reagent, added to pre-coated wells, washed, and detected using goat anti-mouse HRP-labeled detection antibody, followed by washing and incubation with TMB substrate and stop solution. The endpoint titer is defined as the reciprocal of the highest antibody dilution at which the ELISA signal (absorbance at 450 nm) exceeds 3 standard deviations above the background signal.

[0239] Dual color ELISpot assays are performed as described herein and as known in the art, for example, as described in Cold Spring Harb Protoc 2010 doi:10.1101 / pdb.prot5369. Briefly, splenocytes are isolated on day 42, stimulated with respective prM or E peptide arrays (Biodefense and Emerging Infections Research Resources Repository), and added to pre-prepared ELISpot microplates. Negative (medium) and positive controls (phorbol myristate acetate / ionomycin) are included in the assay. The number of antigen-reactive IFN-γ and IL-4 secreting T cells is counted using an ELISpot reader.

[0240] Finally, the presence of WNV neutralizing antibodies (see above) in mouse sera isolated at different time points is assessed as described herein and in The Journal of Infectious Diseases, Volume 196, Issue 12, 15 December 2007, Pages 1732-1740, and Virology, Volume 346, Issue 1, 1 March 2006, Pages 53-65. Briefly, WNV reporter-virus particles (RVPs) are generated in HEK293T cells by transiently transfecting WNV prM and E proteins (to form virus-like particles also known as subviral particles) and complemented with a transiently transfected reporter replicon (luciferase) and a transiently transfected capsid protein. The isolated RVPs are incubated with mouse serum samples at different serial dilutions, added to pre-plated PHK-21 cells, incubated for 2 days, and then reporter gene activity is measured using a microplate reader. The decrease in reporter gene activity reflects the level of WNV neutralizing antibodies in the mouse serum. Example 13: Expression and immunogenicity of additional polynucleotide constructs

[0241] The construction of plasmids encoding viral proteins from other viruses, such as influenza virus proteins (e.g., HA, NA, M1, M2, or any combination thereof), Hepatitis B virus proteins (e.g., sAg (S protein), sAg (M protein), sAg (L protein), preS1, preS2, cAg (core antigen), or any combination thereof), human papillomavirus (e.g., HPV6 L1 protein, HPV11 L1 protein, HPV16 L1 protein, HPV18 L1 protein, or any combination thereof) is performed using the methods described in Example 6. The expression of these proteins in different combinations in HEK293T cells and the isolation of VLPs is performed using the methods described in Examples 7, 8, 10, and 11. Finally, the immunogenicity of the plasmids encoding these proteins is tested using the methods described in Examples 9 and 12. Incorporation by Reference

[0242] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an acknowledgment or any form of suggestion that they constitute available prior art or form part of the common general knowledge anywhere in the world. Numbered embodiments

[0243] Embodiment 1. A vector for use as a vaccine comprising an expression cassette comprising a polynucleotide encoding a viral protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof.

[0244] Embodiment 2. The vector of embodiment 1, wherein the amino acid sequence of the enhancer protein has at least 95% identity to SEQ ID NO:1 or at least 95% identity to SEQ ID NO:2.

[0245] Embodiment 3. The vector of embodiment 1 or embodiment 2, wherein the amino acid sequence of the enhancer protein is SEQ ID NO:1 or SEQ ID NO:2.

[0246] Embodiment 4. A vector according to any one of embodiments 1 to 3, wherein the polynucleotide encoding the enhancer protein is operably linked to a polynucleotide encoding an internal ribosome entry site (IRES).

[0247] Embodiment 5. The vector of embodiment 4, wherein the polynucleotide encoding the IRES is SEQ ID NO: 24.

[0248] Embodiment 6. The vector of any one of embodiments 1 to 5, wherein the viral protein is a viral antigen.

[0249] Embodiment 6.1. The vector according to any one of embodiments 1 to 6, wherein the viral protein is derived from a virus selected from the group consisting of coronavirus, influenza virus, hepatitis B virus, human papillomavirus (HPV), West Nile virus, and human immunodeficiency virus (HIV).

