Treatment of COVID-19 and its methods

Abstract

Vaccine compositions for inducing immunity to coronavirus in a subject include recombinant nucleic acids encoding N-ETSD, a modified nucleocapsid protein containing an endosomal targeting sequence, and / or S-fusions, a modified spike protein with improved surface expression. Vaccines may be formulated as recombinant nucleic acids, recombinant yeast, and / or recombinant viruses such as adenoviruses, and can be administered via injection and / or mucosal delivery.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 988,328, filed on March 11, 2020; U.S. Provisional Patent Application No. 63 / 009,960, filed on April 14, 2020; U.S. Provisional Patent Application No. 63 / 010,010, filed on April 14, 2020; U.S. Provisional Patent Application No. 63 / 059,975, filed on August 1, 2020; U.S. Provisional Patent Application No. 63 / 064,157, filed on August 11, 2020; U.S. Provisional Patent Application No. 63 / 117,460, filed on November 24, 2020; U.S. Provisional Patent Application No. 63 / 117,847, filed on November 24, 2020; U.S. Provisional Patent Application No. 63 / 117,922, filed on November 24, 2020; U.S. Provisional Patent Application No. 63 / 118,697, filed on November 26, 2020; U.S. Provisional Patent Application No. 63 / 135,380, filed on January 8, 2021; and U.S. Provisional Patent Application No. 16 / 883,263, filed on May 26, 2020, all of which are hereby incorporated by reference in their entirety. This application also claims priority to U.S. Non-Provisional Patent Application No. 102538.0080US3, titled "Anti COVID-19 Therapies targeting nucleocapsid and spike proteins," filed concurrently herewith, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing The content of the ASCII text file of the sequence listing, named 102690-0041PCT_REV003_ST25.txt, having a size of 172 KB, was created on March 4, 2021, and was electronically submitted via EFS-Web together with this application, and the entire content is hereby incorporated by reference.

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

Background Art

[0004] The description of the background art includes information that may be useful for understanding the present disclosure. None of the information provided in this specification, whether it is prior art, related to the invention currently claimed, or any publication specifically or implicitly referenced, is admitted to be prior art.

[0005] All publications and patent applications in this specification 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. If the definition or use of a term in an incorporated reference does not agree with or is contrary to the definition of that term provided in this specification, the definition of that term provided in this specification applies and the definition of that term in the reference does not apply.

[0006] SARS-CoV2 diagnostic tests have become available relatively quickly, but numerous attempts to treat the disease have so far shown conflicting or inconclusive results. Most typically, patients with severe symptoms are treated to maintain respiratory / blood oxygenation. The COVID-19 mortality rate is particularly notable in the elderly, immunocompromised, and individuals with heart disease, lung disease, or diabetes. Despite improvements in acute care, it has become clear that disease containment is extremely important because social distancing and other public health mitigation measures can only provide moderate relief.

[0007] To that end, numerous candidate anti-SARS-CoV2 vaccine compositions target one or more proteins of the virus (see, e.g., FIMMU 2020, 11:602256). For example, Sinovac and Sinopharm are currently testing inactivated virus vaccine preparations. Cansino Biologics, Janssen Pharma, Oxford University, and Gamaleya have developed vaccines based on non-replicating adenovirus vectors encoding one or more viral proteins. Novamax has produced protein subunit-based vaccines. 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 the disease-causing infection, but it is unclear whether the protection is effective over several months and / or whether sufficient immune memory protects vaccinated individuals over the long term. Furthermore, it is unclear whether such vaccines generate a clinically meaningful T cell-based response.

[0008] While certain vaccines have become available, the rapid and worldwide distribution and administration of vaccines has faced considerable difficulties, delaying global distribution. Furthermore, the logistic requirements and the need for medical specialists limit the administration to certain vaccination platforms.

[0009] To overcome such difficulties, solid dosage forms of vaccines are desired. However, such dosage forms (e.g., powders, tablets, capsules) require the disintegration and release of the active ingredient at physiologically relevant sites. Dosage forms loaded with the active ingredient must be stable during transport and storage from manufacture to administration. Mucosal or oral delivery of vaccines would be highly desirable as such administration aids the production of IgA, IgE, and IgM class antibodies, which are important for immunity against mucosal or intestinal infections. Unfortunately, such vaccines are not available.

[0010] Although various vaccine compositions and methods for inducing immunity against SARS-CoV-2 are known in the art, all are plagued by some drawbacks. Therefore, improved vaccine compositions and methods are still needed.

Summary of the Invention

Means for Solving the Problems

[0011] Various vaccine compositions and methods therefor are disclosed herein, wherein a recombinant modified nucleocapsid protein and / or a modified spike protein of a coronavirus induces immunity against the coronavirus in a subject. Most preferably, the recombinant protein is encoded by a recombinant nucleic acid that can be delivered in a lipid formulation as part of a recombinant virus and / or as part of a 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) fused to an endosomal targeting sequence (N-ETSD), the first portion being operably linked to one or more regulatory elements that enable 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 that enable S expression, respectively.

