COVID-19 treatment and its methods

Abstract

The vaccine composition that induces immunity against coronavirus in a subject comprises a recombinant nucleic acid encoding N-ETSD, which is a modified nucleocapsid protein including an endosomal targeting sequence, and / or a recombinant nucleic acid encoding S-fusion, which is a modified spike protein with improved surface expression. The vaccine can be formulated as a recombinant nucleic acid, a recombinant yeast, and / or a recombinant virus, such as an adenovirus, and can be administered via injection and / or mucosal delivery.

Description

[0001] This application claims priority to our co-pending U.S. provisional patent applications having the following serial numbers: 62 / 988,328 filed on March 11, 2020, 63 / 009,960 filed on April 14, 2020, 63 / 010,010 filed on April 14, 2020, 63 / 059,975 filed on January 1, 2020, 63 / 064,157 filed on August 11, 2020, 63 / 117,460 filed on November 24, 2020, 63 / 117,847 filed on November 24, 2020, 63 / 117,922 filed on November 24, 2020, 63 / 118,697 filed on November 26, 2020, 63 / 135,380 filed on January 8, 2021, and U.S. non-provisional patent application 16 / 883,263 filed on May 26, 2020, and entitled “Anti COVID-19 Therapies targeting nucleocapsid and spike Proteins [Anti-COVID-19 Therapeutics Targeting Nucleocapsid and Spike Proteins]" (filed concurrently and with reference number 102538.0080US3), all of which are incorporated herein by reference.

[0002] Sequence Listing

[0003] The contents of an ASCII text file of a sequence listing named 102690-0041PCT_REV003_ST25.txt, 172 KB in size, was created on March 4, 2021, was submitted electronically with this application via EFS-Web, and is incorporated by reference in its entirety. Technical Field

[0004] The field of the disclosure is vaccine compositions and methods for generating immunity against coronaviruses, and in particular coronaviruses involving SARS-CoV-2. Background Art

[0005] The Background Description includes information that may be useful in understanding the present disclosure. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is an admission made that any publication specifically or implicitly referenced is prior art.

[0006] All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0007] While SARS-CoV2 diagnostic tests have become available in a relatively short time, many attempts to treat the disease have so far shown mixed or inconclusive results. Most typically, patients with severe symptoms are treated to maintain breathing / blood oxygenation. COVID19 has a high mortality rate, especially among the elderly, immunocompromised individuals, and those with pre-existing heart disease, lung disease, or diabetes. Despite improvements in acute care, it is clear that controlling the disease is critical, as social distancing and other public health mitigation measures provide only modest relief.

[0008] To this end, many candidate anti-SARS-CoV2 vaccine compositions target one or more proteins of the virus (see, e.g., FIMMU 2020, 11: 602256). For example, Sinovac and Sinopharm are currently testing inactivated virus vaccine formulations. Cansino Biologics, Janssen Pharma, Oxford University, and Garnaleya have developed vaccines based on non-replicating adenoviral vectors encoding one or more viral proteins. Novamax has produced a protein subunit-based vaccine. Recently, RNA-based vaccines from Moderna and Pfizer have been approved in several jurisdictions. Most of these vaccines induce at least some (typically non-sterile) immunity against the infection that causes the disease, but it is unclear whether protection is effective over several months and / or whether sufficient immune memory can protect vaccinated individuals over an extended period of time. In addition, it is unclear whether such vaccines will produce clinically meaningful T-cell-based responses.

[0009] While some vaccines are already available, the rapid and global distribution and administration of vaccines has encountered significant difficulties and slowed global distribution. In addition, logistics requirements and the need for medical professionals have limited the management of specific vaccination infrastructure.

[0010] In order to overcome such difficulties, the solid dosage form of vaccine is needed. However, such dosage forms (such as powders, tablets, capsules) need to disintegrate and release one or more active ingredients in physiologically relevant positions. The dosage form loaded with active ingredients must remain stable during transportation and storage from production to application. The mucosa or oral delivery of vaccine is highly desirable because this application helps to produce IgA-, IgE- and IgM-class antibodies, which are very important for immunity against mucosa or intestinal infection. Unfortunately, there is no such vaccine.

[0011] Although various vaccine compositions and methods for inducing immunity against SARS-CoV-2 are known in the art, they all have some drawbacks. Therefore, there is still a need for improved vaccine compositions and methods. Summary of the invention

[0012] Disclosed herein are various vaccine compositions and methods thereof, wherein recombinant modified nucleocapsid proteins and / or modified spike proteins of coronaviruses induce immunity against coronaviruses in a subject. Most preferably, the recombinant protein is encoded in a recombinant nucleic acid, which can be delivered as part of a recombinant virus and / or as part of a recombinant yeast or yeast lysate in a lipid formulation.

[0013] Also disclosed herein is a recombinant nucleic acid comprising a first portion (N-ETSD) encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) fused to an endosomal targeting sequence, wherein the first portion is functionally coupled to one or more regulatory elements that enable expression of N-ETSD, and a second portion encoding a SARS virus spike protein (S), wherein each of the second portions is functionally coupled to one or more regulatory elements that enable expression of S.

[0014] In some embodiments, the SARS virus is SARS-CoV-2, and / or the endosomal targeting sequence of N-ETSD is encoded at the 5'-end and / or 3'-end of the first portion. In further embodiments, the second portion encodes S optimized for surface expression. The first portion and the second portion can be arranged in a bicistronic sequence. For example, N-ETSD can have an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO:1. In certain embodiments, the first portion can have a nucleotide sequence of SEQ ID NO:2. In another example, the S protein or S-fusion protein can have an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO:3 (e.g., SEQ ID NO:3) or an amino acid sequence that is at least 90% identical to SEQ ID NO:4 (e.g., SEQ ID NO:4). In certain embodiments, the second portion can have a nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0015] In further embodiments, the recombinant nucleic acid may further comprise a third portion encoding a co-stimulatory molecule or an immunostimulatory cytokine. The recombinant nucleic acid may also be incorporated into a viral or yeast expression vector (e.g., an adenovirus expression vector with a deletion of the E1 gene region and a deletion of the E2b gene region, and / or a yeast expression vector for Saccharomyces cerevisiae). Most typically, but not necessarily, the nucleic acid is deoxyribonucleic acid (DNA).

[0016] The present invention describes a recombinant replication-deficient adenovirus comprising an E1 gene deletion, an E2b gene deletion, and a recombinant nucleic acid comprising a first portion encoding the SARS coronavirus N-ETSD. The first portion is functionally coupled to one or more regulatory elements that enable the expression of N-ETSD. The adenovirus also comprises a second portion encoding the SARS S protein. Each of the second portions is functionally coupled to one or more regulatory elements that enable the expression of S.

[0017] In further embodiments, the adenovirus may further comprise a third portion encoding a co-stimulatory molecule or an immunostimulatory cytokine, and / or the recombinant adenovirus may have a deletion of the E3 and / or E4 gene regions.

[0018] Alternatively or additionally, disclosed herein is a recombinant yeast comprising a recombinant nucleic acid comprising a first portion encoding SARS coronavirus N-ETSD, wherein the first portion is functionally coupled to one or more regulatory elements that enable expression of N-ETSD, and a second portion encoding SARS S protein, wherein each of the second portions is functionally coupled to one or more regulatory elements that enable expression of S. The yeast may be Saccharomyces cerevisiae. In certain embodiments, the yeast may be lysed.

[0019] In yet further embodiments, disclosed herein are vaccine compositions comprising a recombinant nucleic acid provided herein, and the recombinant nucleic acid may be encapsulated in lipid nanoparticles.

[0020] In yet other embodiments, disclosed herein are vaccine compositions comprising aragonite particles admixed with a recombinant replication-defective adenovirus provided herein, wherein the recombinant replication-defective adenovirus is lyophilized. Most typically, the aragonite particles have an average particle size between 100 nm and 1 mm.

[0021] The present invention provides a recombinant nucleic acid for producing a vaccine against SARS virus and / or for inducing immunity against SARS virus. Similarly, the present invention discloses a recombinant replication-deficient adenovirus or recombinant yeast for inducing immunity against SARS virus.

[0022] Various objects, features, aspects, and advantages of the subject matter disclosed herein will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings, in which like reference numerals represent like components. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic illustration of various crystalline forms of calcium carbonate.

[0024] Figure 2 Three photographs (from right to left: 1, 2, 3) of a bivalent human adenovirus serotype 5 COVID-Spike and nucleocapsid antigen vaccine (hAD5-COVID-S / N) in an uncoated aragonite capsule (sample #6) in 0.1 M hydrochloric acid (HCL) are shown, wherein wrinkles, swelling, or holes in the capsules are observed, as shown: 1): 2 minutes after HCL acid exposure; 2): 2 hours after HCL acid exposure; and 3) 2 hours after HCL acid exposure and drying.

[0025] Figure 3 Three photographs (from right to left: 1, 2, 3) showing hAD5-COVID-S / N in uncoated aragonite capsules (sample #7 or #8) in 0.1M HCL, where swelling, distortion, or holes in the capsules were observed as shown: 1): Sample #7 2 hours after HCL acid exposure; 2): Sample #8 2 hours after HCL acid exposure; 3) 2 hours after HCL acid exposure and drying.

[0026] Figure 4 Two photographs (from right to left: 1, 2) showing hAD5-COVID-S / N in uncoated lactose capsules (sample #3 or #4) in 0.1M HCL, where the swelling of the capsules was observed as shown: 1): Sample #3 2 hours after HCL acid exposure; 2): Sample #4 2 hours after HCL acid exposure.

[0027] Figure 5 Two photographs (from right to left: 1, 2) showing hAD5-COVID-S / N in coated aragonite capsules (sample #1 or #5) in 0.1M HCL, where the swelling of the capsules was observed as shown: 1): Sample #1 2 hours after HCL acid exposure; 2): Sample #5 2 hours after HCL acid exposure.

[0028] Figure 6 The infectious units per gram (IFU / g) (y-axis) for the indicated hAD5-COVID-S / N capsule types are shown, as indicated.

[0029] Figure 7 The percent (%) virus recovery for each hAD5-COVID-S / N capsule type is shown (y-axis), as indicated.

[0030] Figure 8 The IFU / g and corresponding pH for each hAD5-COVID-S / N capsule type are shown, as indicated.

[0031] Fig. 9 The percent (%) of virus recovery for each hAD5-COVID-S / N capsule type is shown, as indicated.

[0032] Fig.10 The percent virus recovery for each hAD5-COVID-S / N capsule type is shown, as indicated, with acid treatment indicated for those with shading.

[0033] Fig.11 The IFU / g for each hAD5-COVID-S / N capsule type is shown, as indicated, with acid treatment indicated for those with shading.

[0034] Fig.12 Describes a conceptual diagram of an ideal vaccine that would elicit durable and effective immunity through multiple routes.

[0035] Fig.13 Described are the results of an exemplary expression experiment in which SARS-CoV2 N was overexpressed in Saccharomyces cerevisiae.

[0036] Fig.14 Shown are the results of an ELISA detecting IgG serological reactivity against SARS-CoV-2 spike in serum samples extracted from immunized rhesus macaques.

[0037] Fig.15 Sera that inhibit SARS-CoV-2 infectivity are depicted. Panel A shows that sera from vaccinated Group 1 rhesus monkeys inhibit SARS-CoV-2 infectivity in vitro. Panel B shows sera from vaccinated Group 2 rhesus monkeys. The dashed line indicates 20% inhibition.

[0038] Fig.16 Describes the changes in nasopharyngeal viral load over time in non-human primates (NHPs). Panel A shows the viral load (qPCR) in nasal swabs of rhesus macaques after SC+SC+oral vaccination. Panel B shows the viral load (qPCR) in nasal swabs after SC+oral+oral vaccination.

[0039] Fig.17 Describes the changes in viral load in the lung over time in NHP. Panel A shows the viral load (qPCR) in the BAL of Group 1 rhesus monkeys after SC+SC+oral vaccination, and Panel B shows the viral load (qPCR) in the BAL of Group 1 rhesus monkeys after SC+oral+oral vaccination.

[0040] Fig.18 IgG and IgM serum reactivity against SARS-CoV-2 spike in serum samples from human patients immunized with various experimental anti-SARS-CoV-2 vaccines is described.

[0041] Fig.19 ELISpot results from Th1 N reactive patients are shown.

[0042] Fig. 20 ELISpot results from patient 4 (N-unreactive) and patient 10 (weak Th1 N-reactive) are shown.

[0043] Fig.21 Exemplary results of humoral responses and neutralizing capacity of sera from hAd5 S-fusion + N-ETSD vaccinated NHPs are depicted. The following anti-Spike IgG levels are shown: (A) individual SC oral-oral NHPs (ELISA; OD450nm) together with (B) geometric mean and (C) inhibition in alternative assays. These data are also shown in SC-SC-oral NHPs: (D) individual anti-S IgG, (E) geometric mean and (F) inhibition in alternative assays. Inhibition of more than 20% (dashed line) at a serum dilution of 1:30 is associated with neutralization of SARS-CoV-2 infection. NHPs received vaccinations on days 0, 14, and 28 (black arrows).

[0044] Fig. 22 Viral loads (gRNA) in the nasal passages and lungs of SC-oral-oral and SC-SC-oral vaccinated NHPs after challenge are depicted. (A) Geometric means of individual viral gRNAs (RT qPCR) and (B) nasal swab samples; and (C) geometric means of gRNAs and (D) bronchoalveolar lavage (BAL) samples from SC-oral-oral NHPs. (E) Geometric means of individual gRNAs and (F) nasal swab samples; and (G) geometric means of gRNAs and (H) BAL samples from SC-SC-oral NHPs. SARS-CoV-2 challenge occurred on day 56 (black arrow). The level of detection (LOD; dashed line) was 54 gene copies / mL (GC / mL) for gRNA and 119 GC / mL for sgRNA. For values ​​below the LOD, half of the LOD value (27 GC / mL for gRNA and 59 GC / mL for sgRNA) was used to plot individual values ​​and calculate geometric means.

[0045] Fig.23Viral replication (sgRNA) in the nasal passages and lungs of SC-oral-oral and SC-SC-oral vaccinated NHPs after challenge is depicted. (A) Geometric means of individual viral sgRNAs (RT qPCR) and (B) nasal swab samples; and (C) sgRNAs and (D) bronchoalveolar lavage (BAL) samples from SC-oral-oral NHPs. (E) Geometric means of individual sgRNAs and (F) nasal swab samples; and (G) sgRNAs and (H) geometric means of BAL samples from SC-SC-oral NHPs. SARS-CoV-2 challenge occurred on day 56 (black arrow). LOD (dashed line) was 54 GC / mL for gRNA and 119 GC / mL for sgRNA. For values ​​below the LOD, half of the LOD value was used to plot individual values ​​and calculate geometric means.

[0046] Fig.24 T cell responses to vaccination and the neutralizing capacity of sera following SARS-CoV-2 challenge are depicted. (A) Interferon-g (IFN-γ) and (B) interleukin-4 (IL-4) secretion by PBMC-derived T cells from SC-oral-oral vaccinated NHPs in response to spike (S) and nucleocapsid (N) peptides, as measured by ELISpot, and (C) IFN-γ / IL-4 ratio are shown. The “infinity” ratio due to undetectable IL-4 is indicated as an open circle. Cells pulsed with PMA-ionomycin were used as a positive control. Data are plotted with mean and SEM. (D) MNs throughout the study are shown. 50 (dilution factor at which SARS-CoV-2 infection of Vero E6 cells is inhibited by 50%); unpaired two-tailed Student's t-test was used to compare MN in vaccinated NHPs and placebo NHPs at day 70 50 Nasal gRNA (E) and sgRNA (F) at days 57, 63, and 70, and lung gRNA (G) and sgRNA at days 57 and 63 are presented. The same data are shown for SC-SC-oral NHP, including T cell responses (IJ), MN 50 (L), nasal gRNA (M) and sgRNA (N), and lung gRNA (O) and sgRNA (P). The detection level was 54 GC / mL for gRNA and 119 GC / mL for sgRNA. Half of the LOD was used to plot data below the LOD.

[0047] Fig.25The hAd5 platform and hAd5 S-fusion + N-ETSD construct are schematically depicted. Panel A shows a human adenovirus serotype 5 vaccine platform lacking the E1, E2b, and E3 regions (*). The vaccine construct is inserted into the E1 region (arrow). Panel B shows a dual antigen vaccine comprising S-fusion and N-ETSD delivered by the hAd5 [E1-, E2b-, E3-] platform, which is under the control of the cytomegalovirus (CMV) promoter and has a C-terminal SV40 polyadenylation sequence.

[0048] Fig.26 Exemplary constructs for cloning into adenovirus are described.

[0049] Fig. 27 is a Western blot showing in vitro construct expression and S and N detection.

[0050] Fig.28 Other exemplary constructs for cloning into adenovirus are described.

[0051] Fig.29 Depicts antibody responses to N with a Th1 phenotype. Shows humoral immune responses T H 1Compared to T H 2 Related isoform analysis.

[0052] Fig.30 Cell-mediated immune (CMI) responses to the N-focal phenotype are described - IFN-γ and IL-2 ELISpot.

[0053] Fig.31 Depicted are enhanced cell surface expression of RBD in the case of S fusions and in the case of S fusion + N combination constructs compared to S-WT.

[0054] Fig.32 Recognition of antigens by recovered COVID-19 patient plasma is described. Antigens include RBD-ETSD and fusion S / N-ETSD constructs.

[0055] Fig.33SARS-CoV-2 virus, spike, hAd5[E1-,E2b-,E3-] vector, and candidate vaccine constructs are depicted. (a) Trimeric S protein is displayed on the viral surface; N protein is associated with viral RNA. (b) RBD is within the S1 region, followed by other functional regions, namely the transmembrane domain (TM) and the C-terminus (CT) within the virus. (c) The second generation human adenovirus serotype 5 (hAd5) vector used has deleted E1, E2b, and E3 regions. Constructs of (d) S wild type (S-WT), (e) S-RBD with enhanced T cell stimulatory domain (SRBD-ETSD), (f) S-fusion, (g) N-ETSD, and (h) bivalent hAd5 S-fusion+N-ETSD are shown; LP-leader peptide.

[0056] Fig.34 Transfection of HEK293T with hAd5 S-fusion + ETSD results in enhanced RBD surface expression. Flow cytometric analysis of cells transfected with the constructs with anti-RBD antibodies shows no detectable RBD surface expression in cells transfected with S-WT or (b) S-WT + N-ETSD. Surface RBD expression is high for SRBD-ETSD and SRBD-ETSD + N-ETSD (c, d). (e) Expression is low in cells transfected with S-fusion. Cell surface expression of RBD is high in cells transfected with (f) S-fusion + N-ETSD, especially on days 1 and 2. (g) No expression is detected for the N-ETSD negative control. Y-axis scale is normalized to mode (NM).

[0057] Fig.35 Depicted are immunoblot analyses of S expression. Cell surface RBD expression in HEK 293T cells for (a) hAd5 S-WT, S-fusion, and (c) S-fusion + N-ETSD is highly correlated with (d) S expression in immunoblots of HEK293T cell lysates probed with anti-full-length (S2) antibody. Y-axis scale normalized to mode (NM).

[0058] Fig.36Depicted is the binding of RBD expressed on the surface of recombinant ACE2-Fc HEK293T cells after transfection, confirming native protein folding. Flow cytometric analysis of binding between recombinant ACE2-Fc (with which the spike RBD interacts in vivo to initiate infection) and cell surface antigens expressed after transfection of HEK293T cells with (a) hAd5 S-WT, (b) hAd5 S-fusion, (c) hAd5 S-fusion+N-ETSD, (d) hAd5 SRBD-ETSD, or (e) hAd5 SRBD-ETSD+N-ETSD constructs revealed that the binding of ACE-Fc and anti-RBD specific antibodies (fj) was highest after transfection with bivalent S-fusion+N-ETSD. Two constructs containing SRBD-ETSD also showed binding. The Y-axis scale is normalized to mode (NM).

[0059] Fig.37 Depicts the localization of N expressed from hAd5 N-ETSD to endosomal / lysosomal compartments. In HeLa cells infected with N-ETSD, (a) N (red) colocalizes with the endosomal marker CD71 (b), as indicated by the arrow in (c). In transfected HeLa cells, (d) N-ETSD also colocalizes with the lysosomal marker Lamp1, while (e) N wild type (N-WT) does not, instead showing a diffuse cytoplasmic distribution.

[0060] Fig.38 Figure 2 shows ICS assays of cytokine-expressing splenocytes from CD-1 mice vaccinated at day 28 with hAd5 S-fusion + N-ETSD in response to peptide pools. (a) Highest CD8β + Splenocyte IFN-γ responses in splenocytes of hAd5 S-fusion + N-ETSD-vaccinated mice exposed to S peptide pool 1 (S-peptide pool 1); splenocytes from these mice also expressed IFN-γ in response to the N peptide pool (N-peptide pool). (b) CD4+ splenocytes from hAd5 S-fusion + N-ETSD-vaccinated mice expressed only IFN-γ in response to the N peptide pool. (c) CD8β+ splenocytes from hAd5 S-fusion + N-ETSD-vaccinated mice expressed only IFN-γ in response to the N peptide pool. + The IFN-γTNF-α responses of splenocytes were very similar to those in (a); (d) CD4+ splenocytes to the N peptide pool were also very similar to those in (b). N=5 mice / group. All data sets are plotted as mean and SEM, and all statistics are performed using the Mann-Whitney test, with *<0.05, **<0.01, ***<0.001, and ****<0.0001.

[0061] Fig.39Anti-spike and anti-nucleocapsid antibody responses in sera from hAd5 S-fusion + N-ETSD vaccinated mice are depicted. Based on absorbance, (a) anti-S antibodies and (c) anti-nucleocapsid antibodies were significantly produced. (b, d) ng equivalents are shown. Sera were diluted 1:30 (for anti-spike antibodies) and 1:90 (for anti-nucleocapsid antibodies). Data are presented as mean and SEM. Statistical analysis was performed using an unpaired two-tailed Student's t-test, with *<0.05, **<0.01, ***<0.001, and ****<0.0001.

[0062] Fig.40 cPass and Vero E6 cell SARS-CoV-2 confirmed antibody neutralization are depicted. (a) In the cPass assay, inhibition of the S RBD interaction with ACE2 was significant in 1:20 and 1:60 dilutions of sera from hAd5 S-fusion + N-ETSD vaccinated mice. (b) Vero E6 cell SARS-CoV-2 virus infection results for mice showing S-specific antibodies by ELISA also showed high neutralization for mice and very high neutralization for pooled sera (G4 pool, blue line), even compared to COVID-19 convalescent sera. G4 pool - mice with S-specific antibodies; M1, M2, M3, M4 - mouse ID; +C - convalescent sera; and medium - medium only negative control.

[0063] Fig.41 Isotypes of anti-spike and anti-nucleocapsid antibodies are depicted. Panels A and C show that hAd5 S-fusion + N-ETSD mice have significantly increased anti-spike and anti-nucleocapsid antibodies of the IgG2a and IgG2b isotypes compared to hAd5 null mice. Panels B and D show the ng equivalents of the antibody isotypes. Data are presented as mean and SEM. Statistical analysis was performed using an unpaired two-tailed Student's t-test, with * < 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001.

[0064] Fig.42 ELISpots of secreted cytokines are depicted. (a) IFN-γ secretion by hAd5 S-fusion+N-ETSD splenocytes was significantly higher than hAd5 null in response to S peptide pool 1 and N peptide pool; however (b) IL-4 was secreted only in the hAd5 S-fusion+N-ETSD case in response to the N peptide pool (one high outlier in hAd5 null was removed). N=5 mice / group. All data sets are plotted as mean and SEM, and all statistics were performed using the Mann-Whitney test, with *<0.05, **<0.01, ***<0.001, and ****<0.0001.

[0065] Fig.43 The ratio of T cell and humoral responses is depicted, showing Th1 dominance. (a) The ratio of total Th1 (IFN-γ) to Th2 (IL-4) spot forming units is shown for the response to the combined S pool and to the N pool. (b) The Th1 / Th2 ratios for antibodies against S and N are shown. For both (a) and (b), the dashed line indicates a ratio of 1 or a balance of Th1 and Th2 (no dominance).

[0066] Fig.44 Schematic illustration of the hAd5 vector, SARS-CoV-2, spike, and constructs. (A) Human adenovirus serotype 5 (hAd5[E1-, E2b-,E3-]) lacking the E1, E2b, and E3 regions is shown. (B) The SARS-CoV-2 virus displays the spike (S) protein as a trimer on the viral surface. The S protein includes the N-terminus (NT), the S1 region (including the RBD, S2, and TM regions), and the C-terminus (CT); other functional regions are not labeled. (C) Spike wild type (SWT), (D) spike fusion (S-fusion), (E) nucleocapsid without ETSD, mainly cytoplasmic localization (N); (F) N with enhanced T cell stimulation domain (N-ETSD), and (G) a bivalent S-fusion + N-ETSD construct is shown.

[0067] Fig.45 Micrographs that determined the localization of N-ETSD to endosomes, lysosomes, and autophagosomes are depicted. MoDCs were infected with Ad5 N-ETSD or N without ETSD and co-labeled with anti-flag (N, N-ETSD has a flag tag here) and anti-CD71 (endosomal marker), anti-Lamp 1 (lysosomal marker), or anti-LC3a / b antibodies. (A) N-ETSD, (B) CD71, and (C) overlay. (D) N, (E) CD71, and (F) overlay. (G) N-ETSD, (H) Lamp-1, and (I) overlay. (J) N, (K) Lamp-1, and (L) overlay. (M) N-ETSD, (N) LC3a / b, and (O) overlay. N / N-ETSD is in red, other markers are in green, yellow arrows indicate co-localization, and white arrows indicate lymphocytes.

[0068] Fig.46Demonstration of patient plasma antibodies recognizing SARS-CoV-2 antigens expressed by MoDC after hAd5 S-fusion + N-ETSD infection. (A) MoDC from two normal individuals were infected with hAd5 vaccine overnight and then exposed to plasma from a previously infected patient from a single individual; antibody binding to the DC cell surface was detected by flow cytometry. Flow histograms of hAd5 S-WT, S-fusion, S-fusion + N-ETSD, and null are shown for MoDC from two sources (B) MoDC1 and (C) MoDC2. (D) The DMFI (difference in MFI for binding to uninfected and infected cells) of hAd5 S-WT, S-fusion, S-fusion + N-ETSD, and null-infected MoDC are plotted.

[0069] Fig.47 Describes exemplary results of T cell responses (previously SARS-CoV-2 infected patients and virus-uninfected T cells) to MoDC pulsed with SARS-CoV-2 peptides. T cells from all four previously SARS-CoV-2 infected patients (Pt) showed significant IFN-γ responses to MoDC pulsed with S1, S2, and N peptide pools compared to "none". T cells from virus-uninfected (unexposed, UnEx) control individuals showed much lower responses. Statistical analysis of each patient's samples using one-way ANOVA and Tukey post hoc analysis was compared only to "none", with *p<=0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as mean and SEM; n=3-4.