[0250] Embodiment 6.2. The vector of embodiment 6.1, wherein the viral protein is derived from a coronavirus.

[0251] Embodiment 7. The vector of any one of embodiments 1 to 6.2, wherein the coronavirus is a betacoronavirus.

[0252] Embodiment 8. The vector of embodiment 7, wherein the betacoronavirus is a severe acute respiratory syndrome (SARS) virus.

[0253] Embodiment 9. The vector of embodiment 8, wherein the SARS virus is a SARS-CoV-2 virus.

[0254] Embodiment 10. The vector of embodiment 7, wherein the betacoronavirus is Middle East Respiratory Syndrome (MERS) virus.

[0255] Embodiment 11. The vector of any one of embodiments 1 to 10, wherein the coronavirus protein is a coronavirus spike protein.

[0256] Embodiment 12. The vector of embodiment 11, wherein the spike protein shares at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:13.

[0257] Embodiment 13 The vector of embodiment 12, wherein the spike protein is SEQ ID NO: 13.

[0258] Embodiment 13.1 The vector of embodiment 11, wherein the spike protein is a mutant spike protein.

[0259] Embodiment 13.2. The vector of embodiment 13.1, wherein the mutant spike protein comprises the amino acid substitutions R682G, R683S, R685S, K986P, and V987P in SEQ ID NO:13.

[0260] Embodiment 13.3. The vector of embodiment 13.1, wherein the mutant spike protein comprises the amino acid sequence of SEQ ID NO:51.

[0261] Embodiment 14. A vector according to any one of embodiments 1 to 13.3, wherein the coronavirus protein is a coronavirus membrane (M) protein.

[0262] Embodiment 15. The vector of embodiment 14, wherein the M protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:33.

[0263] Embodiment 16. The vector of embodiment 14 or embodiment 15, wherein the M protein is SEQ ID NO: 33.

[0264] Embodiment 17. The vector of any one of embodiments 1 to 16, wherein the coronavirus protein is a coronavirus envelope (E) protein.

[0265] Embodiment 18. The vector of embodiment 17, wherein the E protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:22.

[0266] Embodiment 19. A vector described in embodiment 17 or embodiment 18, wherein the E protein is sequence number 22.

[0267] Embodiment 20. The vector of any one of embodiments 1 to 19, wherein the coronavirus protein is a coronavirus nucleocapsid (N) protein.

[0268] Embodiment 21. The vector of embodiment 20, wherein the N protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:20.

[0269] Embodiment 22. A vector according to embodiment 20 or embodiment 21, wherein the N protein is SEQ ID NO: 20.

[0270] Embodiment 23. A vector according to any one of embodiments 1 to 22, wherein the coronavirus proteins form a virus-like particle (VLP).

[0271] Embodiment 23.1. The vector of embodiment 6.1, wherein the viral protein is derived from West Nile Virus.

[0272] Embodiment 23.2. The vector of embodiment 23.1, wherein the viral protein is a precursor membrane protein (preM), an envelope glycoprotein (E), or a combination thereof.

[0273] Embodiment 24. A vector for use as a vaccine comprising an expression cassette comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO:30.

[0274] Embodiment 25. The vector of embodiment 24, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 30.

[0275] Embodiment 26. A vector for use as a vaccine comprising an expression cassette comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO:31.

[0276] Embodiment 27. The vector of embodiment 26, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 31.

[0277] Embodiment 27.1. A vector for use as a vaccine comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35-49 and 55.

[0278] Embodiment 28. A vector according to any one of embodiments 1 to 27.1, wherein the vector is a naked polynucleotide.

[0279] Embodiment 29. The vector according to any one of embodiments 1 to 28, wherein the vector is a deoxyribonucleic acid (DNA) polynucleotide.

[0280] Embodiment 30. A vector according to any one of embodiments 1 to 28, wherein the vector is a ribonucleic acid (RNA) polynucleotide.

[0281] Embodiment 31. The vector of any one of embodiments 1 to 30, wherein the vector comprises a plasmid.