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

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

[0015] Recombinant replication-deficient adenoviruses are described herein that comprise a recombinant nucleic acid comprising a deletion in the E1 gene, a deletion in the E2b gene, and a first portion encoding the SARS coronavirus N-ETSD. The first portion is operably linked to one or more regulatory elements that permit N-ETSD expression. The adenovirus also comprises a second portion encoding the SARS S protein. The second portion is operably linked to one or more regulatory elements that permit S expression, respectively.

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

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

[0018] In yet further embodiments, a vaccine composition is disclosed herein that includes the recombinant nucleic acid presented herein, and the recombinant nucleic acid can be encapsulated in lipid nanoparticles.

[0019] In still other embodiments, a vaccine composition is disclosed herein that includes calcite particles mixed with the recombinant replication-deficient adenovirus presented herein, and the recombinant replication-deficient adenovirus is lyophilized. Most typically, the calcite particles have an average particle size of 100 nm to 1 mm.

[0020] Recombinant nucleic acids are presented herein for generating a vaccine against SARS virus and / or for inducing immunity against SARS virus. Similarly, recombinant replication-deficient adenoviruses or recombinant yeast for inducing immunity against SARS virus are disclosed herein.

[0021] The various objects, features, aspects and advantages of the subject matter disclosed herein will become more apparent from the following detailed description of the preferred embodiments, together with the accompanying drawings in which like numerals represent like components. BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

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Mode for Carrying Out the Invention

[0023] Various vaccine compositions and methods for inducing immunity against the SARS-CoV-2 virus and viruses and mutant forms closely related to the SARS-CoV-2 virus not only promote the production of therapeutically effective antibodies but also induce robust T cell responses. These vaccine compositions can be administered via different routes including intramuscular, subcutaneous, oral, and mucosal routes (alone or in combination) and can also be used as an oral boost after currently known RNA-based vaccines.

[0024] In a particularly contemplated embodiment, the recombinant construct comprises a modified nucleocapsid protein and / or a modified spike protein. Preferably, the modified nucleocapsid protein comprises a transport sequence that sends the modified nucleocapsid protein to the endosome / lysosome intracellular compartment, thereby utilizing an important antigen presentation pathway to stimulate CD4+ T cells, which in turn activate naive CD8+ cytotoxic T cells in dendritic cells. Similarly, the modified spike protein preferably has a modification that enhances the surface expression of the modified spike protein, thereby making the immune response to the spike antigen more robust. Indeed, the vaccines disclosed herein (e.g., the hAd5[E1-,E2b-] vaccine) exemplify the use of an N antigen tagged with an ETSD peptide, but in principle any antigen can be advantageously redirected to the endosome / lysosome intracellular compartment.Exemplary antigens tagged with ETSD for use in this manner in an adenovirus or yeast vaccine vector include CEA, human epidermal growth factor receptor 1 (HER1), HER2 / neu, HER3, HER4, prostate specific antigen (PSA), PSMA, folate receptor alpha, WT1, p53, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, MART-1, MC1R, Gp100, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, BRCA1, brachyury, brachyury (TIVS7-2, polymorphism), brachyury (IVS7 T / C polymorphism), T brachyury, hTERT, hTRT, iCE, MUC1, MUC1 (VNTR polymorphism), MUC1c, MUC1n, MUC2, PRAME, P15, RU1, RU2, SART-1, SART-3, AFP, β-catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, ETV6 / AML, LDLR / FUT, Pml / RARα, HPV E6, HPV E7, and TEL / AML1.

[0025] "N-ETSD" refers to a modified nucleocapsid protein of the SARS-CoV-2 virus that contains an endosomal targeting sequence. 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 having an HA tag. 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 having increased surface expression. Exemplary S-fusions have 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. Exemplary N proteins have the amino acid sequence of SEQ ID NO: 7.

[0029] "ETSD" refers to an endosome targeting sequence. Exemplary ETSDs have the amino acid sequence of SEQ ID NO: 8.

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

[0031] "Soluble ACE2 protein" refers to mutants and cleaved forms of ACE that are soluble under physiological conditions. 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 the treatment and / or prevention of coronavirus disease. In one aspect, replication-deficient adenoviruses are disclosed herein, where the adenovirus has a deletion in the E1 gene region; a deletion in the E2b gene region; a deletion in the E3 gene region, a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid protein, a CoV2 nucleocapsid protein (N-ETSD) fused to an endosome targeting sequence, 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 include a sequence having at least 80% identity to SEQ ID NO: 1. 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%. It is further contemplated that the N-ETSD fusion protein can include a linker between the N-ETSD domain and the nucleocapsid protein. For example, this linker can be a 16-amino acid linker having the sequence (G3S)4. In certain embodiments, methods for enhancing the immunogenicity of an intracellular antigen are disclosed herein, the method including tagging the antigen with ETSD and expressing the tagged antigen in an antigen-presenting cell (e.g., dendritic cell).