[0070] Fig.48 Determination of peptide-pulsed MoDC from patients previously infected with SARS-CoV-2 stimulates autologous patient T cells to secrete IFN-γ. (A) MoDC derived from previously infected SARSCoV-2 patients Pt4 and Pt3 were pulsed overnight with a SARS-CoV-2 peptide mixture (S1, S2, or N) and then incubated with autologous CD4+ (A, B) or CD8+ (C, D) T cells. IFN-γ levels were determined by ELISpot. Statistical analysis was performed using one-way ANOVA and Dunnett post hoc multiple comparison analysis to compare each peptide pool with vehicle (shown above the bar) or between peptide pools (above the line), with *p<0.05, **p<0.01, and ****p<0.00001. Data are presented as mean and SEM; n=3.

[0071] Fig.49It is shown that MoDCs expressing in response to N-ETSD have greater IFN-γ secretion from T cells of patients previously infected with SARS-CoV-2 than N. (A) Experimental design. (BD) IFN-γ secretion by autologous CD3+ T cells in response to MoDCs expressing hAd5-N-ETSD- and hAd5 N is shown. (EG) IL-4 secretion by CD3+ cells in response to infected MoDCs is shown on the same scale as IFN-γ for each. IFN-γ secretion by (HJ) CD4+ and (KM) CD8+ T cells in response to hAd5-N-ETSD compared to null is shown. Statistical analysis was performed using one-way ANOVA and Tukey post hoc multiple comparison analysis, with *p<=0.05; **p<0.01, ***p<0.001, and ****p<0.0001. Comparisons to null are shown above the bars, and comparisons between N-ETSD and N are shown above the lines. Data are presented as mean and SEM; n=3-4.

[0072] Fig.50 Shown are the responses of T cells from patients previously infected with SARS-CoV-2 to the bivalent vaccine and its individual components, revealing different antigen specificities of T cell populations. (AC) CD3+ T cell IFN-γ responses in three patients. (DF) CD3+ T cell IL-4 responses. (GI) CD4+ IFN-γ responses. (JL) CD8+ IFN-γ responses. Statistical analysis was performed using one-way ANOVA with Tukey post hoc multiple comparisons analysis to compare each antigen-containing construct to the null construct, with *p<0.05 and ****p<0.00001. Above the bars are comparisons to the null only; above the lines are comparisons between vaccines expressing the antigens. Data are presented as mean and SEM; n=3-4. DETAILED DESCRIPTION

[0073] Various vaccine compositions and methods for inducing immunity against SARS-CoV-2 virus and closely related viruses as well as mutated forms of SARS-CoV-2 virus not only promote the production of therapeutically effective antibodies but also induce robust T cell responses. These vaccine compositions can be administered via different routes, including intramuscular, subcutaneous, oral, and mucosal routes (alone or in combination), and can even be used as an oral booster after currently known RNA-based vaccines.

[0074] In particularly contemplated embodiments, the recombinant construct comprises a modified nucleocapsid protein and / or a modified spike protein. Preferably, the modified nucleocapsid protein comprises a transport sequence, thereby transporting the modified nucleocapsid protein to the endosomal / lysosomal subcellular compartment, thereby stimulating CD4+T cells using a key antigen presentation pathway, which in turn activates naive CD8+ cytotoxic T cells by dendritic cells. Similarly, it is preferred that the modified spike protein has a modification that enhances the surface expression of the modified spike protein, thereby making the immune response more robust to the spike antigen. In fact, although the vaccines disclosed herein (e.g., hAd5[E1-, E2b-] vaccines) are exemplified using N antigens with ETSD peptide tags, in principle any antigen can be advantageously redirected to the endosomal / lysosomal subcellular compartment. Exemplary antigens to be tagged with ETSD for use in adenoviral or yeast vaccine vectors in this manner include CEA, human epidermal growth factor receptor 1 (HER1), HER2 / neu, HER3, HER4, prostate specific antigen (PSA), PSMA, folate receptor alpha, WT1, p53, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, MART-1, MC1R, Gp100, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, BRCA1, Brachyury, Brachyury (TIVS7-2, polymorphism), Brachyury (IVS7 T / C polymorphism), T Brachyury, hTERT, hTRT, iCE, MUC1, MUC1 (VNTR polymorphism), MUC1c, MUC1n, MUC2, PRAME, P15, RU1, RU2, SART-1, SART-3, AFP, β-catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, ETV6 / AML, LDLR / FUT, Pml / RARα, HPV E6, HPV E7, and TEL / AML1.

[0075] "N-ETSD" refers to a modified nucleocapsid protein of the SARS-CoV-2 virus that includes an endosomal targeting sequence. An exemplary N-ETSD has an amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2.

[0076] "S-HA" or "Spike" or "S" refers to the spike protein of the SARS-CoV-2 virus with an HA tag. An exemplary S-HA has an amino acid sequence of SEQ ID NO: 3 and a nucleotide sequence of SEQ ID NO: 5.

[0077] "S-fusion" or "spike-fusion" refers to a modified spike protein of the SARS-CoV-2 virus with increased surface expression. An exemplary S-fusion has an amino acid sequence of SEQ ID NO: 4 and a nucleotide sequence of SEQ ID NO: 6.

[0078] "N" or "N-wt" or "nucleocapsid" refers to the nucleocapsid protein of the SARS-CoV-2 virus. An exemplary N protein has the amino acid sequence of SEQ ID NO:7.

[0079] "ETSD" refers to endosomal targeting sequence. An exemplary ETSD has the amino acid sequence of SEQ ID NO:8.

[0080] “ACE2” refers to angiotensin-converting enzyme 2. An exemplary (human) ACE2 has the amino acid sequence of SEQ ID NO:9.

[0081] "Soluble ACE2 protein" refers to mutant and truncated forms of ACE that are soluble under physiological conditions. An exemplary soluble ACE has the amino acid sequence of SEQ ID NO: 10.

[0082] Several modified spike and nucleocapsid constructs and methods

[0083] Recombinant viruses and yeast are disclosed herein. The viruses and yeast disclosed herein can be used for a variety of purposes, such as treating and / or preventing coronavirus diseases. In one aspect, a replication-deficient adenovirus is disclosed herein, wherein the adenovirus comprises a deletion of the E1 gene region; a deletion of the E2b gene region; a deletion of the E3 gene region, a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid protein, a CoV2 nucleocapsid protein fused to an endosomal targeting sequence (N-ETSD), and a nucleic acid encoding a CoV2 spike protein sequence optimized for cell surface expression (S-fusion).

[0084] In one embodiment, the N-ETSD polypeptide may comprise a sequence having at least 80% identity to SEQ ID NO: 1. In other embodiments, the identity value is at least 85%. In yet other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. It is further contemplated that the N-ETSD fusion protein contains a linker between the N-ETSD domain and the nucleocapsid protein. For example, the linker can be a 16 amino acid linker having the sequence (G3S)4. In certain embodiments, disclosed herein are methods for enhancing the immunogenicity of an intracellular antigen, the method comprising tagging the antigen with ETSD and expressing the tagged antigen in an antigen presenting cell (e.g., a dendritic cell).

[0085] In some embodiments, the fusion protein comprising N-ETSD and CoV-2 nucleocapsid protein can be encoded by a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0086] The CoV-2 spike protein is expected to have at least 85% identity with SEQ ID NO:3. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. The nucleic acid encoding the CoV-2 spike protein is expected to have at least 85% identity with SEQ ID NO:5. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0087] The CoV-2 spike fusion protein is expected to have at least 85% identity with SEQ ID NO:4. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. The nucleic acid encoding the CoV-2 spike fusion protein is expected to have at least 85% identity with SEQ ID NO:6. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0088] In a second aspect of the disclosure, provided herein is a recombinant yeast comprising a nucleic acid encoding a protein selected from the group consisting of a coronavirus 2 (CoV-2) nucleocapsid protein, a CoV2 N-ETSD protein, a CoV2 spike protein, a CoV2 spike-fusion protein, and a combination thereof. In addition, each of these encoded proteins can be further modified, as described in more detail below. Preferably, the recombinant yeast is Saccharomyces cerevisiae.

[0089] In some embodiments of this second aspect, the CoV-2 nucleocapsid protein or variant thereof comprises a sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 7. In other embodiments, the identity value is at least 85%. In yet other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0090] In some embodiments of this second aspect, the CoV-2 spike protein or spike fusion protein comprises a sequence that is at least 80% identical to SEQ ID NO:3 or SEQ ID NO:4. In other embodiments, the identity value is at least 85%. In yet other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0091] In some embodiments, the nucleic acid encoding the CoV-2 spike protein or spike fusion protein comprises a sequence having at least 80% identity to SEQ ID NO:5 or SEQ ID NO:6. In other embodiments, the identity value is at least 85%. In yet other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0092] Adenovirus and yeast disclosed herein may further comprise nucleic acids encoding transport sequences, costimulatory molecules and / or immunostimulatory cytokines. The costimulatory molecules are selected from the group consisting of: CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1 and LFA3. Immunostimulatory cytokines may be selected from the group consisting of: IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1 and LMP1. Additionally or alternatively, the vaccine disclosed herein may also encode SARS-CoV-2M protein with or without ETSD tag. Additionally or alternatively, the adenovirus and / or yeast can be administered in combination with one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, Norvegian interleukin α-imbakicept, IL-21, IPS1, and LMP1). "Combination" herein means that the one or more immunostimulatory cytokines are administered within 24 hours of the adenovirus and / or yeast. That is, the adenovirus and / or yeast can be administered to a patient (e.g., a patient over 50 years old), and then within the next 24 hours, one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, Norvegian interleukin α-imbakicept, IL-21, IPS1, and LMP1) can be administered to the same patient. Additionally or alternatively, one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, Norveglin α-imbakicept, IL-21, IPS1, and LMP1) can be administered to a patient (e.g., a patient over 50 years old), and then within the next 24 hours, adenovirus and / or yeast can be administered to the same patient.

[0093] Over the age of 40, the ability of patients to generate T cell responses to vaccines gradually decreases. Therefore, the administration of adenovirus and / or yeast described herein in combination with one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, Norgeleukin α-imbakicept, IL-21, IPS1, and LMP1) may be particularly useful for elderly patients. As used herein, "elderly" refers to patients over the age of 50, e.g., over the age of 55, over the age of 60, over the age of 65, over the age of 70, over the age of 75, over the age of 80, over the age of 85, or over the age of 90.

[0094] Most preferably, the recombinant virus is administered via subcutaneous or subdermal injection. However, in other contemplated aspects, administration may also be intravenous injection. Alternatively or additionally, antigen presenting cells may be isolated or grown from the patient's cells, infected in vitro, and then infused into the patient.

[0095] In one aspect of any of the embodiments described above or elsewhere herein, the composition is formulated in a pharmaceutically acceptable excipient suitable for administration to a subject.

[0096] It is also further considered that the recombinant virus and yeast considered herein may further include a sequence encoding at least one of a costimulatory molecule, an immunostimulatory cytokine and a protein that interferes or lowers the inhibition of a checkpoint. For example, suitable costimulatory molecules include CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1 and / or LFA3, and suitable immunostimulatory cytokines include IL-2, IL-12, IL-15, IL-15 superagonist (N803), IL-21, IPS1 and / or LMP1, and / or suitable proteins for interfering include antibodies or antagonists for CTLA-4, PD-1, TIM1 receptors, 2B4 and / or CD160.

[0097] It should be understood that all of the above-mentioned co-stimulatory genes are well known in the art, and sequence information for these genes, isoforms and variants can be retrieved from various public resources, including sequence databases accessible at NCBI, EMBL, GenBank, RefSeq, etc. In addition, while the above-mentioned exemplary stimulatory molecules are preferably expressed in full-length form as expressed in humans, modified and non-human forms are also considered suitable, as long as such forms contribute to the stimulation or activation of T cells. Therefore, truncated and chimeric forms of mutant proteins are clearly contemplated herein.

[0098] The immunotherapeutic compositions disclosed herein can be "preventive" or "therapeutic". When provided prophylactically, the compositions disclosed herein are provided before the development of coronavirus disease or the detection of its development, with the purpose of preventing, inhibiting or delaying the development of coronavirus disease; and / or preventing or inhibiting the progression of coronavirus disease in individuals in general. Thus, the prophylactic composition can be administered to individuals who appear to be free of coronavirus disease (healthy or normal individuals), or to individuals in whom coronavirus has not yet been detected. Individuals at high risk of developing coronavirus disease can be treated prophylactically with the compositions disclosed herein.

[0099] When provided therapeutically, the immunotherapy composition is provided to an individual diagnosed with coronavirus disease with the goal of ameliorating or curing the coronavirus disease; increasing the survival of the individual; preventing, inhibiting, reversing or delaying the development of coronavirus disease in the individual.

[0100] In yet another embodiment, disclosed herein is a vaccine composition comprising an adenovirus or yeast as disclosed above, and wherein the composition is formulated for injection. The vaccine composition can be used to induce immunity against CoV-2 in a patient in need thereof by administering the vaccine composition to the patient in need thereof.

[0101] Also disclosed herein is a method for preventing and / or treating coronavirus disease, especially COVID-19. Preferably, the method comprises using a virus or yeast vector encoding a wild-type or modified form of a nucleocapsid protein and / or a wild-type or modified form of a spike protein of a coronavirus in an immunogenic composition, which is administered to a subject individual. The virus and / or yeast vaccine so administered will infect the individual with a wild-type or modified form of a nucleocapsid or spike protein of CoV-2. In this case, the individual will have an immune response to it and be vaccinated. It is worth noting that since the nucleocapsid protein and the spike protein are relatively conserved polypeptides, an immune response can be elicited against multiple members of the coronavirus family.

[0102] When the recombinant vector is an adenovirus, the adenovirus vector can be modified to encode a wild-type or modified form of a nucleocapsid protein and / or a spike protein. Similarly, in the case of yeast, the yeast vector can also be modified to encode a wild-type or modified form of a nucleocapsid protein and / or a spike protein. As shown in more detail below, after administering an immunogenic composition comprising a virus and / or a yeast vector to a patient in need, a positive immune response was obtained in terms of cell-mediated immunity. Therefore, in one embodiment, the present disclosure contemplates the generation of a coronavirus spike to be expressed on the surface of yeast. In such an embodiment, yeast acts as a surrogate coronavirus to stimulate B cells, which then leads to humoral immunity.

[0103] As disclosed herein are next generation bivalent human adenovirus serotype 5 (hAd5) vaccines capable of inducing immunity in patients with pre-existing adenovirus immunity, comprising an S sequence optimized for cell surface expression (S-fusion) and a conserved nucleocapsid (N) antigen designed for trafficking to endosomal subcellular compartments, with the potential to generate durable immune protection. As further described herein, such bivalent vaccines have been found to be optimized for immunogenicity, as evidenced by the following findings:

[0104] 1) The optimized S-fusions exhibited improved cell surface expression of the S receptor binding domain (RBD) compared to S-WT, where little surface expression was detected;

[0105] 2) RBD expressed from S-fusion retains conformational integrity and is recognized by ACE2-Fc;

[0106] 3) viral N protein modified with enhanced T cell stimulatory domain (ETSD) localizes to endosomal / lysosomal subcellular compartments for MHC I / II presentation; and

[0107] 4) These optimizations of S and N (S-fusion and N-ETSD) generated enhanced de novo antigen-specific B cell and CD4+ and CD8+ T cell responses in antigen-naive preclinical models.

[0108] Both T cell and antibody immune responses to the S and N components show a T helper 1 (Th1) bias. As demonstrated by independent SARS-CoV-2 neutralization assays, the antibody response is neutralizing. Therefore, in one embodiment, the next generation of bivalent hAd5 S-fusion + N-ETSD vaccines provides robust, durable cell-mediated and humoral immunity against SARS-CoV-2 infection. In addition, as further described in more detail below, the vaccine construct can be administered orally, intranasally or sublingually. Therefore, in one embodiment, the present disclosure also provides vaccine constructs in oral, intranasal and sublingual formulations in addition to injectable formulations (e.g., SC or IM) to induce mucosal immunity in addition to cell-mediated immunity and humoral immunity. From another perspective, a large amount of immunity can be generated by injection, oral / mucosal administration alone or in combination. In one embodiment, the COVID-19 vaccine disclosed herein produces long-term T and B cell memory.

[0109] Coronavirus and its vaccine

[0110] Coronaviruses are found in birds and mammals. They are morphologically and chemically similar to each other: for example, human and bovine coronaviruses are antigenically related. However, there is no evidence that human coronaviruses can be transmitted from animals. In animals, various coronaviruses invade many different tissues and cause various diseases in humans. One such disease is the severe acute respiratory syndrome (SARS) coronavirus disease, which spread to several countries in Asia, Europe, and North America in late 2002 / early 2003. Another such disease is the 2019 novel coronavirus disease (COVID 19), which has spread to several countries around the world. The pathogen was quickly identified as a novel coronavirus and was named SARS-coronavirus 2 (SARS-CoV-2). The disease it causes is called COVID-19 and has rapidly become a global pandemic that has disrupted socioeconomic life and, as of late October 2020, has resulted in more than 32 million infections and more than 1.1 million deaths worldwide.

[0111] COVID 19 typically begins with a fever above 38°C. Initial symptoms may also include cough, sore throat, malaise, and mild respiratory symptoms. Within two days to a week, patients may develop difficulty breathing. Patients in more advanced stages of COVID 19 develop pneumonia or respiratory distress syndrome. Public health interventions, such as surveillance, travel restrictions, and quarantines, are being used to contain the spread of COVID 19. However, it is unclear whether these draconian containment measures can be sustained with each emergence of COVID19 in humans. In addition, the potential of this new and sometimes deadly CoV as a bioterrorism threat is clear.

[0112] Coronavirus virions are spherical to pleomorphic envelope particles. The envelope is covered with protruding glycoproteins and surrounds a core composed of matrix proteins, in which is a single-stranded positive-sense RNA (Mr 6×10 6 ). In this regard, it should be noted that the terms "nucleocapsid protein", "nucleoprotein" and "nucleocapsid" are used interchangeably in this disclosure. The coronavirus nucleocapsid (N) is a structural protein found in all coronaviruses, including COVID 19. The nucleocapsid protein forms a complex with the genomic RNA, interacts with the viral membrane protein during the assembly of the virion, and plays a key role in increasing the efficiency of viral transcription and assembly.

[0113] Another protein found in all coronavirus virions is the viral spike (S) protein. Coronaviruses are large positive-strand RNA viruses that typically have a broad host range. Like other enveloped viruses, CoV enters target cells through fusion between the viral and cellular membranes, and this process is mediated by the viral spike (S) protein.

[0114] SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA beta coronavirus, composed mainly of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope, as well as viral membrane and genomic RNA. Among them, S is the largest and N is the most common. The S glycoprotein is displayed on the surface of the virus as a trimer ( Fig.33 , sub-figure A), while N is located inside the virus particle. The schematic diagram of the primary structure of S is shown in Fig.33 , panel B. The sequence of SARS-CoV-2 has been published and compared with those of previous coronaviruses. This was followed by the report of the crystal structure of the S protein. The virus uses the S protein to enter host cells through the interaction of the S receptor binding domain (S RBD) with angiotensin-converting enzyme 2 (ACE2), an enzyme expressed on various cell types in the nose, mouth, intestines, and lungs, as well as other organs (importantly on alveolar epithelial cells, where infection is primarily manifested). Fig.33 , as shown in panel B, S RBD is located in the S1 region of the spike polypeptide.

[0115] The methods and compositions disclosed herein target nucleoproteins and spike proteins that are conserved in all types of coronaviruses. In one embodiment, the present disclosure provides a vaccine formulation comprising a recombinant entity, wherein the recombinant entity comprises a nucleic acid encoding a nucleocapsid protein of coronavirus 2 (CoV2) or a modified form thereof and / or wherein the recombinant entity encodes a spike protein CoV2 or a modified form thereof. Vaccine formulations can be used to treat diseases, such as coronavirus-mediated diseases or infections. Therefore, in another embodiment, a method for treating coronavirus disease is contemplated for patients in need. Such methods will preferably include the step of administering to a subject an immunotherapy composition comprising a recombinant entity, wherein the recombinant entity comprises a nucleic acid encoding a nucleocapsid protein of coronavirus 2 (CoV2) or a modified form thereof and / or a nucleic acid encoding a spike protein of coronavirus 2 (CoV2) or a modified form thereof. The coronavirus contemplated herein may be coronavirus disease 2019 (COVID-19) and / or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0116] For example, the present disclosure provides a method for treating coronavirus disease 2019 (COVID-19) and / or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a patient in need thereof, the method comprising: administering to the subject a first immunotherapy composition comprising a recombinant virus, wherein the recombinant virus comprises a nucleic acid encoding a nucleocapsid protein of coronavirus 2 (CoV2) or a modified form thereof, administering to the subject a second immunotherapy composition comprising a recombinant yeast, wherein the recombinant yeast comprises a nucleic acid encoding a spike protein of CoV2. The first and second immunotherapy compositions can be administered to the patient simultaneously or sequentially.

[0117] From a different perspective, contemplated herein are viral vectors (e.g., recombinant adenoviral genomes, optionally with E2b gene deletion or non-functionality) comprising a nucleic acid encoding (a) at least one nucleocapsid protein or a modified form thereof; and (b) at least one spike protein or a modified form thereof. The viral vector may further encode one or more co-stimulatory molecules. Most typically, the nucleic acid will also include a transport signal to direct the peptide product encoded by the nucleic acid to the cytoplasm, endosomal compartment, or lysosomal compartment, and the peptide product may also include a sequence portion that enhances the intracellular turnover of the peptide product.

[0118] Most SARS-CoV-2 vaccines currently in development target S because of the potential to neutralize the virus’s ability to bind to host cells by generating antibodies against the RBD. Support for the RBD as a key antigen was recently demonstrated when it was reported that, in a cohort of 44 hospitalized COVID-19 patients, RBD-specific IgG responses and neutralizing antibody titers were detectable and correlated in all patients by 6 days after PCR-confirmed infection. In addition to humoral responses, the S epitope is also a common target of T cells in patients who have recovered from COVID-19, providing further rationale for incorporating S into preventive immunization strategies.

[0119] While there is an urgent need to rapidly develop a SARS-CoV-2 vaccine, reliance on any one antigenic cargo or immune pathway that emerges in the monovalent vaccines under development is not without risk. Evaluation of nearly 4,000 SARS-CoV-2 genome sequences has identified numerous mutations in S, with the D614G variant having recently emerged as a potentially more contagious strain six months after the original virus was identified.

[0120] In designing the vaccines disclosed herein, an optimized N sequence was added to overcome the risk of new virus strains with mutations in S and to provide additional antigens that can elicit responses against them. The N protein is a highly conserved and antigenic SARS-CoV-2-related protein that has been previously studied as an antigen in SARS-CoV coronavirus vaccine design. N associates with viral RNA within the virus and plays a role in viral RNA replication, viral particle assembly and release. SARS-CoV-2N is a highly antigenic protein and almost all patients infected with SARS-CoV-2 have an antibody response to N. In addition, another study reported that most, if not all, of the COVID-19 survivors tested were shown to have N-specific CD4+ T cell responses.

[0121] Currently, there is a great deal of focus on generating humoral responses to vaccines, with arguably less attention paid to T cell responses. However, the natural history of SARS-CoV-2 infection suggests that a robust T cell response to vaccination is at least as important as the generation of antibodies and should be a key consideration for COVID-19 vaccine efficacy.

[0122] First, humoral and T cell responses were highly correlated, with neutralizing antibody titers proportional to T cell levels, suggesting that T responses are required for effective humoral responses. It is well known that activation of CD4+ T helper cells enhances antibody production by B cells. Second, based on findings from patients who recovered from the closely related SARS-CoV, not only were virus-specific CD4+ and CD8+ T cells widely detected in COVID-19 patients, but such T cells persisted for at least 6-17 years, suggesting that T cells may be an important part of long-term immunity. These T cell responses were primarily directed against N, and it was reported that in all 36 recovered COVID-19 patients they studied, CD4+ and CD8+ T cells that recognized multiple regions of the N protein could be demonstrated. Examination of the blood of 23 individuals who recovered from SARS-CoV revealed that memory T cells acquired 17 years earlier also recognized multiple proteins of SARS-CoV-2. These findings highlight the importance of designing a vaccine with a highly conserved nucleocapsid present in both SARS-CoV and SARS-CoV-2. Third, recovered patients exposed to SARS-CoV-2 have been found to have no seroconversion but evidence of T cell responses. T cell-based responses become even more critical given that at least one study has found that neutralizing antibody titers in some COVID-19 patients decline after about 3 months.

[0123] In one embodiment, the vaccines disclosed herein result in the generation of T cells in addition to humoral responses. A bivalent vaccine comprising multiple antigens (such as S RBD displayed by comprising a full-length S including SD1, S1 and S2 epitopes, and N) is considered and shown to be more effective than constructs with any single antigen in eliciting T cell and antibody-based responses by presenting unique and conserved SARS-CoV-2 antigenic sites to the immune system. The importance of S and N is highlighted by identifying S and N antigens as a priori potential B and T cell epitopes of the SARS-CoV virus that are very similar to SARS-CoV-2 (which is expected to induce both T cell and B cell responses).

[0124] Another consideration in designing an effective vaccine is the possibility of antigen presentation on the surface of cells expressing the recombinant protein, and the possibility of expression in a conformation that reproduces natural viral infection. First, because wild-type N does not have a signaling domain that directs it to endosomal processing and ultimately presents the MHC II class complex to CD4+T cells, the wild-type N sequence is not optimal for inducing a strong CD4+T cell response (which is necessary for cell-mediated and B cell memory). To overcome this limitation, the enhanced T cell stimulation domain (ETSD) of N allows for the necessary processing and presentation. A preferred ETSD polypeptide has the amino acid sequence of SEQ ID NO: 8. Of course, it should be understood that the sequence can be modified while maintaining the desired activity. Therefore, the ETSD sequence can have at least 85%, or at least 90%, or at least 95, or at least 98% identity with SEQ ID NO: 8. Secondly, in order to display the highly antigenic RBD region of S on the cell surface, optimizing the wild-type S protein to an "S-fusion" sequence can increase the possibility of natural folding, increase the stability of RBD and appropriate cell surface expression. Thus, in one embodiment, the vaccine construct comprises an S-fusion sequence and an N-ETSD sequence.

