Treatment of covid-19 and methods therefor

Abstract

To provide a vaccine composition to induce immunity against a coronavirus in a subject.SOLUTION: A vaccine composition comprises a recombinant nucleic acid that encodes N-ETSD, a modified nucleocapsid protein that includes an endosomal targeting sequence, and / or that encodes S-Fusion, a modified spike protein that has improved surface expression. The vaccine may be formulated as a recombinant nucleic acid, recombinant yeast, and / or recombinant virus such as an adenovirus and can be administered via injection and / or mucosal delivery.SELECTED DRAWING: Figure 25

Description

[Technical field]

[0001] This application is a continuation of the inventors' co-pending U.S. Provisional Patent Application Nos. 62 / 988,328, filed March 11, 2020, 63 / 009,960, filed April 14, 2020, 63 / 010,010, filed April 14, 2020, 63 / 059,975, filed August 1, 2020, 63 / 064,157, filed August 11, 2020, 63 / 117,460, filed November 24, 2020, and 63 / 120,460, filed November 24, 2020. This application claims priority to U.S. Provisional Patent Application No. 16 / 883,263, filed May 26, 2020, and the U.S. Provisional Patent Application having the reference number 102538.0080US3, entitled "Anti COVID-19 Therapies targeting nucleocapsid and spike proteins," filed on May 26, 2020, all of which are incorporated herein by reference.

[0002] Sequence Listing The contents of the ASCII text file of the Sequence Listing named 102690-0041PCT_REV003_ST25.txt, having a size of 172KB, was created on March 4, 2021, and was submitted electronically via EFS-Web herewith, and is incorporated by reference in its entirety.

[0003] The field of the disclosure is vaccine compositions and methods for generating immunity against coronaviruses, particularly related to SARS-CoV-2. [Background technology]

[0004] The Background Description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] All publications and patent applications herein are 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. In the event that a definition or use of a term in an incorporated reference is inconsistent with 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.

[0006] Although SARS-CoV2 diagnostic tests have become available in a relatively short time, numerous attempts to treat the disease have so far shown conflicting or inconclusive results. Most typically, patients with severe symptoms are treated to maintain breathing / blood oxygenation. COVID19 mortality is particularly pronounced in the elderly, immunocompromised, and individuals with cardiac disease, pulmonary disease, or diabetes. Despite improvements in acute care, it has become clear that disease containment is crucial, as social distancing and other public health mitigation measures can only provide moderate relief.

[0007] To that end, many candidate anti-SARS-CoV2 vaccine compositions target one or more proteins of the virus (see, for example, FIMMU 2020, 11:602256). For example, Sinovac and Sinopharm are currently testing inactivated viral vaccine preparations. 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. More 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 disease-causing infection, but it is unclear whether protection is effective over months and / or whether sufficient immune memory protects vaccinated individuals over the long term. Furthermore, it is unclear whether such vaccines generate clinically meaningful T cell-based responses.

[0008] Although certain vaccines have become available, rapid distribution and administration of the vaccine on a global scale has faced significant challenges, slowing global distribution. Additionally, logistical requirements and the need for medical expertise limit administration to certain vaccination infrastructures.

[0009] To overcome these difficulties, solid dosage forms of vaccines are desirable. However, such dosage forms (e.g., powders, tablets, capsules) require disintegration and release of the active ingredient at physiologically relevant locations. The active ingredient-loaded dosage forms must be stable during transportation and storage from the time of manufacture until administration. Mucosal or oral delivery of vaccines would be highly desirable, as such administration would aid in the generation of IgA, IgE, and IgM class antibodies that are important for immunity against mucosal or intestinal infections. Unfortunately, such vaccines are not available.

[0010] Various vaccine compositions and methods for inducing immunity against SARS-CoV-2 are known in the art, but all suffer from several drawbacks. Thus, there remains a need for improved vaccine compositions and methods. Summary of the Invention [Means for solving the problem]

[0011] Disclosed herein are various vaccine compositions and methods therefor, in which recombinant modified nucleocapsid and / or modified spike proteins of coronavirus induce immunity to coronavirus in a subject. Most preferably, the recombinant proteins are encoded by recombinant nucleic acids that can be delivered in lipid formulations, as part of a recombinant virus, and / or as part of recombinant yeast or yeast lysate.

[0012] Also disclosed herein is a recombinant nucleic acid comprising a first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) (N-ETSD) fused to an endosomal targeting sequence, the first portion being operably linked to one or more regulatory elements enabling N-ETSD expression, and a second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements enabling S expression.

[0013] In some embodiments, the SARS virus is SARS-CoV-2, and / or the endosomal targeting sequence of the N-ETSD is encoded at the 5' and / or 3' end of the first portion. In further embodiments, the second portion encodes an S optimized for surface expression. The first and second portions may be arranged in a bicistronic arrangement. For example, the N-ETSD may have an amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO:1. The first portion may, in certain embodiments, have the nucleotide sequence SEQ ID NO:2. In another example, the S protein or S-fusion protein may have an amino acid sequence having at least 90% identity to the amino acid sequence SEQ ID NO:3 (e.g., SEQ ID NO:3) or at least 90% identity to SEQ ID NO:4 (e.g., SEQ ID NO:4). The second portion may, in certain embodiments, have the nucleotide sequence SEQ ID NO:5 or SEQ ID NO:6.

[0014] In yet further embodiments, the recombinant nucleic acid may further comprise a third portion encoding a costimulatory molecule or an immunostimulatory cytokine. The recombinant nucleic acid may also be incorporated into a virus or yeast expression vector (e.g., an adenovirus expression vector having an E1 gene region deletion and an E2b gene region deletion, and / or a yeast expression vector of Saccharomyces cerevisiae). Most typically, but not necessarily, the nucleic acid is deoxyribonucleic acid (DNA).

[0015] Described herein is a recombinant replication-deficient adenovirus comprising a recombinant nucleic acid comprising an E1 gene deletion, an E2b gene deletion, and a first portion encoding a SARS coronavirus N-ETSD. The first portion is operably linked to one or more regulatory elements that allow for N-ETSD expression. The adenovirus also comprises a second portion encoding a SARS S protein. The second portions are each operably linked to one or more regulatory elements that allow for S expression.

[0016] In further embodiments, the adenovirus can further comprise a third portion encoding a costimulatory molecule or an immunostimulatory cytokine, and / or the recombinant adenovirus can have a deletion of the E3 and / or E4 gene regions.

[0017] Alternatively or additionally, disclosed herein is a recombinant yeast comprising a recombinant nucleic acid comprising a first portion encoding a SARS coronavirus N-ETSD, the first portion operably linked to one or more regulatory elements enabling N-ETSD expression, and a second portion encoding a SARS S protein, the second portions each operably linked to one or more regulatory elements enabling S expression. The yeast can be S. cerevisiae. In certain embodiments, the yeast can be lysed.

[0018] In yet further embodiments, disclosed herein are vaccine compositions comprising a recombinant nucleic acid as presented herein, which may be encapsulated in a lipid nanoparticle.

[0019] In yet another embodiment, disclosed herein is a vaccine composition comprising aragonite particles mixed with a recombinant replication-deficient adenovirus as provided herein, the recombinant replication-deficient adenovirus being lyophilized. Most typically, the aragonite particles have an average particle size of 100 nm to 1 mm.

[0020] Provided herein are recombinant nucleic acids for generating vaccines against and / or inducing immunity against the SARS virus. Similarly, disclosed herein are recombinant replication-defective adenoviruses or recombinant yeast for inducing immunity against the SARS virus.

[0021] Various objects, features, aspects and advantages of the subject matter disclosed herein will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like elements and in which: [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows a schematic representation of the various crystalline forms of calcium carbonate. [Diagram 2] Figure 2 shows three photographs (right to left: 1, 2, 3) of bivalent human adenovirus serotype 5 COVID-spike and nucleocapsid antigen vaccine (hAD5-COVID-S / N) in uncoated aragonite capsules (sample #6) in 0.1 M hydrochloric acid (HCL) with wrinkles, swelling, or holes observed in the capsules as indicated: 1): 2 min after HCL acid exposure; 2): 2 hr after HCL acid exposure; and 3) 2 hr after HCL acid exposure and dry. [Diagram 3] Figure 3 shows three photographs (from right to left: 1, 2, 3) of hAD5-COVID-S / N in uncoated aragonite capsules (samples #7 or #8) in 0.1M HCL with swelling, twisting, or holes observed in the capsule as indicated: 1): Sample #7 after 2 hours of HCL acid exposure; 2): Sample #8 after 2 hours of HCL acid exposure; 3) After 2 hours of HCL acid exposure and dried. [Figure 4] Figure 4 shows two photographs (right to left: 1, 2) of hAD5-COVID-S / N in uncoated lactose capsules (samples #3 or #4) in 0.1M HCL, with capsule swelling observed as indicated: 1): Sample #3 after 2 hours of HCL acid exposure; 2): Sample #4 after 2 hours of HCL acid exposure. [Diagram 5] Figure 5 shows two photographs (right to left: 1, 2) of hAD5-COVID-S / N in coated aragonite capsules (sample #1 or #5) in 0.1 M HCL, with capsule swelling observed as indicated: 1): Sample #1 after 2 hours of HCL acid exposure; 2): Sample #5 after 2 hours of HCL acid exposure. [Figure 6] FIG. 6 shows infectious units per gram (IFU / g) (y-axis) for the indicated hAD5-COVID-S / N capsule types, as indicated. [Figure 7]Figure 7 shows the percentage (%) of viral recovery for each hAD5-COVID-S / N capsule type (y-axis), as indicated. [Figure 8] Figure 8 shows the IFU / g and corresponding pH for each hAD5-COVID-S / N capsule type, as indicated. [Figure 9] Figure 9 shows the percentage (%) of viral recovery for each hAD5-COVID-S / N capsule type, as indicated. [Figure 10] FIG. 10 shows the percent of virus recovered for each hAD5-COVID-S / N capsule type, as indicated, with acid treatment shown as shaded. [Figure 11] FIG. 11 shows the IFU / g for each hAD5-COVID-S / N capsule type, as indicated, with acid treatment shown with shading. [Figure 12] FIG. 12 shows a schematic diagram of an ideal vaccine that induces durable and effective immunity across multiple routes. [Figure 13] FIG. 13 shows the results of an exemplary expression experiment in which SARS-CoV2 N is overexpressed in S. cerevisiae. [Figure 14] Figure 14 shows the ELISA results detecting IgG seroreactivity to the SARS-CoV-2 spike in serum samples taken from immunized rhesus macaques. [Figure 15] Figure 15 shows sera that inhibit SARS-CoV-2 infectivity. Panel A shows sera from vaccinated Group 1 rhesus macaques that inhibit SARS-CoV-2 infectivity in vitro. Panel B shows sera from vaccinated Group 2 rhesus macaques. The dotted line indicates 20% inhibition. [Figure 16]Figure 16 shows non-human primate (NHP) nasopharyngeal viral load over time. Panel A shows viral load (qPCR) in nasal swabs from rhesus macaques after SC+SC+oral vaccination. Panel B shows viral load (qPCR) in nasal swabs after SC+oral+oral vaccination. [Figure 17] Figure 17 shows the viral load over time in lungs in NHPs. Panel A shows the viral load (qPCR) in BAL from Group 1 rhesus macaques after SC+SC+oral vaccination, and Panel B shows the viral load (qPCR) in BAL from Group 1 rhesus macaques after SC+oral+oral vaccination. [Figure 18] FIG. 18 shows IgG and IgM seroreactivity to the SARS-CoV-2 spike in serum samples from human patients immunized with various experimental anti-SARS-CoV-2 vaccines. [Figure 19] FIG. 19 shows ELISpot results from a Th1 N-responsive patient. [Figure 20] FIG. 20 shows ELISpot results from patient 4 (N-non-responsive) and patient 10 (weak Th1 N-responsive). [Figure 21] Figure 21 shows exemplary results for humoral responses and neutralization capacity of sera from hAd5 S-fusion + N-ETSD vaccinated NHPs. Anti-spike IgG levels (ELISA; OD 450 nm) are shown for (A) individual SC oral-oral NHPs, along with (B) geometric mean and (C) inhibition in surrogate assays. These data are also shown for SC-SC-anti-NHPs: (D) individual anti-S IgG, (E) geometric mean, and (F) inhibition in surrogate assays. Inhibition of more than 20% at a serum dilution of 1:30 (dashed line) correlates with neutralization of SARS-CoV-2 infection. NHPs were vaccinated on days 0, 14, and 28 (black arrows). [Figure 22]Figure 22 shows viral load (gRNA) in the nose and lungs of SC-oral-oral and SC-SC-oral vaccinated NHPs after challenge. (A) Individual viral gRNA (RT qPCR) and (B) geometric mean of nasal swab samples; and (C) gRNA and (D) geometric mean of bronchoalveolar lavage (BAL) samples from SC-oral-oral NHPs. (E) Individual gRNA and (F) geometric mean of nasal swab samples; and (G) gRNA and (H) geometric mean of BAL samples from SC-SC-oral NHPs. SARS-CoV-2 challenge was performed on day 56 (black arrow). 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 (or 27 GC / mL for gRNA and 59 GC / mL for sgRNA) was used for graphing individual values ​​and calculating the geometric mean. [Figure 23] Figure 23 shows viral replication (sgRNA) in the nose and lungs of SC-oral-oral and SC-SC-oral vaccinated NHPs after challenge. (A) Individual viral sgRNA (RT qPCR) and (B) geometric mean of nasal swab samples; and (C) sgRNA and (D) geometric mean of bronchoalveolar lavage (BAL) samples from SC-oral-oral NHPs. (E) Individual sgRNA and (F) geometric mean of nasal swab samples; and (G) sgRNA and (H) geometric mean of BAL samples from SC-SC-oral NHPs. SARS-CoV-2 challenge was performed 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 for graphing individual values ​​and calculating geometric means. [Figure 24]Figure 24 shows T cell responses to vaccination and neutralizing capacity of serum after SARS-CoV-2 challenge. (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 determined by ELISpot, and (C) the ratio IFN-γ / IL-4 ratio. Ratios of "infinity" with undetectable IL-4 are represented as open circles. PMA-ionomycin pulsed cells were used as positive control. Data graphed with mean and SEM. (D) MN50 (dilution factor at which SARS-CoV-2 infection of Vero E6 cells is inhibited by 50%) is shown throughout the study period, comparing MN50 of vaccinated and placebo NHPs at day 70 using unpaired two-tailed Student's t-test. Nasal gRNA (E) and sgRNA (F) on days 57, 63, and 70, and lung gRNA (G) and sgRNA on days 57 and 63 are presented. The same data are shown for SC-SC-oral NHPs, including T cell responses (I-J), MN50 (L), nasal gRNA (M) and sgRNA (N), and lung gRNA (O) and sgRNA (P). The detection level for gRNA was 54 GC / mL, and for sgRNA was 119 GC / mL. Half the LOD was used for graphing data below the LOD. [Diagram 25] Figure 25 shows a schematic of the hAd5 platform and the hAd5 S-fusion + N-ETSD construct. Panel A shows the human adenovirus serotype 5 vaccine platform with deletions (*) of the E1, E2b, and E3 regions. The vaccine construct is inserted into the E1 region (arrow). Panel B shows that the dual antigen vaccine contains both the S-fusion and N-ETSD under the control of the cytomegalovirus (CMV) promoter and the C-terminal SV40 polyA sequence delivered by the hAd5[E1-, E2b-, E3-] platform. [Figure 26] FIG. 26 shows an exemplary construct for cloning into adenovirus. [Figure 27]FIG. 27 is a Western blot showing expression of the in vitro constructs and detection of S and N. [Figure 28] FIG. 28 shows further exemplary constructs for cloning into adenovirus. [Figure 29] Figure 29 shows the antibody response to N with a Th1 phenotype. Humoral immune response TH1 vs. TH2 associated isotype analysis. [Diagram 30] FIG. 30 shows cell mediated immune (CMI) responses against the N focus phenotype - IFN-γ and IL-2 ELISpot. [Diagram 31] FIG. 31 shows enhanced cell surface expression of RBD using S-fusion and S-fusion+N combination constructs compared to S-WT. [Diagram 32] Figure 32 shows antigen recognition by recovered COVID-19 patient plasma. Antigens include RBD-ETSD and fusion S / N-ETSD constructs. [Diagram 33] Figure 33 shows the SARS-CoV-2 virus, spike, hAd5 [E1-, E2b-, E3-] vector and vaccine candidate constructs. (a) The trimeric S protein is displayed on the viral surface and the N protein is associated with the viral RNA. (b) The RBD is in the S1 region, followed by other functional regions, the transmembrane domain (TM) and the C-terminus (CT), which are in the virus. (c) The second generation human adenovirus serotype 5 (hAd5) vector used is deleted for the E1, E2b and E3 regions. (d) S wild type (S-WT), (e) S-RBD with enhanced T cell stimulatory domain (S RBD-ETSD), (f) S-fusion, (g) N-ETSD, and (h) bivalent hAd5 S-fusion + N-ETSD; LP-leader peptide constructs are shown. [Diagram 34]FIG. 34 shows HEK293T transfection with hAd5 S-fusion + ETSD resulting in enhanced RBD surface expression. Flow cytometry analysis of anti-RBD antibody with construct-transfected cells reveals no detectable RBD surface expression in either S-WT or (b) S-WT + N-ETSD transfected cells. Surface RBD expression was high in S RBD-ETSD and S RBD-ETSD + N-ETSD (c, d). Expression was low in (e) S-fusion transfected cells. Cell surface expression of RBD was high in (f) S-fusion + N-ETSD transfected cells, especially on days 1 and 2. No expression was detected in (g) N-ETSD negative control. Y-axis scale is normalized to mode (NM). [Diagram 35] Figure 35 shows immunoblot analysis of S expression. Cell surface RBD expression in HEK 293T cells using (a) hAd5 S-WT, S-fusions, and (c) S-fusions + N-ETSD correlated highly with (d) expression of S in immunoblots of HEK 293T cell lysates probed with anti-full length (S2) antibody. Y-axis scale is normalized to mode (NM). [Diagram 36]Figure 36 shows that binding of recombinant ACE2-Fc HEK293T cell surface RBD after transfection confirms native protein folding. Flow cytometry analysis of binding between recombinant ACE2-Fc (by which 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 S RBD-ETSD, or (e) hAd5 S RBD-ETSD + ETSD constructs reveals that the highest binding of both ACE-Fc and anti-RBD specific antigens (fj) is seen after transfection with bivalent S-fusion + N-ETSD. Both S RBD-ETSD containing constructs also showed binding. Y-axis scale is normalized to mode (NM). [Figure 37] Figure 37 shows that N expressed from hAd5 N-ETSD localizes 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, whereas (e) N wild type (N-WT) does not, instead showing a diffuse cytoplasmic distribution. [Figure 38]Figure 38 shows ICS detection of cytokine-expressing splenocytes from hAd5 S-fusion + N-ETSD vaccinated day 28 CD-1 mice in response to peptide pools. (a) The highest CD8β+ splenocyte IFN-γ response was in hAd5 S-fusion + N-ETSD vaccinated mouse splenocytes exposed to S peptide pool 1 (S-pep pool 1); splenocytes from these mice also expressed IFN-γ in response to the N peptide pool (N-pep pool). (b) CD4+ splenocytes from hAd5 S-fusion + N-ETSD vaccinated mice expressed only IFN-γ in response to the N peptide pool. (c) The IFN-γ TNF-α response of CD8β+ splenocytes from hAd5 S-fusion + N-ETSD vaccinated mice was very similar to that in (a); so was the CD4+ splenocyte response to the (d) N peptide pool for (b). N=5 mice per group. All data sets were graphed as means with SEM and all statistics were performed using the Mann-Whitney test. *<0.05, **<0.01, ***<0.001, and ****<0.0001. [Figure 39] Figure 39 shows anti-spike and anti-nucleocapsid antibody responses in serum from hAd5 S-fusion + N-ETSD vaccinated mice. Based on absorbance, there was significant production of both (a) anti-S and (c) anti-nucleocapsid antibodies. (b, d) ng equivalents are shown. Serum was diluted 1:30 for anti-spike and 1:90 for anti-nucleocapsid antibodies. Data are graphed as mean and SEM. Statistical analysis was performed using unpaired two-tailed Student's t-test. *<0.05, **<0.01, ***<0.001, and ****<0.0001. [Diagram 40]Figure 40 shows cPass and Vero E6 cell SARS-CoV-2 confirm neutralization by antibodies. (a) In the cPass assay, inhibition of S RBD interaction with ACE2 was significant at both 1:20 and 1:60 dilutions of serum from hAd5 S-fusion + N-ETSD vaccinated mice. (b) Vero E6 cell SARS-CoV-2 virus infection results for mice that showed S-specific antibodies by ELISA also showed high neutralization for mice and very high neutralization for pooled serum (G4 pool, blue line) compared to COVID-19 convalescent sera. G4 pool - mice with S-specific antibodies; M1, M2, M3, M4 - mouse ID; +C - convalescent serum; and media - negative control with media only. [Diagram 41] Figure 41 shows the isotypes of anti-spike and anti-nucleocapsid antibodies. Panels A and C show that IgG2a and IgG2b isotype anti-spike and anti-nucleocapsid antibodies were significantly increased for hAd5 S-fusion + N-ETSD mice compared to hAd5 null mice. Panels B and D show ng equivalents for antibody isotypes. Data are graphed as mean and SEM. Statistical analysis was performed using unpaired two-tailed Student's t-test. *<0.05, **<0.01, ***<0.001, and ****<0.0001. [Diagram 42] Figure 42 shows ELISpot of secreted cytokines. (a) IFN-γ secretion by hAd5 S-fusion + N-ETSD splenocytes was significantly higher than hAd5 null in response to both S peptide pool 1 and N peptide pool, whereas (b) IL-4 was only secreted by hAd5 S-fusion + N-ETSD in response to N peptide pool (one high outlier in hAd5 null was removed). N=5 mice per group. All data sets were graphed as mean with SEM and all statistics were performed using Mann-Whitney test. *<0.05, **<0.01, ***<0.001, ****<0.0001. [Diagram 43]Figure 43 shows that ratios of T cell and humoral responses reveal Th1 dominance. (a) Ratios of total Th1 (IFN-γ) to Th2 (IL-4) spot forming units are shown for responses to combined S and N pools. (b) Th1 / Th2 ratios of antibodies to S and N are shown. For both (a) and (b), the dashed line indicates a ratio of 1, or a balance (no dominance) of Th1 and Th2. [Diagram 44] Figure 44 shows a schematic of the hAd5 vector, SARS-CoV-2, spike, and constructs. (A) Human adenovirus serotype 5 with deleted E1, E2b, and E3 regions (hAd5[E1-, E2b-, E3-]) is shown. (B) SARS-CoV-2 virus displays spike (S) protein as a trimer on the viral surface. The S protein includes the N-terminus (NT), the S1 region including the RBD, the 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 and with predominantly cytoplasmic localization (N); (F) N with enhanced T cell stimulatory domain (N-ETSD), and (G) bivalent S-fusion + N-ETSD constructs are shown. [Diagram 45] Figure 45 shows micrographs demonstrating that N-ETSD localizes to endosomes, lysosomes, and autophagosomes. MoDCs were infected with Ad5 N-ETSD or N without ETSD and co-labeled with anti-Flag (N, N-ETSD here has a Flag tag) and anti-CD71 (endosomal marker), anti-Lamp1 (lysosomal marker), or anti-LC3a / b antibodies. (A) N-ETSD, (B) CD71, and (C) overlay. (D) N, (E) CD71, and (F) overlay. (G) NETSD, (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, co-localization is indicated by yellow arrows, white arrows indicate lymphocytes. [Diagram 46]Figure 46 demonstrates that patient plasma antibodies recognize SARS-CoV-2 antigens expressed by MoDCs after hAd5 S-fusion + N-ETSD infection. (A) MoDCs from two normal individuals were infected overnight with hAd5 vaccine and then exposed to previously infected patient plasma 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 MoDCs from two sources (B) MoDC1 and (C) MoDC2. (D) DMFI (difference in MFI for binding to non-infected and infected cells) is graphed for hAd5 S-WT, S-fusion, S-fusion + N-ETSD, and Null infected MoDCs. [Figure 47] Figure 47 shows exemplary results of T cell responses (of previously SARS-CoV-2 infected patients and virus naive T cells) to moDCs pulsed with SARS-CoV-2 peptides. T cells from all four previously SARS-CoV-2 infected patients (Pt) show significant IFN-γ responses to S1, S2, and N peptide pool pulsed moDCs compared to "None". T cells from virus naive (unexposed, UnEx) control individuals showed much lower responses. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc analysis for samples from each patient compared to "None" only (*p≦0.05, **p<0.01, ***p<0.001, ****p<0.0001). Graphed as mean and SEM; n=3-4. [Figure 48]Figure 48 demonstrates that peptide-pulsed MoDCs from patients previously infected with SARS-CoV-2 stimulate autologous patient T cells to secrete IFN-γ. (A) MoDCs from previously infected SARSCoV-2 patients Pt4 and Pt3 were pulsed overnight with SARS-CoV-2 peptide mixtures (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's post hoc multiple comparison analysis to compare each peptide pool to Veh (shown above the bars) or between peptide pools (above the lines) (*p<0.05, **p<0.01 and ****p<0.00001). Data are graphed as mean and SEM: n=3. [Figure 49] FIG. 49 demonstrates that IFN-γ secretion by T cells from patients previously infected with SARS-CoV-2 is greater in response to moDC expression of N-ETSD compared to N. (A) Experimental design. (B-D) Secretion of IFN-γ by autologous CD3+ T cells in response to hAd5-N-ETSD- and hAd5 N-expressing moDCs is shown. (E-G) Secretion of IL-4 by CD3+ cells in response to infected moDCs is shown on the same scale as IFN-γ for each. IFN-γ secretion by (H-J) 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's post hoc multiple comparison analysis (*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 graphed as mean and SEM; n=3-4. [Figure 50]Figure 50 shows that T cell responses of previously SARS-CoV-2 infected patients to the bivalent vaccine and its individual components reveal distinct antigen specificities of T cell populations. (A-C) CD3+ T cell IFN-γ responses for three patients. (D-F) CD3+ T cell IL-4 responses. (GI) CD4+ IFN-γ responses. (J-L) CD8+ IFN-γ responses. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc multiple comparison analysis to compare each antigen-containing construct to a null construct (*p<0.05 and ****p<0.00001). Comparisons to null are above the bars only; comparisons between antigen-expressing vaccines are above the lines. Data are graphed as mean and SEM: n=3-4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Various vaccine compositions and methods for inducing immunity against the SARS-CoV-2 virus and closely related and mutant forms of the SARS-CoV-2 virus not only promote the generation 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 also be used as an oral boost after currently known RNA-based vaccines.