[0282] Embodiment 32. A vector according to any one of embodiments 1 to 30, wherein the vector comprises linear DNA.

[0283] Embodiment 33. A vector according to any one of embodiments 1 to 32, wherein the expression cassette comprises a promoter operably linked to each of the polynucleotide sequences of the expression cassette.

[0284] Embodiment 33.1 A vector according to any one of embodiments 1 to 33, wherein the vector comprises a DNA polynucleotide, said DNA polynucleotide encoding a viral packaging signal.

[0285] Embodiment 33.2. The vector of embodiment 33.1, wherein the viral packaging signal is an RNA polynucleotide.

[0286] Embodiment 33.3. The vector of embodiment 33.2, wherein the viral packaging signal is derived from a coronavirus.

[0287] Embodiment 34. A vaccine composition comprising a vector according to any one of embodiments 1 to 33.4 and a pharma- ceutically acceptable carrier.

[0288] Embodiment 35. The vaccine composition of embodiment 34, wherein the vaccine composition comprises an adjuvant.

[0289] Embodiment 36 The vaccine composition of embodiment 35, wherein the adjuvant is alum.

[0290] Embodiment 37. The vaccine composition of embodiment 35, wherein the adjuvant is monophosphoryl lipid A (MPL).

[0291] Embodiment 38. A method for expressing a viral antigen in a eukaryotic cell, comprising contacting the cell with a vector according to any one of embodiments 1 to 33.4.

[0292] Embodiment 39. The method of embodiment 38, wherein contacting the cell with the vector results in (i) expression of the antigen at a higher expression level, and / or (ii) expression of the antigen for a longer period of time, and / or (iii) expression of the antigen with better protein quality than a vector lacking the enhancer protein.

[0293] Embodiment 40. The method of embodiment 38 or embodiment 39, wherein contacting the cell with the vector results in (i) expression of a virus-like particle (VLP) comprising the antigen at a higher expression level, and / or (ii) expression of a VLP comprising the antigen for a longer period of time, and / or (iii) expression of a VLP comprising the antigen with better protein quality than a vector lacking an enhancer protein.

[0294] Embodiment 40.1 The method of embodiment 40, wherein the vector comprises a DNA polynucleotide encoding a viral packaging signal, and contacting the cell with the vector results in expression of the viral packaging signal, and the VLPs encapsidate the viral packaging signal.

[0295] Embodiment 40.2. The method of embodiment 40.1, wherein the vector results in the formation of a greater number of VLPs compared to a control vector lacking a DNA polynucleotide encoding a viral packaging signal.

[0296] Embodiment 41. A method for inducing an immune response in a subject, comprising administering to the subject an effective amount of a vaccine composition according to any one of embodiments 34 to 37.

[0297] Embodiment 42. The method of embodiment 41, wherein the subject's tissue at the administration site expresses the antigen and / or a VLP comprising the antigen.

[0298] Embodiment 43. The method of embodiment 42, wherein the tissue at the subject's administration site (i) expresses the antigen and / or the VLP comprising the antigen at a higher expression level, and / or (ii) expresses the antigen and / or the VLP comprising the antigen for a longer period of time, and / or (iii) expresses the antigen and / or the VLP comprising the antigen with better protein quality than when a vector lacking the enhancer protein is administered.

[0299] Embodiment 43.1 The method of any one of embodiments 41 to 43, wherein the vector comprises a DNA polynucleotide encoding a viral packaging signal, the tissue at the site of administration of the subject expresses the viral packaging signal, and the VLP encapsidates the viral packaging signal.

[0300] Embodiment 43.2 The method of embodiment 43 or 43.1, wherein the vector results in the expression of a greater number of VLPs compared to a control vector lacking a DNA polynucleotide encoding a viral packaging signal.

[0301] Embodiment 43.3 The method of any one of embodiments 43 to 43.2, wherein the VLP that encapsidates the viral packaging signal is more immunogenic than a control VLP that contains the antigen but lacks the viral packaging signal.