[0125] The vaccine platform used here is a next-generation recombinant human adenovirus serotype 5 (hAd5) vector with deletions in the E1, E2b, and E3 gene regions (hAd5[E1-,E2b-,E3-]). This hAd5[E1-,E2b-,E3-] vector ( Fig.33, Panel C) differs primarily from other first generation [E1-, E3-] recombinant Ad5 platforms in having additional deletions in the early gene 2b (E2b) region (which removes the viral DNA polymerase (pol) and in the pre-terminal protein (pTP) gene, as well as its propagation in the E.C7 human cell line. The removal of these E2b regions confers favorable immunological properties by minimizing immune responses to Ad5 viral proteins such as viral fibers, thereby eliciting effective immune responses to specific antigens in patients with pre-existing adenovirus (Ad) immunity. As an additional benefit of these deletions, this vector has an expanded gene carrying / cloning capacity compared to first generation Ad5[E1-, E3-] vectors. Compared to Ad5[E1-, E3-] based platforms, this next generation hAd5[E1-, E2b-, E3-] vaccine The platform does not promote activity that inhibits innate immune signaling, allowing for improved vaccine efficacy and an excellent safety profile independent of previous Ad immunity. Because these deletions make the hAd5 platform effective even in the presence of existing Ad immunity, the platform is able to achieve relatively long-term antigen expression without significantly inducing anti-vector immunity. Therefore, unlike first-generation Ad platforms that face limitations of pre-existing and vaccine-induced Ad immunity, the same vector / construct can also be used for homologous prime-boost treatment regimens. Importantly, this next-generation Ad vector has demonstrated safety in more than 125 patients with solid tumors. In these Phase I / II studies, CD4+ and CD8+ antigen-specific T cells were successfully generated against a variety of somatic antigens (CEA, MUC1, brachyury), even in the presence of pre-existing Ad immunity.

[0126] The present disclosure provides the following findings that demonstrate enhanced cell surface expression and physiologically relevant folding of the expressed S RBD from the S-fusion by ACE2-Fc binding. The N-ETSD protein is successfully localized to the endosomal / lysosomal subcellular compartments for MHC presentation and thus generates CD4+ and CD8+ T cell responses. Immunization of CD-1 mice with the hAd5 S fusion + N-ETSD vaccine elicits both humoral and cell-mediated immune responses to the vaccine antigens. CD8+ and CD4+ T cell responses were observed for both S and N. Statistically significant IgG responses were observed for antibodies against S and N. Effective neutralization of SARS-CoV-2 by sera from mice immunized with hAd5 S fusion + N-ETSD was confirmed by two independent SARS-CoV-2 neutralization assays: the cPass assay measures competitive inhibition of RBD binding to ACE2,44, and in a live SARS-CoV-2 virus assay with infected Vero E6 cells. Analysis of T cell responses to S and N as well as humoral responses favored a Th1-specific response.

[0127] Together, these findings suggest that the hAd5 S-fusion + N-ETSD vaccine combination is particularly effective against SARS-CoV-2.

[0128] Recombinant virus

[0129] With regard to recombinant viruses, all known ways of preparing recombinant viruses are considered to be applicable herein, however, particularly preferred viruses are those that have been established in therapy, including adenovirus, adeno-associated virus, alphavirus, herpes virus, lentivirus, etc. Among other suitable choices, adenovirus is particularly preferred.

[0130] In addition, it is generally further preferred that the virus is a replication-deficient and non-immunogenic virus. For example, suitable viruses include genetically modified alpha viruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like. However, adenoviruses are particularly preferred. For example, genetically modified replication-deficient adenoviruses are preferred, which are not only suitable for multiple vaccinations, but also suitable for vaccination in individuals with pre-existing immunity to adenoviruses (see, for example, WO 2009 / 006479 and WO2014 / 031178, which are incorporated herein by reference in their entirety). In some embodiments, the replication-deficient adenoviral vector includes a replication-deficient adenoviral 5 vector. In some embodiments, the replication-deficient adenoviral vector comprises a deletion in the E2b region. In some embodiments, the replication-deficient adenoviral vector further comprises a deletion in the E1 region. In this regard, it should be noted that the E2b gene and other late proteins are deleted in the genetically modified replication-deficient adenovirus to reduce immunogenicity. Moreover, due to these specific deletions, such genetically modified viruses are replication-deficient and allow for use in relatively large recombinant cargoes.

[0131] For example, WO 2014 / 031178 describes the use of such genetically modified viruses to express CEA (colorectal embryonic antigen) to provide an immune response against colon cancer. Moreover, as has been reported, relatively high titers of recombinant viruses can be achieved using genetically modified human 293 cells (e.g., J Virol. [Journal of Virology] 1998 Feb; 72(2): 926-933).

[0132] The E1-deleted adenoviral vector Ad5[E1-] is constructed so that the transgene replaces only the E1 region of the gene. Typically, about 90% of the wild-type Ad5 genome is retained in the vector. Ad5[E1-] vectors have reduced replication capacity and cannot produce infectious virus after infecting cells that do not express the Ad5 E1 gene. Recombinant Ad5[E1-] vectors are propagated in human cells, allowing Ad5[E1-] vector replication and packaging. Ad5[E1-] vectors have many positive attributes; one of the most important is that they are relatively easy to scale up and produce cGMP. Currently, more than 220 human clinical trials have used Ad5[E1-] vectors, and more than two thousand subjects have received the virus sc, im, or iv. In addition, Ad5 vectors do not integrate; their genome remains episomal. In general, the risk of insertional mutagenesis and / or germline transmission is very low for vectors that are not integrated into the host genome. Traditional Ad5[E1-] vectors have a carrying capacity of nearly 7 kb.

[0133] One obstacle to using first generation (E1 deleted) Ad5 based vectors is the high frequency of pre-existing neutralizing antibodies against adenovirus type 5. An attempt to overcome this immunity is described in WO 2014 / 031178, which is incorporated herein by reference. Specifically, a novel recombinant Ad5 platform has been described that has a deletion in the early 1 (E1) gene region and other deletions in the early 2b (E2b) gene region (Ad5[E1-, E2b-]). The deletion of the E2b region (encoding DNA polymerase and pre-terminal protein) results in reduced viral DNA replication and reduced late viral protein expression. E2b deleted adenoviral vectors provide improved Ad based vectors that are safer, more effective and more versatile than first generation adenoviral vectors.

[0134] In yet another embodiment, the adenoviral vectors contemplated for use in the present disclosure include adenoviral vectors having a deletion in the E2b region of the Ad genome, optionally a deletion in the E1, E3 region, and optionally an E4 region that is partially or completely removed. In yet another embodiment, the adenoviral vectors used herein have deleted E1 and / or deleted the front end protein function of the E2b region. In some cases, such vectors have no other deletions. In another embodiment, the adenoviral vectors used herein have deleted E1, DNA polymerase and / or front end protein function.

[0135] As used herein, the term "E2b deletion" refers to a specific DNA sequence that is mutated in a manner that prevents the expression and / or function of at least one E2b gene product. Thus, in certain embodiments, "E2b deletion" is used with respect to a specific DNA sequence that is deleted (removed) from the Ad genome. An E2b deletion or "deletion included in the E2b region" refers to a deletion of at least one base pair within the E2b region of the Ad genome. Thus, in certain embodiments, more than one base pair is deleted, and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted. In another embodiment, the deletion is a deletion of more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within the E2b region of the Ad genome. The E2b deletion can be a deletion that prevents the expression and / or function of at least one E2b gene product, and thus encompasses deletions within exons that encode portions of the E2b-specific protein and deletions within promoter and leader sequences. In certain embodiments, the E2b deletion is a deletion that prevents the expression and / or function of one or both of the DNA polymerase and pre-terminal proteins of the E2b region. In further embodiments, "E2b deletion" refers to one or more point mutations in the DNA sequence of that region of the Ad genome, rendering one or more encoded proteins inoperative. Such mutations include residues replaced by different residues, resulting in changes in the amino acid sequence that lead to inoperative proteins.

[0136] As will be readily appreciated, the desired nucleic acid sequence (for expression from virally infected cells) is under the control of appropriate regulatory elements well known in the art. In view of the foregoing, it will be appreciated that the proposed compositions and methods are not only suitable for directing virally expressed antigenic specificity to one or the other (or both) MHC systems, but will also provide increased stimulation of CD8+ and / or CD4+ by including various co-stimulatory molecules (e.g., ICAM-1 (CD54), ICOS-L, LFA-3 (CD58), and at least one of B7.1 (CD80) and B7.2 (CD86)) and via secretion or membrane-bound presentation of checkpoint inhibitors.

[0137] With respect to viral expression and vaccination systems, it is contemplated that all therapeutic recombinant viral expression systems are considered suitable for use herein, so long as such viruses are capable of resulting in expression of the recombinant payload in infected cells.

[0138] Regardless of the type of recombinant virus, it is contemplated that the virus can be used to infect patient (or non-patient) cells in vitro or in vivo. For example, the virus can be injected subcutaneously or intravenously, or can be administered intranasally or via inhalation, so as to infect patient cells and especially antigen presenting cells. Alternatively, the immune competent cells (e.g., NK cells, T cells, macrophages, dendritic cells, etc.) of the patient (or from allogeneic sources) can be infected in vitro, and then infused to the patient. Alternatively, immunotherapy does not have to rely on viruses, but can be achieved by nucleic acid transfection or using RNA or DNA or other recombinant vectors that cause new epitopes (e.g., as single peptides, tandem minigenes, etc.) to be expressed in desired cells, especially immune competent cells, to be inoculated. Such nucleic acids are typically delivered in association with lipid formulations to protect nucleic acids from degradation and promote nucleic acid uptake into target cells.

[0139] As described above, the desired nucleic acid sequence (for expression from virally infected cells) is under the control of suitable regulatory elements well known in the art. For example, suitable promoter elements include constitutive strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoters), but inducible promoters are also considered suitable for use herein, particularly under induction conditions typical for tumor microenvironments. For example, inducible promoters include those promoters that are sensitive to hypoxia and promoters that are sensitive to TGF-β or IL-8 (e.g., via TRAF, JNK, Erk or other response element promoters). In other examples, suitable inducible promoters include tetracycline inducible promoters, myxovirus resistance protein 1 (Mx1) promoters, and the like.

[0140] A replication-defective adenovirus comprising a deletion of the E1 gene region, a deletion of the E2b gene region, and a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid protein and / or a CoV2 spike protein as disclosed herein can be administered to a patient in need thereof to induce immunity against CoV2. The route and frequency of administration and dosage of the therapeutic compositions described herein can vary with the individual and the severity of the disease and can be readily established using standard techniques. In some embodiments, administration comprises delivering 4.8-5.2×10 11 replication-defective adenoviral particles, or 4.9-5.1 × 10 11 replication-defective adenoviral particles, or 4.95-5.05 × 10 11 replication-defective adenoviral particles, or 4.99-5.01×10 11 Replication-defective adenoviral particles.

[0141] Administration of the viral particles can be carried out by a variety of suitable delivery routes. A preferred route contemplated herein is by injection, such as intradermal injection, intramuscular injection, intravenous injection or subcutaneous injection. In some embodiments, subcutaneous delivery may be preferred.

[0142] Recombinant yeast

[0143] With regard to yeast expression and vaccination systems, all known yeast strains are considered suitable for use herein. However, preferably, the yeast is a recombinant yeast strain that is genetically modified with a nucleic acid construct encoding a group consisting of a coronavirus 2 (CoV2) nucleocapsid protein, a CoV2 spike protein, and a combination thereof, thereby initiating an immune response against a CoV2 viral disease. In one aspect of any embodiment of the disclosure described above or elsewhere herein, the yeast vehicle is a whole yeast. In one aspect, the whole yeast is killed. In one aspect, the whole yeast is heat-inactivated. In a preferred embodiment, the yeast is a whole, heat-inactivated yeast from Saccharomyces cerevisiae.

[0144] The use of yeast-based therapeutic compositions is disclosed in the art. For example, WO 2012 / 109404 discloses a yeast composition for treating chronic hepatitis B infection.

[0145] It should be noted that any yeast strain can be used to produce the yeast vehicle of the present disclosure. Yeast is a unicellular microorganism belonging to one of the following three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. A consideration for selecting the type of yeast used as an immunomodulator is the pathogenicity of the yeast. In a preferred embodiment, the yeast is a non-pathogenic strain (e.g., Saccharomyces cerevisiae) because the non-pathogenic yeast strain minimizes any adverse effects on the individual to which the yeast vehicle is applied. However, if the pathogenicity of the yeast can be eliminated by drug intervention, pathogenic yeast can be used.

[0146] For example, suitable yeast strains include Saccharomyces cerevisiae, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect, the yeast genus is selected from Saccharomyces cerevisiae, Candida, Hansenula, Pichia or Schizosaccharomyces, and in a preferred aspect, Saccharomyces cerevisiae is used. Species of yeast strains that can be used include Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica.

[0147] It should be further understood that many of these species include various subspecies, types, subtypes, etc. intended to be included in the above-mentioned species. On the one hand, the yeast species used in the present disclosure include Saccharomyces cerevisiae (S.cerevisiae), Candida albicans (C.albicans), Hansen yeast (H.polymorpha), Pichia pastoris (P.pastoris) and Schizosaccharomyces pombe (S.pombe). Saccharomyces cerevisiae is useful because it is relatively easy to operate and is used as a food additive and is "generally recognized as safe" or "GRAS" (GRAS, FDA recommended rules 62FR18938, April 17, 1997). Therefore, it is particularly contemplated herein that plasmids can be replicated to yeast strains (e.g., Saccharomyces cerevisiae circular (cir) strains) of particularly high copy numbers. Saccharomyces cerevisiae strains are such strains that can support expression vectors that allow one or more target antigens and / or antigen fusion proteins and / or other proteins to be expressed at high levels. Furthermore, any mutant yeast strain may be used, including those that exhibit reduced expression of the target antigen or other post-translational modifications of other proteins, such as mutations in enzymes that extend N-linked glycosylation.

[0148] The expression of the envisioned peptide / protein in yeast can be accomplished using techniques known to those skilled in the art. Most typically, a nucleic acid molecule encoding at least one protein is inserted into an expression vector so that the nucleic acid molecule is operably linked to a transcription control sequence, which, when transformed into a host yeast cell, can affect constitutive or regulated expression of the nucleic acid molecule. As will be readily appreciated, nucleic acid molecules encoding one or more proteins can be on one or more expression vectors operably linked to one or more expression control sequences. Particularly important expression control sequences are those that control transcription initiation (e.g., promoters and upstream activation sequences).

[0149] Any suitable yeast promoter may be used in the methods and compositions of the present disclosure, and a variety of such promoters are known to those of skill in the art and are generally discussed above. Promoters for expression in Saccharomyces cerevisiae include promoters of genes encoding the following yeast proteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), translation elongation factor EF-1α (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as TDH3 for triosephosphate dehydrogenase), galactokinase (GAL1), galactose-1-phosphate uracil transferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome c1 (CYC1), Sec7 protein (SEC7), and acid phosphatase (PHO5), including hybrid promoters such as the ADH2 / GAPDH and CYC1 / GAL10 promoters, and including the ADH2 / GAPDH promoter (induced when the glucose concentration in the cell is low (e.g., about 0.1% to about 0.2%)), as well as the CUP1 promoter and the TEF2 promoter. Similarly, many upstream activation sequences (UAS) (also referred to as enhancers) are known. Upstream activation sequences for expression in Saccharomyces cerevisiae include UASs of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7, and GAL10, as well as other UASs activated by the GAL4 gene product, wherein the ADH2 UAS is used on the one hand. Since the ADH2 UAS is activated by the ADR1 gene product, when a heterologous gene is operably linked to the ADH2 UAS, it may be more preferable to overexpress the ADR1 gene. Transcription termination sequences for expression in Saccharomyces cerevisiae include termination sequences of α-factor, GAPDH, and CYC1 genes. Transcription control sequences for expressing genes in methylotrophic yeast include transcription control regions of genes encoding alcohol oxidase and formate dehydrogenase.

[0150] Similarly, according to the present disclosure, nucleic acid molecules can be transfected into yeast cells by any method in which nucleic acid molecules are applied to cells, including diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption and protoplast fusion. The nucleic acid molecules of transfection can be integrated into yeast chromosomes using technology known to those skilled in the art, or maintained on extrachromosomal vectors. As discussed above, it is also possible to produce antigens therein by transfecting complete yeast microorganisms or yeast spheroplasts with desired nucleic acid molecules, and then using technology known to those skilled in the art to further process these microorganisms or spheroplasts, to produce cytoplasm, shells or subcellular yeast membrane extracts or their fractions containing desired antigens or other proteins, recombinant production of yeast cytoplasm, yeast shells and yeast membrane particles or cell wall preparations. Other exemplary yeast expression systems, methods and conditions suitable for use in this article are described in US20100196411 A1, US 2017 / 0246276 or US 2017 / 0224794 and US 2012 / 0107347.

[0151] The recombinant viruses and yeast so produced can then be used alone or in combination as therapeutic vaccines in pharmaceutical compositions, typically with 10 4 -10 13 Virus or yeast particles, or more preferably 10 per dosage unit. 9 -10 12 The invention relates to a method for preparing a sterile injectable composition by combining individual virus or yeast particles. Alternatively, the virus or yeast can be used to infect patient cells in vitro, and the cells thus infected are then transfused to the patient. However, alternative formulations are also considered to be suitable for use herein, and all known routes and modes of administration are contemplated herein.

[0152] In further contemplated embodiments, the second generation hAd5[E1-, E2b-, E3-] based vaccines disclosed herein overcome pre-existing anti-Ad5 immunity. To avoid the Ad immune barrier and circumvent the disadvantages of the first generation Ad5[E1-E3-] vector, an advanced second generation human adenovirus (hAd5) vector was constructed with two (2) additional deletions in the E2b region, removing the DNA polymerase and pre-terminal protein genes [E1-, E2b-, E3-]. (The old names of adenovirus vectors in the literature are Ad5, ETBX)

[0153] Compared to the 7kb capacity of the first-generation Ad5[E1-] vector, the E2b-deleted hAd5 vector has a gene-carrying capacity of up to 12-14kb, providing space for multiple genes if needed. The hAd5[E1-, E2b-, E3-]-based recombinant vectors were produced using the human E.C7 cell line. The deletion of the E2b region also confers these novel Ad vectors with favorable immune properties, eliciting effective immune responses to specific non-viral antigens while minimizing immune responses to Ad viral proteins.

[0154] Even in the presence of Ad immunity, hAd5[E1-, E2b-, E3-] vectors induce potent cell-mediated immune (CMI) responses and Abs against vector-loaded antigens. Compared with Ad5[E1-] vectors, hAd5[E1-, E2b-, E3-] vectors also reduce the occurrence of adverse reactions, particularly hepatotoxicity and tissue damage. In one embodiment, the reduced inflammatory response to viral proteins of the hAd5[E1-, E2b-, E3-] vector and the resulting escape from pre-existing Ad immunity increase the ability of the hAd5[E1-, E2b-, E3-] vector to infect dendritic cells (DCs), resulting in greater immunity of the vaccine. In addition, increased infection of other cell types provides the high levels of antigen presentation required for effective CD8+ and CD4+ T cell responses, leading to memory T cell development. In one embodiment, the hAd5[E1-, E2b-, E3-] vector is superior to the Ad5[E1-] vector in terms of immunogenicity and safety and will be the best platform for developing COVID-19 vaccines in a rapid and efficient manner. In one embodiment, by utilizing this new hAd5 vector system for testing preventive vaccines against COVID-19, the vector system overcomes the barriers of other Ad5 systems and allows for immunization of people who have been previously exposed to Ad5.

[0155] Track record of rapid vaccine development using second generation human (hAd5) adenoviral platforms during pandemic treatment: 2009 H1N1 experience. To address emerging pathogen threats, especially during pandemics, it is critical to deploy modern vaccine technologies. These technologies will leverage the power of genome sequencing, rapid transfection in well-established vaccine vectors to rapidly identify constructs with high immunogenicity.

[0156] Vaccines against emerging pathogens, such as the 2009 H1N1 pandemic virus, can benefit from current technologies, such as rapid genome sequencing, to construct the most biologically relevant vaccines. A novel platform (hAd5[E1-, E2b-, E3-]) has been used to induce immune responses to various antigenic versions. This vector platform expresses the hemagglutinin (HA) and neuraminidase (NA) genes from the 2009 H1N1 pandemic virus. The insert is a consensus sequence designed from viral isolation sequences, and the vaccine is rapidly constructed and produced. Vaccination induces H1N1 immune responses in mice, which provides protection from lethal viral challenge. In ferrets, vaccination prevents disease development and significantly reduces viral titers in nasal washes. H1N1 cell-mediated immunity and antibody induction are associated with preventing disease symptoms and reducing viral replication. Therefore, hAd5[E1-, E2b-, E3-] has demonstrated the ability to rapidly develop effective vaccines against infectious diseases.

[0157] For at least these reasons, it is generally preferred that contemplated vaccine compositions, when based on adenoviral vectors, will utilize a recombinant hAd5[E1-, E2b-, E3-] platform to generate recombinant nucleic acids for therapeutic use in humans.

[0158] Example 1: Selected hAd5 vaccine constructs and results

[0159] Constructs that have been constructed and tested are disclosed herein, in particular, hAd5-COVID-19 vaccine constructs E1-, E2b-, E3-hAd5 vectors ( Fig.26 The construct has been tested in preclinical experiments, including in vitro expression ( Fig. 27 ) and small animal immunogenicity. Multiple COVID-19 constructs included RBD alone, S1 alone, S1 fusion protein, and combinations of RBD, S1, and S1 fusion with N. Preliminary in vitro studies showed that these constructs ( Fig.28 ) identifies convalescent serum antibodies and may serve as an alternative vaccine.

[0160] Rationale for Inclusion of Nucleocapsid (N) in hAd5 Constructs for COVID-19: The nucleocapsid (N) protein of SARS-CoV-2 is highly conserved and highly expressed. Previous studies on related coronaviruses that cause SARS have shown that the N protein is immunogenic when integrated with intracellular trafficking structures. To date, all vaccine strategies under development have involved developing immunogenicity against the spike (S) protein. However, recent evidence from patients who have recovered from COVID-19 suggests that Th1 immunity is generated against the nucleocapsid (N). Other reports have further confirmed that T and B cell epitopes of the spike glycoprotein and nucleoprotein are the highest in predictive bioinformatics models. The present disclosure demonstrates that by combining S with N, long-term cell-mediated immunity with a Th1 phenotype can be induced. In fact, given the mutations that occur in S and the finding that the structural N protein is highly conserved across the coronavirus family, this combination vaccine has great potential as a long-term “universal” COVID-19 vaccine.

[0161] Example 2: Immunogenicity study (small animal model):

[0162] Homologous prime-boost immunogenicity in BALB-c mice. Mice have been treated with 1, 2 or 3 doses of hAd5COVID-19 vaccine and serum and splenocyte samples are being tested for SARS-CoV-2 antigen-specific immune responses. Sera are tested for anti-spike and anti-nucleocapsid antibody responses by ELISA. Splenocytes are tested for spike- and nucleocapsid-specific cell-mediated immune responses by ELISPOT and intracellular cytokine mimic assays.

[0163] The results showed promising immunogenic activity. In one embodiment, hAd5[E1-, E2b-, E3-]N-ETSD (a vaccine comprising a SARS-CoV-2 nucleocapsid and an enhanced T cell stimulatory domain (ETSD)) alters T cell responses to the nucleocapsid. Mice were immunized subcutaneously (SC) twice at a dose of 1010VP at an interval of 7 days. Blood was collected at several time points and spleens were collected after sacrifice for immunogenicity experiments. Splenocytes were isolated and tested for cell-mediated immunity (CMI) responses. The results showed that SARS-CoV-2 nucleocapsid antigen-specific CMI responses were detected in the spleens of all mice immunized with the hAd5[E1-, E2b-, E3-]N-ETSD vaccine by ELISpot and flow cytometry analysis, but not in the spleens of mice immunized with the vector control (hAd5[E1-, E2b-, E3-] without). Furthermore, antibody responses were detected in all mice immunized with the hAd5[E1-,E2b-,E3-]-N-ETSD vaccine but not in mice immunized with the vector control (Ad5[E1-,E2b-,E3-]-None) ( Fig.29and Figure 30).

[0164] Example 3: Enhanced RBD cell surface expression:

[0165] Further evidence of the enhanced immunogenicity value of N when combined with S is the following unexpected finding: Fig.31 Enhanced surface expression of the RBD protein in 293 cells transfected with the N-ETSD+S construct as seen in Figure 2. The expression and presentation of the RBD appears to be very important, as evidenced by a recent report by others showing that rare but recurrent RBD-specific antibodies with potent antiviral activity were found in all individuals tested who recovered from COVID-19 infection. This finding of enhanced RBD expression when the N and S-fusions were combined was confirmed in a study using plasma from patients who recovered from COVID-19 infection ( Fig.32 ). Alternative structures of RBD-ETSD could serve as alternative vaccines.

[0166] In summary, based on the enhanced RBD protein expression and exposure with S fusion and S fusion + N constructs, both were tested in hAd5 vectors. In addition, based on recent clinical data in recovered COVID-19 patients and the fact that N constructs can induce long-lasting CD4 + Based on the confirmed preclinical data on Th1 cell-mediated immunity, this combination of S fusion + N construct could provide long-lasting immunity beyond the short-term neutralizing antibodies.

[0167] Example 4: Immunogenicity testing of candidate COVID-19 vaccine constructs

[0168] Two adenovirus-based COVID-19 vaccine constructs will be tested in preclinical experiments, including in vitro expression; small animal immunogenicity; and non-human primate immunogenicity and efficacy.

[0169] Construct Description: Two (2) hAd5-based second-generation COVID-19 vaccine constructs were evaluated. The first was an hAd5 vector with SARS-CoV-2 and spike protein inserts (see Fig.26 The second is an E1-, E2b-, E3-hAd5 vector with a SARS-CoV-2 wild-type spike protein (S) insert and a nucleocapsid protein (N) insert containing an endosomal targeting domain sequence (ETSD) in the same vector backbone.

[0170] Immunogenicity studies: 1, 2 or 3 doses of Fig.26Mice will be treated with candidate adenovirus vaccines listed in to examine homologous prime-boost immunogenicity in mice, and serum and splenocyte samples will be tested for SARS-CoV-2 antigen-specific immune responses. Sera are being tested for anti-spike and anti-nucleocapsid antibody responses by ELISA. Splenocytes will be tested for spike- and nucleocapsid-specific cell-mediated immune responses by ELISPOT and intracellular cytokine simulation assays. Data from these studies are disclosed throughout this disclosure.

[0171] SARS-CoV-2 Virus Neutralization Studies: Sera from mice immunized during the above immunogenicity studies will be sent to the testing laboratory to conduct SARS-CoV-2 neutralization studies in their ABSL-3 facility. The sera will be tested for COVID 19 virus neutralization activity by mixing various dilutions of sera with COVID 19 virus, incubating the mixture, and then exposing the mixture to Vero cells to detect cytopathic effect (CPE). The last dilution that prevents CPE will be considered the endpoint neutralization titer.

[0172] Immunogenicity and efficacy evaluation in nonhuman primates: Fig.26 Rhesus macaques will be treated with the three doses of the candidate adenoviral vaccine listed in the study. SARS-CoV-2 antigen-specific immune responses in serum and PBMCs will be monitored by ELISA, ELISPOT, and ICS throughout the therapy. Four weeks after the last vaccination, animals will receive a SARS-CoV-2 challenge and monitor disease characteristics and viral shedding.

[0173] Example 5: Phase Ib clinical trial testing of hAd5[E1-,E2b-,E3-]CoV-2 vaccine.