[0024] 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 trafficking sequence that directs the modified nucleocapsid protein to the endosomal / lysosomal intracellular compartment, thereby utilizing a critical antigen presentation pathway to stimulate CD4+ T cells, which in turn direct dendritic cells to activate naive CD8+ cytotoxic T cells. Similarly, the modified spike protein preferably has a modification that enhances surface expression of the modified spike protein, thereby making the immune response to the spike antigen more robust. Indeed, while the vaccines disclosed herein (e.g., hAd5[E1-,E2b-] vaccines) exemplify the use of N antigens tagged with ETSD peptides, in principle any antigen can be advantageously redirected to the endosomal / lysosomal intracellular compartment.Exemplary antigens that may be tagged with ETSD for such use in adenovirus or yeast vaccine vectors 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, BA, BA, BA-1, BA-2, BA-3, BA-4, BA-5, BA-6, BA-7, BA-8, BA-9, BA-10, BA-11, BA-12, BA-13, BA-14, BA-15, BA-16, BA-17, BA-18, BA-19, BA-20, BA-21, BA-22, BA-23, BA-24, BA-25, BA-26, BA-27, BA-28, BA-29, BA-30, BA-31, BA-32, BA-33, BA-34, BA-35, BA-36, BA-37, BA-38, BA-39, BA-40, BA-41, BA-42, BA-43, BA-44, BA-45, BA-46, BA-47, BA-48, BA-49, BA-49, BA-41, BA-45, BA-46, BA-47, BA-48, BA-49, BA-49, BA-41, BA-42, BA-43, BA-44, BA-45, BA-46, BA-47, BA GE, 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 These include 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.

[0025] "N-ETSD" refers to a modified nucleocapsid protein of the SARS-CoV-2 virus that contains an endosomal targeting sequence. An exemplary N-ETSD has the amino acid sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:2.

[0026] "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 the amino acid sequence of SEQ ID NO:3 and the nucleotide sequence of SEQ ID NO:5.

[0027] "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 the amino acid sequence of SEQ ID NO:4 and the nucleotide sequence of SEQ ID NO:6.

[0028] "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.

[0029] "ETSD" refers to an endosomal targeting sequence. An exemplary ETSD has the amino acid sequence of SEQ ID NO:8.

[0030] "ACE2" refers to angiotensin-converting enzyme 2. An exemplary (human) ACE2 has the amino acid sequence of SEQ ID NO:9.

[0031] "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.

[0032] Modified spike and nucleocapsid constructs and methods Recombinant viruses and yeasts are disclosed herein. The viruses and yeasts disclosed herein may be useful for a variety of purposes, such as treating and / or preventing coronavirus disease. In one aspect, a replication-deficient adenovirus is disclosed herein, wherein the adenovirus comprises an E1 gene region deletion; an E2b gene region deletion; an E3 gene region deletion, a coronavirus 2 (CoV2) nucleocapsid protein, a nucleic acid encoding 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).

[0033] In one embodiment, the N-ETSD polypeptide can 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 comprises 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, methods are disclosed herein for enhancing the immunogenicity of intracellular antigens, the methods comprising tagging the antigen with ETSD and expressing the tagged antigen in an antigen-presenting cell (e.g., a dendritic cell).

[0034] In some embodiments, a fusion protein comprising N-ETSD and a CoV-2 nucleocapsid protein may 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%.

[0035] It is contemplated that the CoV-2 spike protein has at least 85% identity to 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 has at least 85% identity to 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%.

[0036] It is contemplated that the CoV-2 spike fusion protein has at least 85% identity to 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 has at least 85% identity to 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%.

[0037] 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 combinations thereof. Additionally, each of these encoded proteins may be further modified, as described in more detail below. Preferably, the recombinant yeast is Saccharomyces cerevisiae.

[0038] 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 still 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%.

[0039] In some embodiments of this second aspect, the CoV-2 spike protein or spike fusion protein comprises a sequence having at least 80% identity to SEQ ID NO:3 or SEQ ID NO:4. In other embodiments, the identity value is at least 85%. In still 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%.

[0040] In some embodiments, the nucleic acid encoding a 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 still 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%.

[0041] The adenoviruses and yeasts disclosed herein may further comprise nucleic acids encoding trafficking 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. The 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 vaccines disclosed herein may also encode a SARS-CoV-2 M protein with or without an ETSD tag. Additionally or alternatively, the adenovirus and / or yeast may be administered in combination with one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-invaxcept, IL-21, IPS1, & LMP1). By "in combination" in this context, it is intended that the immunostimulatory cytokine is administered within 24 hours of the adenovirus and / or yeast. That is, the adenovirus and / or yeast may be administered to a patient (e.g., a patient over 50 years of age) and then within 24 hours, one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-invaxcept, IL-21, IPS1, & LMP1) may be administered to the same patient. Additionally or alternatively, one or more immune stimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-invaxcept, IL-21, IPS1, & LMP1) may be administered to a patient (e.g., a patient over 50 years of age) and then within 24 hours, adenovirus and / or yeast may be administered to the same patient.

[0042] Above the age of 40, the ability of patients to mount a T cell response to a vaccine gradually declines. Thus, administering the adenovirus and / or yeast described herein in combination with one or more immune stimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-invaxcept, IL-21, IPS1, & LMP1) may be particularly useful in elderly patients. As used herein, "elderly" includes patients over 50 years of age, e.g., over 55 years of age, over 60 years of age, over 65 years of age, over 70 years of age, over 75 years of age, over 80 years of age, over 85 years of age, or over 90 years of age.

[0043] Most preferably, the recombinant virus is administered via subcutaneous or subdermal injection. However, in other contemplated embodiments, administration can also be intravenous. Alternatively or additionally, antigen presenting cells can be isolated or grown from the patient's cells, infected in vitro, and then injected into the patient.

[0044] In one aspect of any of the embodiments described above or elsewhere herein, the composition is formulated in a pharma- ceutically acceptable excipient suitable for administration to a subject.

[0045] It is still further contemplated that the recombinant viruses and yeast contemplated herein may further comprise sequences encoding at least one of a costimulatory molecule, an immunostimulatory cytokine, and a protein that prevents or downregulates checkpoint inhibition. 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, while suitable immunostimulatory cytokines include IL-2, IL-12, IL-15, IL-15 superagonist (N803), IL-21, IPS1, and / or LMP1, and / or suitable interfering proteins include antibody agonists or antagonists of CTLA-4, PD-1, TIM1 receptor, 2B4, and / or CD160.

[0046] It should be understood that all of the above costimulatory 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. Furthermore, the above exemplary stimulatory molecules are preferably expressed in full-length form as expressed in humans, although modified and non-human forms are also considered suitable, so long as such forms aid in the stimulation or activation of T cells. Thus, muteins, truncated forms, and chimeric forms are expressly contemplated herein.

[0047] The immunotherapeutic compositions disclosed herein can be either "prophylactic" or "therapeutic." When provided prophylactically, the compositions of the disclosure are provided prior to or upon detection of an outbreak of coronavirus disease, with the goal of preventing, inhibiting, or delaying the onset of coronavirus disease; and / or generally preventing or inhibiting the progression of coronavirus disease in an individual. Thus, prophylactic compositions can be administered to individuals believed to be free of coronavirus disease (healthy, or normal individuals) or individuals in whom coronavirus has not yet been detected. Individuals at high risk of developing coronavirus disease can be treated prophylactically with the compositions of the disclosure.

[0048] When provided therapeutically, the immunotherapeutic composition is provided to an individual diagnosed with a coronavirus disease with the objective of ameliorating or curing the coronavirus disease; increasing the individual's survival rate; or preventing, inhibiting, reversing or delaying the onset of the coronavirus disease in the individual.

[0049] In yet another embodiment, disclosed herein is a vaccine composition comprising the adenovirus or yeast disclosed above, 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.

[0050] Also disclosed herein are methods for preventing and / or treating coronavirus diseases, particularly COVID-19. Preferably, the methods include using a virus or yeast vector encoding a wild-type or modified form of the coronavirus nucleocapsid protein and / or a wild-type or modified form of the spike protein in an immunogenic composition administered to a subject individual. The virus and / or yeast vaccine so administered will infect the individual with the wild-type or modified form of CoV-2, nucleocapsid or spike protein. Once that is in place, the individual will have an immune response against it and will be vaccinated. In particular, since the nucleocapsid protein and the spike protein are relatively conserved polypeptides, an immune response can be elicited against diverse members of the coronavirus family.

[0051] In the case where the recombinant vector is an adenovirus, the adenovirus vector can be modified to encode wild-type or modified forms of nucleocapsid protein and / or spike protein. Similarly, in the case of yeast, the yeast vector can also be modified to encode wild-type or modified forms of nucleocapsid protein and / or spike protein. As shown in more detail below, after administration of an immunogenic composition comprising a virus and / or yeast vector in a patient in need thereof, a positive immune response is obtained for cell-mediated immunity. Thus, in one embodiment, the present disclosure contemplates creating expression of coronavirus spikes on the yeast surface. In such an embodiment, the yeast functions as an avatar coronavirus to stimulate B cells, which then provide humoral immunity.

[0052] As disclosed herein, a next-generation bivalent human adenovirus serotype 5 (hAd5) vaccine capable of inducing immunity in patients with pre-existing adenovirus immunity, which contains both an S sequence optimized for cell surface expression (S-fusion) and a conserved nucleocapsid (N) antigen designed to be trafficked to the endosomal intracellular compartment, has the potential to generate durable immune protection. As further described herein, such a bivalent vaccine has been found to be optimized for immunogenicity as evidenced by the following findings: 1) The optimized S-fusions showed improved S receptor binding domain (RBD) cell surface expression compared to S-WT, which had barely detectable surface expression; 2) the expressed RBD from the S-fusions retained conformational integrity and recognition by ACE2-Fc; 3) viral N proteins modified with enhanced T cell stimulatory domains (ETSDs) localized to endosomal / lysosomal intracellular compartments for MHC I / II presentation; and 4) Optimization of these S and N (S-fusions and N-ETSDs) generated enhanced de novo antigen-specific B cell and CD4+ and CD8+ T cell responses in antigen-native preclinical models.

[0053] Both T cell and antibody immune responses to the S and N components showed a T-helper 1 (Th1) bias. The antibody responses were neutralizing as shown by an independent SARS-CoV-2 neutralization assay. Thus, in one embodiment, the next generation bivalent hAd5 S-fusion + N-ETSD vaccine provides robust and durable cell-mediated and humoral immunity against SARS-CoV-2 infection. Additionally, and as further described in more detail below, the vaccine constructs can be administered orally, intranasally, or sublingually. Thus, 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 and humoral immunity. Viewed from another perspective, substantial immunity can be generated by injection, oral / mucosal administration, alone or in combination. In one embodiment, the COVID-19 vaccine disclosed herein generates long-term T and B cell memory.

[0054] Coronavirus and vaccines for it Coronaviruses are found in avian and mammalian species. They are similar to each other in morphology and chemical structure: for example, human and cattle coronaviruses are antigenically related. However, there is no evidence that human coronaviruses can be transmitted by objects. In animals, various coronaviruses invade many different tissues and cause diverse diseases in humans. One such disease is the severe acute respiratory syndrome (SARS) coronavirus disease that spread in 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. In December 2019, reports emerged from Wuhan, China, of a new infectious respiratory disease with high morbidity and mortality rates1-3 indicative of human-to-human transmission. The causative agent was quickly identified as a novel coronavirus and named SARS-coronavirus 2 (SARS-CoV-2). The resulting disease, termed COVID-19, rapidly became a global pandemic that disrupted socio-economic life and caused over 32 million infections and over 1.1 million deaths worldwide as of late October 2020.

[0055] COVID 19 usually starts with a fever above 38°C. Early symptoms may also include cough, sore throat, fatigue, and mild respiratory symptoms. Within 2 days to a week, patients may have difficulty breathing. Patients with more advanced stages of COVID 19 develop either pneumonia or respiratory distress syndrome. Public health interventions, such as surveillance, travel restrictions, and quarantine, have been used to contain the spread of COVID 19. However, it is not known whether these strict containment measures can withstand each emergence of COVID 19 in humans. Furthermore, the potential of this new and sometimes deadly CoV as a bioterrorism threat is clear.

[0056] Coronavirus virions are spherical to polymorphic enveloped particles. The envelope is studded with protruding glycoproteins and surrounds a core of matrix protein that contains positive-sense RNA (Mr 6×10) associated with nucleocapsid proteins. 6 In that context, it should be noted that the terms "nucleocapsid protein", "nucleoprotein" and "nucleocapsid" are used interchangeably throughout this disclosure. Coronavirus nucleocapsid (N) is a structural protein found in all coronaviruses, including COVID 19. Nucleocapsid protein forms a complex with genomic RNA and interacts with viral membrane proteins during virion assembly, playing an important role in increasing the efficiency of viral transcription and assembly.

[0057] Another protein found throughout all coronavirus virions is the viral spike (S) protein. Coronaviruses are typically large positive-stranded RNA viruses with a broad host range. Like other enveloped viruses, CoVs enter target cells by fusion of the viral and cellular membranes, a process mediated by the viral spike (S) protein.

[0058] SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA betacoronavirus composed primarily of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope, as well as viral membrane and genomic RNA. Of these, S is the largest and N is the most predominant. The S glycoprotein is displayed as a trimer on the viral surface (Figure 33, Panel A), while N is located in the viral particle. A schematic of the primary structure of S is shown in Figure 33, Panel B. The sequence of SARS-CoV-2 was published and compared to previous coronaviruses. Shortly after this, the crystal structure of the S protein was reported. The virus uses the S protein to enter host cells by interaction of the S receptor binding domain (S RBD) with angiotensin-converting enzyme 2 (ACE2), an enzyme expressed on diverse cell types in the nose, oral cavity, intestine, and lungs, as well as other organs, and importantly, on alveolar epithelial cells in the lungs, where infection primarily manifests. As depicted in Figure 33, panel B, the S RBD is found within the S1 region of the spike polypeptide.

[0059] The methods and compositions disclosed herein target the nucleoprotein and spike protein, which are conserved across all types of coronaviruses. In one embodiment, the disclosure provides a vaccine formulation comprising a recombinant, wherein the recombinant comprises a nucleic acid encoding the nucleocapsid protein of coronavirus 2 (CoV2) or a modified form thereof, and / or the recombinant encodes the spike protein of CoV2 or a modified form thereof. The vaccine formulation may be useful for treating a disease, such as a coronavirus-mediated disease or infection. Accordingly, in another embodiment, a method of treating a coronavirus disease for a patient in need thereof is contemplated. Such a method preferably comprises administering to a subject an immunotherapeutic composition comprising a recombinant, wherein the recombinant would comprise a nucleic acid encoding the nucleocapsid protein of coronavirus 2 (CoV2) or a modified form thereof, and / or a nucleic acid encoding the 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).

[0060] For example, the disclosure provides a method of 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 immunotherapeutic composition comprising a recombinant virus, the recombinant virus comprising a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid protein or a modified form thereof, and administering to the subject a second immunotherapeutic composition comprising a recombinant yeast, the recombinant yeast comprising a nucleic acid encoding a CoV2 spike protein. The first and second immunotherapeutic compositions may be administered to the patient simultaneously or sequentially.

[0061] From a different perspective, contemplated herein is a viral vector (e.g., a recombinant adenovirus genome, optionally with a deleted or non-functional E2b gene) 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 costimulatory molecules. Most typically, the nucleic acid also comprises a trafficking signal that directs the peptide product encoded by the nucleic acid to the cytoplasm, an endosomal compartment, or a lysosomal compartment, and the peptide product may also comprise a sequence portion that enhances the intracellular turnover of the peptide product.

[0062] The majority of current SARS-CoV-2 vaccines in development target S because the generation of antibodies against the RBD may neutralize the virus' ability to bind to host cells. Support for the RBD as a critical antigen was recently confirmed when it was reported that in 44 hospitalized COVID-19 patients, RBD-specific IgG responses and neutralizing antibody titers were detectable in all patients by 6 days after PCR confirmation of infection, and the two correlated. In addition to humoral responses, S epitopes are also frequent targets of T cells in patients who have recovered from COVID-19, providing further justification for the inclusion of S in preventive immunization strategies.

[0063] Despite the urgent need for rapid development of a SARS-CoV-2 vaccine, reliance on any one of the antigenic cargoes or immunological pathways resulting in monovalent vaccines under development is not without risk. Evaluation of nearly 4000 SARS-CoV-2 genome sequences has identified numerous mutations in S, with the D614G variant recently emerging as a potentially more infectious strain, 6 months after identification of the original virus.

[0064] In the design of the vaccine disclosed herein, an optimized N sequence was added to overcome the risk of emergence of new strains of the virus with mutations in S and to provide an additional antigen against which a response can be elicited. The N protein is an antigenic SARS-CoV-2-associated protein that is highly conserved and has been previously studied as an antigen in coronavirus vaccine design against SARS-CoV. N is associated with viral RNA within the virus and has a role in viral RNA replication, viral particle assembly, and release. SARS-CoV-2 N is a highly antigenic protein, and nearly all patients infected with SARS-CoV-2 have an antibody response against N. Furthermore, another study reported that most, but not all, COVID-19 survivors tested showed N-specific CD4+ T cell responses.

[0065] Currently, there is strong interest in generating humoral responses to vaccines, and perhaps less attention has been 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 antibody production and should be a critical consideration for COVID-19 vaccine efficacy.

[0066] First, humoral and T cell responses are highly correlated, and titers of neutralizing antibodies are proportional to T cell levels, suggesting that T responses are necessary for effective humoral responses. It is well established that activation of CD4+ T helper cells enhances B cell production of antibodies. Second, virus-specific CD4+ and CD8+ T cells are not only widely detected in COVID-19 patients, but based on findings from patients who recovered from the closely related SARS-CoV, such T cells persist for at least 6-17 years, suggesting that T cells may be an important part of long-term immunity. These T cell responses are primarily directed against N, and it has been reported that in all 36 convalescent COVID-19 patients in their study, the presence of CD4+ and CD8+ T cells recognizing multiple regions of the N protein could be demonstrated. Blood from 23 individuals who had recovered from SARS-CoV was examined, and memory T cells acquired 17 years earlier were also found to recognize multiple proteins of SARS-CoV-2. These findings highlight the importance of designing vaccines using the highly conserved nucleocapsid present in both SARS-CoV and SARS-CoV-2.Third, recovered patients exposed to SARS-CoV-2 were found to have no seroconversion and evidence of T cell responses.T-cell-based responses take on added importance in light of the finding that, in at least one study, neutralizing antibody titers decline in some COVID-19 patients after approximately 3 months.

[0067] In one embodiment, the vaccines disclosed herein result in the production of T cells in addition to humoral responses. A bivalent vaccine comprising the S RBD, represented by the inclusion of the full-length S, which contains many antigens, SD1, S1 and S2 epitopes along with N, has been contemplated and shown to be more effective at eliciting both T cell and antibody-based responses than constructs using either antigen alone, by presenting both unique and conserved SARS-CoV-2 antigenic sites to the immune system. The importance of both S and N was highlighted by identifying that both S and N antigens, as a priori potential B and T cell epitopes of the SARS-CoV virus, are highly similar to SARS-CoV-2, which is predicted to induce both T and B cell responses.

[0068] Additional considerations for the design of an effective vaccine are the possibility of antigen presentation on the surface of recombinant protein-expressing cells and expression in a conformation that recapitulates natural virus infection. First, wild-type N does not have the signaling domain that directs it to endosomal processing and ultimately MHC class II complex presentation to CD4+ T cells, so the wild-type N sequence is not optimal for inducing vigorous CD4+ T cell responses necessary for both cell-mediated and B cell memory. To overcome this limitation, an enhanced T cell stimulatory domain (ETSD) for N allows for the necessary processing and presentation. One preferred ETSD polypeptide has the amino acid sequence of SEQ ID NO:8. Of course, it should be understood that this sequence can be modified while maintaining the desired activity. Thus, the ETSD sequence can have at least 85%, or at least 90%, or at least 95, or at least 98% identity to SEQ ID NO:8. Second, to display the highly antigenic RBD of S on the cell surface, optimization of the wild-type S protein into an "S-fusion" sequence increases the likelihood of native folding, increased stability, and proper cell surface expression of the RBD. Thus, in one embodiment, the vaccine construct comprises an S-fusion sequence and an N-ETSD sequence.

[0069] The vaccine platform used herein 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 (Figure 33, panel C) is distinguished from other first-generation [E1-, E3-] recombinant Ad5 platforms primarily by having additional deletions in the early gene 2b (E2b) region that eliminate expression of the viral DNA polymerase (pol) and in the pre-terminal protein (pTP) gene, as well as by its propagation in the E.C7 human cell line. These deletions of the E2b region confer advantageous immune properties in patients with pre-existing adenovirus (Ad) immunity by minimizing immune responses against Ad5 viral proteins, such as viral fiber, thereby eliciting potent immune responses against specific antigens. As an added benefit of these deletions, the vector has an expanded gene carrying / cloning capacity compared to the first generation Ad5[E1-, E3-] vector. This next generation hAd5[E1-, E2b-, E3-] vaccine platform, in contrast to Ad5[E1-, E3-] based platforms, does not promote activity that suppresses innate immune signaling, thereby allowing improved vaccine efficacy and a superior safety profile independent of prior Ad immunity. Because these deletions allow the hAd5 platform to be effective even in the presence of pre-existing Ad immunity, this platform allows for relatively long-term antigen expression without a significant decrease in anti-vector immunity. Thus, unlike the first generation Ad platform, which faces the limitations of pre-existing and vaccine-induced Ad immunity, it is also possible to use the same vector / construct in an allogeneic prime-boost treatment regimen. Importantly, this next generation Ad vector has been proven safe in over 125 patients with solid tumors. In these Phase I / II trials, CD4+ and CD8+ antigen-specific T cells successfully produced multiple somatic antigens (CEA, MUC1, Brachyury) even in the presence of pre-existing Ad immunity.