[0302] Embodiment 44. The method of any one of embodiments 41 to 43, wherein the method induces an antibody response in the subject.

[0303] Embodiment 45 The method of embodiment 44, wherein the antibody response is a neutralizing antibody response.

[0304] Embodiment 46 The method of any one of embodiments 41 to 43, wherein the method induces a cellular immune response.

[0305] Embodiment 47. The method of any one of embodiments 41 to 46, wherein the method induces a prophylactic, protective, and / or therapeutic immune response in the subject.

[0306] Embodiment 48. The method of any one of embodiments 41 to 47, wherein the administration is intradermal, intramuscular, subcutaneous, or intranasal.

[0307] Embodiment 49. A polynucleotide comprising an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof.

[0308] Embodiment 50. The polynucleotide of embodiment 49, wherein the amino acid sequence of the enhancer protein has at least 95% identity to SEQ ID NO:1 or at least 95% identity to SEQ ID NO:2.

[0309] Embodiment 51. A polynucleotide described in embodiment 49 or embodiment 50, wherein the amino acid sequence of the enhancer protein is SEQ ID NO:1 or SEQ ID NO:2.

[0310] Embodiment 52. A polynucleotide according to any one of embodiments 49 to 51, wherein the polynucleotide encoding an enhancer protein is operably linked to a polynucleotide encoding an internal ribosome entry site (IRES).

[0311] Embodiment 53 The polynucleotide of embodiment 52, wherein the polynucleotide encoding the IRES is SEQ ID NO: 24.

[0312] Embodiment 54 The polynucleotide of any one of embodiments 49 to 53, wherein the coronavirus protein is a coronavirus antigen.

[0313] Embodiment 55. A polynucleotide according to any one of embodiments 49 to 54, wherein the coronavirus is a betacoronavirus.

[0314] Embodiment 56 The polynucleotide of embodiment 55, wherein the betacoronavirus is a severe acute respiratory syndrome (SARS) virus.

[0315] Embodiment 57. The polynucleotide of embodiment 56, wherein the SARS virus is a SARS-CoV-2 virus.

[0316] Embodiment 58. The polynucleotide of embodiment 55, wherein the betacoronavirus is Middle East Respiratory Syndrome (MERS) virus.

[0317] Embodiment 59. The polynucleotide of any one of embodiments 49 to 58, wherein the coronavirus protein is a coronavirus spike protein.

[0318] Embodiment 60. The polynucleotide of embodiment 59, wherein the spike protein shares at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 13.

[0319] Embodiment 61 The polynucleotide of embodiment 59 or embodiment 60, wherein the spike protein is sequence number 13.

[0320] Embodiment 62. A polynucleotide according to any one of embodiments 49 to 61, wherein the coronavirus protein is a coronavirus membrane (M) protein.

[0321] Embodiment 63. The polynucleotide of embodiment 62, wherein the M protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:33.

[0322] Embodiment 64. A polynucleotide described in embodiment 62 or embodiment 63, wherein the spike protein is sequence number 33.

[0323] Embodiment 65. The polynucleotide of any one of embodiments 49 to 64, wherein the coronavirus protein is a coronavirus envelope (E) protein.

[0324] Embodiment 66. The polynucleotide of embodiment 65, wherein the E protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:22.

[0325] Embodiment 67. The polynucleotide of embodiment 65 or embodiment 66, wherein the spike protein is SEQ ID NO: 22.

[0326] Embodiment 68. The polynucleotide of any one of embodiments 49 to 67, wherein the coronavirus protein is a coronavirus nucleocapsid (N) protein.

[0327] Embodiment 69. The polynucleotide of embodiment 68, wherein the N protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:20.

[0328] Embodiment 70. The polynucleotide of embodiment 68 or embodiment 69, wherein the N protein is SEQ ID NO: 20.

[0329] Embodiment 71. A polynucleotide according to any one of embodiments 49 to 70, wherein the coronavirus proteins form a virus-like particle (VLP).