[0174] Study Design: This is a Phase 1b open-label study in adult healthy subjects. The clinical trial is designed to evaluate the safety, reactogenicity, and immunogenicity of hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines. hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines are targeted vaccines based on the hAd5[E1-,E2b-,E3-] vector, encoding the SARS-CoV-2 spike (S) protein alone or together with the SARS-CoV-2 nucleocapsid (N) protein. The hAd5[E1-,E2b-,E3-] vector is a platform technology for targeted vaccines, and to date, the hAd5[E1-,E2b-,E3-] vector has been used in up to 5×10 11 Its safety has been demonstrated in more than 125 cancer patients at a dose of 5×10 viral particles / dose. 11 Virus particles / dose (1.5×10 12Co-administration of three different hAd5[E1-, E2b-,E3-] vector-based vaccines (100 total viral particles) was also shown to be safe.

[0175] COVID-19 infection causes significant morbidity and mortality in the global population. The hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines are designed to induce humoral and cellular responses, even in individuals with pre-existing adenoviral immunity. Therefore, hAd5-COVID-19-S and hAd5-COVID-19-S / N have the potential to induce immunity against COVID-19 and prevent or mitigate the health effects of COVID-19 infection in healthy subjects.

[0176] Phase 1b Safety Analysis: In the initial safety analysis of Phase 1b, a total of 40 healthy subjects will be divided into 4 dosing cohorts (cohorts 1A, 1B, 2A, 2B; n = 10 in each cohort):

[0177] Cohort 1A-hAd5-COVID-19-S, 5×10 10 Virus particles (VP) / dose (n = 10),

[0178] Cohort 1B-hAd5-COVID-19-S, 1×10 11 VP / dose (n=10),

[0179] Cohort 2A-hAd5-COVID-19-S / N, 5×10 10 VP / dose (n=10),

[0180] Cohort 2B-hAd5-COVID-19-S / N, 1×10 11 VP / dose (n=10).

[0181] Each subject will receive a subcutaneous (SC) injection of hAd5-COVID-19-S or hAd5-COVID-19-S / N on Days 1 and 22 (i.e., 2 doses). This dosing schedule is consistent with hAd5[E1-,E2b-,E3-] vector-based vaccines currently in clinical trials. Cohorts 1-2 will enroll in parallel and may open simultaneously or in a staggered manner, depending on study product supply. Subjects in Cohorts 1A and 2A will first complete the low-dose vaccination schedule. After all subjects in Cohorts 1A and 2A have completed at least a single dose and subsequent assessments during the toxicity assessment period through Study Day 8, enrollment will continue if the Safety Review Committee (SRC) and at least one qualified infectious disease physician, independent of the Sponsor and the trial, confirm that there are no safety concerns. Subjects will then be enrolled in the higher-dose Cohorts 1B and 2B and vaccinated. For all subjects, follow-up study visits will occur on Days 8, 22, 29, 52 and 3, 6, and 12 months after the last vaccination. Additional follow-up for safety information will be conducted via telephone contact as described in the event protocol. Initial Safety The primary objectives of the Phase 1b trial are to evaluate the preliminary safety and reactogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines. Secondary objectives are to evaluate the extended safety and immunogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines.

[0182] Example 6: Expanded Phase 1b: Safety and Immunogenicity of Construct Selection

[0183] If the SRC determines that a Phase 1b expansion is safe based on its review of safety data from the Phase 1b safety assessment, it will move forward to do so. In the Phase 1b expansion, a total of 60 healthy subjects will be divided into 4 dosing cohorts (Cohorts 1A, 1B, 2A, 2B; n=15 per cohort):

[0184] Cohort 1A-hAd5-COVID-19-S, 5×10 10 VP / dose (n=15),

[0185] Cohort 1B-hAd5-COVID-19-S, 1×10 11 VP / dose (n=15),

[0186] Cohort 2A-hAd5-COVID-19-S / N, 5×10 10 VP / dose (n=15)

[0187] Cohort 2B-hAd5-COVID-19-S / N, 1×10 11 VP / dose (n=15)

[0188] Each subject will receive a SC injection of hAd5-COVID-19-S or hAd5-COVID-19-S / N on days 1 and 22 (i.e., 2 doses). For all subjects, follow-up study visits will be conducted on days 8, 22, 29, 52 and at 3, 6, and 12 months after the last vaccination. Additional follow-up for safety information will be conducted by telephone contact as described in the event protocol. The primary objective of the expanded Phase 1b is to select the most immunogenic construct between hAd5-COVID-19-S and hAd5-COVID-19-S / N and to determine the dose level based on changes in humoral and cellular immunogenicity indices. Secondary objectives are to evaluate the safety and reactogenicity of hAd5-COVID-19-S and hAd5-COVID-19-S / N.

[0189] Embodiments of the present disclosure are further described in the following examples. The examples are merely illustrative and do not in any way limit the scope of the claimed invention.

[0190] Example 7: hAd5[E1-, E2b-, E3-] Platform and Constructs

[0191] For the examples presented here, a next generation hAd5[E1-, E2b-, E3-] vector ( Fig.33 , Figure C) to create candidate viral vaccine constructs. Fig.33 As shown in panel DH, various constructs were created: Fig.33 , Panel D: S WT: S protein contains 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2); Fig.33 , Panel E: S RBD-ETSD: S receptor binding domain with enhanced T cell stimulatory domain (ETSD); Fig.33 , Panel F: S fusion: S is optimized to enhance the surface expression and display of RBD; Fig.33 , Sub-figure G: N-ETSD: nucleocapsid (N) sequence with ETSD; Fig.33 , Panel H: bivalent S-fusion + N-ETSD; S-WT + N-ETSD and S RBD-ETSD + N-ETSD constructs were also generated but not shown.

[0192] Example 8: Enhanced surface expression of RBD in HEK 293T cells after transfection with Ad5 S-fusion + N-ETSD

[0193] like Fig.34 As shown, anti-RBD specific antibodies did not detect hAd5 S-WT (Figure 34, panel A) or hAd5 S-WT+N-ETSD ( Fig. 9b) RBD on the surface of HEK 293T cells transfected with the construct, while hAd5 S-fusion alone was slightly higher ( Fig.34 , Panel E). As expected, both constructs with RBD, hAd5 RBD-ETSD and RBD-ETSD+N-ETSD, showed high binding of anti-RBD antibodies ( Fig.34 , panels C and D). Notably, high cell surface expression of RBD was detected after transfection with the bivalent hAd5 S-fusion + N-ETSD ( Fig.34 , Panel F). These findings support the proposition that the hAd5 S-fusion+N-ETSD construct (comprising a large and diverse number of antigens provided by optimized S and N with proper folding, full length) results in enhanced expression and cell surface display of the RBD in the vaccine construct.

[0194] Example 9: Immunoblot correlation of enhanced S expression with hAd5 S-fusion + N-ETSD

[0195] Immunoblotting analysis of S expression was associated with enhanced S expression ( Fig.35 ), again indicating that the bivalent hAd5 S-fusion + N-ETSD construct enhanced the expression of S compared to S-fusion alone. Fig.35 Depicted are immunoblot analyses of S expression. Cell surface RBD expression in HEK 293T cells for (a) hAd5 S-WT, S-fusion, and (c) S-fusion + N-ETSD correlates well with (d) S expression in immunoblots of HEK 293T cell lysates probed with anti-full-length (S2) antibody. Y-axis scale normalized to mode (NM).

[0196] Example 10: Confirmation of native folding of enhanced surface RBD after hAd5 S-fusion + N-ETSD transfection

[0197] Assays for recombinant ACE2-Fc binding were performed to confirm the native, physiologically relevant folding of the S RBD after expression from the hAd5 S-fusion + N-ETSD vaccine candidate. The S RBD binds to ACE2 during SARS-CoV-2 infection, and an effective neutralizing antibody blocks this interaction and thereby blocks infection. Such neutralizing antibodies are more likely to be effective if they are produced in response to S presented in the correct conformation. In addition to enhancing cell surface expression, the optimized S also allows for proper protein folding. It was found that ACE2-Fc binding to the S RBD expressed from hAd5S-fusion + N-ETSD was significantly enhanced ( Figure 36 , panels A and B, respectively) compared to hAd5 S-WT or hAd5 S-fusion ( Figure 36 , panels A and B, respectively). Fig.36 , Panel C). Anti-RBD antibody binding studies performed using the same assay ( Fig.36, Panels FJ) confirmed the enhanced surface expression findings noted for ACE2-Fc binding. Based on these findings of conformationally correct and enhanced SRBD expression (which is important for generating neutralizing antibodies), the hAd5 S-fusion + N-ETSD vaccine candidate was selected for clinical trials.

[0198] Example 11: hAd5 N-ETSD successfully directs N to the endosomal / lysosomal compartment

[0199] The ETSD design successfully transferred N to the endosomal subcellular compartment. After infection of HeLa cells with N-ETSD, N colocalized with the endosomal marker 45 transferrin receptor (CD71), such as Fig.37 As shown in Figure C, it also co-localizes with the lysosomal marker Lamp1 ( Fig.37 , panel D), indicating that N-ETSD translocates to lysosomes throughout the endosomal pathway, thereby enabling processing for MHCII presentation. Compared with N-ETSD, N-wild type (N-WT) showed a diffuse cytoplasmic distribution and did not colocalize with lysosomal markers ( Fig.37 , Panel E). These findings confirm a role for ETSD in directing N to the endosomal / lysosomal compartment, which would lead to increased MHC II presentation and CD4+ activation by N.

[0200] Example 12: In vivo hAd5 S-fusion + N-ETSD vaccine immunogenicity study

[0201] Based on the evidence that S-fusion + N-ETSD resulted in enhanced expression of the physiologically relevant RBD and successful translocation of N-ETSD to endosomal / lysosomal compartments, the bivalent hAd5 S-fusion + N-ETSD vaccine was chosen for vaccination of 7-week-old female CD-1 mice. The unique properties of this construct will result in CD8+ and CD4+ T cell responses and the production of neutralizing antibodies. As described in the methods, mice received a primary injection on day 0 and a second injection on day 21. Serum was collected at the end of the study on days 0 and 28 for antibody and neutralization analysis. Splenocytes were also collected on day 28 for intracellular cytokine staining (ICS) and ELISpot analysis. All age- and sex-matched animals assigned to this study appeared normal, and no site reactions and weight loss were observed throughout the dosing period, which is consistent with previous observations using the hAd5 [E1-, E2b-, E3-] platform.

[0202] Example 13: hAd5 S-fusion + N-ETSD generates CD8β+ and CD4+ T cell responses

[0203] S and N activated CD8+: CD8β+ splenocytes from hAd5 S-fusion + N-ETSD-vaccinated mice exposed to S peptide pool 1 (containing RBD and S1) showed significantly higher IFN-γ expression compared with hAd5-null mice ( Fig.38, panel A); splenocytes from these mice also expressed intracellular IFN-γ in response to the N peptide pool. Assessment of simultaneous IFN-γ / TNF-α expression from CD8β+ splenocytes ( Fig.38 , Figure C) reflects those when IFN-γ alone is expressed. These results indicate that both S and N activate CD8+ T cells.

[0204] N activated CD4+: Although CD8+ cytotoxic T cells mediate killing of virus-infected cells, CD4+ T cells are required for sustained cytotoxic T lymphocyte (CTL) activity. Therefore, CD4+ T cells in vaccinated animals were evaluated. Compared with CD8β+ splenocytes, only the N peptide pool stimulated CD4+ splenocytes from hAd5 S-fusion + N-ETSD-vaccinated mice to express IFN-γ (Figure 38, panel B) or IFN-γ / TNF-α (Figure 39, panel B) at significantly higher levels than the hAd5 null control. Fig.38 , Panel D). The contribution of N to CD4+ T cell responses is critical for an effective immune response to candidate vaccines.

[0205] Example 14: hAd5 S-fusion + N-ETSD generates antibody responses to both S and N antigens

[0206] The main target of coronavirus vaccines currently under development is neutralizing antibodies against the spike. In mice vaccinated with the bivalent vaccine, anti-S (Figure 39, panel A) and anti-N (Figure 39, panel B) were significantly produced in sera from CD-1 mice vaccinated with hAd5 S-fusion + N-ETSD on day 28 of the study. Fig.39 , Panel C) Antibodies. Anti-N antibodies were higher in serum compared to anti-S antibodies, as the serum dilution factor for anti-N antibody analysis was 1:90 and the dilution factor for anti-S antibody analysis was 1:30.

[0207] A standard curve for IgG was generated and the absorbance values ​​were then converted to mass equivalents of anti-S and anti-N antibodies ( Fig.39 , panels B and D). These values ​​were used to calculate the geometric mean of 5.8 μg S-specific IgG and 42 μg N-specific IgG / mL serum generated by hAd5 S-fusion + NETSD vaccination, thus the relative μg amounts of anti-N antibodies were higher compared to anti-S antibodies, reflecting the strong contribution of N to the production of anti-SARS-CoV-2 antibodies.

[0208] Example 15: hAd5 S-fusion + N-ETSD vaccine produces potent neutralizing antibodies, as demonstrated by cPass and live virus Neutralization assay evaluated

[0209] Neutralizing antibody activity was assessed using a cell-free assay (cPass) as well as in vitro live virus infection. Fig.40As seen in panel A, the cPass assay showed that S RBD:ACE2 binding was inhibited for all mice at dilutions of 1:20 and 1:60, with inhibition rates of about 100% in two mice. The results of the VeroE6 neutralization assay for four mice showing S-specific antibodies by ELISA are shown. High sustained neutralization was seen even at high dilution factors, suggesting the interesting possibility that the hAd5 S-fusion + N-ETSD vaccine produces a bivalent, multi-antigenic, multi-epitope vaccine that can lead to synergy in neutralizing immune responses at epitopes other than those associated with RBD-ACE2 binding ( Fig.40 , sub-figure B). Fig.40 As can be seen in panel B, for the G4 serum pool from mice showing S-specific antibodies, a 50% neutralization value (IC50) at a serum dilution of 1:10,000 was presented, which was 10 times that of the convalescent serum at a dilution of 1:1,000. The effective neutralization confirmed by both assays supports the expected efficacy of the hAd5 S-fusion + ETSD candidate vaccine and its progress in clinical trials.

[0210] Example 16: hAd5 S-fusion + N-ETSD produces Th1-dominant responses in both humoral and T cell immunity

[0211] Th1 dominance of antibodies in response to N and S: IgG2a, IgG2b, and IgG3 represent Th1 dominance; whereas IgG1 represents Th2 dominance. Fig.41 , sub-figure A) and anti-N ( Fig.41 , Panel C) Antibodies, IgG2a and IgG2b isotypes predominated and were significantly higher compared to the hAd5 null control. These data show a Th1 predominance of antibody production in response to the hAd5 S-fusion + N-ETSD vaccine.

[0212] T cell Th1 dominance in response to N and S: IFN-γ production is associated with CTL activity 47 (Th1 dominance), while IL-4 leads to delayed viral clearance 48 (Th2 dominance). An IFN-γ to IL-4 ratio of 1 is balanced, and a ratio greater than 1 indicates Th1 dominance. ELISpots from animals immunized with the bivalent S plus N vaccine showed that IFN-γ secretion was significantly higher for hAd5 S-fusion + N-ETSD than for hAd5 null splenocytes in response to both S peptide pool 1 and N peptide pool ( FIG. 42 , panel A), but IL-4 was secreted at significantly higher levels only for hAd5 S-fusion + N-ETSD in response to the N peptide pool ( FIG. 42 , panel A). Fig.42 , sub-figure B).

[0213] The Th1 predominance was also seen when considering the ratio of IFN-γ to IL-4 based on spot forming units in response to the combined S-peptide pool and the N-peptide pool ( Fig.43 , panel A). Th1 dominance was again seen in the humoral response, with ratios of ng-equivalent Th1-associated antibodies (IgG2a, IgG2b, and IgG3) to Th2-associated antibodies (IgG1) greater than 1 in all mice for both anti-S and anti-N antibodies ( Fig.43 , sub-figure B).

[0214] This Th1-dominant profile of the hAd5 S-fusion+N-ETSD vaccine candidate provides further rationale for hAd5 S-fusion+N-ETSD to be a prime candidate for clinical testing.

[0215] The hAd5 S-fusion + N-ETSD vaccine is designed to overcome the risks of S-only vaccines and elicit T cell immunity and neutralizing antibodies, taking advantage of the important role that T cells play in generating durable antibody responses and direct killing of infected cells. Both CD4+ and CD8+ T cells are multifunctional, and the induction of such multifunctional T cells by the vaccine is associated with better protection against infection. The enhanced CD4+ T cell response and Th1 dominance resulting from the expression of the S antigen optimized for surface display and the N antigen optimized for localization in endosomal / lysosomal subcellular compartments and, therefore, MHC I and II presentation, lead to increased dendritic cell presentation, cross-presentation, B cell activation, and ultimately high neutralizing capacity. Furthermore, the potent neutralizing capacity of pooled sera from mice vaccinated with hAd5 S-fusion + N-ETSD at high dilutions, combined with the Th1 dominance of antibodies generated in response to both S and N antigens, supports the goal of this vaccine design.

[0216] Simultaneous presentation of antigen by both MHC I and MHC II of antigen-presenting cells activates both CD4+ and CD8+ T cells and is optimal for generating memory B and T cells. A key finding of this construct is that N-ETSD is directed to the endosomal / lysosomal compartment. N-ETSD elicits CD4+ responses, which is necessary for the induction of memory T cells and helper cells for B cell antibody production. The importance of lysosomal localization for eliciting the strongest T cell IFN-γ and CTL responses compared to native N has also been reported by others. 50,51

[0217] T cell responses to S and N antigens expressed by hAd5 S-fusion+N-ETSD were multicytokine, including IFN-γ and TNF-α, consistent with successful antimicrobial immunity in bacterial and viral infections. Multicytokine T cell responses following vaccination have been shown to correlate with vaccine efficacy, including those when viral vectors are used. Highly relevant, multicytokine T cell responses to SARS-CoV-2 N protein were consistent with those of recovered COVID-19 patients, suggesting that the bivalent hAd5S-fusion+N-ETSD vaccine will provide vaccine recipients with greater protection against SARS-CoV-2.

[0218] Compared to N, the S protein (expressed here as S-fusion with enhanced RBD cell surface expression and conformational integrity as demonstrated by high ACE2-Fc binding) predominantly generated CD8+ T cells. Our results confirm the vaccine design goals, showing that S-fusion induced elevated levels of antigen-specific T cell responses against S compared to S-WT. Vaccination with optimized S and N antigens is necessary to ensure MHC presentation at both MHC I (for CD8+ T cell activation) and MHC II (for CD4+ T cell activation) to generate this coordinated response.

[0219] Neutralization data for live SARS-CoV-2 virus demonstrated the potency of the antibody response generated following vaccination with hAd5 S-fusion + N-ETSD, with evidence of high neutralization even at high dilutions. Furthermore, a significant synergistic effect of the combined sera was evident, with potent neutralization even greater than control convalescent sera at dilutions ≥1:1,000.

[0220] The hAd5 S-fusion + N-ETSD construct described above was delivered by the next-generation hAd5 [E1-, E2b-, E3-] platform, in which only the E2b deletion (pol) enabled prolonged transgene production and allowed homologous vaccination in the setting of pre-existing adenoviral immunity (prime and boost formulations were identical). 38 In addition to the generation of cellular and humoral immunity by subcutaneous injection of hAd5 S-fusion + N-ETSD, the same vaccine in an oral or sublingual formulation could also induce IgA mucosal immunity.

[0221] Example 17: Methods and Constructs

[0222] hAd5[E1-,E2b-,E3-] platform and constructs

[0223] For the studies presented here, the second generation hAd5[E1-, E2b-, E3-] vector ( Fig.44, panel A) to create candidate viral vaccine constructs. The hAd5[E1-, E2b-, E3-] backbone containing SARS-CoV-2 antigen expression inserts and viral particles was generated as described previously. Briefly, high-titer adenoviral stocks were generated by serial propagation in an E.C7 packaging cell line expressing E1 and E2b, followed by CsCl2 purification and dialysis into storage buffer (2.5% glycerol, 20mM Tris pH 8, 25mM NaCl) (performed by ViraQuest Inc. (North Liberty, Iowa)). Viral particle counts were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280nm and Adeno-X TM Virus titers were determined using a rapid titration kit (Takara Bio). Constructs created included:

[0224] S-WT: S protein contains 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234) and cytoplasmic (1235-1273) (Unitprot P0DTC2); S RBD-ETSD: S receptor binding domain (S RBD) with ETSD (SEQ ID NO: 11); N-ETSD: nucleocapsid (N) with ETSD; S-WT+ N-ETSD: S-WT with enhanced T cell stimulation domain (ETSD); S-RBD-ETSD+N-ETSD; S-fusion: S optimized to enhance surface expression and display of RBD; and bivalent S-fusion+N-ETSD;

[0225] Transfection of HEK 293T cells with hAd5 constructs

[0226] To determine the surface expression of RBD epitopes by candidate vaccine constructs, we transfected HEK 293T cells with hAd5 construct DNA and quantified surface RBD by flow cytometry using anti-RBD antibodies. Seven constructs were tested: S-WT, S-WT+N-ETSD, S RBD-ETSD, S RBD-ETSD+N-ETSD, S-fusion, S-fusion+N-ETSD, and N-ETSD. HEK 293T cells (2.5x10 5Cells / well in 24-well plates) were grown in DMEM (Gibco Cat#11995-065) containing 10% FBS and 1X PSA (100 units / mL penicillin, 100 μg / mL streptomycin, 0.25 ug / mL amphotericin B). Cells were transfected with 0.5 μg hAd5 plasmid DNA using JetPrime transfection reagent (Polyplus Catalog No. 89129-924) according to the manufacturer's instructions. Cells were harvested 1, 2, 3, and 7 days after transfection by gently pipetting into culture medium and labeled with anti-RBD monoclonal antibody (clone D003 Sino Biological Catalog #40150-D003) and F(ab')2-goat anti-human IgG-Fc secondary antibody conjugated to R-phycoerythrin (ThermoFisher Catalog No. H10104). Labeled cells were acquired using a Thermo-Fisher Attune NxT flow cytometer and analyzed using Flowjo software.

[0227] Immunocytochemical labeling of hAd5-infected HeLa cells

[0228] To determine the subcellular localization of N after infection or transfection of HeLa cells with hAd5 N-wild type (WT) or hAd5 N-ETSD (each with a flag tag to allow labeling), cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.4% Triton X100 in PBS for 15 minutes at room temperature 48 hours after infection or transfection. To label N, cells were then incubated with anti-flag monoclonal (anti-Flag M2 raised in mouse, Sigma cat# F1804) antibody at 1:1000 in phosphate-buffered saline containing 3% BSA at 4°C overnight, then washed in PBS and incubated with goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 555 (Life Technologies, Cat# A32727) at 1:500 for 1 hour. For colocalization studies, cells were also incubated overnight at 4°C with sheep anti-Lamp1 Alexa Fluor 488-conjugated (lysosomal marker) antibody (R&D systems, Cat# IC7985G) at 1:10 or with rabbit anti-CD71 (transferrin receptor, endosomal marker) antibody (Thermo Fisher Cat# PA5-83022) at 1:200. After removal of primary antibodies, washing twice in PBS and three times in PBS containing 3% BSA, cells were incubated with fluorophore-conjugated secondary antibodies when applicable at 1:500 (goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488, Life technologies, A-11034) at room temperature for 1 hour. After brief washing, cells were mounted with Vectashield antifade mounting medium with DAPI (Fisher Scientific, Cat# NC9524612) and immediately imaged using a Keyence integrated fluorescence microscope camera and Keyence software.

[0229] Immunoblot analysis of S antigen expression

[0230] HEK 293T cells transfected with hAd5 S-WT, S-fusion or S-fusion + N-ETSD constructs were cultured and transfected as described in the main manuscript and harvested 3 days post-transfection in 150 mL RIPA lysis buffer containing 1X final protease inhibitor cocktail (Roche). After protein determination, equivalent amounts of total protein were loaded and run on 4 to 12% gradient polyacrylamide gels (type) and transferred to nitrocellulose membranes using a semi-dry transfer apparatus. Anti-Spike S2 ​​(SinoBiological Cat#40590-T62) was used as the primary antibody, 800CW goat anti-rabbit IgG (H+L) (Li-Cor, 925-32211) was used as the secondary antibody, using the Ibind Flex platform. The antibody-specific signal was detected using an infrared Licor Odyssey instrument.

[0231] ACE2-IgG1Fc binds to hAd5-transfected HEK 293T cells

[0232] HEK 293T cells were cultured at 37°C under the above conditions for transfection with hAd5 S-WT, S-fusion, S-fusion+N-ETSD, SRBD-ETSD or SRBD-ETSD+N-ETSD and incubated for 2 days and harvested for ACE2-Fc binding analysis. Recombinant ACE2-IgG1Fc protein was produced in CHO-S cells cultured for 14 days using Maxcyte transfection. ACE2-IgG1Fc was then purified using a MabSelect SuRe affinity column on an AKTA Explorer.

[0233] Purified ACE2-IgG1Fc was dialyzed into 10mM HEPES (pH7.4), 150mM NaCl and concentrated to 2.6mg / mL. For binding studies, ACE2-IgG1Fc was used at a concentration of 1μg / mL for binding. Cells were incubated with ACE2-Fc for 20 minutes and then, after a wash step, labeled with a 1:100 dilution of PE-conjugated F(ab')2-goat anti-human IgG Fc secondary antibody, incubated for 20 minutes, washed and acquired on a flow cytometer. Histograms are based on the normalized mode (NM) of cell counts - counts of cells with positive signals in the PE channel.

[0234] Vaccination of CD-1 mice with hAd5 S-fusion + N-ETSD candidate vaccines

[0235] Seven-week-old female CD-1 mice (Charles River Laboratories) were used for immunological studies at the animal care facility of Omeros Inc. (Seattle, WA). After the initial blood draw, 1 × 10 10 Mice were injected with hAd5 null (negative control) or candidate vaccine hAd5 S-fusion + N-ETSD at a dose of 100 viral particles (VP). There were 5 mice in each group. Mice received the second vaccine dose on days 21 and 28, blood was collected from isoflurane-anesthetized mice via the submandibular vein to separate serum, and then the mice were euthanized to collect spleen and other tissues.

[0236] Splenocyte collection and intracellular cytokine staining (ICS)

[0237] The spleen was removed from each mouse and placed in 5 mL sterile culture medium with RPMI (Gibco Cat#22400105), HEPES (Hyclone Cat#SH30237.01), 1X Pen / Strep (Gibco Cat#15140122) and 10% FBS (Gibco Cat#16140-089). Splenocytes were isolated within 2 hours of collection. ICS was used for flow cytometry to detect IFN-γ and IFN-γ / TNFα+ production associated with CD8β+ and CD4+ T cells in response to stimulation with S and N peptide pools.