[0070] This disclosure provides confirmation of enhanced cell surface expression of S RBD expressed from S-fusions upon ACE2-Fc binding, and insight into physiologically relevant folding. N-ETSD protein successfully localized to endosomal / lysosomal intracellular compartments for MHC presentation, resulting in the generation of both CD4+ and CD8+ T cell responses. Immunization of CD-1 mice with hAd5 S fusions + N-ETSD vaccines elicited both humoral and cell-mediated immune responses to the vaccine antigen. CD8+ and CD4+ T cell responses were observed for both S and N. Statistically significant IgG responses were seen for antibody production against S and N. Potent neutralization of SARS-CoV-2 by sera from hAd5 S fusions + N-ETSD-immunized mice was confirmed by two independent SARS-CoV-2 neutralization assays: a cPass assay measuring competitive inhibition of RBD binding to ACE2,44 and a live SARS-CoV-2 virus assay using infected Vero E6 cells. T cell responses and humoral responses to S and N were biased towards Th1 specific responses.

[0071] Collectively, these findings indicate that the hAd5 S-fusion + N-ETSD vaccine composition may be particularly effective against SARS-CoV-2.

[0072] Recombinant viruses With respect to recombinant viruses, it is contemplated that all known methods for producing recombinant viruses are suitable for use herein, however, particularly preferred viruses are those already established in therapy, including adenoviruses, adeno-associated viruses, alphaviruses, herpes viruses, lentiviruses, etc. Among other suitable options, adenoviruses are particularly preferred.

[0073] Moreover, in general, it is further preferred that the virus is a replication-deficient and non-immunogenic virus. For example, suitable viruses include recombinant alphaviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like. However, adenoviruses are particularly preferred. For example, recombinant replication-deficient adenoviruses suitable for multiple vaccinations as well as vaccination of individuals with pre-existing immunity to adenoviruses are preferred (see, for example, WO 2009 / 006479 and WO 2014 / 031178, which are incorporated herein by reference in their entirety). In some embodiments, the replication-deficient adenovirus vector comprises a replication-deficient adenovirus 5 vector. In some embodiments, the replication-deficient adenovirus vector comprises a deletion in the E2b region. In some embodiments, the replication-deficient adenovirus vector further comprises a deletion in the El region. In that regard, it should be noted that the deletion of the E2b gene and other late proteins in the recombinant replication-deficient adenovirus reduces immunogenicity. Moreover, due to these specific deletions, such recombinant viruses are replication-deficient and capable of carrying relatively large recombinant cargoes.

[0074] For example, WO 2014 / 031178 describes the use of such recombinant viruses expressing CEA (colorectal embryonic antigen) to provide an immune response against colon cancer. Furthermore, relatively high titers of recombinant viruses can be achieved using recombinant human 293 cells, as reported (e.g., J Virol. 1998 Feb;72(2):926-933).

[0075] The El deleted adenoviral vector Ad5[E1-] has been constructed such that the transgene replaces only the El 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 are unable to generate infectious virus after infection of cells that do not express the Ad5 El gene. Recombinant Ad5[E1-] vectors grow in human cells, allowing Ad5[E1-] vector replication and packaging. Ad5[E1-] vectors have many positive attributes; the most important of which is their relative ease of scale-up and cGMP production. Currently, well over 220 human clinical trials have used Ad5[E1-] vectors, with over 2000 subjects receiving the virus sc, im, or iv. Furthermore, Ad5 vectors do not integrate; their genome remains episomal. In general, vectors that do not integrate into the host genome have very low, if any, risks of insertional mutagenesis and / or germline transmission. Conventional Ad5[E1-] vectors have a carrying capacity approaching 7 kb.

[0076] One barrier to the use of first generation (El-deleted) Ad5-based vectors is the high frequency of pre-existing anti-adenovirus type 5 neutralizing antibodies. Attempts to overcome this immunity are described in WO 2014 / 031178, incorporated herein by reference. In particular, a novel recombinant Ad5 platform with a deletion in the early 1 (El) gene region and an additional deletion in the early 2b (E2b) gene region (Ad5[E1-, E2b-]) is described. The deletion of the E2b region (encoding DNA polymerase and pre-terminal proteins) results in reduced viral DNA replication and late viral protein expression. E2b-deleted adenoviral vectors provide an improved Ad-based vector that is safer, more effective, and more versatile than first generation adenoviral vectors.

[0077] In further embodiments, adenoviral vectors contemplated for other uses include adenoviral vectors having a deletion in the E2b region of the Ad genome, and optionally a deletion in El, E3, and optionally a partial or complete removal of the E4 region. In further embodiments, the adenoviral vectors used herein have a deletion of El and / or pre-terminal protein function of the E2b region. In some cases, such vectors have no other deletions. In another embodiment, the adenoviral vectors used herein have a deletion of El, DNA polymerase, and / or pre-terminal protein function.

[0078] The term "E2b deletion" as used herein refers to a specific DNA sequence that has been mutated to prevent the expression and / or function of at least one E2b gene product. Thus, in certain embodiments, "E2b deletion" is used in reference to a specific DNA sequence that has been deleted (removed) from the Ad genome. An E2b deletion or "comprising a deletion in the E2b region" refers to a deletion of at least one base pair in 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 of more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs in the E2b region of the Ad genome. An E2b deletion may be a deletion that prevents expression and / or function of at least one E2b gene product, and thus encompasses deletions within exons coding for E2b-specific proteins, as well as deletions within promoter and leader sequences. In a particular embodiment, an E2b deletion is a deletion that prevents expression and / or function of both the DNA polymerase and the pre-terminal proteins of the E2b region. In a further embodiment, an "E2b deletion" refers to one or more point mutations in the DNA sequence of this region of the Ad genome, such that one or more encoded proteins are non-functional. Such mutations include residues replaced with different residues, leading to a change in the amino acid sequence resulting in a non-functional protein.

[0079] As can be easily understood, the desired nucleic acid sequence (for expression from virus-infected cells) is under the control of suitable regulatory elements well known in the art. In view of the above, it should be understood that the compositions and methods presented are not only suitable for specifically directing virus-expressed antigens to one or another (or both) MHC system, but also provide increased stimulatory effect on CD8+ and / or CD4+ cells by including various costimulatory molecules (e.g., ICAM-1 (CD54), ICOS-L, LFA-3 (CD58), and at least one of B7.1 (CD80) and B7.2 (CD86)) and by secretion or membrane-bound presentation of checkpoint inhibitors.

[0080] 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 effecting expression of the recombinant payload in infected cells.

[0081] Regardless of the type of recombinant virus, it is contemplated that the virus can be used to infect patient (or non-patient) cells ex vivo or in vivo. For example, the virus can be injected subcutaneously or intravenously, or administered intranasally or via inhalation, to infect the patient's cells, particularly antigen-presenting cells. Alternatively, the patient's (or from an allogeneic source) immune-competent cells (e.g., NK cells, T cells, macrophages, dendritic cells, etc.) can be infected in vitro and then injected into the patient. Alternatively, immunotherapy does not have to rely on viruses, but can be achieved by nucleic acid transfection or vaccination using RNA or DNA, or other recombinant vectors that result in the expression of neo-epitopes (e.g., single peptides, tandem minigenes, etc.) in desired cells, particularly immune-competent cells. Such nucleic acids are typically delivered in association with lipid formulations to protect the nucleic acid from degradation and to facilitate uptake of the nucleic acid into target cells.

[0082] As mentioned above, the desired nucleic acid sequence (for expression from virus-infected cells) is under the control of suitable regulatory elements known in the art. For example, suitable promoter elements include constitutive strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoters), although inducible promoters are also considered suitable for use in the present invention, especially when the induction conditions are typical for the tumor microenvironment. For example, inducible promoters include those 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 1 (Mx1) promoters, and the like.

[0083] Replication-deficient adenoviruses comprising an E1 gene region deletion, an E2b gene region deletion, and a nucleic acid encoding coronavirus 2 (CoV2) nucleocapsid protein and / or a CoV2 spike protein, as disclosed herein, can be administered to patients in need of inducing immunity against CoV2. The route and frequency of administration of the therapeutic compositions described herein, as well as the dosage, can vary between individuals and with disease severity, and can be readily established using standard techniques. In some embodiments, administration is at a dose of 4.8-5.2x10 11 Replication-deficient adenovirus particles, or 4.9-5.1x10 11 Replication-deficient adenovirus particles, or 4.95-5.05x10 11 Replication-deficient adenovirus particles, or 4.99-5.01x10 11 The present invention relates to a method for the delivery of a replication-deficient adenoviral particle.

[0084] Administration of the viral particles can be via a variety of suitable routes for delivery. One preferred route contemplated herein is by injection, such as intradermal, intramuscular, intravenous or subcutaneous injection. In some embodiments, subcutaneous delivery may be preferred.

[0085] Recombinant yeast In the context of the yeast expression and vaccination system, it is contemplated that all known yeast strains are deemed suitable for use herein. However, it is preferred that the yeast is a recombinant Saccharomyces strain genetically modified with a nucleic acid construct encoding a protein selected from the group consisting of coronavirus 2 (CoV2) nucleocapsid protein, CoV2 spike protein, and combinations thereof, thereby initiating an immune response against CoV2 viral disease. In one aspect of any of the disclosed embodiments described above or elsewhere herein, the yeast vehicle is whole yeast. The whole yeast is killed in one aspect. In one aspect, the whole yeast is heat inactivated. In a preferred embodiment, the yeast is heat inactivated whole yeast from Saccharomyces cerevisiae.

[0086] The use of yeast-based therapeutic compositions has been disclosed in the art, for example WO 2012 / 109404 discloses yeast compositions for the treatment of chronic Hepatitis B infection.

[0087] It is noted that any yeast strain can be used to generate the yeast vehicle of the present disclosure. Yeasts are unicellular microorganisms that belong to one of three classes: Ascomycetes, Basidiomycetes, and Fungi Imperfecti. One consideration regarding the selection of a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In a preferred embodiment, the yeast is a non-pathogenic strain, such as Saccharomyces cerevisiae, since a non-pathogenic yeast strain will minimize any adverse effects on the individual to whom the yeast vehicle is administered. However, pathogenic yeasts can also be used if the pathogenicity of the yeast can be negated using pharmaceutical intervention.

[0088] For example, suitable genera of yeast strains include Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one embodiment, the yeast genus is selected from Saccharomyces, Candida, Hansenula, Pichia or Schizosaccharomyces, and in a preferred embodiment, Saccharomyces is used. Species of yeast strains that may 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, and the like. lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica.

[0089] It should be further understood that these species numbers include various subspecies, types, subtypes, etc., that are intended to be included in the aforementioned species. In one aspect, yeast species used in the present disclosure include S. cerevisiae, C. albicans, H. polymorpha, P. pastoris, and S. pombe. S. cerevisiae is useful because it is relatively easy to manipulate, and is "Generally Recognized As Safe" or "GRAS" for use as a food additive (GRAS, FDA proposed rule 62FR18938, Apr. 17, 1997). Thus, yeast strains that can replicate plasmids to particularly high copy numbers, such as S. cerevisiae cir strains, are specifically contemplated herein. S. cerevisiae strains are among the strains that can support expression vectors that provide high level expression of one or more target antigens and / or antigen fusion proteins and / or other proteins. Additionally, any mutant yeast strain can be used, including those that exhibit reduced post-translational modification of the expressed target antigen or other protein, such as mutations in enzymes that extend N-linked glycosylation.

[0090] Expression of the intended peptide / protein in yeast can be achieved 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 such that the nucleic acid molecule is operably linked to a transcription control sequence such that, upon transformation into a host yeast cell, constitutive or regulated expression of the nucleic acid molecule can be achieved. As will be readily understood, a nucleic acid molecule encoding one or more proteins can be present 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, such as promoters and upstream activating sequences.

[0091] Any suitable yeast promoter can be used in the disclosed methods and compositions, 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-1. alpha (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also called TDH3 for triosephosphate dehydrogenase), galactokinase (GAL1), galactose-1-phosphate uridyl-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, which is induced when the glucose concentration in the cell is low (e.g., about 0.1 to about 0.2 percent), as well as the CUP1 promoter and the TEF2 promoter. Similarly, numerous upstream activating sequences (UAS), also called enhancers, are known. Upstream activation sequences for expression in Saccharomyces cerevisiae include the UAS of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7, and GAL10, as well as other UAS activated by the GAL4 gene product, where the ADH2 UAS is used in one embodiment. Since the ADH2 UAS is activated by the ADR1 gene product, it may be preferable to overexpress the ADR1 gene when a heterologous gene is operably linked to the ADH2 UAS. Transcription termination sequences for expression in Saccharomyces cerevisiae include the termination sequences of the α-factor, GAPDH, and CYC1 genes.Transcriptional control sequences for expressing genes in methylotrophic yeast include the transcriptional control regions of the genes encoding alcohol oxidase and formate dehydrogenase.

[0092] Similarly, transfection of a nucleic acid molecule into a yeast cell according to the present disclosure can be accomplished by any method by which the nucleic acid molecule is administered into the cell, including nucleic acid, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. The transfected nucleic acid molecule is integrated into the yeast chromosome or maintained on an extrachromosomal vector using techniques known to those of skill in the art. As discussed above, yeast cytoplasts, yeast ghosts, and yeast membrane particles or cell wall preparations can also be produced recombinantly by transfecting an intact yeast microorganism or yeast spheroplast with the desired nucleic acid molecule, producing the antigen therein, and then further manipulating the microorganism or spheroplast using techniques known to those of skill in the art to produce a cytoplast, ghost, or intracellular yeast membrane extract or fraction thereof that contains the desired antigen or other protein. Further exemplary yeast expression systems, methods, and conditions suitable for use in the present invention are described in U.S. Patent Application Publication Nos. 20100196411A1, 2017 / 0246276, or 2017 / 0224794, and 2012 / 0107347.

[0093] The recombinant viruses and yeasts so produced can then be used, individually or in combination, as therapeutic vaccines in pharmaceutical compositions, typically containing 10 4 ~10 13 Virus or yeast particles, or more preferably 10 per dosage unit. 9 ~10 12 The virus or yeast particles are formulated as a sterile injectable composition with the virus. Alternatively, the virus or yeast may be used to infect patient cells ex vivo, and the infected cells are then injected into the patient. However, alternative formulations are also contemplated as suitable for use herein, and all known routes and modes of administration are contemplated herein.

[0094] In further contemplated embodiments, the second generation hAd5[E1-, E2b-, E3-] based vaccine disclosed herein overcomes pre-existing anti-Ad5 immunity. To avoid the Ad immunization barrier and to avoid adverse conditions for 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 former name of the adenovirus vector was Ad5, ETBX in the literature.)

[0095] E2b-deleted hAd5 vectors have a gene carrying capacity of up to 12-14 kb, compared with the 7-kb capacity of first generation Ad5[E1-] vectors, providing space for multiple genes if needed. hAd5[E1-, E2b-, E3-]-based recombinant vectors are generated using the human E.C7 cell line. The deletion of the E2b region also confers favorable immunological properties to these novel Ad vectors, eliciting strong immune responses against certain non-viral antigens while minimizing immune responses against Ad viral proteins.

[0096] hAd5[E1-, E2b-, E3-] vectors induce strong cell-mediated immune (CMI) responses and Abs against vectored antigens, even in the presence of Ad immunity. hAd5[E1-, E2b-, E3-] vectors also have reduced incidence of adverse reactions, particularly hepatotoxicity and tissue damage, compared to Ad5[E1-] vectors. In one embodiment, the reduced inflammatory response to hAd5[E1-, E2b-, E3-] vector viral proteins, and the resulting evasion of pre-existing Ad immunity, enhances the ability of hAd5[E1-, E2b-, E3-] vectors to infect dendritic cells (DCs), enhancing vaccine immunity. Furthermore, increased infection of other cell types provides the high levels of antigen presentation required for strong CD8+ and CD4+ T cell responses, leading to the development of memory T cells. 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 a COVID-19 vaccine in a fast and efficient manner. In one embodiment, a prophylactic vaccine will be tested against COVID-19 by utilizing this new hAd5 vector system that overcomes the barriers found in other Ad5 systems, allowing immunization of individuals previously exposed to Ad5.

[0097] A proven track record of rapid vaccine development utilizing second generation human (hAd5) adenovirus platforms during pandemic threats: the H1N1 experience in 2009. It is critical to use modernized vaccine technologies to address emerging pathogen threats, especially in times of pandemics. These technologies will harness the power of genome sequencing, rapid transfection in established vaccine vectors to rapidly identify constructs with high immunogenicity.

[0098] Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from modern 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 against various antigenic targets. This vector platform expressed the hemagglutinin (HA) and neuraminidase (NA) genes from the 2009 H1N1 pandemic virus. The inserts were consensus sequences designed from viral isolate sequences, and vaccines were rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice that provided protection from lethal virus challenge. In ferrets, vaccination prevented disease development and significantly reduced virus titers in nasal washes. H1N1 cells mediated antibody induction that correlated with immunity and prevention of disease symptoms and reduced virus replication. Thus, hAd5[E1-, E2b-, E3-] demonstrated potential for the rapid development of effective vaccines against infectious diseases.

[0099] For at least these reasons, when a contemplated vaccine composition is based on an adenoviral vector, it is generally preferred to use the recombinant hAd5[E1-, E2b-, E3-] platform to generate recombinant nucleic acids for therapeutic use in humans. EXAMPLES

[0100] Example 1: Selected hAd5 Vaccine Constructs and Results Constructs constructed and tested, specifically hAd5-COVID-19 vaccine constructs E1-, E2b-, E3-hAd5 vectors with SARS-CoV-2 (S / N) protein inserts are disclosed herein (Figure 26). The constructs were tested in preclinical experiments including in vitro expression (Figure 27) and small animal immunogenicity. Multiple COVID-19 constructs include RBD alone, S1 alone, S1 fusion protein, and combinations of RBD, S1 and S1 fusion with N. Preliminary in vitro studies demonstrate that these constructs (Figure 28) recognize convalescent serum antibodies and can function as surrogate vaccines.

[0101] 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 demonstrated that the N protein is immunogenic when integrated with intracellular trafficking constructs. To date, all vaccine strategies in development include development of immunogenicity against the spike (S) protein. However, very recent evidence in patients who have recovered from COVID-19 has demonstrated Th1 immunity generated against the nucleocapsid (N). Further reports have further confirmed in predictive bioinformatics models that T and B cell epitopes are highest for both the spike glycoprotein and nucleoprotein. The present disclosure confirms that by combining S with N, long-term cell-mediated immunity with a Th1 phenotype can be induced. Indeed, in light of the mutations in S and the knowledge that the structural N protein is highly conserved within the coronavirus family, there is significant potential for this combination vaccine to serve as a long-term "universal" COVID-19 vaccine.

[0102] Example 2: Immunogenicity study (small animal model): Homologous prime boost immunogenicity in BALB-c mice. Mice are treated with 1, 2 or 3 doses of hAd5 COVID-19 vaccine and serum and splenocyte samples are tested for SARS-CoV-2 antigen-specific immune responses. Serum is 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 simulation assays.

[0103] The results show promising immunogenic activity. In one embodiment, the vaccine containing SARS-CoV-2 nucleocapsid + enhanced T cell stimulatory domain (ETSD), hAd5[E1-, E2b-, E3-]N-ETSD, alters T cell responses to nucleocapsid. Mice were immunized subcutaneously (SC) twice at 7-day intervals with a dose of 1010VP. Blood was collected at several time points and spleens were collected at sacrifice to perform immunogenicity experiments. Splenocytes were isolated and tested for cell-mediated immune (CMI) responses. The results showed that SARS-CoV-2 nucleocapsid antigen-specific CMI responses were detected by ELISpot and flow cytometry analysis in the spleens of all mice immunized with the hAd5[E1-, E2b-, E3-]N-ETSD vaccine, but not in mice immunized with the vector control (hAd5[E1-, E2b-, E3-] null). 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-]-null) (Figures 29 and 30).

[0104] Example 3: Enhancement of RBD cell surface expression: Further evidence of the potential enhanced immunogenic value of N when combined with S was the surprising finding of enhanced surface expression of RBD protein in 293 cells transfected with the N-ETSD+S construct, as seen in Figure 31. Expression and presentation of the RBD appears to be highly important, as evidenced by recent reports by others showing rare but recurrent RBD-specific antibodies with strong antiviral activity were found in all tested individuals who had recovered from COVID-19 infection. This finding of enhanced expression of the RBD when N was combined with the S-fusion was corroborated in a study using plasma from patients who had recovered from COVID-19 infection (Figure 32). Alternative constructs of RBD-ETSD could serve as alternative vaccines.

[0105] In summary, based on the enhanced expression and exposure of the RBD protein by the S-fusion and S-fusion+N constructs, both were tested in hAd5 vectors. Furthermore, recent clinical data from patients who recovered from COVID-19 and the N-construct demonstrated long-lasting CD4 + Based on supportive preclinical data that S-fusions induce Th1 cell-mediated immunity, this combination of S-fusions plus N-constructs may provide long-lasting immunity that goes beyond short-term neutralizing antibodies.

[0106] Example 4: Immunogenicity testing of candidate COVID-19 vaccine constructs 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.

[0107] Construct Description: Two second generation hAd5-based COVID-19 vaccine constructs were evaluated. The first is a hAd5 vector with a SARS-CoV-2 spike protein insert (see Figure 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.

[0108] Immunogenicity studies: Homologous prime-boost immunogenicity in mice is tested by treating mice with 1, 2 or 3 doses of the adenoviral vaccine candidates listed in Figure 26, and serum and splenocyte samples are tested for SARS-CoV-2 antigen-specific immune responses. Serum is 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 simulation assays. Data from these studies are disclosed throughout this disclosure.

[0109] SARS-CoV-2 Virus Neutralization Study: Serum from mice immunized during the course of the immunogenicity study described above will be used and sent to a testing laboratory for SARS-CoV-2 neutralization studies to be performed in their ABSL-3 facility. The serum will be tested for COVID 19 virus neutralization activity by mixing various dilutions of serum 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 is considered the endpoint neutralization titer.

[0110] Immunogenicity and efficacy evaluation in non-human primates: Rhesus macaques are treated with three doses of the adenoviral vaccine candidates listed in Figure 26. SARS-CoV-2 antigen-specific immune responses are monitored in serum and PBMCs by ELISA, ELISPOT and ICS throughout the course of treatment. Four weeks after the final vaccination, animals are challenged with SARS-CoV-2 and monitored for disease characteristics and viral shedding.

[0111] Example 5: Phase Ib clinical trial of hAd5[E1-, E2b-, E3-] CoV-2 vaccine. Study Design: This is a Phase 1b open-label study in adult healthy subjects. This clinical trial is designed to evaluate the safety, reactogenicity, and immunogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines. The hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines are targeted hAd5[E1-, E2b-, E3-] vector-based vaccines 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 has been shown to be effective in >125 cancer patients to date, with a 5×10 11 It is a platform technology for targeted vaccines that has demonstrated safety at high doses of viral particles. Three different hAd5 [E1-, E2b-, E3-] vector-based vaccines were administered on the same day at 5 × 10 per dose each. 11 Viral particles (1.5 × 10 12 Coadministration of up to 100 mg of total viral particles has also been demonstrated to be safe.

[0112] COVID-19 infection causes significant morbidity and mortality in populations worldwide. The hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines are designed to induce both humoral and cellular responses, even in individuals with pre-existing adenovirus immunity. Thus, there is the potential for hAd5-COVID-19-S and hAd5-COVID-19-S / N to induce anti-COVID-19 immunity in healthy subjects and prevent or reduce the health impact of COVID-19 infection.

[0113] Phase 1b Safety Analysis: In the first safety analysis of Phase 1b, a total of 40 healthy subjects were divided into four dosing cohorts (Cohorts 1A, 1B, 2A, 2B; n=10 for each cohort): Cohort 1A - hAd5-COVID-19-S 5 × 10 10 viral particles (VP) / dose (n=10); Cohort 1B - hAd5-COVID-19-S 1×10 11 VP / dose (n=10), Cohort 2A - hAd5-COVID-19-S / N 5×10 10 VP / dose (n=10), Cohort 2B - hAd5-COVID-19-S / N, 1 × 10 11 VP / dose (n=10).

[0114] Each subject will receive subcutaneous (SC) injections 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 the hAd5[E1-, E2b-, E3-] vector-based vaccine currently in clinical trials. Cohorts 1-2 will be enrolled in parallel and may open simultaneously or staggered depending on the supply of investigational drug. Subjects in cohorts 1A and 2A will first complete the low-dose vaccination regimen. After all subjects in cohorts 1A and 2A have completed at least a single dose and follow-up evaluations during the toxicity evaluation period through study day 8, if the sponsor and independent of the trial, Safety Review Committee (SRC) and at least one qualified infectious disease physician confirm that there are no safety concerns, enrollment will proceed. Subjects will then be enrolled and vaccinated in high-dose cohorts 1B and 2B. For all subjects, follow-up study visits will occur at days 8, 22, 29, 52, and at months 3, 6, and 12 after the last vaccination. Additional follow-up for safety information will occur via telephone contact as described in the event schedule. The primary objective of the initial safety Phase 1b is 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 long-term safety and immunogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S / N vaccines.