[0034] In some embodiments, a fusion protein comprising N-ETSD and a CoV-2 nucleocapsid protein can be encoded by a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0035] The CoV-2 spike protein is intended to have 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] The CoV-2 spike fusion protein is intended to have 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 present 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. Further, each of these encoded proteins can 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 a 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 the CoV-2 spike protein or spike fusion protein comprises a sequence having at least 80% identity to SEQ ID NO: 5 or SEQ ID NO: 6. In other embodiments, the identity value is at least 85%. In 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 can further comprise nucleic acids encoding trafficking sequences, costimulatory molecules, and / or immunostimulatory cytokines. Costimulatory molecules are selected from the group consisting of CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3. Immunostimulatory cytokines may be selected from the group consisting of IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, and LMP1. Additionally or alternatively, the vaccines disclosed herein may also encode an 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-imbakicept, IL-21, IPS1, & LMP1). "In combination" in this context is intended to mean that the immunostimulatory cytokine is administered within 24 hours of the adenovirus and / or yeast. That is, the adenovirus and / or yeast can be administered to a patient (e.g., a patient over 50 years old), and then within 24 hours, one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, & LMP1) can be administered to the same patient. Additionally or alternatively, one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, & LMP1) can be administered to a patient (e.g., a patient over 50 years old), and then within 24 hours, the adenovirus and / or yeast can be administered to the same patient.

[0042] After the age of 40, a patient's ability to initiate a T cell response to a vaccine gradually declines. Thus, administering the adenoviruses and / or yeasts described herein in combination with one or more immunostimulatory cytokines (e.g., IL-2, IL-12, IL-15, nongapendekin alpha - inbaxcept, IL-21, IPS1, & LMP1) can be particularly useful for elderly patients. As used herein, "elderly" includes patients over 50 years of age, e.g., patients over 55, over 60, over 65, over 70, over 75, over 80, over 85, 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 by intravenous injection. Alternatively or additionally, antigen - presenting cells can be isolated or grown from the patient's cells, infected in vitro, and then infused into the patient.

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

[0045] It is further contemplated that the recombinant viruses and yeasts contemplated herein may further comprise a sequence encoding at least one of a costimulatory molecule, an immunostimulatory cytokine, and a protein that interferes with 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 proteins that interfere with include antibody agonists or antagonists of CTLA-4, PD-1, TIM1 receptor, 2B4, and / or CD160.

[0046] All of the above costimulatory genes are well known in the art, and it should be understood that 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 so as to be expressed in humans, although modified and non-human forms are also considered appropriate as long as such forms assist 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 "preventive" or "therapeutic". When provided preventively, the compositions of the disclosure are provided prior to the occurrence of, or upon detection of the occurrence of, a coronavirus disease, with the goal of preventing, inhibiting, or delaying the occurrence of the coronavirus disease; and / or generally preventing or inhibiting the progression of the coronavirus disease in an individual. Accordingly, a preventive composition can be administered to an individual who is thought to be free of (healthy or normal) coronavirus disease, or in whom the coronavirus has not yet been detected. Individuals at high risk of developing coronavirus disease can be prophylactically treated using the compositions of the disclosure.

[0048] When provided therapeutically, the immunotherapeutic composition is provided to an individual diagnosed with a coronavirus disease for the purpose of ameliorating or curing the coronavirus disease; increasing the survival rate of the individual; and / or preventing, inhibiting, reversing, or delaying the occurrence of the coronavirus disease in the individual.

[0049] In yet another further embodiment, a vaccine composition comprising the adenovirus or yeast disclosed above is disclosed herein, 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] Methods for preventing and / or treating coronavirus disease, particularly COVID-19, are also disclosed herein. Preferably, the method involves using a viral or yeast vector encoding a wild-type or modified form of the nucleocapsid protein of coronavirus and / or a wild-type or modified form of the spike protein in an immunogenic composition to be administered to a subject individual. The virus and / or yeast vaccine so administered will infect the individual with a wild-type or modified form of CoV-2, nucleocapsid or spike protein. Once that has occurred, the individual will have an immune response thereto and will be vaccinated. In particular, since the nucleocapsid protein and the spike protein are relatively conserved polypeptides, an immune response can be induced against diverse members of the coronavirus family.