[0238] In a 96-well U-bottom plate, use 10 6 Stimulation assay was performed on live splenocytes. Splenocytes in RPMI medium supplemented with 10% FBS were stimulated for 6 hours by adding a peptide pool of 2 μg / mL / peptide at 37°C and 5% CO2, and the protein transport inhibitor GolgiStop (BD) was added two hours after the start of incubation. The stimulated splenocytes were then stained for lymphocyte surface markers CD8β and CD4, fixed with CytoFix (BD), permeabilized, and stained for intracellular accumulation of IFN-γ and TNF-α. Fluorescent conjugated antibodies against mouse CD8β antibodies (clone H35-17.2, Thermo Fisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD) and TNF-α (clone MP6-XT22, BD) were used and stained in the presence of unlabeled anti-CD16 / CD32 antibodies (clone 2.4G2). Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo software.

[0239] ELISpot assay

[0240] ELISpot assays were used to detect cytokines secreted by splenocytes of vaccinated mice. Fresh splenocytes as well as cryopreserved splenocytes containing lymphocytes were used on the same day. Cells (2-4x10 per well of a 96-well plate) were plated. 5 10 cells) were added to ELISpot plates containing immobilized primary antibodies against IFN-γ or IL-4 (BD) and exposed to various stimuli (e.g., control peptide, target peptide pool / protein) containing 2 μg / mL peptide pool or 10 μg / mL protein for 36-40 hours. After aspiration and washing to remove cells and culture medium, extracellular cytokines were detected by secondary antibodies against cytokines conjugated to biotin (BD). Biotin-conjugated secondary antibodies were detected using streptavidin / horseradish peroxidase conjugates. 2-4x10 cells were counted per well or per well using an ELISpot plate reader. 5 The number of spots per cell.

[0241] ELISA for antibody detection

[0242] For antibody detection in sera of vaccinated mice, ELISAs specific for spike and nucleocapsid antibodies and IgG subtypes (IgG1, IgG2a, IgG2b, and IgG3) were used. Microtiter plates were coated overnight with 100 ng of purified recombinant SARS-CoV-2 S-FTD (full-length S with a fibrin trimerization domain, constructed and purified by ImmunityBio, Inc., 9920 Jefferson Avenue, Culver City, CA 90232), SARS-CoV-2 SRBD (Sino Biological, Beijing, China; Cat#401591-V08B1-100) or purified recombinant SARS-CoV-2 nucleocapsid (N) protein (Sino Biological, Beijing, China; Cat#40588-V08B) in 100 μL coating buffer (0.05 M carbonate buffer, pH 9.6). The wells were washed three times with 250 μL PBS containing 1% Tween 20 (PBST) to remove unbound proteins, and the plate was blocked with 250 μL PBST for 60 minutes at room temperature. After blocking, the wells were washed with PBST, 100 μL of diluted serum samples were added to the wells, and the samples were incubated at room temperature for 60 minutes. After incubation, the wells were washed with PBST and 100 μL of 1 / 5000 diluted anti-mouse IgG HRP (GE Health Care; Cat#NA9310V) or anti-mouse IgG1 HRP (Sigma; Cat#SAB3701171) or anti-mouse IgG2a HRP (Sigma; Cat#SAB3701178) or anti-mouse IgG2b HRP (Sigma; Catalog No. SAB3701185) or anti-mouse IgG3 HRP conjugated antibody (Sigma; Cat#SAB3701192) was added to the wells. For positive controls, 100 μL of 1 / 5000 diluted rabbit anti-N IgG Ab or 100 μL of 1 / 25 diluted mouse anti-S serum (from mice immunized with purified S antigen in adjuvant) was added to the appropriate wells. After incubation for 1 hour at room temperature, the wells were washed with PBS-T and incubated with 200 μL of o-phenylenediamine-dihydrochloride (OPD substrate (Thermo Scientific Cat# A34006)) until the appropriate color appeared. The color reaction was terminated by adding 50 μL of 10% phosphoric acid aqueous solution (Fisher Cat# A260-500) and the wells were analyzed using a microplate reader ( The absorbance at 490 nm was measured using a 4% paraformaldehyde (AFP) kit (Pro, Molecular Devices).

[0243] Calculate relative μg antibody amount

[0244] A standard curve for IgG was generated and absorbance values ​​were converted to mass equivalents of anti-S and anti-N antibodies. hAd5S-fusion + N-ETSD vaccination produced a geometric mean of 5.8 μg S-specific IgG and 42 μg N-specific IgG per ml of serum.

[0245] cPassTM Neutralizing Antibody Test

[0246] GenScript cPass was used according to the manufacturer's instructions. TM Detection of neutralizing antibodies. 44 This kit detects circulating neutralizing antibodies against SARS-CoV-2 that block the interaction between the S RBD and the ACE2 cell surface receptor. It is applicable to all antibody isotypes and can be used in animal models without modification.

[0247] Neutralization assay in Vero E6 cells

[0248] All aspects of the assays using the virus were performed in a BSL3 containment facility in accordance with the ISMMS routine biocontainment facility SOP for SARS-CoV-2 cell culture studies. Vero e6 renal epithelial cells from African green monkeys (Cercopithecus aethiops) (ATCC CRL-1586) were plated at 20,000 cells / well in a 96-well format, and 24 hours later, the cells were incubated with antibodies or heat-inactivated sera that had been serially diluted in 3-fold steps in DMEM containing 2% FBS, 1% NEAA, and 1% Pen-Strep; the diluted samples were mixed 1:1 with SARS-CoV-2 at 10,000 TCID 50 / mL in DMEM containing 2% FBS, 1% NEAA, and 1% Pen-Strep for 1 hour at 37°C, 5% CO2. This incubation did not include cells to allow neutralization activity to occur prior to infection. Samples tested included sera from four mice that showed >20% inhibition of ACE2 binding in cPass, pooled sera from these four mice, sera from patients recovering from COVID-19, and culture medium alone. To detect neutralization, 120 μL of the virus / sample mixture was transferred to Vero E6 cells and incubated for 48 hours before fixation with 4% PFA. Each well received 60 μL of virus or an infectious dose of 600 TCID50. Control wells, including 6 wells on each plate, were used for no virus and virus-only controls. The neutralization percentage was calculated as 100-((target sample-[mean value of "no virus"]) / [mean value of "virus-only"])*100), where staining of CoV-2Np was imaged on a Celigo imaging cytometer (Nexcelom Bioscience).

[0249] Vaccine characterization

[0250] Most prophylactic vaccines currently in development against SARS-CoV-2 are designed to prevent further disease-related mortality and morbidity by targeting the viral spike (S) protein with the goal of generating a neutralizing antibody response in the recipient prior to viral exposure. However, recent characterization of the immune response to SARS-CoV-2 in COVID-19 patients suggests that other immune cells, such as T cells, are critical for clearing the infection and generating long-term immunity to coronavirus infection. Both CD4+ and CD8+ T cells support a durable humoral response, as CD4+ T cells, while not effector cells like CD8+ T cells, are critical for robust and durable immunity generated by antibody-secreting plasma cells and memory cytotoxic CD8+ T cells to eliminate infected cells. The dual-antigen vaccine candidates disclosed today more broadly activate the immune system against SARS-CoV-2 by incorporating modified viral nucleocapsid (N) antigens, potent CD4+ and CD8+ T cell targets, and an optimized S protein (S-fusion) to stimulate a humoral response.

[0251] Human adenovirus serotype 5 (hAd5) E1, E2b and E3 region deleted [E1-, E2b-, E3-] vaccine platform ( Fig.44 , panel A) is superior to the adenoviral platform used in other COVID-19 vaccines currently in clinical trials because it is effective in the presence of pre-existing adenoviral immunity and has a reduced likelihood of generating a host immune response targeting the vector, so it can be used as a prime and boost. Using this platform, the vaccine contains an optimized S surface protein S-fusion to increase cell surface display and humoral responses; and the highly conserved and antigenic N protein found in viral particles, here with a subcellular compartment targeting sequence to enhance antigen presentation. This strategy will be safe and robust in eliciting humoral and T cell responses to SARS-CoV-2. The addition of N addresses the risk of loss of vaccine efficacy in S-only monovalent vaccines due to the emergence of S mutations in the population over time. In contrast, N is highly conserved, has a lower risk of mutation, and is also highly immunogenic. It is a known target antigen for natural immunity, and antibodies and T cells against N have been found in most people who have recovered from SARS-CoV-2 and similar viruses SARS-CoV infection. In the vaccine of this aspect, the enhanced T cell stimulatory domain (ETSD) directs the N protein to the endosomal-lysosomal subcellular compartment after translation to support MHC class II presentation to activate T helper cells and promote CD8+ T cell activation through dendritic cell licensing. Despite the risk of emerging mutations, S remains a key antigen for vaccination due to its role in infection. Spike, displayed on the surface of the virus as a trimer ( Fig.44, panel B), has a receptor binding domain (RBD) that interacts with host angiotensin-converting enzyme 2 (ACE2) to facilitate entry into host cells and proliferation, so antibodies against S are key to neutralizing infection. Antibodies against the S RBD are common in patients who have recovered from COVID-1951, as are antibodies against other S epitopes. In the bivalent vaccine construct, the S-fusion was S-optimized by adding a fusion linker to display the S RBD in a physiologically relevant form on the cell surface, with the goal of improving the production of anti-S RBD antibodies that will neutralize the virus.

[0252] The bivalent hAd5 S-fusion + N-ETSD vaccines disclosed so far generate excellent T cell responses. Vaccines currently in clinical trials focus on generating humoral responses as a means of neutralizing infection. However, given that antibodies, even if successfully generated and fully neutralizing, can wane over time, T cell responses become critical. If T cell responses are absent due to viral-induced lymphopenia, infected individuals are at risk for developing acute symptoms of the disease even in the presence of abundant neutralizing antibodies. While it cannot be ruled out that S (and other viral proteins) can induce T cell responses, evidence in the literature supports a critical role for N. Not only are T cell responses to N found in the majority of patients who have recovered from COVID-19, these responses to N are remarkably durable in patients exposed to the very similar virus SARS-CoV. Strong evidence for the importance of N in natural T cell immunity can be found in a recent report by Ferretti et al. They found that, through an unbiased genome-wide screen for the precise peptide sequences recognized by memory CD8+ T cells from COVID-19 patients, only 3 of the 29 shared epitopes were from the spike protein, with the highest density of epitopes being located on the nucleocapsid protein. Therefore, in the hAd5 S-fusion + N-ETSD vaccine, N is expected to elicit not only humoral responses but also T cell responses, which better reproduce disease-restricted natural immunity.

[0253] In the initial report, the hAd5 S-fusion + N-ETSD vaccine provided enhanced cell surface expression of the S RBD that was readily recognized by ACE2, reflecting its conformational integrity. N-ETSD with endosomal / lysosomal localization for enhanced antigen presentation generated neutralizing antibodies and CD4+ / CD8+ T cell-mediated responses in vaccinated mice, with a predominance of Th1. The present disclosure extends these findings using plasma and monocyte-derived dendritic cells (MoDCs) from patients previously infected with SARS-CoV-2 to confirm native S antigen expression. We elucidated the localization of N-ETSD in antigen-presenting MoDCs, showing that it localizes to endosomes, lysosomes, and autophagosomes. Protein processing through this subcellular pathway enhances MHC class II presentation and increases peptide recycling, thereby also enabling MHC class I presentation. Both CD4+ and CD8+ SARS-CoV-2-specific memory T cells were recapitulated from patients previously infected with SARS-CoV-2 by targeting the nucleocapsid protein to the late lysosomal-autophagosomal compartment. Furthermore, in these immune response recall studies, hAd5-infected MoDCs presenting S-fusion and N-ETSD elicited a dominant Th1 response in vitro from autologous memory T cells of patients previously infected with SARS-CoV-2. N specifically drove CD8+ T cell responses in the in vitro recall studies. The hAd5 S-fusion + N-ETSD vaccine's recapitulation of natural infection and immunity to the extent that can be achieved through vaccination makes it a prime candidate for clinical testing of its ability to protect individuals from SARS-CoV-2 infection and COVID-19, and this second-generation vaccine construct has now entered Phase I clinical trials.

[0254] Example 18: hAd5[E1-, E2b-, E3-] Platform and Constructs

[0255] For the studies presented here, the next generation hAd5[E1-, E2b-, E3-] vector ( Fig.44 , Figure A) to create candidate viral vaccine constructs. Various constructs were created: Fig.44 , Panel C: S WT: S protein contains 1274 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2); Fig.44 , Panel D: S-fusion: S is optimized to enhance surface expression and display of RBD; FIG44 , Panel E: N (N without ETSD): nucleocapsid (wild type) sequence with a tag for immunodetection, but without ETSD modification, mainly localized in the cytoplasm. Fig.44, Panel F. N with enhanced T cell stimulation domain (N-ETSD): nucleocapsid with ETSD (to direct lysosomal / endosomal localization) and a tag for immunodetection (wild type); Fig.44 , Panel G: Bivalent hAd5 S-fusion + N-ETSD vaccine.

[0256] Nucleocapsid antigens engineered with enhanced T cell stimulatory domains (ETSDs) direct N to endosomes, lysosomes, and autophagosomes in MoDCs, driving enhanced CD4+ T cell activation:

[0257] The hAd5 bivalent vaccine construct included sequences designed to target N to the MHC class II antigen-loading compartment. To further investigate factors influencing antigen presentation in MoDC, MoDC from healthy subjects were infected with hAd5 N-ETSD or hAd5 N, and localization was determined by immunocytochemistry. N-ETSD was shown to localize to discrete vesicles, some of which were associated with CD71, a marker for circulating endosomes ( Fig.45 , panels A to C) and LAMP-1 (a marker for late endosomes / lysosomes) ( Fig.45 GI), while N was widely and evenly expressed throughout the cytoplasm ( Fig.45 , panels DF and JL). Lysosomes fuse with autophagosomes to enhance peptide processing and MHC class II presentation. N-ETSD also shows some colocalization with autophagosome markers ( Fig.45 , Panel MO). Protein processing in autophagosomes plays a key role in MHC-mediated antigen presentation in DCs, providing a potential mechanism for the N-ETSD-induced enhancement of CD4+ T cells in the vaccine construct. Evidence of such T cell interaction with MoDCs infected with N-ETSDs translocated to autophagosomes (and presumably also endosomes and lysosomes) was seen in phase contrast microscopy of cells co-labeled for N-ETSD and LC3a / b, which revealed an elongated DC morphology in contrast to the spherical morphology of undifferentiated lymphocytes. Lymphocytes, which were also distinguished from those infected with hAd5 N-ETSDs (lymphocytes lack the hAd5 receptor), were also thought to interact with MoDCs expressing N-ETSDs.

[0258] Validation of SARS-CoV-2 antibody and cell-mediated immune responses in patients previously infected with SARS-CoV-2 and those not infected with the virus for memory T cell recall studies:

[0259] For the studies described below, plasma samples were collected from four individuals who had recovered from SARS-CoV-2 infection (confirmed by antibody testing and patient history, as described below). The presence of anti-Spike IgG and neutralizing antibodies was confirmed in all patient samples by cPass66 and live virus assays. Samples were also collected from four individuals who were not infected with the virus and used as controls. In other studies validating immune responses to SARS-CoV-2 antigens, plasma from patients with prior SARS-CoV-2 infection and control individuals who were not infected with the virus was evaluated for binding to human embryonic kidney (HEK) 293T cells transfected with hAd5 S-fusion alone or hAd5 S-fusion + N-ETSD. This binding reflects the presence of antibodies in the plasma that recognize the antigen expressed by the hAd5 vector vaccine. Quantification of the histograms showed that plasma antibodies from patients who were not infected with the virus had little or no binding to cells expressing either construct, and plasma antibodies from patients who were previously infected with SARSCoV-2 had the highest binding to cells expressing the bivalent S-fusion + N-ETSD construct. This could be due to enhanced cell surface expression of S or expression of both S and N antigens found in hAd5 S-fusion + N-ETSD infected HEK 293T cells compared to hAd5 S-fusion alone.

[0260] Example 19: N-ETSD optimizes spike antigen expression: Compared with hAd5 S-fusion or hAd5 S-WT, For MoDCs infected with S-fusion+N-ETSD, binding of plasma antibodies from previously infected SARS-CoV-2 patients was increased. powerful.

[0261] The studies herein focused on the response of T cells from patients previously infected with SARS-CoV-2 to autologous MoDC infected with the hAd5 vaccine construct. DCs are powerful antigen presenting cells that process and present complex antigens acquired by infection or phagocytosis to elicit T cell responses. Therefore, in addition to evaluating the binding of patient plasma antibodies to HEK293T cells expressing the hAd5 vaccine, MoDCs from two healthy individuals were infected overnight with hAd5 S-WT, hAd5 S-fusion, hAd5 S-fusion + N-ETSD, or hAd5 ineffective, and then gene expression was assessed using plasma from patients previously infected with SARS-CoV-2 (Figure 46, panel A). For both MoDC sources, the highest binding of plasma antibodies from previously infected patients to MoDCs was observed after infection with hAd5 S-fusion + N-ETSD (Figure 46, panel A). Fig.46 , sub-figures BD), providing further evidence that antigen expression is optimized in the hAd5 S-fusion + N-ETSD bivalent vaccine. This finding in the highly relevant in vitro system of human MoDC and plasma is not only an important confirmation of the results tested with HEK 293T cells and commercially available anti-SRBD antibodies, it represents a potential method for screening plasma for SARS-CoV-2 antigen reactivity.

[0262] Example 20: SARS-CoV-2 peptide pool immune response:

[0263] T cells from previously infected SARS-CoV-2 patients secreted significant levels of interferon-g (IFN-γ) in response to pools of S1, S2, and N SARSCoV-2 peptides compared to T cells from virus-uninfected controls. To demonstrate the responsiveness of T cells from four previously infected SARS-CoV-2 patients compared to virus-uninfected T cells from four unexposed individuals, T cells from each group were cultured with autologous MoDC pulsed with a mixture of peptides spanning the N and S protein sequences. T cells from previously infected SARS-CoV-2 patients but not unexposed subjects secreted IFNg( Fig.47 ), validating the selective reactivity of T cells from patients previously infected with SARS-CoV-2.

[0264] Example 21: SARS-CoV-2 peptide pool immune response: CD4+ T cells from patients previously infected with SARSCoV-2 Recognizes S and N peptide pool antigens, but CD8+ T cells show stronger recognition of N peptide antigens.

[0265] CD4+ T cells from both patient samples tested responded to both S and N peptide pools, with Pt3 responding more strongly to N ( Fig.48 , Panel B). In contrast, CD8+ T cells from two patients showed highly significant responses to N, but not to S1 or S2 peptide pools ( Fig.48 , panels C and D). These data are consistent with published studies demonstrating T cell responses against multiple antigens, including S and N, in previously infected SARS-CoV-2 patients.

[0266] Example 22: Endosome / lysosome-directed nucleocapsid compared to cytoplasmic nucleocapsid protein (hAd5 N) - ETSD's autologous MoDC elicit higher levels of IFN-γ from CD4+ and CD8+ T cells of previously infected SARS-CoV-2 patients secretion.

[0267] To assess the immunological significance of endosomal / lysosomal localized N-ETSD compared to cytoplasmic N, MoDC were infected with hAd5 constructs (null, N-ETSD, or N) and then incubated with autologous CD3+ and CD4+ or CD8+ selected T cells ( Fig.49 , panel A). In the two patients comparing N-ETSD and N, CD3+ T cells from previously infected SARS-CoV-2 patients secreted significantly more IFN-γ in response to NETSD than ineffective and cytoplasmic N ( Fig.49 , panels C and D). For all patients, relatively few CD3+ T cells secreting interleukin 4 (IL-4) were found ( Fig.49 , sub-figures EG). The IFN-γ responses of CD4+ and CD8+ selected T cell populations to N-ETSD were significantly higher than those to ineffective ( Fig.49 , panels HM), and in two of three patients, N-ETSD was more strongly recognized by CD4+ and CD8+ T cells compared with N. High IFN-γ and low IL4 responses indicated a dominant Th1 cytokine response to N / N-ETSD. Cellular expression of NETSD and N was equivalent in 293T HEK cells, suggesting that the elevated T cell response to N-ETSD may be due to epitope processing and MHC loading.

[0268] Example 23: hAd5S-fusion + N-ETSD infection with autologous MoDC from a patient previously infected with SARS-CoV-2 Inducing Th1-dominant SARS-CoV-2-specific CD4+ and CD8+ memory T cell recall against nucleocapsid and spike antigens.

[0269] N-ETSD was more effective than N in eliciting cytokine responses in patient T cells. For total T cells (CD3+), IFN-γ responses were similar for S-fusion+N-ETSD and N-ETSD, with responses to S-fusion being relatively lower ( Fig.50 , panels AC). For all, the number of IL-4-secreting T cells was very low ( Fig.50 , panels DF). Based on the increased expression of S in the bivalent vaccine compared to the monovalent S-fusion, the increased T cell responses could be explained by T cell recognition of the presence of increased S or N. Importantly, these T cell responses were characterized by a predominance of IFN-γ (Th1) over IL-4 (Th2). CD4+ T cells from all three patients recognized all three constructs significantly more strongly compared to the null ( Fig.50 , sub-figure GI). Although some individuals responded more strongly to specific constructs, overall responses to S-fusion, S-fusion + N-ETSD, and N-ETSD were similar. CD8+ T cells from all three patients recognized the bivalent vaccine and N-ETSD vaccine at levels significantly higher than the ineffective vaccine; only in two of the three patients did CD8+ T cells recognize S-fusion at a significantly higher level than the ineffective vaccine ( Fig.50 , subfigures JL). These data demonstrate that T cells from previously infected SARS-CoV-2 patients are reactive and have immune memory recall against both vaccine antigens (S and N) in the vaccine vector.

[0270] A feature of S-fusions is the higher expression of the S RBD compared to S-WT. This was a goal in vaccine design based on early findings from cryo-electron micrographs of the structure of S that showed that the RBD epitope was largely unavailable for immune detection. Combining S with N in a bivalent vaccine would provide further advantage through the ability of N to enhance immune detection of S, a phenomenon that has been generally observed by others for gene expression. Vaccine-expressed N proteins traffic to the endosomal / lysosomal subcellular compartment, a key antigen presentation pathway for stimulation of CD4+ T cells, so that they can license dendritic cells to activate naive CD8+ CTLs. N-ETSD localizes to endosomes and lysosomes in MoDCs, as well as to autophagosomes. Both endosomal and lysosomal targeting are required for enhanced antigen presentation and CD4+ T cell activation. Lysosomes can fuse with acidic autophagosomes, facilitating protein processing; this has important implications for effective immune stimulation through modulation of MHC class II presentation. T cells from previously infected SARS-CoV-2 patients recognize N-ETSD more readily than N. The data presented here strongly support the potential for enhanced efficacy of vaccine constructs that specifically express modified N-ETSD.

[0271] T cells are essential for the elimination of SARS-CoV infection. 36,74-78 Here, hAd5 expressing S and N elicited strong antigen-specific IFN-γ but little IL-4 secretion from T cells of patients previously infected with SARS-CoV-2, suggesting a Th1 predominance. Antiviral Th1 cytokine responses eliminate a wide range of viruses in infected hosts79, including those closely related to SARS-CoV-2 and SARS-CoV. 80 These data are also consistent with studies in preclinical models. Importantly, the data suggest that both S and N are targets of CD4+ T cells that contribute to B cell antibody production and CD8+ T cell memory, which work together to kill virally infected targets. Recognition of these vaccine antigens by T cell subsets is consistent with immune control of the pathogen. Interestingly, the hAd5 S-fusion+N-ETSD T cell-biased vaccine has the potential to not only provide protection to uninfected patients, but also be used as a therapeutic in infected patients to induce rapid clearance of the virus by activating T cells to kill virus-infected cells, thereby reducing viral replication and lateral spread. Importantly, T cell recall in N-ETSD was shown to be Th1-dominant, as indicated by a strong interferon-g response and a low IL-4 response.

[0272] Methods and constructs used above

[0273] Example 25: hAd5[E1-, E2b-, E3-] Platform and Constructs

[0274] For the studies presented here, the second generation hAd5[E1-, E2b-, E3-] vector ( Fig.44 , sub-figure A) to create candidate viral vaccine constructs. The hAd5[E1-, E2b-, E3-] backbone containing SARSCoV-2 antigen expression inserts and viral particles was generated as described previously. Briefly, high titer adenovirus stocks were generated by serial propagation in an E.C7 packaging cell line expressing E1 and E2b, followed by CsCl2 purification and dialysis into storage buffer (2.5% glycerol, 20mM Tris pH 8, 25mM NaCl) (performed by ViraQuest Inc. (North Liberty, Iowa)). Viral particle counts were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280nm. Adeno-X TM The virus titer was determined by a rapid titer kit (Takara Biotech Co., Ltd.). The constructed constructs included: iS-WT: S protein contains 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234) and cytoplasmic (1235-1273) (Unitprot P0DTC2); ii. S fusion: S is optimized to enhance the surface expression and display of RBD; iii. N: nucleocapsid (N) wild-type sequence protein with a tag for immunodetection; iv. N-ETSD: N with an enhanced T cell stimulatory domain (ETSD) and a tag for immunodetection; and v. bivalent S-fusion + N-ETSD;

[0275] Plasma and PBMCs were collected from patients with confirmed prior SARS-CoV-2 infection and from volunteers without the virus: Blood was collected via venipuncture with informed consent from volunteers who had not been exposed to (UNEX) SARS-CoV-2 as confirmed by ELISA and multiple negative SARSCoV-2 tests or had recovered from COVID-19 as indicated by recent medical history and positive SARS-CoV-2 antibody tests (patients, Pt). A third source of whole blood was apheresis from healthy subjects from a commercial source (HemaCare). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation and plasma was collected after density gradient centrifugation.

[0276] As previously described, monocyte-derived dendritic cells (MoDC) were differentiated from PBMC using GM-CSF (200 U / ml) and IL-4 (100 U / ml) 86. In short, monocytes were enriched by adhering to plastic, while non-adherent cells were preserved and frozen as a source of lymphocytes, particularly T cells. Adherent cells were differentiated into dendritic cells (3-5 days in RPMI containing 10% FBS) and then frozen in liquid nitrogen for later use. T cells were enriched from the non-adherent fraction of PBMC using MojoSort (BioLegend CD3 enrichment). CD4+ and CD8+ T cells were enriched using a similar kit from the same manufacturer. The efficiency of cell separation was assessed by flow cytometry.