[0115] Example 6: Expanded Phase 1b: Safety and Immunogenicity for Construct Selection If the SRC determines it is safe to do so, based on a review of the safety data from the Phase 1b safety evaluation, it will proceed to Phase 1b expansion. Phase 1b expansion will divide a total of 60 healthy subjects into four dosing cohorts (Cohorts 1A, 1B, 2A, 2B; n=15 for each cohort): Cohort 1A - hAd5-COVID-19-S 5 × 10 10 VP / dose (n=15) Cohort 1B - hAd5-COVID-19-S 1×10 11 VP / dose (n=15) Cohort 2A - hAd5-COVID-19-S / N 5×10 10 VP / dose (n=15) Cohort 2B - hAd5-COVID-19-S / N 1×10 11 VP / dose (n=15)

[0116] Each subject will receive an 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 occur on days 8, 22, 29, 52, and 3, 6, and 12 after the last vaccination. Additional follow-up for safety information will be conducted by telephone contact as described in the event schedule. 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 the dose level as determined by 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.

[0117] Embodiments of the present disclosure are further described in the following examples, which are merely illustrative and do not in any way limit the scope of the invention as claimed.

[0118] Example 7: hAd5[E1-, E2b-, E3-] Platform and Constructs For the examples presented here, next generation hAd5[E1-, E2b-, E3-] vectors were used (Figure 33, panel C) to generate viral vaccine candidate constructs. As shown in Figure 33, panels D-H, various constructs were generated: Figure 33, panel D: S WT: S protein containing 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2); Figure 33, panel E: S RBD-ETSD: S receptor binding domain with enhanced T cell stimulatory domain (ETSD); Figure 33, panel F: S fusions: S optimized to enhance surface expression and display of RBD; Figure 33, panel G: N-ETSD: nucleocapsid (N) sequence with ETSD; and Figure 33, panel H: bivalent S fusions + N-ETSD; S-WT + N-ETSD and S RBD-ETSD + N-ETSD constructs were also generated but are not shown.

[0119] Example 8: Enhanced HEK 293T cell surface expression of RBD following transfection with Ad5 S fusions + N-ETSD As shown in Figure 34, anti-RBD specific antibodies did not detect RBD on the surface of HEK 293T cells transfected with hAd5 S-WT (Figure 34, panel A) or hAd5 S-WT + N-ETSD (Figure 9b) constructs, while only hAd5 S-fusion was slightly higher (Figure 34, panel E). As expected, both constructs carrying RBD, hAd5 RBD-ETSD and RBD-ETSD + N-ETSD, showed high binding of anti-RBD antibodies (Figure 34, panels C and D). Notably, high cell surface expression of RBD was detected after transfection with bivalent hAd5 S-fusion + N-ETSD (Figure 34, panel F). These findings support the proposal that the hAd5 S-fusion + N-ETSD construct, which contains a large number of diverse antigens provided by both full-length optimized S and N with proper folding, results in enhanced expression and cell surface display of RBD in vaccine constructs.

[0120] Example 9: Enhanced S expression and immunoblot correlation with hAd5 S-fusions + N-ETSD Immunoblot analysis of S expression correlates with enhanced S expression (Figure 35), again showing that the bivalent hAd5 S-fusion + N-ETSD construct enhances expression of S compared to S-fusion alone. Figure 35 shows immunoblot analysis of S expression. Cell surface RBD expression in HEK 293T cells using (a) hAd5 S-WT, S-fusion, and (c) S-fusion + N-ETSD was highly correlated with expression of S (d) in immunoblots of HEK 293T cell lysates probed with anti-full length (S2) antibody. Y-axis scale is normalized to mode (NM).

[0121] Example 10: Confirmation of native folding of enhanced surface RBD following hAd5 S-fusion + N-ETSD transfection Recombinant ACE2-Fc binding determinations 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 the course of SARS-CoV-2 infection, and effective neutralizing antibodies prevent this interaction and thus infection. Such neutralizing antibodies are likely to be more effective if produced in response to S presented in the correct conformation. In addition to enhanced cell surface expression, an optimized S allows for proper protein folding. A clear enhancement of ACE2-Fc binding to the S RBD expressed from hAd5 S-fusion + N-ETSD was found (Figure 36, panel C) compared to either hAd5 S-WT or hAd5 S-fusion (Figure 36, panels A and B, respectively). Anti-RBD antibody binding studies performed in the same experiment (Figure 36, panels F-J) confirmed the enhanced surface expression findings indicated by ACE2-Fc binding. The hAd5 S-fusion + N-ETSD vaccine candidate was selected for clinical trials based on these findings of conformationally correct and enhanced S RBD expression, which is critical for the production of neutralizing antibodies.

[0122] Example 11: hAd5 N-ETSD successfully targets N to the endosomal / lysosomal compartment The ETSD design successfully translocated N to the endosomal intracellular compartment. After infection of HeLa cells with N-ETSD, N colocalized with the endosomal marker 45 transferrin receptor (CD71) as shown in Figure 37, panel C, and also colocalized with the lysosomal marker Lamp1 (Figure 37, panel D), demonstrating that N-ETSD translocates through the endosomal pathway to lysosomes to allow processing for MHC II presentation. Compared to N-ETSD, N-wild type (N-WT) exhibits a diffuse cytoplasmic distribution and does not colocalize with lysosomal markers (Figure 37, panel E). These findings confirm the role of ETSD in directing N to the endosomal / lysosomal compartment, leading to increased MHC II presentation and CD4+ activation by N.

[0123] Example 12: In vivo hAd5 S-fusion + N-ETSD vaccine immunogenicity study Based on evidence that S-fusion + N-ETSD resulted in enhanced expression of physiologically relevant RBD and that N-ETSD successfully translocated to endosomal / lysosomal compartments, a bivalent hAd5 S-fusion + N-ETSD vaccine was selected for inoculation of 7-week-old female CD-1 mice. The unique properties of this construct resulted in the generation of both CD8+ and CD4+ T cell responses and neutralizing antibodies. As described in the methods, mice received a first 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 the study did not have site reactions, did not lose weight throughout dosing, and appeared normal, consistent with previous observations with the hAd5[E1-, E2b-, E3-] platform.

[0124] Example 13: hAd5 S-fusion + N-ETSD generates both CD8β+ and CD4+ T cell responses CD8+ activation by both S and N: 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 to hAd5 null mice (Figure 38, Panel A); splenocytes from these mice also expressed intracellular IFN-γ in response to the N peptide pool. Assessment of concurrent IFN-γ / TNF-α expression from CD8β+ splenocytes (Figure 38, Panel C) mirrored that for IFN-γ expression alone. These results indicate that both S and N activate CD8+ T cells.

[0125] CD4+ activation by N: CD8+ cytotoxic T cells mediate killing of virus-infected cells, but CD4+ T cells are required for sustained cytotoxic T lymphocyte (CTL) activity. Therefore, CD4+ T cells in vaccinated animals were evaluated. In contrast to CD8β+ splenocytes, only N peptide pools 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 38, panel D) at levels substantially higher than hAd5 null controls. N contribution of the CD4+ T cell response is essential for an effective immune response to a candidate vaccine.

[0126] Example 14: hAd5 S-fusion + N-ETSD generates an antibody response against both S and N antigens The primary goal of coronavirus vaccines currently under development is neutralizing antibodies against the spike. In mice vaccinated with the bivalent vaccine, there was significant production of both anti-S (Figure 39, Panel A) and anti-N (Figure 39, Panel C) antibodies in serum from CD-1 mice vaccinated with hAd5 S-fusion + N-ETSD on day 28 of the study. Compared to anti-S antibodies, anti-N antibodies were higher in serum given the serum dilution factor of 1:90 for anti-N antibody assay and 1:30 for anti-S antibody assay.

[0127] A standard curve for IgG was generated and absorbance values ​​were then converted to mass equivalents for both anti-S and anti-N antibodies (Figure 39, panels B and D). Using these values, it was calculated that hAd5 S-fusion + NETSD vaccination produced a geometric mean of 5.8 μg S-specific IgG and 42 μg N-specific IgG per mL of serum, thus the relative μg amount of anti-N antibodies was higher than that of anti-S antibodies, reflecting a strong contribution of N to anti-SARS-CoV-2 antibody production.

[0128] Example 15: hAd5 S-fusion + N-ETSD vaccine generates potent neutralizing antibodies as assessed by both cPass and live virus neutralization assays Neutralizing antibody activity was assessed using a cell-free assay (cPass) and live virus infection in vitro. As seen in Figure 40, panel A, the cPass assay showed inhibition of S RBD:ACE2 binding for all mice and approximately 100% inhibition for two mice at both 1:20 and 1:60 dilutions. Vero E6 neutralization assay results are shown for four mice that showed S-specific antibodies by ELISA. The high and persistent neutralization seen even at high dilution factors suggests the intriguing possibility that bivalent multi-antigen multi-epitope generation by the hAd5 S-fusion + N-ETSD vaccine may result in synergistic neutralizing immune responses at epitopes in addition to those associated with RBD-ACE2 binding (Figure 40, panel B). As seen in Figure 40, panel B, 50% neutralization (IC50) values ​​are present at 1:10,000 serum dilution for the G4 pool of sera from mice that exhibited S-specific antibodies, 10-fold higher than the convalescent serum at 1:1,000 dilution. The potent neutralization confirmed by the two assays supports the predicted efficacy of the hAd5 S-fusion + ETSD vaccine candidate and its progression into clinical trials.

[0129] Example 16: hAd5 S-fusion + N-ETSD generates Th1-dominant responses in both humoral and T cell immunity Antibody Th1 dominance in response to N and S: IgG2a, IgG2b, and IgG3 represent Th1 dominance; whereas IgG1 represents Th2 dominance. For both anti-S (Figure 41, panel A) and anti-N (Figure 41, panel C) antibodies in sera from hAd5 S-fusion + N-ETSD vaccinated mice, IgG2a and IgG2b isotypes were predominant and significantly higher compared to hAd5 null controls. These data indicate a Th1 dominance of antibody production in response to the hAd5 S-fusion + N-ETSD vaccine.

[0130] T cell Th1 dominance in response to N and S: IFN-γ production correlates with CTL activity47 (Th1 dominance), whereas IL-4 induces delayed viral clearance48 (Th2 dominance). An IFN-γ to IL-4 ratio of 1 is balanced, whereas a ratio above 1 indicates Th1 dominance. ELISpots from animals immunized with the bivalent S+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 the N peptide pool (Figure 42, panel A), whereas IL-4 was secreted at significantly higher levels for hAd5 S-fusion + N-ETSD in response to only the N peptide pool (Figure 42, panel B).

[0131] Th1 type dominance is also seen when considering the ratio of IFN-γ to IL-4 based on spot forming units in response to combined S and N peptide pools (Figure 43, Panel A). Th1 dominance is again seen in the humoral response, where the ratio of Th1 associated antibodies (IgG2a, IgG2b, and IgG3) to Th2 associated antibodies (IgG1) based on ng equivalents was greater than 1 in all mice for both anti-S and anti-N antibodies (Figure 43, Panel B).

[0132] This Th1-dominant profile of the hAd5 S-fusion + N-ETSD vaccine candidate provides further justification for hAd5 S-fusion + N-ETSD as a lead candidate for clinical trials.

[0133] The hAd5 S-fusion + N-ETSD vaccine was designed to overcome the risks of S-only vaccines and induce both T cell immunity and neutralizing antibodies, leveraging the important role that T cells play in generating long-lasting antibody responses and directly killing infected cells. Both CD4+ and CD8+ T cells are polyfunctional, and the induction of such polyfunctional T cells by the vaccine correlated with better protection against infection. The enhanced CD4+ T cell response and Th1 dominance resulting from expression of S antigens optimized for surface display and N antigens optimized for endosomal / lysosomal intracellular compartment localization, and thus MHC I and II presentation, led to increased dendritic cell presentation, cross-presentation, B cell activation, and ultimately high neutralizing capacity. Furthermore, the strong neutralizing capacity at high dilutions seen for pooled sera from hAd5 S-fusion + N-ETSD vaccinated mice, combined with the Th1 dominance of antibodies generated in response to both S and N antigens, supports the objectives of this vaccine design.

[0134] Simultaneous MHC I and MHC II presentation of antigen by antigen presenting cells simultaneously activates CD4+ and CD8+ T cells, optimal for the generation of memory B and T cells. A key finding of the construct is that N-ETSD is targeted to the endosomal / lysosomal compartment. N-ETSD induces 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 has also been reported, compared to native N.50,51

[0135] T cell responses to the S and N antigens expressed by hAd5 S-fusions + N-ETSD were polycytokine, including IFN-γ and TNF-a, consistent with successful antibacterial immunity in bacterial and viral infections. Polycytokine T cell responses following vaccination have been shown to correlate with vaccine efficacy, including that of viral vectors. Highly relevant here is that polycytokine T cell responses to the SARS-CoV-2 N protein were consistent with recovered COVID-19 patients, suggesting that the bivalent hAd5 S-fusions + N-ETSD vaccine provides greater protection against SARS-CoV-2 in vaccine subjects.

[0136] In contrast to N, the S protein, expressed here as an S fusion, confirmed enhanced RBD cell surface expression and conformational integrity as evidenced by high ACE2-Fc binding, and generated primarily CD8+ T cells. Our results confirmed the vaccine design goal, showing that the S-fusion induced elevated levels of antigen-specific T cell responses to S compared to S-WT. To ensure MHC presentation to both MHC I (for CD8+ T cell activation) and MHC II (for CD4+ T cell activation), it is necessary to vaccinate with both S and N antigens optimized to generate this coordinated response.

[0137] Neutralization data with live SARS-CoV-2 virus demonstrated the potency of the antibody responses generated after vaccination with hAd5 S-fusion + N-ETSD, with evidence of high neutralization even at high dilution factors. Furthermore, a significant synergistic effect of pooled sera was evident, with even more potent neutralization than control convalescent sera at dilutions of ≥1:1,000.

[0138] The hAd5 S-fusion + N-ETSD construct described above is delivered by the next generation hAd5 [E1-, E2b-, E3-] platform, where the E2b deletion (pol) alone allows for extended transgene production and allows for homologous vaccination (prime and boost formulations are the same) in the presence of pre-existing adenoviral immunity.38 In addition to generating cellular and humoral immunity by subcutaneous injection of hAd5 S-fusion + N-ETSD, the same vaccine in an oral or sublingual formulation can also induce IgA mucosal immunity.

[0139] Example 17: Methods and Constructs hAd5[E1-, E2b-, E3-] platform and constructs For testing herein, a second generation hAd5[E1-, E2b-, E3-] vector was used (Figure 44, Panel A) to generate a viral vaccine candidate construct. The hAd5[E1-, E2b-, E3-] backbone containing SARS-CoV-2 antigen-expressing inserts and viral particles was generated as previously described. Briefly, high titer adenovirus stocks were generated by serial propagation in E1- and E2b-expressing E.C7 packaging cell lines, followed by CsCl2 purification and dialysis into storage buffer (2.5% glycerol, 20 mM Tris pH 8, 25 mM NaCl) by ViraQuest Inc. (North Liberty, IA). Viral particle numbers were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280 nm, and viral titers were determined using the Adeno-X™ Rapid Titer Kit (Takara Bio). The constructs made included:

[0140] S-WT: S protein containing 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2); S-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 stimulatory domain (ETSD); S-RBD-ETSD + N-ETSD; S-fusions: S optimized to enhance surface expression and display of RBD; and bivalent S-fusions + N-ETSD;

[0141] Transfection of HEK 293T cells with hAd5 constructs To determine surface expression of the RBD epitope by vaccine candidate constructs, we transfected HEK 293T cells with hAd5 construct DNA and quantified surface RBD by flow cytometric detection with an anti-RBD antibody. There were seven constructs 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 (2.5 × 10 in 24-well plates) were transfected with hAd5 construct DNA and quantified surface RBD by flow cytometric detection with an anti-RBD antibody. There were seven constructs tested: S-WT, S-WT + N-ETSD, S RBD-ETSD, S RBD-ETSD + N-ETSD, S-fusion, S-fusion + N-ETSD, and N-ETSD. 5Cells / well) were grown at 37° C. in DMEM (Gibco Cat# 11995-065) with 10% FBS and 1× PSA (100 units / mL penicillin, 100 μg / mL streptomycin, 0.25 ug / mL amphotericin B). Cells were transfected with 0.5 μg of hAd5 plasmid DNA using JetPrime transfection reagent (Polyplus Cat# 89129-924) according to the manufacturer's instructions. At 1, 2, 3, and 7 days after transfection, cells were harvested by gently pipetting them 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 with R-phycoerythrin (ThermoFisher Catalog # H10104). Labeled cells were acquired using a Thermo-Fisher Attune NxT flow cytometer and analyzed using Flowjo Software.

[0142] Immunocytochemical labeling of hAd5-infected HeLa cells 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 carrying a Flag tag to enable labeling), 48 hours after infection or transfection, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.4% 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, Sigma cat# F1804) antibody at 1:1000 in phosphate buffered saline containing 3% BSA overnight at 4°C, followed by washing in PBS and incubation 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 with sheep anti-Lamp1 Alexa Fluor 488-conjugated (lysosomal marker) antibody (R&D systems, Cat# IC7985G) at 1:10 or rabbit anti-CD71 (transferrin receptor, endosomal marker) antibody (ThermoFisher Cat# PA5-83022) at 1:200 overnight at 4° C. After removing the primary antibody and washing twice in PBS and three times in PBS containing 3% BSA, cells were incubated with fluorescently conjugated secondary antibody (goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488, Life technologies, A-11034) at 1:500 for 1 h at room temperature, if applicable. After a brief wash, cells were mounted with Vectashield Antifade mounting medium containing DAPI (Fisher Scientific, Cat. No. NC9524612) and immediately imaged using a Keyence all-in-one Fluorescence microscope camera and Keyence software.

[0143] Immunoblot analysis of S antigen expression HEK 293T cells transfected with hAd5 S-WT, S-fusion, or S-fusion + N-ETSD constructs were cultured and transfected as described in this manuscript and harvested 3 days after transfection in 150 ml RIPA lysis buffer containing 1X final Protease Inhibitor cocktail (Roche). After protein assay, equal amounts of total protein were loaded onto 4-12% gradient polyacrylamide gels (type), poured, and transferred to nitrocellulose membranes using a semi-dry transfer apparatus. Anti-Spike S2 ​​(SinoBiological Cat#40590-T62) was used as the primary antibody and IRDye® 800CW Goat Anti-Rabbit IgG (H+L) (Li-Cor, 925-32211) was used as the secondary antibody using the Ibind Flex platform. Antibody-specific signals were detected with an infrared Licor Odyssey instrument.

[0144] Binding of ACE2-IgG1Fc to hAd5-transfected HEK 293T cells HEK 293T cells were cultured at 37°C under the conditions described above for transfection with hAd5 S-WT, S-fusion, S-fusion + N-ETS, RBD-ETSD or S RBD-ETSD + N-ETSD, incubated for 2 days and harvested for ACE2-Fc binding analysis. Recombinant ACE2-IgG1Fc protein was produced using Maxcyte transfection in CHO-S cells cultured for 14 days. ACE2-IgG1Fc was then purified using a MabSelect SuRe affinity column on an AKTA Explorer.

[0145] Purified ACE2-IgG1Fc was dialyzed into 10 mM HEPES, pH 7.4, 150 mM NaCl and concentrated to 2.6 mg / mL. For binding studies, ACE2-IgG1Fc was used at a concentration of 1 μg / mL for binding. Cells were incubated with ACE2-Fc for 20 min and, after a washing step, were labeled with PE-conjugated F(ab')2-goat anti-human IgG Fc secondary antibody at a 1:100 dilution, incubated for 20 min, washed and acquired on a flow cytometer. Histograms are based on normalized mode of cell number (NM) - the number of cells positive for the signal in the PE channel.

[0146] Vaccination of CD-1 mice with hAd5 S-fusions + N-ETSD vaccine candidates Seven-week-old CD-1 female mice (Charles River Laboratories) were used for immunological studies, which were performed in the vivarium facility of Omeros Inc. (Seattle, WA). On day 0 after the first bleed, mice were injected with 1 × 10 10 Mice were injected with a dose of viral particles (VP). There were 5 mice per group. Mice received a second vaccination on days 21 and 28, blood was collected from isoflurane-anesthetized mice via the submandibular vein for serum isolation, and the mice were then euthanized for collection of spleen and other tissues.

[0147] Spleen cell harvesting and intracellular cytokine staining (ICS) Spleens were removed from each mouse and placed in 5 mL of sterile medium: 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 for flow cytometric detection of CD8β+ and CD4+ T cell-associated IFN-γ and IFN-γ / TNFα+ production in response to stimulation with S and N peptide pools.

[0148] Stimulation assays were performed in 96-well U-bottom plates with 10 6 The study was performed with live splenocytes. Splenocytes in RPMI medium supplemented with 10% FBS were stimulated by adding the peptide pool at 2 μg / mL / peptide for 6 h at 37 °C in 5% CO2, and the protein transport inhibitor, GolgiStop (BD), was added 2 h after the start of incubation. 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. Fluorescently conjugated antibodies against mouse CD8β antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), and TNF-α (clone MP6-XT22, BD), as well as staining was performed in the presence of unlabeled anti-CD16 / CD32 antibody (clone 2.4G2). Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software.

[0149] ELISpot assay ELISpot assay was used to detect cytokines secreted by splenocytes from inoculated mice. Fresh splenocytes were used on the same day as the cryopreserved splenocytes containing lymphocytes. Cells (2–4 × 10 per well of a 96-well plate) were used at 10–20 °C for 1 h. 5 10 cells) were added to ELISpot plates containing immobilized primary antibodies against either 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 h. After aspiration and washing to remove cells and media, extracellular cytokines were detected by secondary antibodies against cytokines conjugated to biotin (BD). Biotin-conjugated secondary antibodies were detected using streptavidin / horseradish peroxidase conjugates. 2–4 × 10 cells were added per well or 2–4 × 10 cells were added per well. 5The number of spots per cell was counted using an ELISpot plate reader.

[0150] ELISA for detection of antibodies For antibody detection in serum from inoculated mice, ELISAs specific for spike and nucleocapsid antibodies, as well as IgG subtype (IgG1, IgG2a, IgG2b, and IgG3) antibodies were used.Microtiter plates were coated overnight with 100 ng of either purified recombinant SARS-CoV-SS-FTD (full-length S with fibritin trimerization domain, ImmunityBio, Inc., 9920 Jefferson Blvd, Culver City, CA 90232), SARS-CoV-2 S RBD (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 of coating buffer (0.05 M carbonate buffer, pH 9.6). Wells were washed three times with 250 μL of PBS containing 1% Tween 20 (PBST) to remove unbound proteins and plates were blocked with 250 μL of PBST for 60 minutes at room temperature. After blocking, wells were washed with PBST and 100 μL of diluted serum samples were added to the wells and the samples were incubated for 60 minutes at room temperature. After incubation, wells were washed with PBST and 100 μL of 1 / 5000 dilution of 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; Cat# SAB3701185), or anti-mouse IgG3 HRP conjugated antibody (Sigma; Cat# SAB3701192) was added to the wells. For positive controls, 100 μL of a 1 / 5000 dilution of rabbit anti-N IgG Ab or 100 μL of a 1 / 25 dilution of mouse anti-S serum (from mice immunized with purified S antigen in adjuvant) were added to appropriate wells.After 1 h incubation 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 appropriate color development. The color reaction was stopped by adding 50 μL of 10% phosphoric acid solution (Fisher Cat# A260-500) in water, and the absorbance at 490 nm was determined using a microplate reader (SoftMax® Pro, Molecular Devices).

[0151] Calculating relative μg amount of antibody A standard curve for IgG was constructed and absorbance values ​​were converted to mass equivalents for both anti-S and anti-N antibodies. hAd5 S-fusion + N-ETSD vaccination yielded a geometric mean of 5.8 μg S-specific IgG and 42 μg N-specific IgG per mL of serum.

[0152] cPass™ Neutralizing Antibody Detection GenScript cPass™ for detection of neutralizing antibodies was used according to the manufacturer's instructions. The 44 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 suitable for all antibody isotypes and is suitable for use in animal models without modification.