[0051] When the recombinant vector is an adenovirus, the adenovirus vector can be modified to encode a wild-type or modified form of the nucleocapsid protein and / or the spike protein. Similarly, in the case of yeast, the yeast vector can also be modified to encode a wild-type or modified form of the nucleocapsid protein and / or the spike protein. As shown in more detail below, in patients in need thereof, a positive immune response is obtained against cell-mediated immunity after administration of an immunogenic composition containing a virus and / or yeast vector. Thus, in one embodiment, the present disclosure contemplates generating the expression of coronavirus spike on the surface of yeast. In such an embodiment, the yeast functions as an avatar coronavirus to stimulate B cells and then results in 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 comprises both an S sequence (S-fusion) optimized for cell surface expression and a conserved nucleocapsid (N) antigen designed to be transported to the endosomal intracellular compartment, and has the potential to generate a durable immune defense. As further described herein, such bivalent vaccines have been found to be optimized for immunogenicity, as demonstrated by the following findings: 1) The optimized S-fusion showed improved cell surface expression of the S receptor-binding domain (RBD) compared to S-WT with little detectable surface expression; 2) The expressed RBD from the S-fusion retained conformational integrity and recognition by ACE2-Fc; 3) The viral N protein modified with an enhanced T cell-stimulating domain (ETSD) localized to the endosomal / lysosomal intracellular compartment for MHC I / II presentation; and 4) Optimization to these S and N (S-fusion and N-ETSD) generated enhanced de novo antigen-specific B cell as well as CD4+ and CD8+ T cell responses in antigen-naïve preclinical models.

[0053] Both the T cell and antibody immune responses to the S and N components showed a T-helper 1 (Th1) bias. The antibody response was 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. Further, as described in more detail below, the vaccine construct can be administered orally, intranasally, or sublingually. Thus, in one embodiment, the present disclosure also provides the vaccine construct 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. 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 Therefor Coronaviruses are found in avian and mammalian species. They are morphologically and chemically similar to each other: for example, human and bovine coronaviruses are antigenically related. However, there is no evidence that human coronaviruses can be transmitted by animals. In animals, various coronaviruses invade many different tissues and cause a variety of 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 the second half of 2002 / first half of 2003. Another such disease is the Coronavirus Disease 2019 (COVID-19) that spread in several countries around the world in 2019. In December 2019, reports emerged from Wuhan, China, about a new infectious respiratory disease with a high morbidity and mortality rate 1-3 indicating human-to-human transmission. The causative agent was quickly identified as a novel coronavirus and named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The disease caused by this is called COVID-19 and has rapidly become a global pandemic, disrupting socio-economic life and resulting in more than 32 million infections and more than 1.1 million deaths worldwide as of the end of October 2020.

[0055] COVID-19 usually starts with a fever above 38°C. Initial symptoms can also include cough, sore throat, fatigue, and mild respiratory symptoms. Within 2 to 1 week, patients can develop difficulty breathing. Patients in more advanced stages of COVID-19 develop either pneumonia or respiratory distress syndrome. Public health interventions, such as surveillance, travel restrictions, and quarantine, are being 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 lethal CoV as a bioterror threat is clear.

[0056] Coronavirus virions are spherical to pleomorphic enveloped particles. The envelope is studded with protruding glycoproteins and surrounds a core consisting of a matrix protein, within which is encapsulated a single strand of plus-sense RNA (Mr 6×10 6 ) associated with the nucleocapsid protein. In this context, it should be noted that the terms "nucleocapsid protein", "nuclear protein" and "nucleocapsid" are used interchangeably throughout this disclosure. The coronavirus nucleocapsid (N) is a structural protein found in all coronaviruses, including COVID 19. The nucleocapsid protein forms a complex with genomic RNA, interacts with viral membrane proteins during virion assembly, and plays an important role in improving 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 plus-strand RNA viruses with a broad host range. Like other enveloped viruses, CoVs enter target cells by fusion of the virus with the cell membrane, and this process is mediated by the viral spike (S) protein.

[0058] SARS-CoV-2 is an enveloped positive-sense single-stranded RNA β-coronavirus mainly composed of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope, as well as the viral membrane and genomic RNA. Among these, S is the largest and N is the most abundant. The S glycoprotein is displayed as a trimer on the virus surface (Figure 33, panel A), while N is located within the virus particle. A schematic of the primary structure of S is shown in Figure 33, panel B. The sequence of SARS-CoV-2 has been published and compared to previous coronaviruses. Soon after this, the crystal structure of the S protein was reported. The virus uses the S protein to interact with the angiotensin-converting enzyme 2 (ACE2), an enzyme expressed on various cell types in the nose, mouth, intestine, and lung, and importantly, on the alveolar epithelial cells of the lung where infection mainly appears, via the S receptor-binding domain (S RBD). As shown 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 conserved across all types of coronaviruses. In one embodiment, the present disclosure provides a vaccine formulation comprising a recombinant, where 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 the treatment of diseases such as coronavirus-mediated diseases or infectious diseases. Thus, 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 the subject an immunotherapeutic composition comprising a recombinant, the recombinant comprising 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 coronaviruses contemplated herein can be coronavirus disease 2019 (COVID-19) and / or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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

[0061] From a different perspective, viral vectors (e.g., recombinant adenoviral genomes, optionally having a deleted or non-functional E2b gene) are contemplated herein that contain nucleic acids encoding (a) at least one nucleocapsid protein or a modified form thereof; and (b) at least one spike protein or a modified form thereof. The viral vector may further encode one or more co-stimulatory molecules. Most typically, the nucleic acid also includes a transport signal that directs the peptide product encoded by the nucleic acid to the cytoplasm, the endosomal compartment, or the lysosomal compartment, and the peptide product can also include a sequence portion that enhances the intracellular turnover of the peptide product.