[0330] Embodiment 72. A polynucleotide comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO:30.

[0331] Embodiment 73. The polynucleotide of embodiment 72, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 30.

[0332] Embodiment 74. A polynucleotide comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO:31.

[0333] Embodiment 75. The polynucleotide of embodiment 74, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 31.

[0334] Embodiment 76. A polynucleotide according to any one of embodiments 49 to 75, wherein the polynucleotide is a naked polynucleotide.

[0335] Embodiment 77. The polynucleotide of any one of embodiments 49 to 76, wherein the polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide.

[0336] Embodiment 78. A polynucleotide according to any one of embodiments 49 to 76, wherein the polynucleotide is a ribonucleic acid (RNA) polynucleotide.

[0337] Embodiment 79. A polynucleotide described in any one of embodiments 49 to 71 and 76 to 78, wherein the expression cassette comprises a promoter operably linked to each of the polynucleotide sequences of the expression cassette.

[0338] Embodiment 80. A kit comprising a vector, the vector comprising an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof.

[0339] Embodiment 81. The kit described in embodiment 80, wherein the amino acid sequence of the enhancer protein has at least 95% identity to SEQ ID NO:1 or at least 95% identity to SEQ ID NO:2.

[0340] Embodiment 82. A kit described in embodiment 80 or embodiment 81, wherein the polynucleotide encoding the enhancer protein is operably linked to a polynucleotide encoding an internal ribosome entry site (IRES).

[0341] Embodiment 83 The kit of embodiment 82, wherein the polynucleotide encoding the IRES is SEQ ID NO: 24.

[0342] Embodiment 84. A kit according to any one of embodiments 80 to 83, wherein the coronavirus protein is a coronavirus antigen.

[0343] Embodiment 85. A kit according to any one of embodiments 80 to 84, wherein the coronavirus is a betacoronavirus.

[0344] Embodiment 86 The kit of embodiment 85, wherein the betacoronavirus is a severe acute respiratory syndrome (SARS) virus.

[0345] Embodiment 87. The kit of embodiment 86, wherein the SARS virus is a SARS-CoV-2 virus.

[0346] Embodiment 88. The kit of embodiment 85, wherein the betacoronavirus is Middle East Respiratory Syndrome (MERS) virus.

[0347] Embodiment 89. A kit according to any one of embodiments 80 to 88, wherein the coronavirus protein is a coronavirus spike protein.

[0348] Embodiment 90. The kit of embodiment 89, wherein the spike protein shares at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:13.

[0349] Embodiment 91 The kit of embodiment 90, wherein the spike protein is SEQ ID NO: 13.

[0350] Embodiment 92. A kit according to any one of embodiments 80 to 91, wherein the coronavirus protein is a coronavirus membrane (M) protein.

[0351] Embodiment 93. The kit of embodiment 92, wherein the M protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:33.

[0352] Embodiment 94. The kit of embodiment 92 or embodiment 93, wherein the M protein is SEQ ID NO: 33.

[0353] Embodiment 95. A kit according to any one of embodiments 80 to 94, wherein the coronavirus protein is a coronavirus envelope (E) protein.

[0354] Embodiment 96. The kit of embodiment 95, wherein the E protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:22.

[0355] Embodiment 97. A kit according to embodiment 95 or embodiment 96, wherein the E protein is SEQ ID NO: 22.

[0356] Embodiment 98. A kit according to any one of embodiments 80 to 97, wherein the coronavirus protein is a coronavirus nucleocapsid (N) protein.

[0357] Embodiment 99. The kit described in embodiment 98, wherein the N protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:20.

[0358] Embodiment 100. The kit of embodiment 98 or embodiment 99, wherein the N protein is SEQ ID NO:20.

[0359] Embodiment 101. The kit of embodiment 80, wherein the expression cassette comprises a polynucleotide comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 30.

[0360] Embodiment 102. The kit of embodiment 80, wherein the expression cassette comprises a polynucleotide comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 31.

[0361] Embodiment 103. A kit according to any one of embodiments 80 to 102, wherein the kit comprises a pharma- ceutically acceptable carrier.