[0153] Vero E6 cell neutralization assay All aspects of the virus-based assays were performed in a BSL3 containment facility following the ISMMS Conventional Biocontainment Facility SOP for SARS-CoV-2 cell culture testing. Vero e6 kidney epithelial cells from Savannah monkeys (Cercopithecus aethiops) (ATCC CRL-1586) were plated at 20,000 cells / well in a 96-well format, and after 24 hours, the cells were incubated with antibodies or heat-inactivated serum pre-diluted in 3-fold steps in DMEM with 2% FBS, 1% NEAA, and 1% Pen-Strep; diluted samples were mixed 1:1 with SARS-CoV-2 in DMEM with 2% FBS, 1% NEAA, and 1% Pen-Strep at 10,000 TCID50 / mL for 1 hour at 37°C, 5% CO2. This incubation did not include cells to allow for neutralizing activity to develop prior to infection. Samples for testing included serum from four mice that showed >20% inhibition of ACE2 binding in cPass, pooled serum from these four mice, serum from COVID-19 convalescent patients, and medium alone. For detection of neutralization, 120 μL of virus / sample mixture was transferred to Vero E6 cells and incubated for 48 h before fixing with 4% PFA. Each well received 60 μL of virus or an infectious dose of 600 TCID50. For no virus and virus only controls, control wells were used, including six wells on each plate. Percent neutralization was calculated as 100-((sample of interest-[mean of "no virus"]) / [mean of "virus only"]*100) using staining of CoV-2 Np imaged on a Celigo Imaging Cytometer (Nexcelom Bioscience).

[0154] Vaccine characterization The majority of current prophylactic anti-SARS-CoV-2 vaccines in development 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 COVID-19 patient immune responses to SARS-CoV-2 indicates that other immune cells, such as T cells, are important in clearing the infection and generating long-term immunity against coronavirus infection. Both CD4+ and CD8+ T cells support durable humoral responses, as CD4+ T cells are not effector cells like CD8+ T cells, but are important in generating robust and sustained immunity provided by antibody-secreting plasma cells and in the elimination of infected cells by memory cytotoxic CD8+ T cells. The dual antigen candidate vaccine of the present disclosure more broadly activates the immune system to combat SARS-CoV-2 by including a modified viral nucleocapsid (N) antigen, which is a potent CD4+ and CD8+ T cell target, along with an optimized S protein (S-fusion) to stimulate humoral responses.

[0155] The human adenovirus serotype 5 (hAd5) E1, E2b, and E3 region deleted [E1-, E2b-, E3-] vaccine platform (Figure 44, Panel A) is superior to the adenovirus platforms currently used in other COVID-19 vaccines in clinical trials because it is effective in the presence of pre-existing adenovirus immunity and is less likely to generate vector-targeted host immune responses, and therefore can be used as both a prime and boost. Using this platform, the vaccine contains an optimized S surface protein, S-fusions, for increased cell surface display and humoral responses; as well as the highly conserved antigenic N protein found within the viral particle, which now has an intracellular compartment targeting sequence for enhanced antigen presentation. This strategy is safe and robust in eliciting humoral and T cell responses against SARS-CoV-2. The addition of N addresses the risk of loss of vaccine efficacy for S-only monovalent vaccines due to the emergence of S mutations over time in the population. In contrast, N is highly conserved and has a lower risk of mutation, while being highly immunogenic. It is a known target antigen of natural immunity, and antibodies and T cells against N are found in the majority of individuals who have recovered from SARS-CoV-2 and similar virus SARS-CoV infections. In the present vaccine, the enhanced T cell stimulatory domain (ETSD) post-translationally directs the N protein to the endosomal-lysosomal intracellular compartment to support MHC class II presentation for T helper cell activation and promotion of CD8+ T cell activation by dendritic cell licensing. Despite the risk of emerging mutations, S remains an important antigen for vaccination due to its role in infection. The spike (Figure 44, Panel B), displayed as a trimer on the viral surface, has a receptor binding domain (RBD) that interacts with the host angiotensin-converting enzyme 2 (ACE2) to facilitate entry and proliferation in host cells, and thus antibodies against S are key to neutralizing infection. Antibodies against the S RBD, as well as antibodies against other S epitopes, are commonly found in patients who have recovered from COVID19 51.In bivalent vaccine constructs, S-fusions optimize S by the addition of a fusion linker and display the S RBD in a physiologically relevant form on the cell surface, with the goal of improving the generation of virus-neutralizing anti-S RBD antibodies.

[0156] The bivalent hAd5 S-fusion + N-ETSD vaccine of the present disclosure generates a superior T cell response. Vaccines currently in clinical trials are focused on generating humoral responses as a means to neutralize infection. However, T cell responses become important considering that even if antibodies are successfully generated and sufficiently neutralizing, they may decline over time. In the absence of T cell responses due to virus-induced lymphopenia, infected individuals are at risk of developing acute symptoms of the disease, even in the presence of large amounts of neutralizing antibodies. Although it cannot be excluded that S (and other viral proteins) can induce T cell responses, evidence in the literature supports a critical role for N. Not only were T cell responses against N found in the majority of patients who recovered from COVID-19, but these responses against N in patients exposed to the very similar virus SARS-CoV are remarkably durable. Compelling evidence for the importance of N in natural T cells can be found in a recent report by Ferretti et al., who used an unbiased genome-wide screen for the exact peptide sequences recognized by memory CD8+ T cells from COVID-19 patients and found that only 3 of the 29 shared epitopes were from the spike protein, whereas the highest density of epitopes was located in the nucleocapsid protein. Thus, in the hAd5 S-fusions + N-ETSD vaccine, N is expected to not only induce humoral responses but also T cell responses that better recapitulate disease-limiting innate immunity.

[0157] In the first report, hAd5 S-fusion + N-ETSD vaccine provided enhanced cell surface expression of S RBD that is readily recognized by ACE2, reflecting its conformational integrity. N-ETSD with endosomal / lysosomal localization for enhanced antigen presentation generated both neutralizing antibodies and CD4+ / CD8+ T cell-mediated responses with Th1 dominance in vaccinated mice. This disclosure extends these findings, confirming native S antigen expression using plasma and monocyte-derived dendritic cells (MoDCs) from patients previously infected with SARS-CoV-2. We elucidated N-ETSD localization in antigen-presenting MoDCs and showed that it localizes to endosomes, lysosomes, and autophagosomes. Protein processing through this intracellular pathway enhances MHC class II presentation and increases peptide recycling, allowing for MHC class I presentation as well. By localizing the nucleocapsid protein to the late lysosomal-autophagosome compartment, both CD4+ and CD8+ SARS-CoV-2-specific memory T cells are recalled from patients previously infected with SARS-CoV-2. Moreover, in these immune response recall studies, in vitro, hAd5-infected moDCs presenting S-fusions and N-ETSD induce predominantly Th1 responses from autologous memory T cells from patients previously infected with SARS-CoV-2. N specifically drives CD8+ T cell responses in in vitro recall studies. The recapitulation of natural infection and immunity to the extent achievable by vaccination with a hAd5 S-fusion + N-ETSD vaccine makes it a prime candidate for clinical trials of its ability to protect individuals against SARS-CoV-2 infection and COVID-19, and this second-generation vaccine construct is currently in phase I clinical trials.

[0158] Example 18: hAd5[E1-, E2b-, E3-] Platform and Constructs For the studies here, next generation hAd5[E1-, E2b-, E3-] vectors were used (Figure 44, Panel A) to generate viral vaccine candidate constructs. Various constructs were generated: Figure 44, Panel C: S WT: S protein containing 1274 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2); Figure 44, Panel D: S fusions: S optimized to enhance surface expression and display of RBD; Figure 44, Panel E: N (N without ETSD): nucleocapsid (wild type) sequence with tag for immunodetection but without ETSD modification and with predominantly cytoplasmic localization. Figure 44, Panel F. N with enhanced T cell stimulatory domain (N-ETSD): nucleocapsid (wild type) with ETSD to direct lysosomal / endosomal localization and a tag for immunodetection; and Figure 44, panel G: bivalent hAd5 S-fusion + N-ETSD vaccine.

[0159] Nucleocapsid antigen engineered with an enhanced T cell stimulatory domain (ETSD) targets N to endosomes, lysosomes, and autophagosomes in MoDCs and drives enhanced CD4+ T cell activation:

[0160] The hAd5 bivalent vaccine construct contains sequences designed to target N to MHC class II antigen-loading compartments. To further investigate factors influencing antigen presentation in moDCs, moDCs from healthy subjects were infected with hAd5 N-ETSD or hAd5 N and localization was determined by immunocytochemistry. N-ETSD showed localization to discrete vesicles with some consistency with CD71, a marker of recycling endosomes (Figure 45, panels A-C), and LAMP-1, a marker of late endosomes / lysosomes (Figure 45, panels G-I), whereas N was diffusely and uniformly expressed throughout the cytoplasm (Figure 45, panels D-F and J-L). Lysosomes fuse with autophagosomes to enhance peptide processing and MHC class II presentation. N-ETSD also showed some colocalization with autophagosomal markers (Figure 45, panels M-O). Protein processing in autophagosomes plays an important role in MHC-mediated antigen presentation in DCs, providing a potential mechanism for enhanced CD4+ T cell induction induced by N-ETSD in vaccine constructs. Evidence of this T cell interaction with moDCs infected with N-ETSD translocated to autophagosomes (and possibly endosomes and lysosomes) is seen in this phase contrast microscopy of N-ETSD and LC3a / b co-labeled cells, which reveals an elongated DC morphology in contrast to the spherical morphology of undifferentiated lymphocytes. Lymphocytes, also distinguished by the absence of infection with hAd5 N-ETSD (lymphocytes lack the hAd5 receptor), were also seen to interact with N-ETSD-expressing moDCs.

[0161] Validation of SARS-CoV-2 antibody and cell-mediated immune responses from previously SARS-CoV-2 infected and virus-naive patients for memory T cell recall testing:

[0162] For the studies described below, plasma samples were collected from four individuals who had recovered from SARS-CoV-2 infection, as confirmed by antibody assays and patient medical history as described below. The presence of anti-spike IgG by both cPass 66 and live virus assays, and the presence of neutralizing antibodies, were confirmed in all patient samples. Samples were also collected from four virus-naive individuals and used as controls. Additional studies to validate immune responses to SARS-CoV-2 antigens assessed binding of plasma from patients previously infected with SARS-CoV-2 and virus-naive control individuals to human embryonic kidney (HEK) 293T cells transfected with either hAd5 S-fusion alone or hAd5 S-fusion + N-ETSD. This binding reflects the presence of antibodies in the plasma that recognize the antigens expressed by the hAd5 vectored vaccine. Quantification of the histograms showed little or no binding of virus-naive plasma antibodies to cells expressing either construct, and highest binding of plasma antibodies from patients previously infected with SARSCoV-2 to cells expressing the bivalent S-fusion + N-ETSD construct, due to either enhanced 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.

[0163] Example 19: N-ETSD optimizes spike antigen expression: Binding of plasma antibodies from previously infected SARS-CoV-2 patients is enhanced for hAd5 S-fusion + N-ETSD infected MoDCs compared to hAd5 S-fusion or hAd5 S-WT. The study here focuses on the response of T cells from patients previously infected with SARS-CoV-2 to autologous MoDCs infected with hAd5 vaccine constructs. DCs are potent antigen-presenting cells for processing and presenting complex antigens acquired via infection or phagocytosis to induce T cell responses. Therefore, in addition to evaluating the binding of patient plasma antibodies to hAd5 vaccine expressing HEK 293T cells, MoDCs from two healthy individuals were infected overnight with hAd5 S-WT, hAd5 S-fusion, hAd5 S-fusion + N-ETSD, or hAd5 null, and then gene expression was evaluated 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 seen following hAd5 S-fusion + N-ETSD infection (Figure 46, Panels B-D), providing further evidence that antigen expression is optimized in the hAd5 S-fusion + N-ETSD bivalent vaccine. This highly relevant finding in vitro with human MoDCs and plasma is not only an important confirmation of the results from studies with HEK 293T cells and a commercially available anti-RBD antibody, but also represents a potential method to screen plasma for SARS-CoV-2 antigenic reactivity.

[0164] Example 20: SARS-CoV-2 peptide pool immune response: T cells from previously infected SARS-CoV-2 patients secrete significant levels of interferon-g (IFN-γ) in response to S1, S2, and N CoRSSAN-2 peptide pools compared to T cells from virus-naive controls. To demonstrate reactivity of T cells from four previously infected SARS-CoV-2 patients with virus-naive T cells from four unexposed individuals, T cells from each group were cultured with autologous MoDCs pulsed with a peptide mixture spanning the sequence of the N and S proteins. T cells from previously infected SARS-CoV-2 patients secreted IFNg in response to SARS-CoV-2 antigens, whereas T cells from unexposed subjects did not (Figure 47), confirming the selective reactivity of T cells from patients previously infected with SARS-CoV-2.

[0165] Example 21: SARS-CoV-2 peptide pool immune response: CD4+ T cells from previously infected SARSCoV-2 patients recognize S and N peptide pool antigens, whereas CD8+ T cells show greater recognition of the N peptide antigen. CD4+ T cells from the two patient samples tested responded to both S and N peptide pools, with a higher response to N with Pt3 (Figure 48, Panel B). In contrast, CD8+ T cells from both patients responded significantly to N, but not to S1 or S2 peptide pools (Figure 48, Panels C and D). These data are consistent with published studies demonstrating T cell responses to multiple antigens, including S and N, in previously infected SARS-CoV-2 patients.

[0166] Example 22: Autologous MoDCs infected with endo / lysosomotropic nucleocapsid-ETSD induce higher levels of IFN-γ secretion from CD4+ and CD8+ T cells from previously infected SARS-CoV-2 patients compared to cytoplasmic nucleocapsid protein (hAd5 N). To assess the immunosignificance of endo / lysosomal localized N-ETSD versus cytoplasmic N, MoDCs were infected with hAd5 constructs (null, N-ETSD, or N) and then incubated with autologous CD3+ and CD4+ or CD8+ selected T cells (Figure 49, Panel A). CD3+ T cells from previously infected SARS-CoV-2 patients showed significantly greater IFN-γ secretion in response to NETSD than both null and cytoplasmic N in two patients where N-ETSD and N were compared (Figure 49, Panels C and D). For all patients, there were relatively few interleukin-4 (IL-4) secreting CD3+ T cells (Figure 49, Panels E-G). Both CD4+ and CD8+ selected T cell populations showed significantly greater IFN-γ responses to N-ETSD than null (FIG. 49, panels H-M), and in two of three patients, CD4+ and CD8+ T cells showed greater recognition of N-ETSD compared to N. High IFN-γ and low IL4 responses indicate a predominantly Th1 cytokine response to N / N-ETSD. Cellular expression of NETSD and N was comparable in 293T HEK cells, suggesting that epitope processing and MHC loading are likely the reasons for the enhanced T cell response to N-ETSD.

[0167] Example 23: Th1-dominated SARS-CoV-2-specific CD4+ and CD8+ memory T cell recall against nucleocapsid and spike antigens is induced by hAd5 S-fusion + N-ETSD infection of autologous MoDCs from previously SARS-CoV2-infected patients. N-ETSD is more effective than N in eliciting T cell cytokine responses in patients. For total T cells (CD3+), IFN-γ responses were similar for S-fusions + N-ETSD and N-ETSD, with responses to S-fusions being relatively low (Figure 50, panels A-C). The number of IL-4 secreting T cells was very low for all (Figure 50, panels D-F). Based on the increased expression of S in the bivalent vaccine compared to the monovalent S fusions, the increased T cell responses can be explained by either increased S or T cells recognizing the presence of N. Importantly, these T cell responses were characterized by a predominance of IFN-γ (Th1) compared to IL-4 (Th2). CD4+ T cells from all three patients showed significantly higher recognition of all three constructs compared to null (Figure 50, panels G-I). While some individuals had higher responses to certain constructs, the overall responses to S-fusion, S-fusion + N-ETSD, and N-ETSD were similar. CD8+ T cells from all three patients recognized the bivalent and N-ETSD vaccines at levels significantly higher than null; only two of the three patients recognized the S-fusion to a significantly higher extent than null (Figure 50, panels J-L). These data indicate that T cells from previously infected SARS-CoV-2 patients have reactivity and immune memory recall to both vaccine antigens (S and N) in the vaccine vector.

[0168] One feature of the S-fusions is the high expression of the S RBD compared to S-WT. This was a goal of vaccine design based on findings from earlier studies of the S cryo-EM structure suggesting that RBD epitopes are largely unavailable for immune detection. An additional benefit arises from combining S with N in a bivalent vaccine through the ability of N to enhance immune detection of S, a phenomenon that has been observed by others with gene expression in general. Vaccine-expressed N proteins are transported to endosomal / lysosomal intracellular compartments, a critical antigen presentation pathway for stimulating CD4+ T cells, so that they can license dendritic cells to activate naive CD8+ CTLs. N-ETSD localizes to endosomes and lysosomes, as well as autophagosomes, in MoDCs. Targeting of both endosomes and lysosomes is desirable for enhanced antigen presentation and CD4+ T cell activation. Lysosomes can fuse acidic autophagosomes and promote 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 more readily recognized N-ETSD than N. The data presented herein strongly support the potential for enhanced efficacy of vaccine constructs specifically expressing the modified N-ETSD.

[0169] T cells are important for the clearance of SARS-CoV infection. 36,74-78 Here, hAd5 expressed S and N and induced strong antigen-specific IFN-γ but virtually no IL-4 secretion from T cells of previously infected SARS-CoV-2 patients, pointing to Th1 dominance. Antiviral Th1 cytokine responses clear diverse viruses, including SARSCoV-2, a virus closely related to SARS-CoV, from infected hosts. 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 aid in both B cell and antibody production from CD8+ T cell memory, which together function to kill virus-infected targets. Recognition of these vaccine antigens by T cell subsets is consistent with immune control of pathogens. Interestingly, the hAd5 S-fusion + N-ETSD T cell biased vaccine has the potential to not only protect uninfected patients, but also be utilized as a therapeutic for already 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 of N-ETSD was shown to be Th1-dominant, as indicated by robust interferon g and low IL-4 responses.

[0170] Methods and constructs used above Example 25: hAd5[E1-, E2b-, E3-] Platform and Constructs For testing herein, a second generation hAd5[E1-, E2b-, E3-] vector was used (Figure 44, Panel A) to generate a viral vaccine candidate construct. The hAd5[E1-, E2b-, E3-] backbone containing SARSCoV-2 antigen-expressing inserts and viral particles was generated as previously described. Briefly, high titer adenovirus stocks were generated by serial propagation in E1- and E2b-expressing E.C7 packaging cell lines, followed by CsCl2 purification and dialysis into storage buffer (2.5% glycerol, 20 mM Tris pH 8, 25 mM NaCl) by ViraQuest Inc. (North Liberty, IA). Viral particle numbers were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280 nm. Viral titers were determined using the Adeno-X™ Rapid Titer Kit (Takara Bio). The constructs generated were: i. S-WT: S protein containing 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2); ii. S-fusions: S optimized to enhance surface expression and display of RBD; iii. N: nucleocapsid (N) wild type sequence protein containing a tag for immunodetection; iv. N-ETSD: N with an enhanced T cell stimulatory domain (ETSD) along with a tag for immunodetection; and v. bivalent S-fusions + N-ETSD;

[0171] Collection of plasma and peripheral blood mononuclear cells from patients with confirmed prior SARS-CoV-2 infection and virus-naive volunteers: Blood was collected with informed consent via venipuncture from volunteers who had not been exposed to SARS-CoV-2 (UNEX) as confirmed by ELISA, and from volunteers who had been confirmed by multiple negative SARSCoV-2 tests, or had recovered from COVID-19 as indicated by recent medical history and a positive SARS-CoV-2 antibody test (Pts). The third source of whole blood was apheresis of 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.

[0172] Monocyte-derived dendritic cells (MoDCs) were differentiated from PBMCs using GM-CSF (200 U / ml) and IL-4 (100 U / ml) as previously described. Briefly, monocytes were enriched by adherence on plastic, whereas non-adherent cells were preserved and frozen as a source of lymphocytes, especially 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 PBMCs 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.

[0173] Infection of moDCs with hAd5 N-WT or N-ETSD and labeling 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 4Cells / well were plated and transduced with hAd5 N-ETSD or hAd5 N at MOI 5000 1 h after plating. Slides were incubated o / n at 37°C, fixed in 4% paraformaldehyde for 15 min, then permeabilized with 1% Triton X100 in PBS for 15 min 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) containing 3% BSA, 0.5% Triton X100 and 0.01% saponin overnight at 4C, followed by washing three times in PBS and incubation with goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 555 (Life Technologies) at 1:500 for 1 h. For colocalization studies, cells were also incubated overnight at 4C with rabbit anti-CD71 (transferrin receptor, recycling / sorting endosome marker) antibody (ThermoFisher) 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) used at 1:100. After removing the primary antibody, washing twice in PBS and three times in PBS with 3% BSA, cells were incubated with fluorescently conjugated secondary antibody at 1:500 (goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488; 1:500 dilution) for 1 h at room temperature. After a brief wash, cells were mounted with Vectashield Antifade mounting medium containing DAPI (Fisher Scientific) and immediately imaged using a Keyence all-in-one Fluorescence microscope camera and Keyence software.

[0174] Binding of plasma antibodies from patients previously infected with SARS-CoV-2 to antigens expressed by vaccine-infected MoDCs: Binding of plasma antibodies from previously infected subjects to antigens expressed on the surface of MoDCs was determined by differentiating MoDCs from peripheral blood mononuclear cells (PBMCs) into DCs, infecting MoDCs, incubating with previously infected patient plasma, and detecting binding to infected and non-infected MoDCs by flow cytometry.

[0175] MoDCs were infected with hAd5 S-WT, S-fusion, S-fusion + N-ETSD or a "null" construct expressing green fluorescent protein (GFP) at MOI 5000 (0.5x106 / well in 12-well plates). One day after infection, MoDCs were detached with EDTA (0.5mM), gently pipetted and transferred for incubation with previously infected patient plasma from a single patient (Pt4) at 1:100 dilution for 30 min (4°C) and plasma antibodies were detected on the MoDC surface by goat anti-human IgG (phycoerythrin conjugated). For flow cytometry analysis, cells were acquired as described above. Data were graphed as DMFI, i.e., difference in binding between infected and non-infected MoDCs.

[0176] Selected CD4+ and CD8+ T cell secretion of IFN-γ in response to T cells from previously SARS-CoV-2-infected patients and virus-naive individuals, as well as to MoDCs pulsed with SARS-CoV-2 peptide antigen pools

[0177] The ability of T cells from previously infected patients used in these studies to recognize SARS-CoV-2 antigens in vitro was verified, and then a similar analysis was performed on selected CD4+ and CD8+ T cells. Briefly, MoDCs (2 × 10 4) were pulsed with SARS-CoV-2 peptide antigen (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 then incubated with autologous T cells (1 × 10 5 ) was added to concentrated RPMI (10% human AB serum). Cells were cultured overnight (37°C) in microtiter plates (Millipore) containing immobilized primary antibodies targeting IFN-γ, and then IFN-γ spot-forming cells were counted by ELISpot. For ELISpot detection, after aspiration and washing to remove cells and medium, IFN-γ was detected by a secondary antibody against the cytokine conjugated to biotin. A streptavidin / horseradish peroxidase conjugate was used to detect the biotin-conjugated secondary antibody. The number of spots (1 × 10) per well was determined by the following formula: 5 Cells) were counted using an ELISpot plate reader. IL-4 was measured by ELISpot using a kit (MabTech) with wells pre-coated with anti-IL-4 antibody, following the manufacturer's instructions. The remaining steps for IL-4 detection were identical to those for IFN-γ, but with alkaline phosphatase detection instead of peroxidase.

[0178] Determination of T cell reactivity from patients previously infected with SARS-CoV-2 against autologous hAd5 vaccine-infected MoDCs: MoDCs were infected with hAd5 S-fusion, S-fusion + N-ETSD, N-ETSD, N or GFP / null constructs and incubated overnight at 37°C. Infected MoDCs were cultured overnight with CD3+, CD4+ or CD8+ T cells from the same individual. Antigen-specific T cell responses were counted using ELISpot as described above.

[0179] AdV / yeast combinations and yeast lysate preparations In addition to viral constructs expressing N-ETSD and / or S-fusions (or proteins having similarity to N-ETSD and / or S-fusions), N-ETSD and / or S-fusions (or proteins having similarity to N-ETSD and / or S-fusions) can also be expressed in yeast, either on yeast cells that present the antigen, or within yeast cells that can be prepared as a lysate to further enhance immunogenicity, as discussed in more detail below.