[0062] Most of the current SARS-CoV-2 vaccines in development target S because the generation of antibodies against the RBD may neutralize the virus's ability to bind to host cells. Support for the RBD as an important antigen has recently been confirmed, and 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 were reported to be correlated. In addition to the humoral response, S epitopes are also targets of a high frequency of T cells in patients recovered from COVID-19, providing further justification for including S in prophylactic immunization strategies.

[0063] Despite the urgent need for the rapid development of SARS-CoV-2 vaccines, there is no risk-free reliance on any one of the antigen cargos or immunological pathways in the monovalent vaccines in development. Evaluation of nearly 4000 SARS-CoV-2 genomic sequences has identified numerous mutations in S, and 6 months after the identification of the original virus, the D614G variant recently emerged as a potentially more infectious strain.

[0064] In the design of the vaccines 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 additional antigens against which a response can be elicited. The N protein is a highly conserved antigenic SARS-CoV-2-related protein that has been previously studied as an antigen in coronavirus vaccine design against SARS-CoV. N associates with viral RNA within the virus and has roles in viral RNA replication, viral particle assembly, and release. SARS-CoV-2 N is a highly antigenic protein, and almost all patients infected with SARS-CoV-2 have an antibody response against N. Furthermore, another study reported showing that most, but not all, of the COVID-19 survivors tested had an N-specific CD4+ T cell response.

[0065] Currently, there is intense interest in generating a humoral response to vaccines, and perhaps less attention has been paid to T cell responses. However, the natural history of SARS-CoV-2 infection suggests that robust T cell responses to vaccination are at least as important as antibody production and are a critical consideration for COVID-19 vaccine efficacy.

[0066] First, the humoral response and the T cell response are highly correlated, suggesting that the titer of neutralizing antibodies is proportional to the T cell level and that a T response is required for an effective humoral response. The activation of CD4+ T helper cells to enhance antibody production by B cells has been well established. Second, virus-specific CD4+ and CD8+ T cells have not only been widely detected in COVID-19 patients, but also, based on findings from patients who recovered from the closely related SARS-CoV, such T cells persist for at least 6 to 17 years, suggesting that T cells may be an important part of long-term immunity. These T cell responses are mainly against N, and it has been reported that the presence of CD4+ and CD8+ T cells recognizing multiple regions of the N protein could be demonstrated in all 36 convalescent COVID-19 patients in their study. Blood samples from 23 individuals who recovered from SARS-CoV were examined, and memory T cells acquired 17 years ago 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 but evidence of a T cell response. T cell-based responses become even more important considering the finding in at least one study that the neutralizing antibody titer declines in some COVID-19 patients after about three months.

[0067] In one embodiment, the vaccines disclosed herein result in the production of T cells in addition to the humoral response. A bivalent vaccine is contemplated that includes many antigens, an S RBD represented by the inclusion of full-length S containing the SD1, S1, and S2 epitopes along with N, and has been shown to be more effective at inducing both T cell- and antibody-based responses than constructs using any antigen alone by presenting both unique and conserved SARS-CoV-2 antigenic sites to the immune system. The importance of both S and N is emphasized by identifying that both the S and N antigens as empirically potential B and T cell epitopes of the SARS-CoV virus are very 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 potential for antigen presentation on the surface of recombinant protein-expressing cells and the potential for expression in a conformation that mimics natural viral infection. First, wild-type N does not have a signaling domain that leads 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 the robust CD4+ T cell responses necessary for both cell-mediated and B cell memory. To overcome this limitation, an enhanced T cell stimulation domain (ETSD) for N enables 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 to the "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 includes an S-fusion sequence and an N-ETSD sequence.

[0069] The vaccine platform used in this specification 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 mainly by having additional deletions in the early gene 2b (E2b) region that mainly removes the expression of viral DNA polymerase (pol), and in the pre-terminal protein (pTP) gene, and by its growth in the E.C7 human cell line. Removal of these E2b regions confers advantageous immunological properties by minimizing the immune response to Ad5 viral proteins such as viral fiber in patients with existing adenovirus (Ad) immunity, thereby inducing a strong immune response to specific antigens. As a further benefit of these deletions, the vector has an expanded gene carrying / cloning capacity compared to first-generation Ad5[E1-, E3-] vectors. This next-generation hAd5[E1-, E2b-, E3-] vaccine platform, in contrast to Ad5[E1-, E3-]-based platforms, does not promote the activity of suppressing innate immune signaling, thereby enabling improved vaccine efficacy and an excellent safety profile independent of previous Ad immunity. These deletions enable the hAd5 platform to be effective even in the presence of existing Ad immunity, allowing for relatively long-term antigen expression without a significant decrease in anti-vector immunity. Thus, unlike first-generation Ad platforms that face the limitations of existing and vaccine-induced Ad immunity, it is also possible to use the same vector / construct in an allogeneic prime-boost treatment regimen. Importantly, the safety of this next-generation Ad vector has been demonstrated in over 125 patients with solid tumors. In these Phase I / II trials, CD4+ and CD8+ antigen-specific T cells were successful in generating multiple somatic cell antigens (CEA, MUC1, brachyury) even in the presence of existing Ad immunity.