[0362] Embodiment 104. A vector comprising an expression cassette, the expression cassette comprising a promoter linked to a target gene, the vector comprising a nucleic acid sequence encoding a viral packaging element.

[0363] Embodiment 105. The vector of embodiment 104, wherein the viral packaging element is an RNA polynucleotide.

[0364] Embodiment 106. The vector of embodiment 104 or 105, wherein the viral packaging elements are derived from a coronavirus.

[0365] Embodiment 107. The vector of embodiment 106, wherein the viral packaging elements are derived from SARS-CoV2.

[0366] Embodiment 108. A vector described in any one of embodiments 104 to 107, wherein the nucleic acid sequence encoding the viral packaging element has at least about 70% identity to the nucleic acid sequence of SEQ ID NO: 34.

[0367] Embodiment 109. A method for expressing a target protein in a eukaryotic cell, comprising contacting the cell with a vector according to any one of embodiments 104 to 108.

[0368] Embodiment 110. The method of embodiment 109, wherein contacting the cell with the vector results in the formation of a virus-like particle (VLP) comprising the target protein.

[0369] Embodiment 111. The method of embodiment 110, wherein contacting the cell with the vector results in the formation of a greater number of virus-like particles (VLPs) comprising the target protein, compared to a control vector comprising the expression cassette but lacking a nucleic acid sequence encoding a viral packaging element.

[0370] Embodiment 112. A vector described in any one of embodiments 33.1 to 33.3, or a method described in any one of embodiments 40.1, 40.2, 43.1 to 43.3, wherein the nucleic acid sequence encoding the viral packaging element has at least about 70% identity to the nucleic acid sequence of SEQ ID NO: 34.

[0371] Embodiment 113. A vector for use as a vaccine comprising an expression cassette comprising, in 5' to 3' order, the following elements: a promoter, a polynucleotide encoding SEQ ID NO:33 (M protein), a polynucleotide encoding a first proteolytic cleavage site, a polynucleotide encoding SEQ ID NO:20 (N protein), a polynucleotide encoding a second proteolytic cleavage site, a polynucleotide encoding SEQ ID NO:13 (S protein), a polynucleotide encoding a third proteolytic cleavage site, a polynucleotide encoding SEQ ID NO:22 (E protein), a polynucleotide encoding SEQ ID NO:24 (IRES), and a polynucleotide encoding SEQ ID NO:2 (Enhancer L protein).

[0372] Embodiment 114. A vector for use as a vaccine comprising an expression cassette comprising, in 5' to 3' order, the following elements: a promoter, a polynucleotide encoding SEQ ID NO:33 (M protein), a polynucleotide encoding a first proteolytic cleavage site, a polynucleotide encoding SEQ ID NO:13 (S protein), a polynucleotide encoding a second proteolytic cleavage site, a polynucleotide encoding SEQ ID NO:22 (E protein), a polynucleotide encoding SEQ ID NO:24 (IRES), a polynucleotide encoding SEQ ID NO:2 (enhancer L protein), a polynucleotide encoding SEQ ID NO:20 (N protein), and a polynucleotide encoding SEQ ID NO:34 (viral packaging signal).

[0373] Embodiment 115. A vector for use as a vaccine comprising an expression cassette comprising the following elements in the order of 5' to 3': a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises an amino acid sequence of SEQ ID NO: 33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises an amino acid sequence of SEQ ID NO: 20 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, wherein the S protein comprises an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 52, or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein comprises an amino acid sequence of SEQ ID NO: 22 or at least 95% identical thereto, a polynucleotide encoding an IRES, wherein the IRES sequence comprises a polynucleotide sequence of SEQ ID NO: 24 or at least 95% identical thereto, and a polynucleotide encoding an enhancer L protein, wherein the L protein comprises an amino acid sequence of SEQ ID NO: 2 or at least 95% identical thereto.