[0180] Figure 12 shows a schematic of an ideal vaccine that induces durable and effective immunity across multiple routes. As can be readily appreciated, the vaccine composition is preferably (but not necessarily) comprised 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).

[0181] With respect to the yeast component, the yeast is most typically preferably a recombinant yeast expressing one or more antigens of interest from recombinant nucleic acid, and possibly additional DAMP or PAMP or STING pathway signals. The recombinant yeast thus produced is typically heat inactivated, and after heat inactivation, the yeast is lysed. Most preferably, the yeast is lysed using pressure homogenization, and the homogenate is then preferably clarified by filtration. However, it should be understood that other lysis methods are also considered suitable, including enzymatic or chemical lysis of cell walls, sonication, flash freezing / thawing, etc. Similarly, clarification of the lysate may also include centrifugation, sedimentation, flocculation, etc.

[0182] As will be readily appreciated, recombinant antigens and other recombinant proteins can be expressed from any suitable promoter using known expression cassettes. For example, Figure 13 shows the results of an exemplary expression experiment in which the SARS CoV nucleocapsid protein (N) is overexpressed in Saccharomyces cerevisiae. As can be seen, significant amounts of recombinant protein were expressed in yeast.

[0183] Using mouse models, lysed tarmogen (yeast with recombinantly expressed antigens) was up to 150 times more immunogenic than intact tarmogen in terms of inducing T cell responses (based on multiple antigen-specific T cell activation studies in hundreds of mice). Advantageously, lysed yeast has a favorable safety profile and 10,000 10 YU doses can be prepared in one small-scale pressure homogenizer in a two-hour period. Whereas intact yeast induces Th1-Th17 and triggers IFNγ, IL2, IL12, IL6, TNFα, GM-CSF, yeast lysate clearly induced a strong Th1 effect and reduced Th17. Thus, intact or lysed yeast induces coronavirus-specific antibodies (see, e.g., 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).

[0184] Formulations for oral / mucosal administration In further embodiments, disclosed herein are compositions of vaccine compositions and methods for producing vaccine compositions that use aragonite to form solid dosage forms (e.g., powders, tablets, or capsules) that are stable during storage, easily administered (e.g., orally), and dissolve (e.g., releasing antigenic or pre-antigenic vaccine molecules) after passing through the stomach of a subject receiving the vaccine.

[0185] In particular, the present disclosure relates to an aragonite composition made of a plurality of aragonite particles loaded with a vaccine active ingredient to provide a solid dose vaccine in the form of a powder, tablet, or capsule. More specifically, the vaccine composition may include a recombinant expression construct in powder form (e.g., lyophilized) for expressing an antigen corresponding to a relevant infection / disease blended with a plurality of aragonite particles and thereby loaded on its surface. In an exemplary embodiment, the vaccine composition immunizes against coronavirus. Preferably, the recombinant expression construct is an adenovirus construct expressing at least one antigenic coronavirus protein or protein fragment.

[0186] In particular, the use of aragonite in the presently contemplated solid dosage forms allows for cost-effective manufacturing and easy administration of a stable vaccine composition. Thus, the presently contemplated vaccine tablets or capsules can be mass-produced and easily transported. Furthermore, the solid dosage forms allow for oral administration that can be self-administered by most people without the need for a medical professional. Tablet forms can also be made with additional excipients and / or additives (e.g., flavors and gelatin) to form lozenges.

[0187] Aragonite (e.g., ovoid aragonite) is one of the purest forms of naturally precipitated calcium carbonate. With reference to FIG. 1, aragonite has an orthorhombic, bipyramidal, characteristic needle-like crystal morphology, and is thus distinct 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 that exploit the mechanical and chemical properties of calcium carbonate minerals. Aragonite particles as disclosed herein are solid materials having regular (e.g., spherical, or ovoid) or irregular shapes. As used herein, aragonite particles have an average particle size of 100 nm to 1 mm. Methods for grinding aragonite particles are described in U.S. Patent Application Publication No. 2020 / 0308015, the entire contents of which are incorporated herein by reference. For example, methods are disclosed for grinding aragonite particles to 2.0 to 3.5 micron size with clean top size. By fine top size is meant that when produced using the disclosed milling methods with a classifier set at the 2.0-3.5 micron or 2.5-3.5 micron size range, very few particles are greater than 3.5 micron size. Thus, aragonite particles as disclosed herein using the methods of U.S. Patent Application Publication No. 2020 / 0308015 have finer top size than conventional GCC.

[0188] The adsorption capacity of aragonite is a function of three parameters: (1) surface charge (also known as "zeta potential"); (2) surface area / void ratio; and (3) particle solubility. By accurately measuring these three parameters, it is possible to determine which materials will adsorb to the aragonite particle surface under given conditions. In particular, the zeta potential of aragonite increases the stability of surfactants such as glycerol and sorbitol.

[0189] Furthermore, aragonite naturally has a large number of measurable pores in the particles with diameters less than 2 nm (i.e., highly "microporous"). See, for example, EP 2719373. Thus, the aragonite platform strongly holds the active ingredient particles together, allowing the loaded aragonite to be formulated into solid dosage forms (e.g., powders, tablets, or capsules).

[0190] Advantageously, untreated aragonite has a neutral pH (7.8-8.2), natural hydrophilicity, electronic charge (zeta potential), and already creates nitrogenous pairings with amino acids and proteins. Without being bound to any one theory, these advantageous properties of aragonite make it metastable under ambient conditions. More specifically, aragonite particles naturally contain about 2-3% amino acid content, the majority of which are aspartic acid (about 25-30%) and glutamic acid (about 8-10%), which make the aragonite surface hydrophilic. See, e.g., Mitterer, 1972, Geochimic et Cosmochimica Acta, 36:1407-1422. Thus, in some embodiments, the vaccine composition (e.g., recombinant adenovirus) is directly attached to the natural untreated surface of the aragonite particles.

[0191] Currently, calcium carbonate available in the market is processed as or from ground calcium carbonate (GCC), precipitated calcium carbonate (PCC) (synthetic), and / or limestone products. The products produced are commodity grades with different attributes. To obtain clean particle size distribution (PSD) top size and low retention, most companies utilize wet milling processes with either high solids or low solids. As used herein, aragonite refers to natural aragonite with orthorhombic, dipyramidal, and characteristic needle-like crystal morphology that is distinct from GCC, PCC, and limestone. For example, aragonite ball milled using the systems and methods disclosed in U.S. Patent Application Publication No. 2020 / 0308015 can produce 2.0-3.5 micron sized aragonite particles with clean top size. By fine top size is meant that when produced using the present systems and methods with a classifier set for the 2.-3.5 micron size range or the 2.0-3.5 micron size range, very few particles are greater than 3.5 micron size. For example, for aragonite produced using the disclosed systems at this set range, only <0.0005% is retained on 325 mesh and only <0.0007% is retained on 500 mesh compared to GCC products with the same median (D50) particle size distribution (PSD). Thus, aragonite produced using the contemplated systems and methods has a finer top size than conventional GCC.

[0192] Advantageously, solid dosage forms made from aragonite provide a solid vaccine form that can be ingested by oral administration. The solid forms of the present disclosure with an enteric coating are ingested and the antigenic molecules loaded in the inner core are not released until after passing through the stomach, thereby allowing the absorption of the antigenic or pre-antigenic molecules into the bloodstream and delivery to immune cells. As used herein, antigenic molecules refer to the desired vaccine active ingredients that are blended with the aragonite particles and loaded onto the surface of the aragonite particles. These antigenic molecules may be antigenic in the form loaded onto the aragonite particles, or they may be molecules (e.g., expression vectors) that can produce (e.g., express) at least one antigenic protein or fragment. Thus, the active ingredients or vaccine active ingredients disclosed herein are referred to as antigenic molecules, including both antigenic and pre-antigenic molecules, unless otherwise specified.

[0193] 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 that completely surrounds the inner core such that the entire outer surface of the inner core is in contact only with the outer core and no surface of the inner core is exposed; and iii) a coating that covers the entire outer surface of the outer core. Preferably, the inner core is made of aragonite particles having a diameter of at least 2 um or more, thereby providing a surface area for capturing and loading specific antigenic molecules thereon. Preferably, the outer core does not contain any antigenic molecules and is made of 90%-100% aragonite. More preferably, the outer core comprises only 100% aragonite. The outer coating of the solid composition can be any suitable coating (e.g., an enteric coating) that is stable in the highly acidic, low pH environment of the stomach (e.g., a pH of about 3) and dissolves at the higher pH of the small intestine (e.g., a pH of about 7-9). 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-tacking agents may also be combined with the enteric coating (e.g., glycerol monostearate).

[0194] In certain embodiments, the inner core is made of aragonite particles having a diameter of at least 2 um or more (e.g., 2-3.5 um) blended with a lyophilized powder of the antigenic molecule. In some embodiments, additional excipients are blended with the aragonite and antigenic molecule. For example, dimethylglycine and / or methylsulfonylmethane (MSM) can be combined with the lyophilized powder of the antigenic molecule and blended with the aragonite particles.

[0195] In further exemplary embodiments, the lyophilized antigenic 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, contemplated solid dosage vaccine compositions disclosed herein may encode at least one antigen of the nucleoprotein (N) protein and spike (S) protein of coronavirus 2 virus (CoV2), both of which are conserved in all types of coronaviruses. In one embodiment, the antigen-encoding molecule is a lyophilized recombinant, the recombinant comprising a nucleic acid encoding the nucleocapsid protein or fragment thereof of CoV2, and / or the recombinant encoding the spike protein or fragment thereof of CoV2. The vaccine formulation may be useful for treating diseases, such as coronavirus-mediated diseases or infections. Thus, in another embodiment, a method for treating a coronavirus disease in a patient in need thereof is disclosed, the method comprising administering to a subject a solid dosage vaccine composition comprising a recombinant comprising a nucleic acid encoding at least a CoV2 N protein or a fragment thereof, preferably encoding both a CoV2 N protein or a fragment thereof and a CoV2 S protein or a fragment thereof. The coronavirus contemplated herein may be coronavirus disease 2019 (COVID-19) and / or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0196] In a further exemplary embodiment, a contemplated solid dosage vaccine composition disclosed herein comprises aragonite blended with a lyophilized recombinant made with a bivalent human adenovirus serotype 5 (hAd5) expression vector. hAd5 can induce immunity in patients with pre-existing adenovirus immunity and expresses antigens for producing antibodies targeting coronavirus 2 spike (S) protein and / or nucleocapsid (N) protein. For example, hAd5 CoV2 can encode a modified nucleocapsid protein with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1. In a more specific embodiment, hAd5 contains both an S sequence optimized for cell surface expression (S-fusion) and a conserved nucleocapsid (N) antigen designed to be transported to the endosomal intracellular compartment, which may generate durable immune protection against CoV2, as disclosed in US Patent Application Serial No. 16 / 883,263, the entire contents of which are incorporated herein by reference. For example, hAd5 CoV2 may encode an S-fusion or S-HA protein with 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.

[0197] Advantageously, the bivalent hAD5 vaccine provides: (i) an optimized S-fusion with improved S receptor binding domain (RBD) cell surface expression compared to S-WT (wild type), which had barely detectable surface expression; (ii) the expressed RBD from the S-fusion retained conformational integrity and recognition by ACE2-Fc; (iii) the viral N protein modified with an enhanced T cell stimulatory domain (ETSD) localized to endosomal / lysosomal intracellular compartments for MHC I / II presentation; and (iv) these optimizations for 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, as shown in more detail below.

[0198] In a preferred embodiment, the lyophilized bivalent hAd5 vaccine comprises a replication-deficient adenovirus with E1, E2b, and E3 gene region deletions, together with a nucleic acid encoding the coronavirus 2 (CoV2) nucleocapsid (N) protein fused to an endosomal targeting sequence (N-ETSD) and a nucleic acid encoding the 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:2. More typically, the CoV2 N-ETSD protein encoded in the hAd5 adenovirus has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. Typically, the nucleic acid encoding the CoV2 S-HA or S-fusion protein in the hAd5 adenovirus has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity 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 has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3 or SEQ ID NO:4.

[0199] In further embodiments, the lyophilized bivalent hAd5 vaccine may also include nucleic acids encoding trafficking sequences, costimulatory molecules, and / or immunostimulatory cytokines. Examples of encoded costimulatory 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.

[0200] From a different perspective, a solid dosage form of a coronavirus vaccine composition (e.g., a lyophilized powder composition comprising a recombinant adenovirus genome, optionally comprising a deleted or non-functional E2b gene) made of aragonite particles blended with a viral vector in lyophilized form, comprising (a) at least a wild-type or modified nucleocapsid protein; and / or (b) a nucleic acid encoding at least one wild-type or modified spike protein is contemplated herein. The viral vector may further comprise a costimulatory molecule. Typically, the nucleic acid will encode a trafficking signal for directing the peptide product encoded by the nucleic acid to the cytoplasm, endosomal compartment, or lysosomal compartment, and the peptide product will further comprise a sequence portion that enhances the intracellular turnover of the peptide product.

[0201] As discussed, aragonite allows for direct binding of freeze-dried vaccine active ingredients, thus enabling the manufacture and preparation of solid dose vaccine compositions. Thus, contemplated methods for making the disclosed solid dosage vaccines include loading (e.g., by mixing) the vaccine active ingredients into ground aragonite particles to form an inner core of aragonite and vaccine active ingredients, as disclosed herein and in US Patent Publication No. 2020 / 0308015, the entire contents of which are incorporated herein by reference. Any suitable method of blending the freeze-dried vaccine active ingredients with the aragonite particles can be used. Loading or mixing the ground aragonite particles (e.g., having a D50 PSD of 2um to 3.5um) with the freeze-dried vaccine active ingredients can be performed by any conventional methodology. Mixing the aragonite with the active ingredients includes mixing the dry weights of both the aragonite and the freeze-dried active ingredients in a closed vessel suitable for rotation / inversion. For example, inversion or rotation of the aragonite and the active ingredients can be performed for about 5 minutes to up to 30 minutes. For example, the vaccine composition can be loaded onto a solid dosage form in a mixer (e.g., a tumbling mixer) or blender. The amount of aragonite can vary depending on the amount of lyophilized active ingredient and the determined potency of the active ingredient to induce an immune response. For example, an effective dose of the lyophilized bivalent hAd5 vaccine disclosed herein is about 1×10 per capsule (or tablet). 9 IU. Therefore, 2.21 × 10 7 A lyophilized bivalent hAd5 vaccine composition with a potency of IU / mg requires approximately 45.25 mg of lyophilized bivalent hAd5 powder per capsule. In some embodiments, the weight of a solid single capsule can be about 300 mg to 600 mg. In certain embodiments, for a 550 mg capsule, approximately 40-60 mg of lyophilized active ingredient (e.g., lyophilized bivalent hAD5 expression of both N and S CoV2 proteins) is mixed with 490-510 mg of aragonite. 2.21×10 7For an active ingredient having a potency of IU / mg, 45.25 mg of lyophilized active ingredient may be blended with approximately 504.75 mg of aragonite. Formation of the mixture into tablets or caplets may be accomplished using any suitable encapsulation method and / or kit known in the art, such as the Capsule Machine Filler (Item# CPM1001 / 2081677).

[0202] In a preferred embodiment, contemplated methods for making solid dose vaccine tablets or capsules also include making an outer core of aragonite that encompasses (e.g., completely encases) the inner core. Typically, the outer core is made mostly (e.g., at least 90%) of aragonite, and more typically, the outer core is made of at least 99% aragonite.

[0203] To deliver antigenic molecules to the bloodstream in a tablet or capsule for oral administration, the antigenic molecules must remain in the inner core, enclosed by the outer core, until they pass through the stomach and are available for absorption in the intestine (e.g., small intestine). Thus, the outer core is coated with an enteric coating that is stable at the low pH of the stomach (e.g., a pH of about 3) and dissolves at the higher pH of the intestine (e.g., a pH of about 7-9). 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). Methods for applying enteric coatings are well known in the art. For example, a coating device or apparatus can be used. A specific example of a coating device includes the ProCoater (manufactured by Torpac).

[0204] Coated (C) and uncoated (NC) capsules made from aragonite particles or lactose mixed with hAD5-COVID-S / N were exposed to acid (HCl) to determine the acid permeability of the various capsules. For example, Figure 2 shows three pictures (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), with wrinkles, swelling, or holes observed in the capsule as indicated: 1): 2 min after HCL acid exposure; 2): 2 h after HCL acid exposure; and 3) 2 h after HCL acid exposure and drying. Figure 3 shows three photographs (from right to left: 1, 2, 3) of hAD5-COVID-S / N in uncoated aragonite capsules (samples #7 or #8) in 0.1 M HCL, with swelling, twisting, or holes observed in the capsule as indicated: 1): Sample #7 after 2 hours of HCL acid exposure; 2): Sample #8 after 2 hours of HCL acid exposure; 3) after 2 hours of HCL acid exposure and drying. Figure 4 shows two photographs (from right to left: 1, 2) of hAD5-COVID-S / N in uncoated lactose capsules (samples #3 or #4) in 0.1 M HCL, with swelling of the capsule observed as indicated: 1): Sample #3 after 2 hours of HCL acid exposure; 2): Sample #4 after 2 hours of HCL acid exposure. Figure 5 shows two pictures (from right to left: 1, 2) of hAD5-COVID-S / N in coated aragonite capsules (samples #1 or #5) in 0.1M HCL, with capsule swelling observed as shown: 1): Sample #1 after 2 hours of HCL acid exposure; 2): Sample #5 after 2 hours of HCL acid exposure. Clearly, the coated capsules were stable in acid.

[0205] Sample capsules were further assayed for infectious units per gram (IFU / gram) and percent virus recovery, as shown in Figures 6-11. More specifically, Figure 6 shows infectious units per gram (IFU / gram) (y-axis) for the indicated hAD5-COVID-S / N capsule types, as shown. Figure 7 shows the percentage (%) virus recovery (y-axis) for each hAD5-COVID-S / N capsule type, as shown. Figure 8 shows the IFU / gram and corresponding pH for each hAD5-COVID-S / N capsule type, as shown. Figure 9 shows the percentage (%) virus recovery for each hAD5-COVID-S / N capsule type, as shown. Figure 10 shows the percent virus recovered for each hAD5-COVID-S / N capsule type, as shown, with acid treatments shown for those with shading. Figure 11 shows infectious units / gram for each hAD5-COVID-S / N capsule type, as shown, with acid treatments shown for those with shading. The results are summarized in Table 2 below.

[0206] [Table 1]

[0207] In an alternative embodiment, the contemplated dosage form comprises an inner core of aragonite impregnated (i.e., bonded) with carbon dioxide (CO2) prior to the addition of the vaccine composition. See, for example, EP 2719373 and US 2020 / 0155458. In a further embodiment, the contemplated dosage form comprises aragonite with a biocompatible polymer and / or 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 both the biocompatible polymer and the disintegrant prior to the addition of the vaccine composition. More typically, the aragonite is impregnated with CO2, mixed with the biocompatible polymer and the disintegrant, and formed into a solid form (e.g., compressed) prior to the addition of the vaccine composition. See, for example, EP 2719373 and US 2020 / 0155458.

[0208] In a further embodiment, the aragonite may be bound with carbon dioxide (CO2) and mixed with at least one biocompatible polymer as described herein. Typically, the weight ratio of the CO2-bound aragonite to the biocompatible polymer is about 95:5 to 5:95. In a further embodiment, the 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 polymers, acrylonitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide / sulfide, polypopylene, Teflon, polylactic acid, aliphatic polyesters such as polyhydroxybutyrate, poly-3-hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconate, poly(dioxanone), and mixtures thereof. Preferably, the biocompatible polymer resin is PLA.

[0209] Additional excipients may be added to the inner and / or outer core of the solid dosage form as dictated by manufacturing and packaging needs, including ion exchange resins, gums, chitin, chitosan, clays, gellan gum, crosslinked polacrilin copolymer, agar, gelatin, dextrin, acrylic acid polymers, sodium / calcium carboxymethylcellulose, hydroxypropylmethylcellulose phthalate, shellac or mixtures thereof, lubricants, internal phase lubricants, external phase lubricants, impact modifiers, plasticizers, waxes, stabilizers, pigments, colorants, flavors, taste masking agents, flavorings, sweeteners, mouthfeel enhancers, binders, diluents, film formers, adhesives, buffers, adsorbents, odor masking agents, and mixtures thereof.

[0210] In an alternative embodiment, the aragonite particle surface can be treated to modify the binding surface. For example, treatment with stearic acid (i.e., octadecanoic acid) provides a hydrophobic surface, as disclosed in US Patent Application No. 16 / 858,548 and PCT / US20 / 29949. For protein loading, treatment of aragonite with phosphoric acid forms a lamellar structure. Further conjugation techniques for binding reactive groups to the amino acid surface of aragonite are known in the art, as disclosed, for example, in Bioconjugate Techniques, Third Edition, Greg T. Hermanson, Academic Press, 2013.

[0211] Oral / Mucosal Vaccines Based at least in part on the above formulations, the present disclosure also provides methods and compositions for administering, monitoring, and assaying vaccines. Contemplated methods include inducing immunity to the virus in a patient, administering the vaccine composition to the patient by delivery to the patient's nasal, oral, and / or alien mucosa. Preferably, the vaccine targets SARS-like coronavirus (SARS-CoV-2). The oral vaccine compositions described herein can serve as a booster vaccination for any initial prime vaccination against SARS-CoV-2 S or N proteins.

[0212] 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) proteins. This booster can even work in patients immunized with anti-S or anti-N vaccines other than those described herein. In certain embodiments, the initial prime vaccine can be a lipid nanoparticle vaccine containing mRNA encoding the S protein, such as vaccines currently being tested by Moderna and Pfizer. In certain embodiments, the boost described herein is administered at least 7 days after the initial prime vaccination, e.g., 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. The boost described herein can effectively improve both antibody production against SARS-CoV-2 and cell-mediated immunity against SARS-CoV-2.

[0213] Preferably, the vaccine administered to induce immunity in the mucosal tissue of the patient is a vaccine against SARS-CoV-2. In an exemplary embodiment, the vaccine is a replication-deficient adenovirus construct comprising an E1 gene region deletion and an E2b gene region deletion. In a specific embodiment, the adenovirus comprises a sequence encoding a SARS-CoV-2 spike fusion protein antigen having 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 to SEQ ID NO:4 (e.g., SEQ ID NO:12). In a specific embodiment, the adenovirus comprises a sequence encoding a SARS-CoV-2 modified spike protein antigen having 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 to SEQ ID NO:11 (e.g., SEQ ID NO:13). In certain embodiments, the adenovirus comprises a sequence encoding a soluble ACE2 protein linked to an immunoglobulin Fc portion to form an ACE2-Fc hybrid construct that may also include a J chain portion, as disclosed in U.S. Patent Application No. 16 / 880,804 and U.S. Patent No. 63 / 016,048, the entire contents of both of which are incorporated herein by reference. In other exemplary embodiments, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) comprises a mutant of a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10) having at least one mutated amino acid residue (e.g., by substitution) that confers increased binding affinity of the ACE2 protein to the RBD protein domain of the SARS-CoV-2 spike protein, as disclosed in U.S. Patent Application No. 63 / 022,146, the entire contents of both of which are incorporated herein by reference.In another exemplary embodiment, the SARS-CoV-2 vaccine (e.g., adenoviral construct) comprises a CoV2 nucleocapsid protein or a CoV2 spike protein fused to an endosomal targeting sequence (NETSD), as disclosed in U.S. Patent Application No. 16 / 883,263 and U.S. Patent No. 63 / 009,960, the entire contents of both of which are incorporated herein by reference. Additionally or alternatively, the SARS-CoV-2 vaccine comprises modified yeast cells (e.g., Saccharomyces cerevisiae) genetically engineered to express coronavirus spike proteins on the yeast cell surface, as disclosed in U.S. Patent Application No. 63 / 010,010, whereby the yeast display cells stimulate B cells (e.g., humoral immunity). Advantageous features of the compositions and methods described herein are further illustrated (but not limited to) by the following examples.

[0214] Example 26: NHP Vaccination Two groups of rhesus macaques (5 per group) were subcutaneously immunized on day 0 with the adenoviral anti-SARS-CoV-2 vaccine described above. Blood was collected from each macaque before immunization. On day 14, one group of rhesus macaques (group 1) received another subcutaneous booster injection of the same vaccine, while the other group (group 2) received the oral vaccine 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 macaques (controls) were sham vaccinated at the indicated time points. Blood was collected on days 14, 21, 28, 35 and 42.