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

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

[0072] Recombinant virus In relation to recombinant viruses, all known methods of producing recombinant viruses are considered suitable for use herein, although particularly preferred viruses are those already established in therapeutic methods, including adenoviruses, adeno-associated viruses, alphaviruses, herpesviruses, lentiviruses, and the like. Among other suitable options, adenoviruses are particularly preferred.

[0073] Furthermore, generally, it is more preferred that the virus is a replication-deficient and non-immunogenic virus. For example, suitable viruses include recombinant alphaviruses, adenoviruses, adeno-associated viruses, herpesviruses, lentiviruses, etc. However, adenoviruses are particularly preferred. For example, recombinant replication-deficient adenoviruses suitable for vaccination not only of multiple vaccinations but also of individuals having existing immunity to adenoviruses are preferred (see, for example, WO 2009 / 006479 pamphlet and WO 2014 / 031178 pamphlet, which are incorporated herein by reference in their entirety). In some embodiments, the replication-deficient adenovirus vector includes a replication-deficient adenovirus type 5 vector. In some embodiments, the replication-deficient adenovirus vector includes a deletion in the E2b region. In some embodiments, the replication-deficient adenovirus vector further includes a deletion in the E1 region. In that regard, it should be noted that the deletion of the E2b gene and other late proteins in recombinant replication-deficient adenoviruses reduces immunogenicity. Furthermore, due to these specific deletions, such recombinant viruses are replication-deficient and allow for a relatively large recombinant cargo.

[0074] For example, WO 2014 / 031178 pamphlet describes the use of such recombinant viruses that express CEA (carcinoembryonic antigen) to provide an immune response against colon cancer. Furthermore, a relatively high titer of the recombinant virus can be achieved using recombinant human 293 cells as reported (see, for example, J Virol. 1998 Feb;72(2):926-933).

[0075] The El-deleted adenovirus vector Ad5[E1-] is constructed such that the transgene replaces only the El region of the gene. Typically, approximately 90% of the wild-type Ad5 genome is retained in the vector. The Ad5[E1-] vector has reduced replication ability and cannot generate infectious virus after infection of cells that do not express the Ad5 El gene. Recombinant Ad5[E1-] vectors can grow in human cells, enabling Ad5[E1-] vector replication and packaging. Ad5[E1-] vectors have many positive traits; among the most important is that their scale-up and cGMP production are relatively easy. Currently, more than 220 human clinical trials are using Ad5[E1-] vectors, and more than 2000 subjects have been administered the virus sc, im, or iv. Furthermore, Ad5 vectors do not integrate; their genomes remain episomal. Generally, for vectors that do not integrate into the host genome, the risk of insertional mutagenesis and / or germline transmission is very low, if any. 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 International Publication No. WO 2014 / 031178, which is incorporated herein by reference. Specifically, a novel recombinant Ad5 platform with deletions in the early 1 (El) gene region and additional deletions in the early 2b (E2b) gene region (Ad5[E1-, E2b-]) is described. Deletions in the E2b region (encoding DNA polymerase and pre-terminal protein) result in reduced viral DNA replication and late viral protein expression. E2b-deleted adenovirus vectors provide an improved Ad-based vector that is safer, more effective, and more versatile than first-generation adenovirus vectors.

[0077] In further embodiments, adenoviral vectors intended for other uses include adenoviral vectors having deletions in the E2b region of the Ad genome, optionally deletions in E1 and E3, and optionally partial or complete removal of the E4 region. In further embodiments, the adenoviral vectors used herein have deletions in the pre-terminal protein function of the E1 and / or E2b regions. In some cases, such vectors have no other deletions. In another embodiment, the adenoviral vectors used herein have deletions in E1, DNA polymerase and / or pre-terminal protein function.

[0078] As used herein, the term "E2b deletion" refers to a specific DNA sequence mutated to prevent the expression and / or function of at least one E2b gene product. Thus, in certain embodiments, "E2b deletion" is used with respect to a specific DNA sequence deleted (removed) from the Ad genome. An E2b deletion or "including a deletion within the E2b region" refers to a deletion of at least one base pair within the E2b region of the Ad genome. Thus, in certain embodiments, more than one base pair is deleted, and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted. In another embodiment, the deletion is of 150, 160, 170, 180, 190, 200, 250, or more than 300 base pairs within the E2b region of the Ad genome. An E2b deletion may be a deletion that prevents the expression and / or function of at least one E2b gene product, and thus includes deletions within the exons of the coding portion of the E2b-specific protein, as well as deletions within the promoter and leader sequences. In certain embodiments, the E2b deletion is a deletion that prevents the expression and / or function of both DNA polymerase and the pre-terminal protein of the E2b region. In further embodiments, "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 of the encoded proteins are non-functional. Such mutations include residues replaced with different residues that lead to changes in the amino acid sequence resulting in non-functional proteins.