[0277] MoDCs were infected with hAd5 N-WT or N-ETSD and labeled with anti-N, anti-CD71, anti-LAMP-1, and anti-LC3a / b antibodies: Freshly thawed MoDCs were plated on 4-well Lab-Tek II CC2 chamber slides at 3 × 10 cells / well. 4cells and transduced at an MOI of 5000 one hour after plating with hAd5 N-ETSD or hAd5 N. Slides were incubated overnight at 37°C, fixed in 4% paraformaldehyde for 15 minutes, and then permeabilized with 1% Triton X100 in PBS for 15 minutes at room temperature. To label N, cells were then incubated with anti-flag monoclonal (anti-Flag M2 raised in mouse) antibody at 1:1000 in phosphate buffered saline (PBS) with 3% BSA, 0.5% Triton X100, and 0.01% saponin at 4C overnight, then washed three times in PBS, and incubated with goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus555 (Life Technologies) at 1:500 for 1 hour. For colocalization studies, cells were also incubated overnight at 4°C with: rabbit anti-CD71 (transferrin receptor, recycling / sorting endosome marker) antibody (Thermo Fisher) at 1:200; sheep anti-Lamp1 Alexa Fluor 488 conjugated (lysosomal marker) antibody (R&D Systems) at 1:10; or rabbit monoclonal anti-human LC3a / b (light chain 3, autophagy marker) antibody (Cell Signaling Tech #12741S) at 1:100. After removal of primary antibodies, washing twice in PBS and three times in PBS containing 3% BSA, cells were incubated with fluorophore-conjugated secondary antibodies at 1:500 (goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488; 1:500 dilution) for 1 hour at room temperature. After brief washing, cells were mounted with Vectashield antifade mounting medium with DAPI (Fisher Scientific) and immediately imaged using a Keyence integrated fluorescence microscope camera and Keyence software.

[0278] Binding of plasma antibodies from patients with previous SARS-CoV-2 infection to antigens expressed by vaccine-infected MoDC: Binding of plasma antibodies from previously infected subjects to antigens expressed on the surface of MoDC was determined by differentiation of MoDC to DC from peripheral blood mononuclear cells (PBMCs), infection of MoDC, incubation with plasma from previously infected patients, and detection of binding to infected and uninfected MoDC by flow cytometry.

[0279] MoDC (0.5x106 / well in 12-well plates) were infected with hAd5 S-WT, S fusion, S-fusion+N-ETSD or a "null" construct expressing green fluorescent protein (GFP) at an MOI of 5000. One day after infection, MoDC were detached using EDTA (0.5mM), gently pipetted and transferred to incubate with previously infected patient plasma from a single patient (Pt4) at a 1:100 dilution for 30 minutes at (4°C), and plasma antibodies were then detected on the surface of MoDC by goat anti-human IgG (phycoerythrin conjugated). Cells were obtained for flow cytometric analysis as described above. Data are plotted as DMFI, the difference in binding between infected and uninfected MoDC.

[0280] IFN-γ secretion by T cells from previously SARS-CoV-2 infected patients and virus-naive individuals and selected CD4+ and CD8+ T cells in response to MoDCs pulsed with a pool of SARS-CoV-2 peptide antigens

[0281] T cells from previously infected patients used in these studies were validated for their ability to recognize SARS-CoV-2 antigens in vitro, and similar analyses were then performed on selected CD4+ and CD8+ T cells. Briefly, MoDCs (2x104) were incubated with SARS-CoV-2 peptide antigens (1 μg / ml, PepMix S, containing S1 and S2 pools PM-WCPV-S-1; and N PM-WCPVNCAP-1, both from JPT Peptide Technologies), and autologous T cells (1x104) enriched from the non-adherent fraction of PBMCs using MojoSort (BioLegend CD3 Enrichment) were then incubated with 5 ) was added to enriched RPMI (10% human AB serum). Cells were cultured in microtiter plates (Millipore) containing fixed primary antibodies to target IFN-γ, overnight (37°C), and cells that formed IFN-γ spots were counted by ELISpot. For ELISpot detection, after aspiration and washing to remove cells and culture medium, IFN-γ was detected by a secondary antibody against cytokines bound to biotin. Biotin-conjugated secondary antibodies were detected using streptavidin / horseradish peroxidase conjugates. The ELISpot reader was used to calculate the number of cells per well (1x10 5 The number of spots (cells) was measured by ELISpot using a kit (MabTech) and following the manufacturer's instructions with wells pre-coated with anti-IL-4 antibodies. The remaining steps for IL-4 detection were the same as those for IFN-γ, but alkaline phosphatase detection was used instead of peroxidase.

[0282] Determine the reactivity of patient-derived T cells from previous SARS-CoV-2 infection in response to autologous hAd5 vaccine-infected MoDC: MoDC were infected with hAd5 S-fusion, S-fusion + N-ETSD, N-ETSD, N, or GFP / null constructs and incubated overnight at 37°C. Infected MoDC were cultured overnight with CD3+, CD4+, or CD8+ T cells from the same individual. Antigen-specific T cell responses were enumerated using ELISpot as described above.

[0283] AdV / yeast combinations and yeast lysate preparations

[0284] In addition to viral constructs expressing N-ETSD and / or S-fusions (or proteins similar to N-ETSD and / or S-fusions), N-ETSD and / or S-fusions (or proteins similar to N-ETSD and / or S-fusions) can also be expressed in yeast, either on yeast cells that present one or more antigens or in yeast cells that can be prepared as lysates to further enhance immunogenicity, as discussed in more detail below.

[0285] Fig.12 A conceptual diagram of an ideal vaccine is described, which will induce long-lasting and effective immunity through multiple pathways. It can be easily seen that the vaccine composition is preferably (but not necessarily) composed of a yeast vaccine composition and a viral vaccine composition, optionally in combination with a macrophage polarizing agent such as RP182 and an immunostimulatory cytokine or cytokine analog (e.g., N-803).

[0286] With respect to the yeast component, it is most typically preferred that the yeast is a recombinant yeast that expresses one or more antigens of interest from a recombinant nucleic acid, and possibly other DAMP or PAMP or STING pathway signals. The recombinant yeast so produced is typically heat-inactivated, and the yeast is lysed after heat inactivation. Most preferably, pressure homogenization is used to lyse the yeast, and the homogenate is then preferably clarified via filtration. However, it should be understood that other lysis methods are also considered suitable, including enzymatic or chemical lysis of the cell wall, sonication, rapid freezing / thawing, and the like. Similarly, clarification of the lysate may also include centrifugation, sedimentation, flocculation, and the like.

[0287] As will be readily appreciated, recombinant antigens and other recombinant proteins may be expressed from any suitable promoter using known expression cassettes. For example, Fig.13 Described are the results of an exemplary expression experiment in which the SARS CoV nucleocapsid protein (N) was overexpressed in Saccharomyces cerevisiae. As can be seen, a large amount of recombinant protein was expressed in yeast.

[0288] Using mouse models, lysed tarmogens (yeast with recombinantly expressed antigens) are up to 150 times more immunogenic than whole tarmogens in inducing T cell responses (based on a large number of antigen-specific T cell activation studies performed on hundreds of mice). Advantageously, lysed yeast has a good safety profile and 10,000 10YU doses can be prepared in 2 hours in a small pressure homogenizer. Whole yeast is Th1-Th17 inducible and triggers IFNγ, IL2, IL12, IL6, TNFα, GM-CSF, while yeast lysate clearly induces a strong Th1 effect and reduces Th17. Thus, whole or lysed yeast will induce coronavirus-specific antibodies (see, for example, doi.org / 10.1016 / j.jaut.2005.01.008, doi.org / 10.1155 / 2016 / 4131324, or sfamjournals.onlinelibrary.wiley.com / doi / pdf / 10.1111 / lam.12188).

[0289] Formulations for oral / mucosal administration

[0290] In further embodiments, disclosed herein are compositions and methods for producing vaccine compositions using aragonite to form solid dosage forms (e.g., powders, tablets, or capsules) that are stable during storage, easy to administer (e.g., orally), and dissolve (e.g., releasing one or more antigenic or pre-antigenic vaccine molecules) after passing through the stomach of a subject receiving the vaccine.

[0291] Specifically, the present disclosure relates to an aragonite composition made of a plurality of aragonite particles loaded with one or more vaccine active ingredients, thereby obtaining a solid dosage vaccine in the form of a powder, tablet or capsule. More specifically, the vaccine composition can include a recombinant expression construct in powder form (e.g., lyophilized) for expressing the corresponding antigen for the relevant infection / disease, which is mixed with a plurality of aragonite particles and thus loaded on the surface of a plurality of aragonite particles. In an exemplary embodiment, the vaccine composition is immunized against coronavirus. Preferably, the recombinant expression construct is an adenovirus construct expressing at least one antigenic coronavirus protein or protein fragment.

[0292] It is noteworthy that the use of aragonite in the solid dosage forms currently considered allows for cost-effective manufacture and easy administration of stable vaccine compositions. Therefore, the vaccine tablets or capsules currently considered can be mass-produced and easily transported. In addition, solid dosage forms allow oral administration, which can be self-administered without the need for healthcare professionals for most people. Tablet forms can also be formed into lozenges with additional excipients and / or additives (e.g., flavoring agents and gelatin).

[0293] Aragonite (e.g., oolitic aragonite) is one of the purest forms of naturally precipitated calcium carbonate. Figure 1 Aragonite has a crystal morphology of rhombic, bipyramidal, characteristic needle-shaped crystals, and is therefore different from calcite and vaterite. Aragonite can be processed to recrystallize and / or reform into various shapes, so that it can be used for various purposes utilizing the mechanical and chemical properties of calcium carbonate minerals. Aragonite particles as disclosed herein are solid materials with regular (e.g., spherical or oval) or irregular shapes. As used herein, aragonite particles have an average particle size of 100nm to 1mm. US 2020 / 0308015 (the entire contents of which are incorporated herein by reference) describes a method for grinding aragonite particles. For example, a method for grinding aragonite particles with a size of 2.0 to 3.5 microns under a regular maximum size (clean top size) is disclosed. The regular maximum size means that when produced using the disclosed grinding method (the classifier is set at a size range of 2.0 to 3.5 microns or 2.5 to 3.5 microns), there are few particles larger than 3.5 microns in size. Thus, the aragonite particles using the method of US2020 / 0308015 as disclosed herein have a more regular maximum dimension than conventional GCC.

[0294] The adsorption capacity of aragonite is a function of three parameters: (1) surface charge (also known as “zeta potential”); (2) surface area / voidage; and (3) particle solubility. By accurately measuring these three parameters, it is possible to determine which materials will adsorb to the surface of aragonite particles under given conditions. Notably, the zeta potential of aragonite increases the stability of surfactants such as glycerol and sorbitol.

[0295] In addition, aragonite has a large number of measurable pores in the particles with a diameter of less than 2 nm (i.e., has a high "microporosity"). See, for example, EP 2719373. Thus, the aragonite platform firmly clamps the active ingredient particles together, allowing the loaded aragonite to be formulated into a solid dosage form - such as a powder, tablet or capsule.

[0296] Advantageously, untreated aragonite has a neutral pH (7.8 to 8.2), natural hydrophilicity, electronic charge (zeta potential), and has produced nitrogenous pairings with amino acids and proteins. Without being bound by any one theory, these favorable properties of aragonite make aragonite metastable under ambient conditions. More specifically, aragonite particles naturally contain about 2%-3% amino acid content, most of which are aspartic acid (about 25% to 30%) and glutamic acid (about 8% to 10%), making the aragonite surface hydrophilic. See, for example, Mitterer, 1972, Geochimic et Cosmochimica Acta [Geochimica et Cosmochimica Acta], 36: 1407-1422. Therefore, in some embodiments, a vaccine composition (e.g., a recombinant adenovirus) is directly coupled to the natural, untreated surface of the aragonite particles.

[0297] At present, calcium carbonate used in the market is processed as or from ground calcium carbonate (GCC), precipitated calcium carbonate (PCC) (synthetic) and / or limestone. The products produced are commercial grades with different properties. In order to obtain a regular particle size distribution (PSD) maximum particle size and low retention, most companies use a wet grinding process (whether high solid or low solid). As used herein, aragonite refers to naturally occurring aragonite, which has a crystalline morphology of orthorhombic, bipyramidal and characteristic needle-shaped crystals different from GCC, PCC and limestone. For example, ball-milled aragonite using the system and method disclosed in US2020 / 0308015 can produce aragonite particles with a size of 2.0 to 3.5 microns under a regular maximum size. The regular maximum size means that when produced using the system and method (the classifier is set in a size range of 2 to 3.5 microns or a size range of 2.0 to 3.5 microns), there are few particles larger than 3.5 microns in size. For example, for aragonite produced using the disclosed system within this setting range, only <0.0005% is retained on 325 mesh and only slightly more than <0.0007% is retained on 500 mesh, as compared to a GCC product having the same median (D50) particle size distribution (PSD). Thus, the aragonite produced using the contemplated system and method has a more regular maximum size than conventional GCC.

[0298] Advantageously, the solid dosage form made of aragonite provides a solid vaccine form that can be taken in by oral administration. The solid form with enteric coating disclosed by the present invention is taken in and the antigen molecules loaded in the kernel are not released until after passing through the stomach, thereby allowing antigenicity or pre-antigenic molecules to be absorbed into the bloodstream and delivered to immune cells. As used herein, antigen molecules refer to one or more required vaccine active ingredients mixed with aragonite particles and loaded on the aragonite particle surface. These antigen molecules can have antigenicity in the form loaded on the aragonite particles, or they can be one or more molecules (such as expression vectors) that can produce (such as express) at least one antigenic protein or fragment. Like this, active ingredient or vaccine active ingredient disclosed herein are referred to as antigenic molecules, which include antigenic molecules and pre-antigenic molecules, unless otherwise indicated.

[0299] In an exemplary embodiment, the aragonite vaccine composition comprises: i) an inner core made of aragonite and a specific antigenic molecule; ii) an outer core of aragonite completely surrounding the inner core, so that the entire outer surface of the inner core is in contact with the outer core only, and the surface of the inner core is not exposed; and iii) a coating covering all outer surfaces of the outer core. Preferably, the inner core is made of aragonite particles having a diameter of at least 2um or greater, thereby providing a surface area for capturing and loading specific antigenic molecules thereon. Preferably, the outer core does not include any antigenic molecules and is made of 90% to 100% aragonite. More preferably, the outer core contains only 100% aragonite. The outer coating of the solid composition can be any suitable coating that is stable (e.g., enteric coating) in the highly acidic, low pH environment of the stomach (e.g., at pH about 3) and dissolves in the higher pH of the small intestine (e.g., at pH about 7 to 9). Suitable examples of enteric coatings include biopolymer dispersions, such as methacrylic acid, ethyl acrylate, and / or plasticizers / stabilizers (e.g., triethyl citrate (TEC)). Additionally, anti-adherent agents may also be used in combination with the enteric coating - for example, glyceryl monostearate.

[0300] In a specific embodiment, the inner core is made of aragonite particles having a diameter of at least 2 um or greater (e.g., 2 to 3.5 um) mixed with a lyophilized powder of the antigenic molecule. In some embodiments, additional excipients are mixed with the aragonite and the antigenic molecule. For example, dimethylglycine and / or methylsulfonylmethane (MSM) can be combined with the lyophilized powder of the antigenic molecule and mixed with the aragonite particles.

[0301] In a further exemplary embodiment, the lyophilized antigen molecule comprises a lyophilized recombinant expression vector having a nucleic acid corresponding to (i.e., encoding) at least one antigenic protein or optimized protein or fragment thereof of SARS-coronavirus 2 (SARS-CoV-2 or CoV2). For example, the contemplated solid dosage vaccine composition disclosed herein may encode an antigen of at least one of the nucleoprotein (N) protein and the spike (S) protein (both of which are conserved in all types of coronaviruses) of the coronavirus 2 virus (CoV2). In one embodiment, the antigen encoding molecule is a lyophilized recombinant entity, wherein the recombinant entity comprises a nucleic acid encoding a nucleocapsid protein or a fragment thereof of CoV2, and / or wherein the recombinant entity encodes a spike protein or a fragment thereof of CoV2. The vaccine formulation can be used to treat a disease, such as a coronavirus-mediated disease or infection. Therefore, in another embodiment, a method of treating a coronavirus disease in a patient in need is disclosed, wherein the method comprises: administering to the subject a solid dosage vaccine composition comprising a recombinant entity comprising a nucleic acid encoding at least a CoV2 N protein or a fragment thereof, preferably a CoV2 N protein or a fragment thereof and a CoV2 S protein or a fragment thereof. The coronavirus considered herein may be coronavirus disease 2019 (COVID-19) and / or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0302] In a further exemplary embodiment, the contemplated solid dosage vaccine compositions disclosed herein comprise aragonite mixed with a lyophilized recombinant entity made from a bivalent human adenovirus serotype 5 (hAd5) expression vector. hAd5 is capable of inducing immunity in patients with pre-existing adenovirus immunity and expressing antigens to produce antibodies against the coronavirus 2 spike (S) protein and / or nucleocapsid (N) protein. For example, hAd5 CoV2 can encode a modified nucleocapsid protein having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1. In more specific embodiments, hAd5 includes an S sequence optimized for cell surface expression (S-fusion) and a conserved nucleocapsid (N) antigen designed for transport to endosomal subcellular compartments, with the potential to generate durable immune protection against CoV2, as disclosed in U.S. Application No. 16 / 883,263, the entire contents of which are incorporated herein by reference. For example, hAd5 CoV2 can encode an S-fusion or S-HA protein having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4 or SEQ ID NO: 3, respectively.

[0303] Advantageously, the bivalent hAD5 vaccine provides: (i) the optimized S-fusion has improved cell surface expression of the S receptor binding domain (RBD) compared to S-WT (wild type) where little surface expression was detected; (ii) the RBD expressed from the S-fusion retains conformational integrity and is recognized by ACE2-Fc; (iii) the viral N protein modified with an enhanced T cell stimulatory domain (ETSD) is localized to the endosomal / lysosomal subcellular compartments for MHC I / II presentation; and (iv) these optimizations of S and N (S-fusion and N-ETSD) generate enhanced de novo antigen-specific B cell and CD4+ and CD8+ T cell responses in antigen-naive preclinical models, as shown in more detail below.

[0304] In a preferred embodiment, the lyophilized bivalent hAd5 vaccine comprises a replication-deficient adenovirus having a deletion of the E1, E2b, and E3 gene regions and a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid (N) protein fused to an endosomal targeting sequence (N-ETSD), and a nucleic acid encoding a CoV2 spike (S) protein sequence optimized for cell surface expression (S-fusion). Typically, the nucleic acid encoding the CoV2 N-ETSD protein in the hAd5 adenovirus is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2. More typically, the CoV2 N-ETSD protein encoded in the hAd5 adenovirus is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. Typically, the nucleic acid encoding the CoV2 S-HA or S-fusion protein in the hAd5 adenovirus is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 6, respectively. More typically, the CoV2 S-HA or S-fusion protein encoded in the hAd5 adenovirus is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3 or SEQ ID NO:4.

[0305] In other embodiments, the lyophilized bivalent hAd5 vaccine may further include nucleic acids encoding transport sequences, co-stimulatory molecules and / or immunostimulatory cytokines. Examples of encoded co-stimulatory molecules include one or more of CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3. Examples of immunostimulatory cytokines include one or more of IL-2, IL-12, IL-15, IL-15 superagonist (N803), IL-21, IPS1, and LMP1.

[0306] From a different perspective, contemplated herein is a solid dosage form of a coronavirus vaccine composition, which is made by mixing aragonite particles with a lyophilized form of a viral vector (e.g., a lyophilized powder composition comprising a recombinant adenovirus genome, optionally wherein the E2b gene is deleted or non-functional), the viral vector comprising a nucleic acid encoding (a) at least a wild-type or modified nucleocapsid protein; and / or (b) at least one wild-type or modified spike protein. The viral vector may also include a co-stimulatory molecule. Typically, the nucleic acid encodes a transport signal to direct the peptide product encoded by the nucleic acid to the cytoplasm, endosomal compartment, or lysosomal compartment, and the peptide product will further include a sequence portion that enhances the intracellular turnover of the peptide product.

[0307] As discussed, the manufacture and preparation of solid dose vaccine compositions are possible because aragonite allows direct coupling of freeze-dried vaccine active ingredients. Therefore, the expected method for preparing the disclosed solid dosage vaccine includes forming the kernel of aragonite and vaccine active ingredients by loading (for example, by mixing) the vaccine active ingredients with ground aragonite particles, as disclosed herein and in US2020 / 0308015, the entire contents of which are incorporated herein by reference. Any suitable method for mixing freeze-dried vaccine active ingredients with aragonite particles can be used. The aragonite particles (for example, with a D50 PSD of 2um to 3.5um) ground with freeze-dried vaccine active ingredients can be loaded or the two can be mixed by any conventional method. The mixing of aragonite with active ingredients includes mixing the aragonite of dry weight and freeze-dried active ingredients into a closed container suitable for rotation / inversion. For example, the inversion or rotation of aragonite and active ingredients can be carried out for about 5 minutes to 30 minutes. For example, the vaccine composition can be loaded onto a solid dosage form in a mixer (e.g., a tumble mixer) or blender. The amount of aragonite can vary depending on the amount of lyophilized active ingredient and the determined titer of the active ingredient used to induce an immune response. For example, an effective dose of a lyophilized bivalent hAd5 vaccine disclosed herein can be about 1×10 9IU / capsule (or tablet). Therefore, the titer is 2.21x10 7 IU / mg of lyophilized bivalent hAd5 vaccine composition requires about 45.25 mg of lyophilized bivalent hAd5 powder / capsule. In some embodiments, the weight of a solid single capsule can be between about 300 mg and 600 mg. In a specific embodiment, for a 550 mg capsule, about 40 to 60 mg of lyophilized active ingredient (e.g., lyophilized bivalent hAD5 expressing both N and S CoV2 proteins) is mixed with 490 to 510 mg of aragonite. For a 550 mg capsule with 2.21 x 10 7 IU / mg titer of active ingredient, 45.25 mg of lyophilized active ingredient can be mixed with about 504.75 mg of aragonite. Tablet or caplet formation of the mixture can be performed using any suitable encapsulation method and / or kit known in the art. For example, capsule machine filler (Cat. No. CPM1001 / 2081677).

[0308] In a preferred embodiment, the expected method for preparing solid dose vaccine tablets or capsules also includes preparing an aragonite outer core that surrounds (e.g., completely surrounds) the inner core. Typically, the outer core is mainly made of (e.g., at least 90%) aragonite, and more typically, the outer core is made of at least 99% aragonite.

[0309] In order to deliver the antigenic molecules to the bloodstream in the form of tablets or capsules for oral administration, the antigenic molecules must be retained in the inner core surrounded by the outer core until they pass through the stomach and are absorbed by the intestine (e.g., small intestine). Therefore, the outer core is coated with an enteric coating that is stable in the low pH of the stomach (e.g., at pH about 3) and dissolved in the higher pH of the intestine (e.g., at pH about 7 to 9). The outer coating of the solid composition can be any suitable enteric coating. Examples of enteric coatings include methacrylic acid and / or ethyl acrylate polymers in triethyl citrate (TEC). The method of applying enteric coatings is well known in the art. For example, a coating device or apparatus can be used. The specific example of a coating device includes ProCoater (manufactured by Torpac).

[0310] Coated (C) and non-coated (NC) capsules made of aragonite particles or lactose mixed with hAD5-COVID-S / N were exposed to acid (HCl) to determine the acid permeability of various capsules. Figure 2Three photographs (from right to left: 1, 2, 3) of a bivalent human adenovirus serotype 5 COVID-Spike and nucleocapsid antigen vaccine (hAD5-COVID-S / N) in an uncoated aragonite capsule (sample #6) in 0.1 M hydrochloric acid (HCL) are shown, wherein wrinkles, swelling, or holes in the capsules were observed as shown: 1): 2 minutes after HCL acid exposure; 2): 2 hours after HCL acid exposure; and 3) 2 hours after HCL acid exposure and drying. Figure 3 Three photographs (from right to left: 1, 2, 3) showing hAD5-COVID-S / N in uncoated aragonite capsules (sample #7 or #8) in 0.1M HCL, where swelling, distortion, or holes in the capsules were observed as shown: 1): Sample #7 2 hours after HCL acid exposure; 2): Sample #8 2 hours after HCL acid exposure; 3) 2 hours after HCL acid exposure and drying. Figure 4 Two photographs (from right to left: 1, 2) showing hAD5-COVID-S / N in uncoated lactose capsules (sample #3 or #4) in 0.1M HCL, where the swelling of the capsules was observed as shown: 1): Sample #3 2 hours after HCL acid exposure; 2): Sample #4 2 hours after HCL acid exposure. Figure 5 Two photos (from right to left: 1, 2) showing hAD5-COVID-S / N in coated aragonite capsules (sample #1 or #5) in 0.1M HCL, where the swelling of the capsules is observed as shown: 1): Sample #1 2 hours after HCL acid exposure; 2): Sample #5 2 hours after HCL acid exposure. Clearly, the coated capsules are stable in acid.

[0311] like Figure 6-11 As shown, the sample capsules were further analyzed for infectious units per gram (IFU / gram) and virus recovery percentage. More specifically, Figure 6 The infectious units per gram (IFU / gram) (y-axis) for the indicated hAD5-COVID-S / N capsule types are shown, as indicated. Figure 7 The percent (%) virus recovery for each hAD5-COVID-S / N capsule type is shown (y-axis), as indicated. Figure 8 The IFU / gram and corresponding pH for each hAD5-COVID-S / N capsule type are shown, as indicated. Fig. 9 The percent (%) of virus recovery for each hAD5-COVID-S / N capsule type is shown, as indicated. Fig.10 The percent virus recovery for each hAD5-COVID-S / N capsule type is shown, as indicated, with acid treatment indicated for those with shading. Fig.11The infectious units / gram for each hAD5-COVID-S / N capsule type are shown, as indicated, with acid treatment indicated for those with shading. The results are also summarized in Table 2 below.

[0312]

[0313] In alternative embodiments, the dosage form contemplated includes an aragonite core impregnated (i.e., coupled) with carbon dioxide (CO2) prior to the addition of the vaccine composition. See, for example, EP 2719373 and US 2020 / 0155458. In further embodiments, the dosage form contemplated includes aragonite with a biocompatible polymer and / or a disintegrant mixed and processed with the aragonite prior to the addition of the vaccine composition. Typically, the aragonite is impregnated with CO2 and mixed with a biocompatible polymer and a disintegrant prior to the addition of the vaccine composition. More typically, the aragonite is impregnated with CO2, mixed with a biocompatible polymer and a disintegrant, and formed (e.g., compressed) into a solid form prior to the addition of the vaccine composition. See, for example, EP 2719373 and US 2020 / 0155458.

[0314] In other embodiments, as described herein, aragonite can be coupled with carbon dioxide (CO2) and mixed with at least one biocompatible polymer. Typically, CO2 coupled aragonite is about 95:5 to 5:95 in weight ratio to biocompatible polymer. In other embodiments, biocompatible polymer is a hot-melt extruded biocompatible polymer. Exemplary biocompatible polymers include polylactic acid (PLA), polyethylene, polystyrene, polyvinyl chloride, polyamide 66 (nylon), polycaprolactam, polycaprolactone, acrylic polymer, acrylonitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide / sulfide, polypropylene, polytetrafluoroethylene, polylactic acid, aliphatic polyesters such as polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymer, poly-(3-hydroxybutyrate-to-3-hydroxyvalerate), polygluconate, poly-(dioxanone) and mixtures thereof. Preferably, biocompatible polymer resin is PLA.