[0215] Serum samples taken from these macaques at the indicated time points were then assessed by ELISA for anti-spike protein IgG and IgM seroreactivity. Briefly, 96-well EIA / RIA plates (ThermoFisher, Cat#07-200-642) were coated with 50 μL / well of a 1 μg / mL solution of purified recombinant SARS-CoV-2 derived spike protein (S-fusion; ImmunityBio, Inc.) suspended in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) and incubated overnight at 4°C. Individual 96-well plates were prepared for each immunoglobulin type (IgG or IgM) by washing three times per well with 150 μL of TPBS solution (PBS + 0.05% Tween 20). Then, 100 μL / well of blocking solution (2% non-fat milk in TPBS) was added and incubated for 1 h at room temperature (RT). Plasma and serum samples were heat inactivated at 56°C for 1 hour before use. Serial dilutions of plasma, serum or antibody samples were prepared in 1% non-fat milk in TPBS. Plates were washed as above and 50 μL / well of each serial dilution was added to the plate and incubated at room temperature for 1 hour. Plates were washed 3 times with 200 μL of TPBS. Dilutions (1:6000) of each goat anti-human IgG (H+L) cross-adsorbed, HRP, polyclonal; or goat anti-human IgM (heavy chain) cross-adsorbed secondary antibody, HRP (ThermoFisher, Cat#62-842-0 or A18841, respectively) were prepared in 1% non-fat milk / TPBS and 50 μL / well of these secondary antibodies were added to separate reactions / plates for each immunoglobulin type (IgG or IgM) and incubated at RT for 1 hour. Plates were washed 3 times with 200 μL of TPBS. One component (3,3',5,5'-tetramethylbenzidine (TMB) substrate, 50 μL / well, VWR, Cat#100359-156) was added to each well and incubated for 10 minutes at room temperature, 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.).The data was analyzed using Prism 8 (GraphPad Software, LLC) and is shown in Figure 14, which shows the ELISA results detecting IgG seroreactivity to the SARS-CoV-2 spike in serum samples collected from immunized rhesus macaques.

[0216] Example 27: NHP Challenge On day 56, the macaques were challenged with respiratory exposure to SARS-CoV-2 virus. Nasal swabs were collected daily from these macaques on days 56-63. Bronchoalveolar lavage (BAL) fluid was collected on days 57, 59, 61, and 63. The ability of sera from collected samples to inhibit SARS-CoV-2 infectivity is shown in Figure 15. Panel A shows the ability of sera from vaccinated group 1 rhesus macaques to inhibit SARS-CoV-2 infectivity in vitro. Panel B shows the ability of sera from vaccinated group 2 rhesus macaques to inhibit SARS-CoV-2 infectivity in vitro. The dotted line indicates 20% inhibition. As can be seen from the figure, sera from both group 1 and group 2 rhesus macaques inhibited infectivity, with sera collected later inhibiting more potently than sera collected earlier. Serum from control rhesus macaques had no inhibitory effect at any of the time points tested. Viral load over time in the nasopharynx is shown in Figure 16. Panel A shows viral load (qPCR) in nasal swabs from macaques in group 1 after SC+SC+oral vaccination, and Panel B shows viral load (qPCR) in nasal swabs from macaques in group 1 after SC+oral+oral vaccination. Viral load over time in the lungs (BAL) is shown in Figure 17. Figure 17. Panel A shows viral load (qPCR) in BAL from macaques in group 1 after SC+SC+oral vaccination, and Panel B shows viral load (qPCR) in BAL from macaques in group 1 after SC+oral+oral vaccination.

[0217] Example 28: Seroreactivity Serum samples from various volunteers who received various experimental anti-SARS-CoV-2 vaccines were collected and assayed for IgG and IgM seroreactivity to the SARS-CoV-2 S protein by ELISA as described above. The results are shown in Figure 18, where the ELISA results detect IgG and IgM seroreactivity to the SARS-CoV-2 spike in serum samples taken from human patients immunized with various experimental anti-SARS-CoV-2 vaccines.

[0218] Example 29: Human Immunization Human volunteers were divided into three cohorts. Cohort 1 (10 individuals) received 5×10 10 Cohort 2 (10 individuals) were immunized by subcutaneous injection with viral particles (E1- / E2b- Ad5 containing SEQ ID NO: 12 or SEQ ID NO: 13). 11 Cohort 3 (15 individuals) were immunized with 10% of the vaccine described herein by subcutaneous injection of viral particles. 11 viral particles (or 5 × 10 if safety concerns dictate a lower dose) 10 Volunteers were immunized by subcutaneous injection of 1000 ribosomal RNA (virion particles) from each of the volunteers. Blood was drawn from each volunteer on the same day that the initial prime vaccination was administered. Blood was drawn again on days 8, 15, and 22. A booster injection of the same vaccine was administered on day 22.

[0219] ELISpot tests were performed on blood drawn on days 1 and 15 to evaluate cell-mediated immunity against SARS-CoV-2. 400,000 viable PBMCs (Cellometer K2w / AO / PI viability staining) from each blood draw well were stimulated with blank medium, SARS-CoV-2 S, SARS-CoV-2 N, SARS-CoV-2 M, CD3 / CD28 / CD2, and CEFT. 48 hours after stimulation, the supernatants were frozen (-80°C) for later testing. Figure 19 shows the results of ELISpot tests from Th1 N-responsive patients 3, 6, and 11. Figure 20 shows the results from patient 4 (N-unresponsive) and patient 10 (weakly Th1 N-responsive). None of these patients showed a Th2 response to N.

[0220] Airway Protection In still further experiments, contemplated vaccine formulations and methods of use provided protection of the nasal and pulmonary airways against SARS-CoV-2 challenge in non-human primates. As shown in more detail below, a dual antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike (S-fusion) protein and the viral nucleocapsid (N) protein with an enhanced T cell stimulatory domain (N-ETSD) increases MHC class I / II responses. The adenovirus serotype 5 platform used, hAd5 [E1-, E2b-, E3-], has previously been demonstrated to be effective in the presence of Ad immunity and can be delivered in an oral formulation that overcomes cold chain limitations. The hAd5 S-fusion + N-ETSD vaccine was evaluated in rhesus macaques and demonstrated that a subcutaneous prime followed by oral challenge elicited humoral and Th1-dominant T cell responses against both S and N, with the upper and lower respiratory tract immunizing to high titers (1x10 6 (A) hAd5 platform and hAd5 S-fusion + N-ETSD constructs with deleted E1, E2b, and E3 regions (B) and (C) were shown to protect against SARS-CoV-2 challenge with 100 TCID50. Notably, viral replication was inhibited within 24 hours of challenge in both the lungs and nasal cavity and was undetectable within 7 days after challenge. Figure 25 shows the hAd5 platform and hAd5 S-fusion + N-ETSD constructs. (A) hAd5 platform with deleted E1, E2b, and E3 regions (C) and (D) hAd5 S-fusion + N-ETSD constructs with deleted E1, E2b, and E3 regions (D) and (C) hAd5 S-fusion + N-ETSD constructs with deleted E1, E2b, and E3 regions (E) and (C) hAd5 S-fusion + N-ETSD constructs with deleted E1, E2b, and E3 regions (E) and (C) hAd5 S-fusion + N-ETSD constructs with deleted E1, E2b, and E3 regions (F ... *) Human adenovirus serotype 5 vaccine platform. The vaccine construct is inserted into the E1 region (red arrow). (B) The dual antigen vaccine contains both the S-fusion and N-ETSD under the control of the cytomegalovirus (CMV) promoter, with a C-terminal SV40 polyA sequence provided by the hAd5[E1-, E2B-, E3-] platform.

[0221] The dual antigen hAd5 S-fusion + N-ETSD vaccine in Figure 25 expresses the viral spike (S) protein fused to a signal sequence (S-fusion), which enhances cell surface expression of the spike receptor binding domain (S RBD) in in vitro studies compared to S wild type, as predicted based on reports of similar sequences and antigens used in the majority of other vaccines being developed. This vaccine also expresses the viral nucleocapsid (N) protein with an enhanced T cell stimulatory domain (N-ETSD) that directs N to the endo / lysosomal intracellular compartment, predicted to enhance MHC class II responses.

[0222] SARS-CoV-2 vaccine antigens are delivered by a recombinant human adenovirus serotype 5 (hAd5) [E1-, E2b-, E3-] vector platform (Figure 25), which allows for rapid generation of vaccines against multiple agents, allowing for the production of multiple doses in a minimal 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 adenovirus vaccine platform technologies in development and allows it to be effective in the presence of pre-existing adenovirus immunity. This platform has generated vaccines against viral antigens such as influenza, HIV-1 and Lassa fever, and has shown induction of both antibody and cell-mediated immunity. In 2009, vaccination of mice with a hAd5[E1-, E2b-, E3-] vector expressing the H1N1 hemagglutinin and neuraminidase genes elicited both cell-mediated immune and humoral responses that protected the animals from lethal viral challenge.

[0223] The vast majority of other SARS-CoV-2 vaccines in development target only wild-type S antigens and are expected to elicit SARS-CoV-2 neutralizing antibody responses. Vaccine development prioritizes activated T cells to enhance respiratory and duration of protective immune responses; the addition of N in particular was predicted to provide a greater opportunity for T cell responses. T cells may provide immune defense at least as important as the production of antibodies. In SARS-CoV-2 convalescent patient studies, virus-specific T cells were found in most patients, including asymptomatic individuals, even those with undetectable antibody responses.

[0224] In preliminary testing of the hAd5 S-fusion + N-ETSD vaccine in mouse models, the vaccine activates T cells as well as elicits T helper cell 1 (Th1)-dominant antibody responses against both S and N. MoDCs from SARS CoV-2 convalescent individuals were transduced with the dual antigen vaccine, and S-fusion and N ETSD-expressing MoDCs were incubated with T cells from those same individuals. The vaccine antigen induces interferon-g (IFN-γ) secretion by both CD4+ and CD8+ T cells. This demonstrates that T cells from SARS-CoV-2 convalescent individuals "recall" the S-fusion and N-ETSD antigens presented by the transduced MoDCs as if they had been re-exposed to the virus itself. This T cell recall of vaccine antigens suggests that, conversely, hAd5 S-fusion + N-ETSD vaccination generates T cells that recognize SARS-CoV-2 antigens upon viral infection and protect vaccinated individuals from disease.

[0225] The generation of T cell responses may be a key feature for a vaccine to be effective against many variants, whose emergence may at least in part be an escape response to antibodies generated by either the first wave virus (28) or antibody-based vaccines. As reported elsewhere, neutralization by 14 of the 17 most potent mRNA vaccine-elicited monoclonal antibodies (mAbs) was either reduced or absent for the variants E484K, N501Y, or the K417N:E484K:N501Y combination. It was also found that these variants were selected when recombinant vesicular stomatitis virus (rVSV) / SARS CoV-2 S was cultured in the presence of these mAbs, strongly suggesting that the presence of these antibodies acts as an evolutionary force driving the emergence of new variants. T cells are not vulnerable to such forces and, if effectively established by vaccination, can provide protection against existing virus strains and circumvent mutants.

[0226] In a next step in the development of a hAd5 S-fusion + N-ETSD vaccine, GMP (Good Manufacturing Practice) grade liquid and oral forms of the vaccine were tested in non-human primates (NHPs). An important objective of the NHP study design was to evaluate the efficacy of a subcutaneous (SC) prime followed by a heat-stable oral boost. The oral boost offers several advantages in SARS-CoV-2 vaccination, including a greater likelihood of generating mucosal immunity, especially in the gastrointestinal tract, one of the main sites of infection. SARS-CoV-2 is a mucosal virus and is rarely detected in the blood, therefore a vaccine that specifically targets mucosal immunity is of interest. A compelling additional advantage of the heat-stable oral boost is the possibility that patients could self-administer the boost at home, which could potentially transform the global distribution of vaccines, especially in developing countries. The hAd5 S-fusion + N-ETSD construct induces both humoral and CMI responses to both antigens and therefore has the potential to serve as a "universal" heterologous booster vaccine for the multiple SARS-CoV-2 vaccines in development.

[0227] One study determined that efficacy of hAd5 S-fusion + N-ETSD vaccine in rhesus macaques maximized T cell responses when delivered as either SC prime and SC and oral boost (SC-SC-Oral; n=5) or SC prime and two oral boosts (SC-Oral-Oral; n=5), both with a regimen of prime on day 0 and boost on days 14 and 28. The objective of this study was to evaluate the immunogenicity of the dual antigen hAd5 vaccine in both SC and oral formulations, as well as the potential of an oral dose to serve as a boost after a single SC prime. Cell-mediated T cell responses after challenge, as well as nasal and pulmonary protection from SARS-CoV-2 infection, and the rate of viral clearance were evaluated.

[0228] Clinical signs, hematology and clinical chemistry: During twice-daily observations for clinical signs of vaccination toxicity, no clinical signs were observed, and 1 x 10 11 Two weeks after a single subcutaneous immunization with vaccine particles (VP), or 1 × 10 10 During the week following oral boost with IU of hAd5-S-fusion + N ETSD, no animals died. Furthermore, 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 as a result of vaccination.

[0229] SC prime with oral boost induces the production of neutralizing, anti-spike antibodies: As shown in FIG. 21, all SC-oral-oral vaccinated NHPs produced anti-S IgG that increased after both oral boosts on days 14 and 28 (panels A and B). Sera from four of the five SC-oral-oral NHPs were collected at baseline and weekly starting on day 14 through day 42 and showed inhibition in a neutralization assay (panel C) that assesses inhibition of binding of S RBD to recombinant angiotensin-converting enzyme 2 (ACE2) and has been reported to correlate with the ability of sera to neutralize SARS-CoV-2 virus. Anti-S IgG production was similar for SC-SC-oral (first boost was SC) hAd5 S fusion + N-ETSD vaccinated NHPs (panels D and E), and sera from all five NHPs in this group showed inhibition in a surrogate assay for virus neutralization (panel F).

[0230] SC prime, oral boost vaccination reduces viral load in the nose and lungs following SARS CoV-2 challenge: RT-qPCR analysis of genomic RNA (gRNA) was performed on nasal swabs and bronchoalveolar lavage (BAL) samples to determine the amount of virus present. SC-oral vaccination of NHPs reduced SARS-CoV-2 gRNA in nasal swab samples compared to placebo control NHPs from day 57, the first day post-challenge (Figure 22, panels A and B). Viral gRNA in this group continued to decrease to very low or below the level of detection (LOD) in all vaccinated animals by day 63, 7 days post-challenge. Placebo controls had moderate to high levels of SARS-CoV-2 present in nasal swab samples throughout the study period (range 2E+09 to 8.4E+03 gene copies / mL).

[0231] gRNA was also rapidly reduced in lungs (bronchoalveolar lavage, BAL) of SC-oral-oral NHPs, with the geometric mean showing an approximately 2 log reduction in vaccinated NHPs compared to placebo NHPs on day 57, just one day after challenge (Figure 22, panels C and D). In the group receiving SC and oral boost (SC-SC-oral), SARS-CoV-2 gRNA in nasal swab samples was also reduced similarly to that seen in SC-oral vaccinated primates, with viral gRNA reduced to levels well below or below the LOD in all vaccinated animals by day 63 (Figure 22, panels E and F). In lungs of SC-SC-oral NHPs, gRNA also showed an approximately 2 log reduction on day 57 (Figure 22, panels G and H).

[0232] SC prime, oral boost vaccination immediately inhibited viral replication188 in the nose and lungs following SARS-CoV-2 challenge: the presence of replicating virus in nasal swab samples was determined by RT qPCR of subgenomic RNA (sgRNA). By day 60, four days after challenge, sgRNA was below the LOD for two SC-oral-oral primates, and beginning on day 61, was below the LOD for all primates that received only the oral boost (Figure 23, panels A and B). In the lungs of SC-oral-oral NHPs, sgRNA was also reduced compared to placebo beginning on day 57, and were all below the LOD by day 63 (Figure 23, panels C and D). Evidence of replicating virus in the nasal cavity also declined rapidly in SC-SC-oral NHPs, below the LOD by day 59 in two primates and below the LOD in all primates by day 63 (Figure 23, panels E and F); in the lungs of this group, sgRNA was reduced by approximately 2 logs compared to placebo controls at day 57, one day after challenge, and sgRNA was below the LOD at day 63 in four of five primates and just above the LOD at day 5 (Figure 23, panels G and H). It is noted that there was not only a rapid decline in both viral load and replicating virus in the nasal cavity and lungs, but also that there was no growth of virus after challenge. This implies the presence of pre-existing humoral and cellular immunity resulting in rapid clearance of the virus upon infection.

[0233] The immediate protection of NHPs from SARS-Cov-2 challenge may be due to the presence of T cells responding to both S and N, and rapid viral clearance upon 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 were determined by ELISpot on day 0 before prime vaccination, day 14 (before boost) and day 35, one week after the second boost on day 28. T cells from SC-oral-oral vaccinated primates secreted interferon-γ (IFN-γ) in response to both S and N peptides on days 14 and 35 (Figure 24, Panel A). Interleukin-4 (IL-4) secretion was very low (Figure 24, Panel B), indicating that the T cell response was T helper cell 1 (Th1) dominated, as reflected by the IFN-γ / IL-4 ratio (Figure 24, Panel C).

[0234] For sera collected in the post-challenge period, a microneutralization assay (see Methods) was used to assess SARS-CoV-2 neutralization capacity as reflected by "MN50", i.e., the serum dilution that correlates with a 50% reduction in viral infectivity compared to serum-free controls. A rapid increase in neutralization capacity of sera for NHPs that received only oral boost was seen over the course of 2 weeks post-challenge, reflecting a reduction in nasal gRNA and sgRNA (Figure 24, panels E and F, respectively) and pulmonary gRNA and sgRNA (Figure 24, panels G and H, respectively) (Figure 24, panel D). Notably, sera from placebo group primates did not show an increase in neutralization capacity after challenge (Figure 24, panels D and L), suggesting the presence of memory B cells in the vaccinated group and the absence of such cells in the non-vaccinated placebo group. Further studies are required to confirm this hypothesis.

[0235] For SC-SC-orally vaccinated NHPs, findings were very similar, including reflection of a reduction in gRNA and sgRNA in the nasal cavity and lungs (Figure 24, panels M-P), for reactive T cells during the pre-challenge vaccination period (Figure 24, panels I-K) and neutralizing capacity during the post-challenge period (Figure 24, panel L).

[0236] The presence of cytotoxic T cells upon vaccination (Figure 24, panels A-C and I-K) led to an almost immediate decrease in viral replication within the first 24 hours after challenge (Figure 24, panels F, H, N, and P), with a continuing decrease over 2 weeks reflecting the increased neutralizing capacity of sera from vaccinated but not placebo NHPs, reflecting the contribution of anti-S-producing memory B cells (Figure 24, panels D and L).

[0237] This study demonstrates that subcutaneous prime and oral boost dual antigen hAd5 S-fusion + N-ETSD vaccination protects both the nasal and pulmonary airways against SARS-CoV-2 challenge in a rhesus macaque NHP model. Inhibition of viral replication in the nasal cavity, as evidenced by a reduction in sgRNA on the first day after viral challenge, was remarkable, with sequential clearance of virus to undetectable levels within 7 days of challenge in all (10 / 10) animals (Figures 22 and 23); and although rhesus macaques are not a model for the assessment of transmission, these rapid reductions in nasal viral replication encourage and support the investigation of the ability of this vaccine to prevent transmission in future studies.

[0238] The ability of hAd5 S fusion + N-ETSD vaccination to elicit virus-neutralizing anti-S antibodies (Figure 21) and T cells responding to both S and N (Figure 23), especially when accompanied by a rapid increase in neutralizing capacity of sera post-challenge, likely indicative of the presence of memory B cells, suggests that the vaccine establishes broad immunity against severe SARS-CoV-2 infection.

[0239] The potential of the hAd5 S-fusion + N-ETSD SC prime, oral boost vaccine to generate cytotoxic T cells is an important feature given the important role T cells play in protection from infection in convalescent COVID-19 patients, where SARS-CoV-2-specific T cells were identified even in the absence of antibody responses. The apparent near-immediate reduction in viral replication with hAd5 S-fusion + N-ETSD vaccination contrasts with findings reported in other adenovirus-vectored S-only vaccine NHP studies, where there was evidence of continued viral replication in some animals for at least 1 day after challenge. Compared to titers of 1 × 10 used in several other NHP vaccine studies, 6 Even when challenged with the relatively high titer of 281 TCID50 / mL, vaccinated animals in the study appeared to be protected from the earliest time points evaluated. This rapid protection and clearance was particularly evident in the lungs, where both viral load and viral replication were approximately 1-2 logs lower than placebo in both vaccinated groups just 1 day after challenge.

[0240] The protection conferred by hAd5 S-fusion + N-ETSD vaccination of NHPs with SC and oral boost doses highlights the potential of this vaccine to be developed for global distribution, especially in light of escape variants that are resistant to antibodies and convalescent plasma and are currently spreading rapidly around the world. Oral hAd5 S-fusion + N-ETSD formulations do not require ultra-low temperature freezing like many COVID vaccines currently in development. Dependence on the cold chain for distribution to geographically remote or underdeveloped areas will likely pose shipping and storage challenges and reduce the accessibility of RNA-based COVID-19 vaccines.

[0241] The thermostable oral hAd5 S-fusion + N-ETSD vaccine also has the potential to act as a "universal" boost to other previously administered vaccines that deliver only the S antigen, due to expression of S and N. This use will also be facilitated by cold chain independence.

[0242] Saliva test In further contemplated aspects, methods and compositions for administering, monitoring, and assaying vaccines are disclosed herein. Contemplated methods include inducing immunity to a virus in a patient, administering a vaccine composition to a patient by administering the vaccine composition to the patient via delivery to the nasal, oral, and / or gastrointestinal mucosa of the patient. Preferably, the vaccine targets SARS-like coronavirus (SARS-CoV-2).

[0243] In particular, the disclosed method also includes obtaining a saliva sample from the patient at a period of time after administering the vaccine. 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 0.10-2.0% weight / volume (w / v) glutaraldehyde, 0.10-1.0% w / v sodium benzoate, and / or 0.025-0.20% w / v citric acid. Further embodiments include analyzing the saliva sample for at least one selected from antibodies targeting the virus or a protein specific for the virus, and in the absence of antibodies in the sample saliva, the method further comprises administering a booster of the vaccine to the patient.

[0244] Additionally, the stabilizing solution further comprises aragonite particle beads having an average particle size of 100 nm to 1 mm. The aragonite particle beads can be bound to immunoglobulin (Ig) proteins, anti-SARS-CoV-2 antibodies, or SARS-CoV-2 viral proteins. In an exemplary embodiment, the aragonite particle beads are bound to recombinant ACE2 protein or recombinant ACE2 alpha helix protein.

[0245] Contemplated subject matter also includes an aragonite composition formulated to bind an immunoglobulin (Ig) protein, an anti-SARS-CoV-2 antibody protein, or a SARS-CoV-2 viral protein, the aragonite composition comprising a plurality of aragonite particle beads having an average particle size between 100 nm and 1 mm, the plurality of aragonite particle beads being functionalized with a moiety capable of binding to the immunoglobulin (Ig) protein, the anti-SARS-CoV-2 antibody protein, and / or the SARS-CoV-2 viral protein.

[0246] In certain embodiments, a 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 may 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.

[0247] Contemplated subject matter includes methods for administering the vaccine to a patient by two or more routes of administration to induce both local and systemic immune responses to the vaccine. Contemplated subject matter also includes compositions and methods for assaying the presence or absence of relevant antibodies (e.g., anti-SARS-CoV-2 antibodies) in patient samples (e.g., saliva, nasal mucosa, gastrointestinal mucosa, or serum). The antibody status in a patient sample can be used to assess the need for additional vaccine doses (e.g., booster doses / shots).

[0248] In addition to the required molecular epitopes presented in the vaccine, the route of administration of the vaccine and the regimen for administering additional (i.e., booster) doses of the vaccine can also influence whether a patient's immune response is sufficiently robust to establish protection.

[0249] For emerging viruses such as SARS-like coronavirus (SARS-CoV-2), the duration of immunity (both humoral and cell-mediated) in patients who have recovered from SARS-CoV-2 infection is not yet fully known, and furthermore, vaccine protocols have yet to be tested across various populations. Given the current SARS-CoV-2 pandemic and the high rate of transmission of the SARS-CoV-2 virus, there is a need for robust vaccination protocols and effective tests for the virus or immunity to the virus (e.g., the presence of anti-SARS-CoV-2 antibodies).