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

[0080] In connection with virus expression and vaccination systems, it is contemplated that all therapeutic recombinant virus expression systems are considered suitable for use herein insofar as such viruses are capable of effecting the expression of recombinant payloads 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 may be injected subcutaneously or intravenously to infect the patient's cells, particularly antigen-presenting cells, or may be administered via intranasal or inhalation routes. Alternatively, immune competent cells (e.g., NK cells, T cells, macrophages, dendritic cells, etc.) from the patient (or from an allogeneic source) may be infected in vitro and then injected into the patient. Alternatively, immunotherapy may be achieved by nucleic acid transfection or vaccination using RNA or DNA, or other recombinant vectors, that result in the expression of neoepitopes (e.g., single peptides, tandem mini-genes, etc.) in the desired cells, particularly immune competent cells, without the need to rely on a virus. Such nucleic acids are typically delivered in association with lipid formulations to protect the nucleic acids from degradation and to facilitate the uptake of the nucleic acids by target cells.

[0082] As described above, a desired nucleic acid sequence (for expression from virus-infected cells) is under the control of appropriate regulatory elements well known in the art. For example, suitable promoter elements include constitutive strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoter), and in particular, in cases where the induction conditions are typical for the tumor microenvironment, inducible promoters are also considered suitable for use in the present invention. For example, inducible promoters include those sensitive to hypoxia and promoters 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 the tetracycline-inducible promoter, the myxovirus resistance 1 (Mx1) promoter, and the like.

[0083] A replication-deficient adenovirus comprising a nucleic acid encoding an E1 gene region deletion, an E2b gene region deletion, and a coronavirus 2 (CoV2) nucleocapsid protein and / or a CoV2 spike protein, as disclosed herein, can be administered to a patient 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 can also vary with the severity of the disease and can be readily established using standard techniques. In some embodiments, administration comprises delivering 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 replication-deficient adenovirus particles.

[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 injection, intramuscular injection, intravenous injection, or subcutaneous injection. In some embodiments, subcutaneous delivery may be preferred.

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

[0086] The use of yeast-based therapeutic compositions is disclosed in the art. For example, WO 2012 / 109404 pamphlet discloses a yeast composition for the treatment of chronic hepatitis B infection.

[0087] It is noted that any yeast strain can be used to generate the yeast vehicles of the present disclosure. Yeast is a unicellular microorganism belonging to one of three classes: Ascomycetes, Basidiomycetes, and Fungi Imperfecti. One consideration regarding the selection of the type of yeast for use as an immunomodulator is the pathogenicity of the yeast. In a preferred embodiment, the yeast is a non-pathogenic strain such as Saccharomyces cerevisiae to minimize any adverse effects on the individual to whom the yeast vehicle is administered. However, pathogenic yeast 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 can be used include Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica.

[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, the 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). Therefore, yeast strains that can replicate plasmids, particularly at a high copy number, such as the S. cerevisiae cir strain, are specifically contemplated herein. S. cerevisiae strains are one of the strains that can support expression vectors that express one or more target antigens and / or antigen fusion proteins and / or other proteins at high levels. Furthermore, any mutant yeast strain can be used, including those that exhibit a decrease in post-translational modification of the expressed target antigen or other protein, such as a mutation in an enzyme that extends N-linked glycosylation.

[0090] The intended expression of peptides / proteins 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 when transformed into a host yeast cell, the nucleic acid molecule is operably linked to transcriptional control sequences to achieve constitutive or regulated expression of the nucleic acid molecule. 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 activation sequences.

[0091] Any suitable yeast promoter can be used in the methods and compositions of the present disclosure, and a variety of such promoters are known to those of skill in the art and are generally discussed above. Promoters for expression in Saccharomyces cerevisiae include the promoters of genes encoding the following yeast proteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), translation elongation factor EF-1α (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referred to as TDH3 in the case of triose phosphate dehydrogenase), galactokinase (GAL1), galactose-1-phosphate uridyl-transferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome c1 (CYC1), Sec7 protein (SEC7), and acid phosphatase (PHO5), (which includes hybrid promoters such as the ADH2 / GAPDH and CYC1 / GAL10 promoters, and includes 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, a number of upstream activation sequences (UAS), also referred to as 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.The 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 nucleic acid molecules into yeast cells according to the present disclosure can be achieved 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 episomal vector using techniques known to those skilled in the art. As discussed above, yeast cytoplasts, yeast ghosts, and yeast membrane particles or cell wall preparations can also be used to transfect intact yeast microorganisms or yeast spheroplasts with the desired nucleic acid molecule, generate an antigen internally, and then further manipulate the microorganism or spheroplast using techniques known to those skilled in the art to generate a cytoplast, ghost, or intracellular yeast membrane extract or fraction thereof containing the desired antigen or other protein, thereby generating recombinantly. 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 10 4 ~10 13 virus or yeast particles, or more preferably 10 9 ~10 12 virus or yeast particles formulated as a sterile injectable composition having the virus. Alternatively, the virus or yeast may be used to infect patient cells ex vivo, and the cells so infected are then injected into the patient. However, alternative formulations are also contemplated and suitable for use herein, and all known routes and modes of administration are contemplated herein.