[0315] Depending on the manufacturing and packaging requirements, additional excipients may also be added to the inner core and / or outer core of the solid dosage form. Other excipients may include ion exchange resins, gums, chitin, chitosan, clay, gellan gum, cross-linked polacrilin copolymers, agar, gelatin, dextrin, acrylic acid polymers, sodium / calcium carboxymethylcellulose, hydroxypropyl methylcellulose phthalate, shellac or mixtures thereof, lubricants, internal lubricants, external lubricants, impact modifiers, plasticizers, waxes, stabilizers, pigments, colorants, flavors, flavor masking agents, flavoring agents, sweeteners, mouthfeel improvers, adhesives, diluents, film formers, adhesives, buffers, adsorbents, odor masking agents and mixtures thereof.

[0316] In an alternative embodiment, the surface of aragonite particles can be treated to modify the binding surface. For example, a hydrophobic surface is provided by treating with stearic acid (i.e., octadecanoic acid), as disclosed in US16 / 858,548 and PCT / US 20 / 29949. For protein loading, aragonite is treated with phosphoric acid to form a layered structure. Other coupling techniques for coupling reactive groups to the amino acid surface of aragonite are known in the art, such as disclosed in Bioconjugate Techniques, the third edition, Greg T. Hermanson, Academic Press, 2013.

[0317] Oral / mucosal vaccines

[0318] At least in part based on the above-mentioned formulation, the disclosure also provides methods and compositions for administering, monitoring and measuring vaccines. The method under consideration includes inducing immunity against the virus in the patient, by administering the vaccine composition to the patient via delivery to the patient's nasal mucosa, oral mucosa and / or digestive tract mucosa to administer the vaccine composition to the patient. Preferably, the vaccine is for SARS-like coronavirus (SARS-CoV-2). The oral vaccine composition described herein can be used as a booster vaccination for any initial primary vaccination against SARS-CoV-2S or N protein.

[0319] The oral vaccine composition described herein can be used as a booster vaccine for any anti-SARS-CoV-2 vaccine against SARS-CoV-2 spike (S) and / or nucleocapsid (N) protein. This booster can even work in patients immunized with anti-S or anti-N vaccines other than those described herein. In a particular embodiment, the initial primary vaccine can be a lipid nanoparticle vaccine containing mRNA encoding the S protein, such as those vaccines currently being tested by Moderna and Pfizer. In certain embodiments, the booster described herein is administered at least 7 days, such as at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 28 days, at least 35 days or at least 42 days after the initial primary vaccination. The booster as described herein can effectively improve the production of antibodies against SARS-CoV-2 and cell-mediated immunity against SARS-CoV-2.

[0320] Preferably, the vaccine administered to induce immunity in the patient's mucosal tissue is a vaccine against SARS-CoV-2. In an exemplary embodiment, the vaccine is a replication-deficient adenovirus construct, including a deletion of the E1 gene region and a deletion of the E2b gene region. In certain embodiments, the adenovirus comprises a sequence encoding a SARS-CoV-2 spike fusion protein antigen (e.g., SEQ ID NO: 12), which has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) primary sequence identity with SEQ ID NO: 4. In certain embodiments, the adenovirus comprises a sequence encoding a modified spike protein antigen of SARS-CoV-2 (e.g., SEQ ID NO: 13), which has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) primary sequence identity with SEQ ID NO: 11. In certain embodiments, the adenovirus includes a sequence encoding a soluble ACE2 protein coupled to an immunoglobulin Fc portion to form an ACE2-Fc hybrid construct that may also include a J-chain portion, as disclosed in US16 / 880,804 and US63 / 016,048, both of which are incorporated herein by reference in their entirety. In other exemplary embodiments, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a mutant variant of a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10), wherein the mutant variant has at least one mutated amino acid residue (e.g., by substitution) that confers an increased binding affinity of the ACE2 protein to the RBD protein domain of the SARS-CoV-2 spike protein, as disclosed in US63 / 022,146 (the entire contents of which are incorporated herein by reference). In another exemplary embodiment, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a CoV2 nucleocapsid protein or a CoV2 spike protein (N-ETSD) fused to an endosomal targeting sequence, as disclosed in US16 / 883,263 and US63 / 009,960, both of which are incorporated herein by reference in their entirety. Additionally or alternatively, the SARS-CoV-2 vaccine includes a modified yeast cell (e.g., Saccharomyces cerevisiae) that is genetically engineered to express a coronavirus spike protein on the surface of the yeast cell to produce yeast presenting cells to stimulate B cells (e.g., humoral immunity), as disclosed in US63 / 010,010. The advantageous features of the compositions and methods described herein are further illustrated by the following examples (but not limited thereto).

[0321] Example 26: NHP vaccination

[0322] Two groups of rhesus monkeys (5 in each group) were immunized subcutaneously with an adenoviral anti-SARS-CoV-2 vaccine as described above on day 0. Blood was drawn from each rhesus monkey before immunization. On day 14, one group of rhesus monkeys (Group 1) received another subcutaneous booster injection of the same vaccine, while the other group (Group 2) received an oral vaccine as described herein (E1- / E2b-Ad5 with SEQ ID NO: 12 or SEQ ID NO: 13). On day 28, both groups received an oral vaccine booster dose. Two rhesus monkeys (control) were vaccinated with a sham vaccine at the specified time points. Blood was drawn on days 14, 21, 28, 35, and 42.

[0323] The anti-spike protein IgG and IgM serum reactivity of serum samples extracted from these rhesus monkeys at the specified time points was then evaluated by ELISA. In brief, 96-well EIA / RIA plates (Thermo Fisher Scientific, Cat#07-200-642) were coated with 50 μL / well of 1 μg / mL purified recombinant SARS-CoV-2 derived spike protein (S-fusion, Immunobiological Company) suspended in coating buffer (0.05M carbonate-bicarbonate, pH 9.6) and incubated overnight at 4 ° C. Each well was washed three times with 150 μL TPBS solution (PBS+0.05% Tween 20) to prepare a separate 96-well plate for each immunoglobulin type (IgG or IgM). Then a volume of 100 μL / well of blocking solution (2% skim milk in TPBS) was incubated at room temperature (RT) for 1 hour. Plasma and serum samples were heat-inactivated at 56 ° C for 1 hour before use. Prepare the serial dilutions of blood plasma, serum or antibody samples in 1% skim milk in TPBS. Wash the plate as described above and add 50 μL / well of each serial dilution to the plate and incubate at room temperature for 1 hour. Wash the plate three times with 200 μL TPBS. Each goat anti-human IgG (H+L) cross-adsorption, HRP, polyclonal dilution (1:6000); or the dilution of goat anti-human IgM (heavy chain) cross-adsorption secondary antibody, HRP (respectively Thermo Fisher Scientific, Cat#62-842-0 or A18841) is prepared in 1% skim milk / TPBS and these secondary antibodies of 50 μL / well are added in a separate reaction / plate by immunoglobulin type (IgG or IgM), and incubate at room temperature for 1 hour. Wash the plate three times with 200 μL TPBS. One component (3,3',5,5'-tetramethylbenzidine (TMB) substrate, 50 μL / well, VWR, Cat#100359-156) was added to each well and incubated at room temperature for 10 minutes, and then the reaction was stopped by adding 50 μL / well of 1N sulfuric acid (H2SO4). The optical density at 450 nm was measured using a Synergy 2 plate reader (BioTek Instruments, Inc). Data were analyzed using Prism 8 (GraphPad Software, LLC) and displayed on Fig.14 In, the results of ELISA for detecting IgG seroreactivity against SARS-CoV-2 spike in serum samples of immunized rhesus macaques are described.

[0324] Example 27: NHP challenge

[0325] On day 56, rhesus macaques were challenged with respiratory exposure to SARS-CoV-2 virus. Nasal swabs were collected from these macaques daily from day 56 to day 63. Bronchoalveolar lavage (BAL) fluid was collected on days 57, 59, 61, and 63. Fig.15 The ability of the sera to inhibit the infectivity of SARS-CoV-2 in the collected samples is shown. Panel A shows the ability of sera from Group 1 vaccinated rhesus monkeys to inhibit SARS-CoV-2 infectivity in vitro. Panel B shows the ability of sera from Group 2 vaccinated rhesus monkeys to inhibit SARS-CoV-2 infectivity in vitro. The dotted line represents 20% inhibition. It can be seen that sera from both Group 1 and Group 2 rhesus monkeys inhibited infectivity, and sera collected later inhibited more strongly than sera collected earlier. Sera from control rhesus monkeys had no inhibitory effect at any time point tested. Fig.16 The viral load in the nasopharynx over time is shown in Figure 2. Panel A shows the viral load (qPCR) in nasal swabs of group 1 rhesus monkeys after SC+SC+oral vaccination, and panel B shows the viral load (qPCR) in nasal swabs of group 1 rhesus monkeys after SC+oral+oral vaccination. Fig.17 Viral load in the lung (BAL) over time is shown. Fig.17 Panel A shows the viral load (qPCR) in the BAL of Group 1 rhesus monkeys after SC+SC+oral vaccination, and Panel B shows the viral load (qPCR) in the BAL of Group 1 rhesus monkeys after SC+oral+oral vaccination.

[0326] Example 28: Serological Reactivity

[0327] Serum samples from various human volunteers who had received various experimental anti-SARS-CoV-2 vaccines were collected and analyzed for IgG and IgM serum reactivity against the SARS-CoV-2 S protein by ELISA as described above. The results are shown in Fig.18 In the study, ELISA results detected IgG and IgM serum reactivity against SARS-CoV-2 spike in serum samples drawn from human patients immunized with various experimental anti-SARS-CoV-2 vaccines.

[0328] Example 29: Human Immunization

[0329] Human volunteers were divided into three cohorts. 5 × 10 10 Cohort 1 (10 individuals) was immunized with 10 viral particles (comprising E1- / E2b- Ad5 of SEQ ID NO: 12 or SEQ ID NO: 13). 11Cohort 2 (10 individuals) was immunized with 10 viral particles of the vaccine described herein by subcutaneous injection. 11 viral particles (or 5 × 10 if safety concerns indicate a lower dose 10 Cohort 3 (15 individuals) was immunized with 100% sera (100% sera) of 100% sera (100% sera). Blood was drawn from each volunteer on the same day as the initial priming vaccine. Blood was drawn again on days 8, 15, and 22. A booster injection of the same vaccine was administered on day 22.

[0330] Blood collected on days 1 and 15 was subjected to ELISpot testing to assess cell-mediated immunity against SARS-CoV-2. 400,000 live PBMCs (Cellometer K2 with AO / PI viability staining) from each blood draw were stimulated per well with empty medium, SARS-CoV-2 S, SARS-CoV-2 N, SARS-CoV-2 M, CD3 / CD28 / CD2, and CEFT. After 48 hours of stimulation, the supernatant was frozen (-80°C) for later testing. Fig.19 ELISpot test results are shown for Th1 N-reactive patients 3, 6 and 11. Figure 20 shows results from patient 4 (N-unreactive) and patient 10 (weak Th1 N-reactive). None of these patients showed a Th2 response to N.

[0331] Airway protection

[0332] In further experiments, the contemplated vaccine formulations and methods of use provided protection against SARS-CoV-2 challenge to the nasal and pulmonary airways in non-human primates. As shown in more detail below, the dual-antigen COVID-19 vaccine, which combines genes for a modified SARS-CoV-2 spike (S-fusion) protein and a viral nucleocapsid (N) protein with an enhanced T cell stimulatory domain (N-ETSD), increased MHC class I / II responses. The adenovirus serotype 5 platform used, hAd5[E1-, E2b-, E3-] (previously shown to be effective in the presence of Ad immunity) can be delivered via an oral formulation that overcomes cold chain limitations. The hAd5 S-fusion + N-ETSD vaccine was evaluated in rhesus macaques, demonstrating that subcutaneous priming followed by oral boosting elicited humoral and Th1-dominant T cell responses to both S and N, protecting the upper and lower respiratory tracts from high titers (1x10 6 Notably, viral replication was suppressed in both the lungs and nasal passages within 24 hours of challenge and became undetectable within 7 days after challenge. Fig.25hAd5 platform and hAd5 S-fusion + N-ETSD constructs are depicted. (A) A human adenovirus serotype 5 vaccine platform lacking the E1, E2b, and E3 regions (*) is shown. The vaccine construct is inserted into the E1 region (red arrow). (B) The dual antigen vaccine comprises S-fusion and N-ETSD delivered by the hAd5 [E1-, E2b-, E3-] platform, which is under the control of the cytomegalovirus (CMV) promoter and has a C-terminal SV40 polyadenylation sequence.

[0333] Fig.25 The dual-antigen hAd5 S-fusion + N-ETSD vaccine expresses the viral spike (S) protein fused to a signal sequence (S-fusion), which, as predicted based on reports of similar sequences, enhances cell surface expression of the spike receptor binding domain (SRBD) in in vitro studies compared to S wild-type (which is the antigen used in most other vaccines under development). The vaccine also expresses the viral nucleocapsid (N) protein with an enhanced T-cell stimulatory domain (N-ETSD), which directs N to the endo / lysosomal subcellular compartments, which is expected to enhance MHC class II responses.

[0334] SARS-CoV-2 vaccine antigens are composed of recombinant human adenovirus serotype 5 (hAd5) [E1-, E2b-, E3-] vector platform ( Fig.25 ) delivery, the platform can rapidly generate vaccines against multiple agents, allowing large numbers of doses to be produced in the shortest possible time frame. The hAd5 platform has unique deletions in the early 1 (E1), early 2 (E2b), and early 3 (E3) regions (hAd5 [E1-, E2b-, E3-]), which distinguishes it from other adenoviral vaccine platform technologies in development and makes it effective in the presence of pre-existing adenoviral immunity. The platform produces vaccines against viral antigens such as influenza, HIV-1, and Lassa fever and has been shown to induce both antibodies and cell-mediated immunity. In 2009, vaccination of mice with hAd5[E1-, E2b-, E3-] vectors expressing the H1N1 hemagglutinin and neuraminidase genes elicited cell-mediated immunity and humoral responses that protected the animals from lethal viral challenge.

[0335] The vast majority of other SARS-CoV-2 vaccines in development target only the wild-type S antigen and are expected to elicit SARS-CoV-2 neutralizing antibody responses. Vaccine development has prioritized activation of T cells to enhance the respiration and duration of protective immune responses; the addition of N, in particular, is expected to provide a greater opportunity for T cell responses. T cells may provide immune protection at least as important as the production of antibodies. In a study of patients recovering from SARS-CoV-2, virus-specific T cells were seen in the majority of patients, including asymptomatic individuals and even those in whom no antibody responses could be detected.

[0336] In preliminary studies of the hAd5 S-fusion + N-ETSD vaccine in a mouse model, the vaccine not only elicited a T helper 1 (Th1) dominant antibody response to both S and N, but also activated T cells. MoDCs from individuals recovering from SARS CoV-2 were transduced with the dual antigen vaccine, and MoDCs expressing S-fusion and N ETSD were incubated with T cells from these same individuals. Vaccine antigens induced CD4+ and CD8+ T cells to secrete interferon-g (IFN-γ). This suggests that T cells from individuals recovering from SARS-CoV-2 "recall" the S-fusion and N-ETSD antigens presented by the transduced MoDCs as if they were re-exposed to the virus itself. This T cell recall of vaccine antigens suggests that, conversely, hAd5 S-fusion + N-ETSD vaccination will generate T cells that will recognize SARS-CoV-2 antigens after viral infection and protect vaccinated individuals from disease.

[0337] The generation of T cell responses may be a key feature of vaccines to be effective against many variants, which may have emerged, at least in part, as an escape response to antibodies generated by the first wave of the virus (28) or by antibody-based vaccines. As reported elsewhere, neutralization by 14 of the 17 most effective mRNA vaccines-elicited monoclonal antibodies (mAbs) reduced or eliminated variants E484K, N501Y, or the K417N:E484K:N501Y combination. They also found that these variants were selected when recombinant vesicular stomatitis virus (rVSV) / SARS CoV-2S was cultured in the presence of these mAbs, highly suggesting that the presence of these antibodies acts as an evolutionary force driving the emergence of new variants. T cells are not susceptible to such forces and, if effectively established through vaccination, could provide protection against both existing virus strains and escape mutants.

[0338] In the next step in the development of the hAd5 S-fusion + N-ETSD vaccine, both GMP (good manufacturing practice) grade liquid and oral forms of the vaccine were tested in non-human primates (NHPs). The key objective of the NHP study design was to evaluate the efficacy of a heat-stable oral boost following a subcutaneous (SC) prime. Oral boosting offers several advantages in SARS-CoV-2 vaccination, including a greater potential to generate mucosal immunity, particularly in the gastrointestinal tract, one of the major sites of infection. SARS-CoV-2 is a mucosal virus that is rarely detected in the blood, so vaccines that specifically target mucosal immunity are of interest. Another compelling advantage of a heat-stable oral booster is that it could transform global distribution of vaccines, particularly in developing countries, and potentially enable patients to self-administer one or more boosters at home. Because the hAd5 S-fusion + N-ETSD construct induces both humoral and CMI responses to both antigens, it also has the potential to serve as a “universal” heterologous booster vaccine for multiple SARS-CoV-2 vaccines under development.

[0339] A study determined the efficacy of the hAd5 S-fusion + N-ETSD vaccine in rhesus macaques as SC prime and SC with oral boost (SC-SC-oral; n=5) or as SC prime and two oral boosts (SC-oral-oral; n=5) (both using a regimen of priming on day 0 and boosting on days 14 and 28 to maximize T cell responses). The purpose of this study was to evaluate the immunogenicity of the dual-antigen hAd5 vaccine in SC and oral formulations, and the potential of the oral dose as a single SC prime followed by a boost. Cell-mediated T cell responses and protection of the nasal passages and lungs from SARS-CoV-2 infection after challenge were assessed, as well as viral clearance.

[0340] Clinical signs, hematology, and clinical chemistry: No clinical signs were found during the twice-daily observations for clinical signs of toxicity due to vaccination, and no clinical signs were observed during the once-s.c. 11 Two weeks after 1×10 vaccine particles (VP) or 10 No animals died one week after an oral boost of 1 IU of hAd5-S-fusion+N ETSD. In addition, no gross pathological effects or adverse events were observed, and there were no significant changes in body weight. Finally, hematology and clinical chemistry showed no abnormalities due to vaccination.

[0341] SC priming together with oral boosting elicits production of neutralizing anti-Spike antibodies: Fig.21As shown, all SC-oral-oral vaccinated NHPs produced anti-S IgG, which increased after oral boosts on days 14 and 28 (panels A and B). Sera from 4 of the 5 SC-oral-oral NHPs (collected at baseline and weekly from day 14 to day 42) demonstrated inhibition in a neutralization assay (panel C) assessing inhibition of S RBD binding to recombinant angiotensin converting enzyme 2 (ACE2), and reportedly correlated with the ability of serum to neutralize SARS-CoV-2 virus. For SC-SC-oral (where the first boost was SC) hAd5 S fusion + N-ETSD vaccinated NHPs (panels D and E), anti-S IgG production was similar, and sera from all five NHPs in this group showed inhibition in an alternative assay for virus neutralization (panel F).

[0342] After SARS CoV-2 challenge, SC primary immunization and oral booster vaccination reduce viral load in the nasal passages and lungs: RT-qPCR analysis of genomic RNA (gRNA) was performed on nasal swabs and bronchoalveolar lavage (BAL) samples to determine the amount of virus present. From day 57, the first day after challenge, SC- oral-oral vaccination of NHPs reduced SARS-CoV-2gRNA in nasal swab samples compared to placebo-controlled NHPs (Figure 22, sub-figures A and B). By day 63, 7 days after challenge, the viral gRNA in this group continued to decrease to very low or below detection levels (LOD) in all vaccinated animals. During the study, placebo controls had moderate to high levels (range 2E+09-8.4E+03 gene copies / mL) of SARS-CoV-2 in nasal swab samples.

[0343] gRNA also declined rapidly in the lungs (bronchoalveolar lavage, BAL) of SC-oral-oral NHPs, with geometric means showing a decrease of approximately 2 log ( 1.3 % vs. 1.3 % in vaccinated NHPs on day 57, just 1 day after challenge. Fig. 22 , panels C and D). In the group that received SC and oral boost (SC-SC-oral), SARS-CoV-2 gRNA in nasal swab samples also decreased similarly to primates vaccinated with SC-oral-oral vaccines, with viral gRNA falling to very low or below LOD levels in all vaccinated animals by day 63 ( Fig. 22 , Panels E and F). In the lungs of SC-SC-oral NHPs, gRNA also showed a decrease of about 2 log on day 57 ( Fig. 22 , Figures G and H).

[0344] SC prime, oral boost vaccination immediately inhibited viral replication in the nasal passages and lungs after SARS-CoV-2 challenge188: The presence of replicating virus in nasal swab samples was determined by RT qPCR of subgenomic RNA (sgRNA). By day 60, 4 days after challenge, sgRNA was below the LOD for both SC-oral-oral primates and, starting on day 61, for all primates that received only oral boost ( Fig.23 , panels A and B). In the lungs of SC-oral-oral NHPs, sgRNA also decreased starting from day 57 compared to placebo and was below the LOD in all animals by day 63 ( Fig.23 , panels C and D). In SC-SC-oral NHPs, evidence of viral replication in the nasal passages also decreased rapidly and was below the LOD by day 59 in two primates and by day 63 in all primates ( Fig.23 , sub-figures E and F); and in the lungs of this group, sgRNA was reduced by about 2 log on day 57 (one day after stimulation) compared with the placebo control, with sgRNA below the LOD in 4 of the 5 primates and slightly above the LOD in the 5th primate on day 63 ( Fig.23 , sub-figures G and H). Not only did the viral load and replicating virus drop rapidly in the nasal passages and lungs, it is noteworthy that there was no viral growth after challenge. This suggests that there is pre-existing humoral and cellular immunity that leads to rapid clearance of the virus after infection.

[0345] Immediate protection of NHPs from SARS-Cov-2 challenge (which may be due to the presence of T cells that respond to both S and N) and rapid viral clearance (due to activation of memory B cell peripheral blood mononuclear cell (PBMC)-derived T cell responses to antigens delivered by hAd5 S-fusion+N+ETSD vaccine, spike, and nucleocapsid) was determined by ELISpot on days 0 before priming, day 14 (before boost), and day 35 (one week after boost on day 28). T cells from primates vaccinated SC-oral-oral secreted interferon-γ (IFN-γ) in response to S and N peptides on days 14 and 35 ( Fig.24 , Panel A). Interleukin 4 (IL-4) secretion is very low ( Fig.24 , panel B), indicating that the T cell response was dominated by T helper 1 (Th1) cells, as reflected by the IFN-γ / IL-4 ratio ( Fig.24 , sub-figure C).

[0346] For sera collected during the post-challenge period, a microneutralization assay (see Methods) was used to assess the SARS-CoV-2 neutralization capacity as reflected by “MN50” (i.e., the serum dilution associated with a 50% reduction in viral infectivity compared to a serum-free control). Within two weeks after challenge, a rapid increase in the neutralization capacity of sera from NHPs that received only the oral boost was observed ( Fig.24 , sub-figure D), which reflects that nasal gRNA and sgRNA ( Fig.24 , Figures E and F, respectively) and lung gRNA and sgRNA ( Fig.24 , panels G and H, respectively). Of note, sera from primates in the placebo group did not show an increase in neutralizing capacity after challenge ( Fig.24 , sub-figures D and L), which indicates the presence of memory B cells in the vaccinated group and the absence of such cells in the placebo group that was not vaccinated. Further studies are needed to confirm this hypothesis.

[0347] For SC-SC-orally vaccinated NHP, the reactive T cells during the pre-priming vaccination period ( Fig.24 , sub-figures IK) and neutralization capacity during the post-challenge period ( Fig.24 ,point Figure L ) showed very similar findings, including a reduction in gRNA and sgRNA in the nasal passages and lungs ( Fig.24 , sub-figure MP).

[0348] Cytotoxic T cells present as a result of vaccination ( Fig.24 , panels AC and IK) resulted in an almost immediate reduction in viral replication within the first 24 hours after challenge ( Fig.24 , panels F, H, N, and P), and continued to decline over the next two weeks, reflecting the increased neutralizing capacity of sera from vaccinated but not placebo NHPs, reflecting the contribution of anti-S-producing memory B cells ( Fig.24 , Figures D and L).

[0349] This study demonstrated that subcutaneous prime and oral boost dual antigen hAd5 S-fusion + N-ETSD vaccination protected the nasal and pulmonary airways against SARS-CoV-2 challenge in a rhesus macaque NHP model. Inhibition of viral replication in the nasal passages was significant, as evidenced by sgRNA reduction on the first day after viral challenge, with sustained viral clearance to below detection levels in all (10 / 10) animals within 7 days of challenge ( Fig. 22 and 23 ); and, while rhesus macaques are not a model for assessing transmission, these rapid reductions in rhinovirus replication are encouraging and support future studies investigating the ability of this vaccine to prevent transmission.

[0350] The ability of hAd5 S-fusion + N-ETSD vaccination to elicit virus-neutralizing anti-S antibodies ( Fig.21 ) and T cells that respond to both S and N ( Fig.23 ), particularly from the rapid increase in neutralizing capacity of sera after challenge (which may indicate the presence of memory B cells), suggesting that the vaccine established broad immunity against severe SARS-CoV-2 infection.

[0351] Given the critical role of T cells in protecting COVID-19 convalescents (where SARS-CoV-2-specific T cells have been identified even in the absence of an antibody response) from infection, the potential of the hAd5 S-fusion + N-ETSD SC prime, oral boost vaccine to generate cytotoxic T cells is a key feature. The apparent almost immediate reduction in viral replication by hAd5 S-fusion + N-ETSD vaccination contrasts with results reported from other adenoviral vector-loaded S-only vaccine NHP studies, where there was evidence of persistent viral replication in some animals for at least one day post-challenge. Compared to the titers used in some other NHP vaccine studies, even with 1 × 10 6 Vaccinated animals appeared protected at the earliest time point evaluated in the study, as evidenced by the relatively high titers of 281 TCID50 / mL. This rapid protection and clearance was particularly evident in the lungs, where viral load and viral replication were approximately 1-2 log lower than placebo in both vaccinated groups just one day after challenge.

[0352] The protection conferred by hAd5 S-fusion + N-ETSD vaccination of NHPs by SC and oral booster administration specifically reveals the potential of this vaccine to be developed for global distribution, especially given the rapid worldwide spread of escape variants resistant to antibodies and convalescent plasma. The oral hAd5 S-fusion + N-ETSD formulation does not require ultracold refrigeration as is required for many COVID vaccines currently in development. Reliance on cold chain distribution to geographically remote or underdeveloped areas creates transportation and storage challenges and could reduce accessibility to RNA-based COVID-19 vaccines.

[0353] The thermostable oral hAd5 S-fusion+N-ETSD vaccine, due to its expression of both S and N, also has the potential to serve as a “universal” booster for other previously administered vaccines that deliver only the S antigen. Cold chain independence would also facilitate such use.