[0250] Contemplated methods disclosed herein for inducing immunity in patients include administering the vaccine at least by oral administration, preferably by oral administration and by injection into the blood supply. Many vaccines are given via the intramuscular (IM) route to optimize immunogenicity and induce systemic immunity with direct delivery of the vaccine into the intramuscular blood supply. IM administration is typically preferred over subcutaneous (SC) injection, which is more likely to have adverse reactions at the injection site than IM injection.

[0251] In addition to IM injection, it has been reported that induction of mucosal immunity is essential to prevent human-to-human transmission of pathogenic microorganisms and limit their proliferation in mucosal tissues. Moreover, for protective immunity against mucosal pathogens (e.g., SARS coronavirus), immune activation in mucosal tissues allows enhanced mucosal immune responses and better local protection, instead of the more common approach of tolerance to maintain mucosal homeostasis. For example, nasal vaccination (delivery of vaccines via nasal administration) induces both mucosal and systemic immunity (see, e.g., Fujikuyama et al., 2012, Expert Rev Vaccines, 11:367-379 and Birkhoff et al., 2009, Indian J.Pharm.Sci., 71:729-731).

[0252] To induce both mucosal and systemic immunity in a patient, embodiments of the present disclosure include providing the vaccine to a patient by administration to at least the patient's nasal, oral, and / or gastrointestinal mucosa. In some embodiments, the route of administration includes administering the vaccine to the patient's nasal, oral, and / or gastrointestinal mucosa together with injection into the blood supply (e.g., intramuscular (IM), intravenous (IV), or subcutaneous (SC)). As used herein, oral administration of a vaccine composition includes nasal injection, nasal inhalation, oral ingestion, and administration to the gastrointestinal mucosa (e.g., inhalation, ingestion, injection). Preferably, the route of administration of the vaccine includes oral administration selected from delivery to the gastrointestinal mucosa, nasal injection, nasal inhalation, oral ingestion, or oral inhalation together with administration by intramuscular (IM) injection.

[0253] In particular, the vaccine administered to induce immunity in the mucosal tissues of the patient is a SARS-CoV-2 vaccine. In an exemplary embodiment, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) comprises a soluble ACE2 protein bound to an immunoglobulin Fc portion, forming an ACE2-Fc hybrid construct, which may also include a J chain portion, as disclosed in US 16 / 880,804 and US 63 / 016,048, the entire contents of both of which are incorporated herein by reference. In another exemplary embodiment, the SARS-CoV-2 vaccine (e.g., an adenoviral construct) comprises a mutant of a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10) having at least one mutated amino acid residue (e.g., by substitution) that confers increased binding affinity of the ACE2 protein to the RBD protein domain of the SARS-CoV-2 spike protein, as disclosed in U.S. Patent Application No. 63 / 022,146, the entire contents of which are incorporated herein by reference. In another exemplary embodiment, the SARS-CoV-2 vaccine (e.g., an adenoviral construct) comprises a CoV2 nucleocapsid protein or a CoV2 spike protein fused to an endosomal targeting sequence (N-ETSD), as disclosed in U.S. Patent Application No. 16 / 883,263 and U.S. Patent Application No. 63 / 009,960, the entire contents of both of which are incorporated herein by reference. Additionally or alternatively, the SARS-CoV-2 vaccine may include modified yeast cells (e.g., Saccharomyces cerevisiae) that have been genetically engineered to express the coronavirus spike protein on the yeast cell surface, such that the yeast display cells stimulate B cells (e.g., humoral immunity), as disclosed in U.S. Pat. No. 63 / 010,010.

[0254] In some embodiments, two or more vaccine compositions disclosed herein may be administered to a patient to induce immunity against SARS-CoV-2. For example, a patient may be administered genetically modified yeast cells expressing the coronavirus spike protein as a single type of vaccine, or the genetically modified yeast cells may be administered together or simultaneously with one or more SARS-CoV-2 adenovirus constructs disclosed herein.

[0255] In further embodiments, a patient's immune response to either a vaccine administered as disclosed herein (e.g., orally and by injection into the blood supply) or infection with a virus can be monitored or evaluated. In particular, compositions and methods are disclosed herein for assessing the continued presence of antibodies in the respiratory and digestive mucosa of a patient following infection with SARS-CoV-2 or following inoculation against SARS-CoV-2 by administration of a SARS coronavirus vaccine.

[0256] To assay samples from patients who have received a vaccine against a pathogenic infection (e.g., targeting SARS-CoV-2) and / or have been infected with a virus (e.g., SARS-CoV-2), the presence of antibodies against the pathogen can be performed using any one of a number of diagnostic tests. In some embodiments, the diagnostic test is a cell viability assay that allows for detection of antibodies in the presence of an antigen. A diagnostic test using a cell viability assay for the detection of anti-SARS-CoV-2 antibodies is disclosed in U.S. Patent No. 62 / 053,691, the entire contents of which are incorporated herein by reference. Cytological diagnostic assays rely on the expression of a target receptor for a given pathogen (e.g., ACE2 for SARS-CoV-2 infection) on the surface of an immune effector cell line (e.g., killer T cells, natural killer cells, NK-92 cells and their derivatives) and the expression of a pathogen ligand (e.g., spike protein for SARS-CoV-2 infection) on the surface of a surrogate cell line (e.g., HEK293 cells or SUP-B15 cells).

[0257] Further diagnostic tests using recombinant protein variants of the ACE2 protein (the human receptor targeted by the SARS-CoV-2 spike protein) are disclosed in U.S. Patent Application No. 16 / 880,804, the entire contents of which are incorporated herein by reference.

[0258] To more easily monitor patients for the presence of anti-pathogen antibodies, assaying saliva samples from patients may allow for rapid sample collection, increased patient participation, and allow patients to obtain their own samples and mail or transport the samples to a laboratory for testing. However, to assay saliva for the presence of neutralizing antibodies to SARS-CoV-2, it may be necessary to stabilize proteins in saliva against degradation during transportation and storage after sample collection prior to testing.

[0259] Once 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). Preservatives for biological samples are disclosed, for example, in Cunningham & al. (2018) report ("Effective Long-term Preservation of Biological Evidence," USDepartment of Justice grant # 2010-DN-BX-K193) and U.S. Pat. No. 6,133,036. For example, a stabilizing 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.

[0260] In certain embodiments, the saliva sample may be mixed with a stabilizing preservative solution of glutaraldehyde to achieve a final glutaraldehyde concentration of between 0.1% (w / v) and 2.0% (w / v), e.g., 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), 1.7% (w / v), about 1.8% (w / v), or about 1.9% (w / v).

[0261] In additional or alternative embodiments, the saliva sample may be mixed with a stabilizing preservative solution of about 0.10% to about 1.00% sodium benzoate (weight / volume of sample) and / or about 0.025% to about 0.20% citric acid (weight / volume of sample). For example, the saliva sample may 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 contains 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) of benzoic acid. and / or mixed with a stabilizing preservative solution of 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, 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 (e.g., sodium benzoate) and "citric acid" is interchangeable with citrate salts (e.g., sodium citrate).

[0262] The saliva sample containing the preservatives described above is stable to storage for at least 1 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) at a temperature between 15° C. and 40° C. Thus, disclosed herein is a method of storing a saliva sample for neutralizing antibody testing, comprising mixing the saliva sample with a stabilizing solution made from one or more of glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, and / or sodium azide, and storing at 15° C. to 25° C. for at least 1 hour, 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 a glutaraldehyde concentration of 0.1% (w / v) to 2.0% (w / v) and the glutaraldehyde-saliva is stored at 15° C. to 25° C. In certain embodiments, the glutaraldehyde-saliva may further comprise citric acid and / or benzoic acid in concentrations disclosed herein.

[0263] Aragonite: In some embodiments, any antibody protein or any specific antibody protein can be captured from a saliva sample using ovo-shaped aragonite particles. For example, the saliva preservation solutions of glutaraldehyde, sodium benzoate and citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, and any combination thereof disclosed herein can also include ovo-shaped aragonite (calcium carbonate, CaCO3) particles. The use of aragonite particles for binding to proteins is disclosed, for example, in U.S. Patent Application Nos. 16 / 858,548 and PCT / US20 / 29949, the entire contents of both of which are incorporated herein by reference. Thus, aragonite particles can be added to capture (e.g., bind to) any antibodies present in a saliva sample or modified to specifically capture antibodies against a particular antigen. For example, aragonite can be functionalized with moieties capable of binding to immunoglobulin (Ig) proteins. Preferably, the Ig protein is an immunoglobulin A (IgA), immunoglobulin G (IgG), or immunoglobulin E (IgE) protein. More preferably, the aragonite is functionalized to bind to an IgA protein. Most preferably, the aragonite particles are functionalized with a moiety capable of binding to a specific antibody. For example, the aragonite particles can be coupled to a moiety specific for an anti-SARS-CoV-2 antibody. Preferably, the aragonite particles are coupled to a recombinant ACE2 protein, for example as disclosed in the above-mentioned US patent application Ser. No. 16 / 880,804. In a typical embodiment, 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.

[0264] In additional or alternative embodiments, the aragonite particles are functionalized (e.g., conjugated) to recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10). For more efficient capture or binding of anti-SARS-CoV-2 antibodies or spike protein of SARS CoV-2, recombinant soluble ACE2 can be mutated to form ACE2 mutants with higher binding affinity to SARS-CoV-2 spike protein (e.g., RBD domain of spike protein). These ACE2 mutants of recombinant soluble ACE2 protein include T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and / or D355L.

[0265] As used herein, the term "functionalized" refers to the coupling or binding of a moiety to an aragonite particle, thereby imparting any functionality of the attached moiety to the aragonite particle. For example, the aragonite particle may be functionalized with a protein moiety. Methods for preparing and using aragonite particle beads are disclosed in U.S. Patent Application No. 16 / 858,548 and PCT / US20 / 29949. In some embodiments, the aragonite composition comprises a plurality of aragonite particle beads. Preferably, the plurality of aragonite particle beads has an average particle size of 100 nm to 1 mm.

[0266] In some embodiments, the protein moiety is directly attached to the native untreated surface of the aragonite particle. The aragonite particle has an amino acid content of about 2-3%, including aspartic acid and glutamic acid, which render the aragonite surface hydrophilic. Thus, in some embodiments, the protein moiety may be directly attached to the surface of the aragonite particle.

[0267] In an alternative embodiment, the aragonite particle surface can be treated to modify the binding surface. For example, as disclosed in US Patent Application No. 16 / 858,548 and PCT / US20 / 29949, treatment with stearic acid (i.e., octadecanoic acid) provides a hydrophobic surface. For protein loading, treatment of aragonite with phosphoric acid forms a lamellar structure. Further conjugation techniques for binding reactive groups to the amino acid surface of aragonite are known in the art, as disclosed, for example, in Bioconjugate Techniques, Third Edition, Greg T. Hermanson, Academic Press, 2013.

[0268] Patients who do not demonstrate sufficient titers (e.g., presence) of neutralizing antibodies in their saliva can be sent an oral dose of the respective vaccine (e.g., a SARS-CoV-2 vaccine 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 above, to a testing facility to confirm that the oral vaccine dose has restored their anti-SARS-CoV-2 antibody (e.g., IgA) titers.

[0269] Thus, in a further embodiment, a kit for collecting a saliva sample from a patient includes a collection container with a saliva preservative solution as disclosed herein. For example, the kit includes a collection container with 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 shipping or mailing. In some embodiments, the kit may also include at least one dose of a vaccine for oral administration.

[0270] immune stimulation In addition to vaccination, or even as an alternative treatment for subjects with a probable or actual diagnosis of coronavirus infection, particularly SARS-CoV-2, immune stimulation with one or more immune stimulatory cytokines may prevent or alleviate the lymphopenia frequently associated with COVID-19. Among other immune stimulatory cytokines, N-803 is specifically contemplated.

[0271] COVID-19 infection causes lymphopenia, especially suppression of NK and CD8 T cells, and severe cases and subsequent deaths are associated with this significant reduction in lymphocytes. Evidence also suggests that COVID-19 infection results in macrophage death and a reduction in natural killer (NK) cells and CD8+ T cells. An analysis of data from 1,099 laboratory-confirmed COVID-19 patients from 552 hospitals across China revealed that 83.2% of patients had lymphopenia at the time of admission. On average, patients with severe disease had more pronounced laboratory abnormalities, e.g., lower lymphocyte counts, compared to patients with non-severe disease. Similarly, another study group showed that median lymphocyte counts were significantly lower in patients admitted to the ICU compared to patients not requiring ICU care.

[0272] At the cellular level, COVID-19 appears to induce immune evasion by the reduction of NK, CD4+ and CD8+ T cells. Although the mechanism of lymphopenia remains unclear, the rapid reduction of both CD4 and CD8 T cells may be associated with adverse outcomes. Lymphopenia and increased viral load during the first 10 days of SARS suggest immune evasion by the SARS coronavirus. A lack of interferon (IFN)-γ responses in SARS-infected cells has been reported in vitro using human primary bone marrow-derived dendritic cells and epithelial 293 cell lines. Other investigators have proposed a mechanism of immune evasion by SARS-CoV in dendritic cells (DCs) based on the findings of low expression of antiviral cytokines (TFN-α, TFN-β, TFN-γ, and IL-12p40), moderate upregulation of inflammatory cytokines (tumor necrosis factor α [TNF-α] and IL-6), and significant upregulation of inflammatory cytokines (MIP-1a, RANTES, IPI 0, and MCP-1). In addition to CD8+ T cell exhaustion, lack of T cell reactivity through insufficient T cell diversity may contribute to the inadequate immune response. This occurs especially in the elderly population.

[0273] N-803 has demonstrated a significant ability to induce increased broad T cell reactivity in both preclinical (mouse) and clinical (breast, lung, and pancreatic cancer) settings. By activating T cell diversity, cross-reactivity to past coronavirus infections may provide immunity to COVTD-19 following N-803 enhancement of T cell reactivity, overcoming immune evasion. N-803 significantly promotes NK cell and CD8- T cell proliferation and activation of peripheral circulation and lymphoid organs in healthy mice and cynomolgus monkeys, as well as in diverse mouse and rodent tumor models, including bladder cancer, lung cancer, melanoma, lymphoma, multiple myeloma, colon cancer, breast cancer, and glioblastoma. Further preclinical evidence exists for the antiviral efficacy of N-803, including in mouse and non-human primate (NHP) models.

[0274] Clinical evidence from early phase studies also supports these preclinical studies showing proliferation and activation of NK cells and CD8+ T cells. N-803 increases the cytotoxicity of these immune cells as shown by upregulation of expression of activation markers including perforin and granzyme B. The phenotypic changes induced in NK and CD8+ T cells by N-803 resulted in enhanced anticancer activity (via antibody-dependent cellular cytotoxicity, direct cytotoxicity, and enhanced tumor-specific cytotoxicity) and prolonged survival in vivo.

[0275] NK cell and CD8+ T cell activation has been demonstrated in healthy volunteers. In a study of healthy volunteers administered N-803 at doses of 10 μg / kg and 20 μg / kg SC, N-803-treated subjects showed a greater than 20-fold increase in Ki-67 stained NK and CD8+ T cells. 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.

[0276] Thus, since preclinical in vitro and in vivo studies, together with clinical data, have demonstrated that N-803 binds with higher affinity to IL-15 receptor-presenting cells, enhances lymphocyte distribution, extends half-life, causes proliferation and activation of effector NK cells and CD8+ memory T cells, resulting in antitumor activity, N-803 may be used as a treatment option to combat COVID-19-associated lymphopenia in a manner similar to N-803 stimulating both NK and CD8 T cells, and to rescue lymphopenia in normal healthy subjects and patients with cancer. Thus, N-803 may be used to reverse or alleviate lymphopenia in patients infected with COVID-19 and to improve disease outcomes. For example, infected subjects may 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 injectable form at a dosage of 10 mcg / kg.

[0277] In some embodiments, the numbers expressing properties such as amounts of ingredients, concentrations, reaction conditions, etc., used to describe and claim certain embodiments of the present invention should be understood as being modified in some instances by the term "about". Thus, in some embodiments, the numerical parameters set forth in the written description and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein.

[0278] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of a pharmaceutical composition or drug, where direct administration of a pharmaceutical composition or drug is typically performed by a medical professional (e.g., a doctor, a nurse, etc.), and indirect administration includes providing or making available a pharmaceutical composition or drug to a medical professional for direct administration (e.g., by injection, infusion, oral delivery, topical delivery, etc.). It should be further noted that the term "predicting" or "predicting" a condition, susceptibility to development of a disease, or response to an intended treatment is intended to cover the act or prediction (but not treatment or diagnosis) of predicting a condition, susceptibility, and / or response, including the rate of progression, improvement, and / or persistence of a condition in a subject.

[0279] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided with respect to specific embodiments herein, or the use of exemplary language (e.g., "etc."), are intended merely to better illustrate the technology disclosed herein, and do not otherwise limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the claimed invention.

[0280] As used throughout this description and the claims that follow, the meanings of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Also, as used in this description, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise. Also, as used herein, unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (wherein the two elements that are coupled to each other are in contact with each other) and indirect coupling (wherein at least one additional element is located between the two elements). Thus, the terms "coupled to" and "coupled with" are used interchangeably.

[0281] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the inventive concept herein. Thus, the subject matter of the present invention should not be limited, except as set forth in the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced element, component, or step may be present, utilized, or combined with other elements, components, or steps not expressly referenced. When the specification or claims refer to at least one selected from the group consisting of A, B, C... and N, the sentence should be interpreted as requiring only one element from the group, not A+N, or B+N, etc.

Claims

1. a. a first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) (N-ETSD) fused to an endosomal targeting sequence, the first portion being operably linked to one or more regulatory elements enabling N-ETSD expression; b. a second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements that allow for S expression; A recombinant nucleic acid comprising:

2. The recombinant nucleic acid of claim 1, wherein the SARS virus is SARS-CoV-2.

3. The recombinant nucleic acid of claim 2, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 5' end of the first portion.

4. The recombinant nucleic acid of claim 2, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 3' end of the first portion.

5. The recombinant nucleic acid of claim 2 , wherein the first and second portions are arranged in a bicistronic sequence.

6. 4. The recombinant nucleic acid of claim 3, wherein the N-ETSD has an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

7. The recombinant nucleic acid of claim 6, wherein the N-ETSD has the amino acid sequence of SEQ ID NO:

1.

8. 8. The recombinant nucleic acid of claim 7, wherein the first portion has the nucleotide sequence of SEQ ID NO:

2.

9. 7. The recombinant nucleic acid of claim 6, wherein the S protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

10. 10. The recombinant nucleic acid of claim 9, wherein the S protein has the amino acid sequence of SEQ ID NO:

3.

11. 10. The recombinant nucleic acid of claim 9, wherein the S protein has the amino acid sequence of SEQ ID NO:

4.

12. 10. The recombinant nucleic acid of claim 9, wherein the second portion has the nucleotide sequence of SEQ ID NO:

5.

13. 10. The recombinant nucleic acid of claim 9, wherein the second portion has the nucleotide sequence of SEQ ID NO:

6.

14. The recombinant nucleic acid of any one of claims 1 to 13, further comprising a third portion encoding a costimulatory molecule or an immunostimulatory cytokine.

15. The recombinant nucleic acid according to any one of claims 1 to 14, which is incorporated into a viral or yeast expression vector.

16. The recombinant nucleic acid of claim 15, wherein the viral expression vector is an adenoviral expression vector having an E1 gene region deletion and an E2b gene region deletion.

17. 16. The recombinant nucleic acid of claim 15, wherein the yeast expression vector is a Saccharomyces cerevisiae expression vector.

18. The recombinant nucleic acid according to any one of claims 1 to 17, wherein the nucleic acid is deoxyribonucleic acid (DNA).

19. E1 gene deletion, E2b gene deletion, and a. a first portion encoding a SARS coronavirus N-ETSD, the first portion being operably linked to one or more regulatory elements that permit expression of the N-ETSD; b. a second portion encoding a SARS S protein, each operably linked to one or more regulatory elements that allow for S expression; A recombinant replication-defective adenovirus comprising a recombinant nucleic acid comprising:

20. The adenovirus of claim 19, wherein the SARS virus is SARS-CoV-2.

21. The adenovirus of claim 20, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 5' end of the first portion.

22. The adenovirus of claim 20, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 3' end of the first portion.

23. 23. The adenovirus of claim 22, wherein the first and second portions are arranged in a bicistronic arrangement.

24. The adenovirus of claim 22, wherein the N-ETSD has an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

25. The adenovirus of claim 24, wherein the N-ETSD has an amino acid sequence having the amino acid sequence of SEQ ID NO:

1.

26. 25. The adenovirus of claim 24, wherein the first portion has the nucleotide sequence of SEQ ID NO:

2.

27. The adenovirus of claim 24, wherein the S protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

28. The adenovirus of claim 27, wherein the S protein has the amino acid sequence of SEQ ID NO:

3.

29. The adenovirus of claim 27, wherein the S protein has the amino acid sequence of SEQ ID NO:

4.

30. 28. The adenovirus of claim 27, wherein the second portion has the nucleotide sequence of SEQ ID NO:

5.

31. 28. The adenovirus of claim 27, wherein the second portion has the nucleotide sequence of SEQ ID NO:

6.

32. The adenovirus according to any one of claims 19 to 31, further comprising a third portion encoding a costimulatory molecule or an immunostimulatory cytokine.

33. The adenovirus according to any one of claims 19 to 32, wherein the recombinant adenovirus has a deletion in the E3 gene region.

34. a. a first portion encoding a SARS coronavirus N-ETSD, the first portion being operably linked to one or more regulatory elements that permit expression of the N-ETSD; b. a second portion encoding a SARS S protein, each operably linked to one or more regulatory elements that allow for S expression; A recombinant yeast comprising a recombinant nucleic acid comprising the

35. The recombinant yeast of claim 34, wherein the SARS virus is SARS-CoV-2.

36. The recombinant yeast of claim 35, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 5' end of the nucleocapsid protein.

37. The recombinant yeast of claim 35, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 3' end of the nucleocapsid protein.

38. 38. The recombinant yeast of claim 37, wherein the first and second portions are arranged in a bicistronic arrangement.

39. 38. The recombinant yeast of claim 37, wherein the N-ETSD has an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

40. 50. The recombinant yeast of claim 49, wherein the N-ETSD has the amino acid sequence of SEQ ID NO:

1.

41. 41. The recombinant yeast of claim 40, wherein the first portion has the nucleotide sequence of SEQ ID NO:

2.

42. 40. The recombinant yeast of claim 39, wherein the S protein has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

43. 43. The recombinant yeast of claim 42, wherein the S protein has the amino acid sequence of SEQ ID NO:

3.

44. 43. The recombinant yeast of claim 42, wherein the S protein has the amino acid sequence of SEQ ID NO:

4.

45. 43. The recombinant yeast of claim 42, wherein the first portion has the nucleotide sequence of SEQ ID NO:

5.

46. 43. The recombinant yeast of claim 42, wherein the first portion has the nucleotide sequence of SEQ ID NO:

6.

47. The recombinant yeast of any one of claims 35 to 46, further comprising a third portion encoding a costimulatory molecule or an immunostimulatory cytokine.

48. The recombinant yeast according to any one of claims 35 to 47, wherein the yeast is Saccharomyces cerevisiae (S. cerevisiae).

49. 49. The recombinant yeast of claim 48, wherein the yeast is a lysing yeast.

50. A vaccine composition comprising a recombinant nucleic acid according to any one of claims 1 to 18.

51. 51. The vaccine composition of claim 50, wherein the recombinant nucleic acid is encapsulated in a lipid nanoparticle.

52. A vaccine composition comprising aragonite particles mixed with a recombinant, replication-deficient adenovirus according to any one of claims 19 to 33, wherein said recombinant, replication-deficient adenovirus is lyophilized.

53. 53. The vaccine composition of claim 52, wherein the aragonite particles have an average particle size of between 100 nm and 1 mm.

54. A recombinant nucleic acid according to any one of claims 1 to 18 for use in the production of a vaccine against the SARS virus.

55. A recombinant nucleic acid according to any one of claims 1 to 18 for use in inducing immunity against SARS virus.

56. A recombinant replication-defective adenovirus according to any one of claims 19 to 33 for use in inducing immunity against SARS virus.

57. The recombinant yeast according to any one of claims 34 to 49 for use in inducing immunity against SARS virus.

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