[0374] Embodiment 116. A vector comprising the following elements in order from 5' to 3': a promoter, a first polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises a polynucleotide sequence of SEQ ID NO:34 or at least 9% identical thereto, a polynucleotide encoding an M protein, wherein the M protein comprises an amino acid sequence of SEQ ID NO:33 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises an amino acid sequence of SEQ ID NO:20 or at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, wherein the S protein comprises an amino acid sequence of SEQ ID NO:51 or SEQ ID NO:52 or at least 95% identical thereto. 23. A vector for use as a vaccine comprising an expression cassette comprising: a polynucleotide comprising an amino acid sequence of SEQ ID NO: 34 or a polynucleotide sequence at least 95% identical thereto; a polynucleotide encoding an IRES, wherein the IRES sequence comprises SEQ ID NO: 24 or a polynucleotide sequence at least 95% identical thereto; a polynucleotide encoding an enhancer L protein, wherein the L protein comprises an amino acid sequence of SEQ ID NO: 2 or a polynucleotide sequence at least 95% identical thereto; and a second polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises a polynucleotide sequence of SEQ ID NO: 34 or a polynucleotide sequence at least 9% identical thereto.

Claims

1. A composition comprising a polynucleotide for use in a vaccine, wherein the polynucleotide encodes a viral antigen protein and an enhancer protein, and the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof.

2. The composition of claim 1, wherein the polynucleotide encodes an enhancer protein amino acid sequence having at least 95% identity to SEQ ID NO:1 or SEQ ID NO:2, or wherein the polynucleotide encodes the enhancer protein amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

2.

3. The composition described in claim 1, wherein the picornavirus L protein is a Theiler's virus L protein or a functional variant thereof.

4. The composition described in claim 1, wherein the picornavirus L protein is an L protein of encephalomyocarditis virus (EMCV) or a functional variant thereof.

5. The composition described in claim 1, wherein the picornavirus L protein or its functional variant is an inhibitor of nucleocytoplasmic transport.

6. The composition described in claim 1, wherein the enhancer protein improves expression of the viral antigen protein.

7. The composition of claim 1 , wherein the polynucleotide encodes an internal ribosome entry site (IRES) operably linked to the enhancer protein.

8. The composition of claim 7, wherein the IRES is encoded by the sequence of SEQ ID NO:

24.

9. The composition of claim 1 , wherein the viral antigen protein is a structural protein.

10. The composition of claim 1, wherein the viral antigen protein is derived from SARS-CoV-1, MERS-CoV, chikungunya virus, African swine fever virus, dengue virus, Zika virus, influenza virus, human immunodeficiency virus (HIV), Ebola virus, hepatitis virus, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), West Nile virus, or human papillomavirus.

11. 2. The composition of claim 1, wherein the viral antigen protein is a spike (S) protein, a membrane (M) protein, an envelope (E) protein, or a nucleocapsid (N) protein.

12. The composition of claim 1 , wherein the viral antigen protein forms a virus-like particle (VLP).

13. The composition of claim 1 , wherein the polynucleotide encodes a viral packaging signal.

14. The composition described in claim 1, wherein the polynucleotide is an RNA molecule.

15. A vector comprising the polynucleotide according to any one of claims 1 to 14.

16. A vaccine composition for inducing an immune response in a subject, comprising the vector described in claim 15 and a pharma- ceutically acceptable carrier and / or adjuvant.

17. 17. The vaccine composition of claim 16, wherein the inclusion of the enhancer protein increases the duration of neutralizing antibodies in the subject compared to a vaccine composition that does not contain an enhancer protein and / or increases a Th1 cell response compared to a vaccine composition that does not contain an enhancer protein.

18. The vaccine composition of claim 16, further comprising a delivery vehicle.

19. The vaccine composition of claim 18, wherein the delivery vehicle comprises one or more of the following: liposomes, nanocapsules, nanoparticles, microparticles, microspheres, lipid particles, vesicles, polymers, or peptides.

20. The vaccine composition of claim 16, wherein the vaccine composition is administered via intradermal, intramuscular, subcutaneous, or intranasal administration.

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