[0354] Saliva test

[0355] In further contemplated aspects, methods and compositions for administering, monitoring and measuring vaccines are disclosed herein. The contemplated methods include inducing immunity against the virus in a patient by administering the vaccine composition to the patient via delivery to the patient's nasal mucosa, oral mucosa and / or digestive tract mucosa. Preferably, the vaccine is for SARS-like coronavirus (SARS-CoV-2).

[0356] It is noteworthy that the disclosed method also includes obtaining a saliva sample from the patient within a period of time after the vaccine is administered. Typically, the saliva sample is stored in a stabilizing solution comprising glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, or any combination thereof. More typically, the stabilizing solution comprises 0.10 to 2.0% weight / volume (w / v) of glutaraldehyde, 0.10 to 1.0% w / v of sodium benzoate, and / or 0.025 to 0.20% w / v of citric acid. Additional embodiments include analyzing at least one of the antibodies selected from the targeted virus or virus-specific protein in the saliva sample, wherein in the absence of antibodies in the sample saliva, the method further comprises administering a booster of the vaccine to the patient.

[0357] In addition, the stabilizing solution further comprises aragonite particle beads having an average particle size of 100 nm to 1 mm. The aragonite particle beads can bind to immunoglobulin (Ig) proteins, anti-SARS-CoV-2 antibodies, or SARS-CoV-2 viral proteins. In an exemplary embodiment, the aragonite particle beads are coupled to recombinant ACE2 protein or recombinant ACE2 alpha helical protein.

[0358] The contemplated subject matter also includes an aragonite composition formulated for binding to an immunoglobulin (Ig) protein, an anti-SARS-CoV-2 antibody protein, or a SARS-CoV-2 viral protein. The aragonite composition includes a plurality of aragonite particle beads having an average particle size between 100 nm and 1 mm, wherein the plurality of aragonite particle beads are functionalized with a moiety capable of binding to an immunoglobulin (Ig), an anti-SARS-CoV-2 antibody protein, and / or a SARS-CoV-2 viral protein.

[0359] In a specific embodiment, the plurality of aragonite particle beads are functionalized with a moiety capable of binding to an anti-SARS-CoV-2 comprising a recombinant ACE2 protein. For example, the moiety capable of binding to an anti-SARS-CoV-2 can be selected from a recombinant ACE2 protein having at least 85% sequence identity to SEQ ID NO: 9, a recombinant α-helical ACE2 protein of SEQ ID NO: 10, or a recombinant α-helical ACE2 protein having at least one mutation selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and / or D355L.

[0360] The subject matter under consideration includes methods of administering a vaccine to a patient by more than one route of administration to induce local and systemic immune responses to the vaccine. The subject matter under consideration also includes compositions and methods for determining whether relevant antibodies (e.g., anti-SARS-CoV-2 antibodies) are present in patient samples (e.g., saliva, nasal mucosa, digestive mucosa, or serum). The antibody status in patient samples can be used to assess the need for additional vaccine doses (e.g., booster doses / injections).

[0361] In addition to the desirable molecular epitopes present in the vaccine, the route of administration of the vaccine and the schedule of administration of additional (ie, booster) doses of the vaccine can also influence whether the patient's immune response is strong enough to establish protection.

[0362] For newly emerging viruses, such as SARS-like coronavirus (SARS-CoV-2), the duration of immunity (humoral and cell-mediated) in patients who have recovered from SARS-CoV-2 infection is not fully understood, and furthermore, vaccine regimens have not been tested in different populations. Considering the current SARS-CoV-2 pandemic and the high transmission rate of the SARS-CoV-2 virus, robust vaccination regimens and validated testing for immunity to or against the virus (presence of anti-SARS-CoV-2 antibodies) are needed.

[0363] Currently disclosed contemplated methods for inducing immunity in patients include administering vaccines by at least oral administration, preferably by oral administration and by injection into the bloodstream. Many vaccines are administered via the intramuscular (IM) route to optimize immunogenicity, wherein the vaccine is delivered directly to the bloodstream in the muscle to induce systemic immunity. IM administration is generally preferred over subcutaneous (SC) injection, which is more likely to produce adverse reactions at the injection site than IM injection.

[0364] In addition to IM injection, it is reported that inducing mucosal immunity is essential for preventing the spread of pathogenic microorganisms between people and limiting their reproduction in mucosal tissues. In addition, for protective immunity against mucosal pathogens (e.g., SARS coronavirus), immune activation in mucosal tissues, rather than the more common method of tolerating and maintaining mucosal homeostasis, can enhance mucosal immune responses and better local protection. For example, nasal vaccination (delivery of vaccines by nasal administration) induces mucosal immunity and systemic immunity (see, e.g., Fujikuyama et al., 2012, Expert Rev Vaccines [vaccine expert review], 11: 367-379 and Birkhoff et al., 2009, Indian J. Pharm. Sci. [Indian Journal of Pharmacy], 71: 729-731).

[0365] In order to induce mucosal immunity and systemic immunity in patients, embodiments of the present disclosure include providing vaccines to patients by at least applying to the nasal mucosa, oral mucosa and / or digestive tract mucosa of the patient. In certain embodiments, the route of administration includes administering the vaccine to the nasal mucosa, oral mucosa and / or digestive tract mucosa of the patient and injecting it into the bloodstream (e.g., intramuscular (IM), intravenous (IV) or subcutaneous (SC)). As used herein, oral administration of vaccine compositions includes nasal injection, nasal inhalation, oral intake and administration to the digestive tract mucosa (e.g., inhalation, intake, injection). Preferably, the route of administering the vaccine includes oral administration, which is selected from delivery to the digestive tract mucosa, nasal injection, nasal inhalation, oral intake or oral inhalation and administration by intramuscular (IM) injection.

[0366] It is noteworthy that the vaccine for inducing immunity in the patient's mucosal tissue is a SARS-CoV-2 vaccine. In an exemplary embodiment, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a soluble ACE2 protein coupled to an immunoglobulin Fc portion to form an ACE2-Fc hybrid construct that can also include a J-chain portion, as disclosed in US16 / 880,804 and US63 / 016,048, the entire contents of which are incorporated herein by reference. In other exemplary embodiments, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a mutant variant of a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10), wherein the mutant variant has at least one mutated amino acid residue (e.g., by substitution), which gives the ACE2 protein an increased binding affinity to the RBD protein domain of the SARS-CoV-2 spike protein, as disclosed in US63 / 022,146 (the entire contents of which are incorporated herein by reference). In another exemplary embodiment, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a CoV2 nucleocapsid protein or a CoV2 spike protein (N-ETSD) fused to an endosomal targeting sequence, as disclosed in US16 / 883,263 and US63 / 009,960, both of which are incorporated herein by reference in their entirety. Additionally or alternatively, the SARS-CoV-2 vaccine includes a modified yeast cell (e.g., Saccharomyces cerevisiae) that is genetically engineered to express a coronavirus spike protein on the surface of the yeast cell to produce yeast presenting cells to stimulate B cells (e.g., humoral immunity), as disclosed in US63 / 010,010.

[0367] In some embodiments, more than one vaccine composition as disclosed herein may be administered to a patient to induce immunity to SARS-CoV-2. For example, genetically modified yeast cells expressing coronavirus spike protein may be administered to a patient as a single type of vaccine, or genetically modified yeast cells may be administered together or simultaneously with one or more SARS-CoV-2 adenovirus constructs disclosed herein.

[0368] In further embodiments, the patient's immune response to a vaccine administered as disclosed herein (e.g., by oral administration and injection into the bloodstream) or viral infection can be monitored or assessed. Specifically, disclosed herein are compositions and methods for assessing the persistence of antibodies in the respiratory and digestive mucosa of a patient after infection with SARS-CoV-2 or after administration of a SAR coronavirus vaccine against SARS-CoV-2.

[0369] For measuring samples from patients who have been vaccinated with vaccines for pathogen infection (e.g., for SARS-CoV-2) and / or infected with viruses (e.g., SARS-CoV-2), any of many diagnostic tests can be used to determine the presence of antibodies against pathogens. In certain embodiments, diagnostic tests are cell viability assays that allow detection of antibodies in the presence of antigens. In US62 / 053,691 (the entire contents of which are incorporated herein by reference), a diagnostic test for detecting anti-SARS-CoV-2 antibodies using cell viability assays is disclosed. Cellular diagnostic assays rely on the expression of target receptors of given pathogens (e.g., ACE2 for SARS-CoV-2) on the surface of immune effector cell lines (e.g., killer T cells, natural killer cells, NK-92 cells and derivatives thereof, etc.) and on the surface of alternative cell lines (e.g., HEK293 cells or SUP-B15 cells) pathogen ligands (e.g., spike proteins for SARS-CoV-2 infection) are expressed.

[0370] Other diagnostic tests using recombinant protein variants of the ACE2 protein (the human receptor targeted by the SARS-CoV-2 spike protein) are disclosed in US 16 / 880,804, the entire contents of which are incorporated herein by reference.

[0371] To more easily monitor patients for the presence of antibodies against pathogens, assaying patients' saliva samples can expedite sample collection, increase patient engagement, and allow patients to obtain their own samples and mail or ship them to a laboratory for testing. However, to assay saliva for the presence of neutralizing antibodies against SARS-CoV-2, proteins in saliva may need to be stabilized to prevent degradation during transport and storage after sample collection prior to testing.

[0372] After the saliva sample is collected, the saliva is placed in a preservative solution to stabilize the components therein (e.g., anti-SARS CoV-2 antibodies or viral spike proteins). For example, preservatives for biological samples are disclosed in the Cunningham & al. (2018) report ("Effective Long-term Preservation of Biological Evidence," U.S. Department of Justice Grant #2010-DN-BX-K193) and US 6,133,036. For example, a stabilized preservative solution for a patient's saliva sample may include any one of glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, and any combination thereof.

[0373] In specific embodiments, the saliva sample can be mixed with a stable preservative solution of glutaraldehyde to achieve a final glutaraldehyde concentration between 0.1% (w / v) and 2.0% (w / v), such as about 0.2% (w / v), about 0.3% (w / v), about 0.4% (w / v), about 0.5% (w / v), about 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 1.0% (w / v), about 1.1% (w / v), about 1.2% (w / v), about 1.3% (w / v), about 1.4% (w / v), about 1.5% (w / v), about 1.6% (w / v), about 1.7% (w / v), about 1.8% (w / v), or about 1.9% (w / v).

[0374] In additional or alternative embodiments, the saliva sample can be mixed with a stable preservative solution of about 0.10% to about 1.00% sodium benzoate (weight / volume sample) and / or about 0.025% to about 0.20% citric acid (weight / volume sample). For example, the saliva sample can be mixed with 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90% or 1.00% w / v sodium benzoate. In further embodiments, the saliva sample is mixed with a stable preservative solution of at least 0.5 mg / mL (e.g., at least 0.6 mg / mL, at least 0.7 mg / mL, at least 0.8 mg / mL, at least 0.9 mg / mL, at least 1 mg / mL, at least 1.5 mg / mL, at least 2 mg / mL, at least 2.5 mg / mL, at least 3 mg / mL, at least 3.5 mg / mL, at least 4 mg / mL, at least 4.5 mg / mL, or even 5 mg / mL) benzoic acid and / or at least 0.2 mg / mL (e.g., at least 0.2 mg / mL, at least 0.25 mg / mL, at least 0.3 mg / mL, at least 0.35 mg / mL, at least 0.40 mg / mL, at least 0.50 mg / mL, at least 0.75 mg / mL, at least 1.0 mg / mL, at least 1.25 mg / mL, at least 1.5 mg / mL, at least 1.75 mg / mL, or even 2.0 mg / mL) citric acid. As used herein, "benzoic acid" is interchangeable with benzoate salts (eg, sodium benzoate), and "citric acid" is interchangeable with citrate salts (eg, sodium citrate).

[0375] The saliva sample with the above preservative can be stably stored at a temperature between 15°C and 40°C for at least one hour (e.g., at least 5 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 48 hours, or even 36 hours). Therefore, disclosed herein is a method for preserving a saliva sample for neutralizing antibody testing, the method comprising mixing the saliva sample with a stabilizing solution made of one or more of glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens and / or sodium azide, and storing it at 15°C and 25°C for at least one hour, and up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours. In some embodiments, the saliva sample is mixed with glutaraldehyde at a concentration between 0.1% (w / v) and 2.0% (w / v), and the glutaraldehyde-saliva is stored between 15° C. and 25° C. In certain embodiments, the glutaraldehyde-saliva may further comprise citric acid and / or benzoic acid at concentrations as disclosed herein.

[0376] Aragonite: In some embodiments, oolitic aragonite particles can be used to capture any antibody protein or any specific antibody protein from a saliva sample. For example, a saliva preservation solution of glutaraldehyde, sodium benzoate and citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, and any combination thereof as disclosed herein can also include oolitic aragonite (calcium carbonate, CaCO3) particles. The use of aragonite particles for binding proteins is disclosed in, for example, US16 / 858,548 and PCT / US 20 / 29949, the entire contents of both of which are incorporated herein by reference. Therefore, aragonite particles can be added to aragonite particles that have been modified to capture (e.g., bind) any antibody present in a saliva sample or specifically capture antibodies against a specific antigen. For example, aragonite can be functionalized with a portion that can bind to an immunoglobulin (Ig) protein. Preferably, the Ig protein is immunoglobulin A (IgA), immunoglobulin G (IgG), or immunoglobulin E (IgE). More preferably, the aragonite is functionalized to bind to IgA protein. Most preferably, the aragonite particles are functionalized with moieties capable of binding to specific antibodies. For example, the aragonite particles can be coupled to moieties specific for antibodies against SARS-CoV-2. Preferably, the aragonite particles are coupled to a recombinant ACE2 protein, such as disclosed in US 16 / 880,804, supra. In typical embodiments, the aragonite particles are coupled to a recombinant human ACE2 protein having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:9.

[0377] In an additional or alternative embodiment, the aragonite particles are functionalized (e.g., coupled to) a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10). In order to more effectively capture or bind to anti-SARS-CoV-2 antibodies or the spike protein of SARS CoV-2, the recombinant soluble ACE2 can be mutated to form an ACE2 variant (e.g., the RBD domain of the spike protein) with a higher binding affinity to the SARS-CoV-2 spike protein. These ACE2 variant mutants of the recombinant soluble ACE2 protein include T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A and / or D355L.

[0378] As used herein, the term "functionalized" refers to the coupling or combination of a moiety with an aragonite particle, thereby imparting any functionality of the coupled moiety to the aragonite particle. For example, the aragonite particle can be functionalized with a protein moiety. Methods for preparing and using aragonite particle beads are disclosed in US16 / 858,548 and PCT / US 20 / 29949. In some embodiments, the aragonite composition includes a plurality of aragonite particle beads. Preferably, the plurality of aragonite particle beads have an average particle size of 100nm to 1mm.

[0379] In some embodiments, the protein moiety is directly coupled to the natural untreated surface of the aragonite particles. Aragonite particles have an amino acid content of about 2%-3%, including aspartic acid and glutamic acid, which makes the aragonite surface hydrophilic. Therefore, in some embodiments, the protein moiety can be directly coupled to the surface of the aragonite particles.

[0380] In an alternative embodiment, the surface of the aragonite particles can be treated to modify the binding surface. For example, a hydrophobic surface is provided by treating with stearic acid (i.e., octadecanoic acid), as disclosed in US16 / 858,548 and PCT / US 20 / 29949. For protein loading, aragonite is treated with phosphoric acid to form a layered structure. Other coupling techniques for coupling reactive groups to the amino acid surface of aragonite are known in the art, such as disclosed in Bioconjugate Techniques, the third edition, Greg T. Hermanson, Academic Press, 2013.

[0381] Patients who do not show adequate titers (e.g., presence) of neutralizing antibodies in their saliva can be sent oral doses of a corresponding vaccine (e.g., a SARS-CoV-2 vaccine as disclosed herein). The patient inhales or ingests these vaccine doses, and then two weeks later sends another saliva sample (prepared and stored in the same manner as described above) to a testing facility to confirm that the oral vaccine dose has restored their anti-SARS-CoV-2 antibody (e.g., IgA) titers.

[0382] Thus, in further embodiments, a kit for collecting a saliva sample from a patient comprises a collection container having a saliva preservative solution as disclosed herein. For example, the kit comprises a collection container having a solution of any one or combination of glutaraldehyde, sodium benzoate and / or citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, and sodium azide. The kit may also include adhesive packaging and / or mailing supplies to secure the collection container with the saliva sample for transport or mailing. In some embodiments, the kit may also include at least one dose of a vaccine for oral administration.

[0383] Immune stimulation

[0384] In addition to vaccination, or even as an alternative treatment for subjects with a possible or actual diagnosis of coronavirus infection, particularly SARS-CoV-2 infection, immune stimulation with one or more immune stimulatory cytokines may prevent or alleviate the lymphocytopenia frequently associated with COVID-19. Among other immune stimulatory cytokines, N-803 is particularly contemplated.

[0385] COVID-19 infection causes lymphocytopenia, particularly suppression of NK and CD8 T cells, and severe cases and subsequent death are associated with this significant decrease in lymphocytes. Evidence also suggests that COVID-19 infection leads to a decrease in macrophage killing and natural killer (NK) cells and CD8+ T cells. Analysis of data from 1,099 laboratory-confirmed COVID-19 patients from 552 hospitals in China found that upon admission, 83.2% of patients had lymphocytopenia. On average, patients with severe disease had more prominent laboratory abnormalities, such as lower lymphocyte counts, compared with patients with non-severe disease. Similarly, another group of researchers showed that patients admitted to the ICU had significantly lower median lymphocyte counts compared with patients who did not require ICU care.

[0386] At the cellular level, COVID-19 appears to induce immune evasion through a reduction in NK, CD4+, and CD8+ T cells. The mechanism of lymphocytopenia is unclear; however, rapid reductions in CD4 and CD8 T cells may be associated with adverse outcomes. Lymphocytopenia and increased viral loads during the first 10 days of SARS suggest that SARS coronavirus can evade immunity. Lack of interferon (IFN)-γ responses in SARS-infected cells have been reported in vitro using human primary bone marrow-derived dendritic cells and the epithelial 293 cell line. Others have proposed immune evasion mechanisms for SARS-CoV in dendritic cells (DCs) based on low expression of antiviral cytokines (TFN-α, TFN-β, TFN-γ, and IL-12p40), moderate upregulation of proinflammatory cytokines (tumor necrosis factor α [TNF-α] and IL-6), and significant upregulation of proinflammatory cytokines (MIP-1a, RANTES, IP-I 0, and MCP-1). In addition to CD8+ T cell exhaustion, insufficient T cell responsiveness due to poor T cell diversity may contribute to poor immune responses, particularly in the elderly population.

[0387] N-803 has shown the ability to significantly induce a broad increase in T cell reactivity in preclinical (mice) and clinical (breast cancer, lung cancer, and pancreatic cancer) settings. It is possible that by activating T cell diversity, cross-reactivity to past coronavirus infections may provide immunity to COVTD-19 after N-803 enhances T cell reactivity and overcomes immune escape. N-803 significantly promotes NK cell and CD8-T cell proliferation and activation in the peripheral circulation and lymphoid organs of healthy mice and cynomolgus monkeys, as well as in various mouse and rodent tumor models including bladder cancer, lung cancer, melanoma, lymphoma, multiple myeloma, colon cancer, breast cancer, and glioblastoma. There is additional preclinical evidence for the antiviral effects of N-803, including in mouse and non-human primate (NHP) models.

[0388] Clinical evidence from early trials also supports these preclinical studies showing proliferation and activation of NK cells and CD8+ T cells. N-803 increases the cytotoxic potential of these immune cells, as shown by upregulation of the expression of activation markers including perforin and granzyme B. The phenotypic changes induced by N-803 in NK and CD8+ T cells result in enhanced anticancer activity (through antibody-dependent cellular cytotoxicity, direct cytotoxicity, and enhanced tumor-specific cytotoxicity) and prolonged survival in vivo.

[0389] In healthy volunteers, NK cell and CD8+T cell activation has been demonstrated. In a study of healthy volunteers administered N-803 at 10 μg / kg and 20 μg / kg SC, N-803 treated subjects demonstrated a greater than 20-fold increase in Ki-67 stained NK and CD8+T cells. The clinical efficacy of N-803 in stimulating lymphopenic responses has been demonstrated in patients with acute myeloid leukemia (AML), breast cancer, lung cancer, and pancreatic cancer.

[0390] Therefore, as preclinical in vitro and in vivo studies and clinical data indicate that N-803 binds to IL-15 receptor presenting cells with higher affinity, enhances lymphoid distribution, prolongs half-life, and leads to proliferation and activation of effector NK cells and CD8+ memory T cells, resulting in anti-tumor activity, N-803 can be used as a therapeutic option to combat COVID-19-related lymphocytopenia and rescue lymphocytopenia in normal healthy subjects and cancer patients in a manner similar to how N-803 stimulates NK and CD8 T cells. Therefore, N-803 can be used to reverse or alleviate lymphocytopenia in patients infected with COVID-19, thereby improving disease outcomes. For example, infected subjects can be treated by receiving subcutaneous (SC) injections of N-803 in the abdomen on days 1 and 15 (if necessary). Most typically, N-803 will be provided in a liquid injection form at a dose of 10mcg / kg.

[0391] In some embodiments, the digits of the amount of the expression composition, characteristic (such as concentration), reaction conditions, etc., used to describe and claim certain embodiments of the present invention should be understood to be modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters listed in the written specification and the appended claims are approximate values, which can vary according to the desired characteristics that a particular embodiment attempts to obtain. The description of the range of values ​​herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise stated in this article, each individual value is incorporated into the specification as if it were individually quoted in this article.

[0392] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of a pharmaceutical composition or drug, wherein direct administration of a pharmaceutical composition or drug is typically performed by a health care professional (e.g., a physician, nurse, etc.), and wherein indirect administration includes the step of providing the pharmaceutical composition or drug to or making available to a health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). It should also be noted that the term "prognosis" or "prediction" of a condition, susceptibility to disease development, or response to a desired treatment is intended to encompass the act of predicting or predicting a condition, susceptibility, and / or response (including the rate of progression, improvement, and / or duration of a condition in a subject) (but not the act of treating or diagnosing).

[0393] Unless otherwise indicated herein or otherwise clearly contradictory to the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended only to better illustrate the technology disclosed herein, and does not limit the scope of the invention originally claimed. No language in this specification should be interpreted as indicating that any unclaimed element is necessary to practice the claimed invention.

[0394] As used in the specification herein and throughout the claims that follow, the meanings of "a", "an", and "the" include plural references unless the context clearly indicates otherwise. Moreover, as used in the specification herein, the meaning of "in" includes "in" and "on", unless the context clearly indicates otherwise. Also as used herein, and unless the context indicates otherwise, the term "coupled to" is intended to include direct coupling (where two elements coupled to each other are in contact with each other) and indirect coupling (where at least one additional element is located between the two elements). Therefore, the terms "coupled to" and "coupled with" are used synonymously.

[0395] It should be clear to those skilled in the art that more modifications other than those described are possible without departing from the inventive concept herein. Therefore, the subject matter of the present invention is limited only to the scope of the appended claims. In addition, when interpreting this specification and claims, all terms should be interpreted in a manner as broad as possible consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components or steps in a non-exclusive manner, thereby indicating that the mentioned elements, components or steps can exist, or use, or combine with other elements, components or steps not explicitly mentioned. When the specification or claim is related to at least one of the groups selected from A, B, C ... and N, the text should be interpreted as requiring only one element in the group, rather than A plus N, or B plus N, etc.

Claims

1. A recombinant nucleic acid comprising: a. encoding a first portion of a severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) nucleocapsid protein (N) (N-ETSD) fused to an endosomal targeting sequence, wherein the first portion encodes a protein having an amino acid sequence of SEQ ID NO: 1; and b. Encoding the second part of the SARS CoV-2 spike protein (S) having the amino acid sequence of SEQ ID NO:

4.

2. The recombinant nucleic acid of claim 1, wherein the first portion has a nucleotide sequence of SEQ ID NO:

2.

3. The recombinant nucleic acid of claim 1, wherein the second portion has a nucleotide sequence of SEQ ID NO:

6.

4. The recombinant nucleic acid of any one of claims 1 to 3, further comprising a third portion encoding a co-stimulatory molecule or an immunostimulatory cytokine.

5. The recombinant nucleic acid of any one of claims 1 to 3, wherein the recombinant nucleic acid is integrated into a viral expression vector.

6. The recombinant nucleic acid of claim 5, wherein the viral expression vector is an adenovirus expression vector having a deletion of the E1 gene region and a deletion of the E2b gene region.

7. The recombinant nucleic acid of any one of claims 1-3, wherein the nucleic acid is deoxyribonucleic acid (DNA).

8. A recombinant replication-deficient adenovirus comprising an E1 gene deletion, an E2b gene deletion and a recombinant nucleic acid, wherein the recombinant nucleic acid comprises: a. encoding a SARS coronavirus 2 (CoV-2) N protein having an amino acid sequence of SEQ ID NO: 7 and a first portion of an endosomal targeting sequence having an amino acid sequence of SEQ ID NO: 8; as well as b. The second part encoding the SARS CoV-2 S protein having the amino acid sequence of SEQ ID NO:

4.

9. The adenovirus of claim 8, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 5' end of the first portion.

10. The adenovirus of claim 8, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 3' end of the first portion.

11. The adenovirus of claim 10, wherein the first portion and the second portion are arranged in a bicistronic sequence.

12. The adenovirus of claim 8, wherein the N-ETSD has the amino acid sequence of SEQ ID NO:

1.

13. The adenovirus of claim 8, wherein the first portion has a nucleotide sequence of SEQ ID NO:

2.

14. The adenovirus according to claim 8, wherein the second part has the nucleotide sequence of SEQ ID NO:

6.

15. The adenovirus of any one of claims 8 to 14, further comprising a third portion encoding a co-stimulatory molecule or an immunostimulatory cytokine.

16. The adenovirus according to any one of claims 8 to 14, wherein the recombinant adenovirus has a deletion in the E3 gene region.

17. A vaccine composition comprising the recombinant nucleic acid according to any one of claims 1 to 7.

18. The vaccine composition of claim 17, wherein the recombinant nucleic acid is encapsulated in lipid nanoparticles.

19. A vaccine composition comprising aragonite particles mixed with the recombinant replication-defective adenovirus of any one of claims 8 to 16, wherein the recombinant replication-defective adenovirus is lyophilized.

20. The vaccine composition of claim 19, wherein the aragonite particles have an average particle size between 100 nm and 1 mm.

21. Use of the recombinant nucleic acid according to any one of claims 1 to 7 in the preparation of a vaccine against SARS virus.

22. Use of the recombinant nucleic acid according to any one of claims 1 to 7 in the preparation of a medicament for inducing immunity against SARS virus.

23. Use of the recombinant replication-deficient adenovirus according to any one of claims 8 to 16 in the preparation of a medicament for inducing immunity against SARS virus.

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