[0094] In a further contemplated embodiment, the second-generation hAd5[E1-, E2b-, E3-]-based vaccine disclosed herein overcomes existing anti-Ad5 immunity. To avoid the Ad immunization barrier and adverse conditions to the first-generation Ad5[E1-E3-] vector, an advanced second-generation human adenovirus (hAd5) vector with two (2) additional deletions in the E2b region, removing the DNA polymerase and pre-terminal protein genes, was constructed [E1-, E2b-, E3-]. (The former name of the adenovirus vector was Ad5, designated ETBX in the literature)

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

[0096] The hAd5[E1-, E2b-, E3-] vector induces a strong cell-mediated immunity (CMI) response and Abs against the vectorized antigen even in the presence of Ad immunity. The hAd5[E1-, E2b-, E3-] vector also has a reduced occurrence of adverse reactions, particularly hepatotoxicity and tissue damage, compared to the Ad5[E1-] vector. In one embodiment, the ability of the hAd5[E1-, E2b-, E3-] vector to infect dendritic cells (DCs) is increased by a reduced inflammatory response to the hAd5[E1-, E2b-, E3-] vector viral proteins and the resulting avoidance of pre-existing Ad immunity, enhancing vaccine-induced immunity. Furthermore, the increased infection of other cell types provides a high level of antigen presentation necessary 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 rapid and efficient manner. In one embodiment, a prophylactic vaccine is tested against COVID-19 by utilizing this new hAd5 vector system that overcomes the barriers found in other Ad5 systems, enabling immunization of individuals previously exposed to Ad5.

[0097] Track record of rapid vaccine development using a second-generation human (hAd5) adenovirus platform during a pandemic threat: The 2009 H1N1 experience. Particularly in the era of pandemics, it is important to use modernized vaccine technologies to address the threat of new pathogens. These technologies will utilize genome sequencing and the power of rapid transfection in established vaccine vectors to quickly identify constructs with high immunogenicity.

[0098] Vaccines against novel pathogens such as the 2009 H1N1 pandemic virus can benefit from modern technologies such as rapid genomic 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 the vaccines were rapidly constructed and produced. Vaccination induced an H1N1 immune response in mice, which provided protection from a lethal viral challenge. In ferrets, vaccination protected against disease onset and significantly reduced viral titers in nasal washes. H1N1 cells mediated antibody induction correlated with immunity and prevention of disease symptoms and reduction of viral replication. Thus, hAd5[E1-, E2b-, E3-] demonstrated the ability for the rapid development of effective vaccines against infectious diseases.

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

Example

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

[0101] Rationale for the 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 causing SARS demonstrated that the N protein is immunogenic when integrated with intracellular transport constructs. To date, all vaccine strategies in development have included the development of immunogenicity against the spike (S) protein. However, very recent evidence in patients recovered from COVID-19 demonstrates Th1 immunity generated against nucleocapsid (N). Further reports have further confirmed that T and B cell epitopes are highest for both the spike glycoprotein and the nucleoprotein in predictive bioinformatics models. 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 finding that mutations in S and the structural N protein are highly conserved in the coronavirus family, there is a significant potential for this combination vaccine to serve as a long-term "universal" COVID-19 vaccine.

[0102] Example 2: Immunogenicity Test (Murine Model): Homologous prime-boost immunogenicity in BALB-c mice. Mice are treated with 1, 2, or 3 doses of the 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, hAd5[E1-,E2b-,E3-]N-ETSD, a vaccine containing SARS-CoV-2 nucleocapsid + enhanced T cell stimulation domain (ETSD), alters the T cell response to nucleocapsid. Mice were immunized subcutaneously (SC) twice at 7-day intervals with a dose of 1010 VP. Blood was collected at several time points, and the spleen was collected at sacrifice for immunogenicity experiments. Splenocytes were isolated and tested for cell-mediated immunity (CMI) responses. The results showed that SARS-CoV-2 nucleocapsid antigen-specific CMI responses were detected 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 immunogenicity value of N when combined with S was the surprising finding of enhanced surface expression of the RBD protein in 293 cells transfected with the N-ETSD+S construct, as seen in Figure 31. The expression and presentation of RBD, as demonstrated by recent reports by others showing that rare but recurring RBD-specific antibodies with potent antiviral activity are found in all individuals of the test subjects who recovered from COVID-19 infection, seems to be very important. This finding of enhanced expression of RBD when N is combined with the S-fusion was corroborated in studies using plasma from patients who recovered from COVID-19 (Figure 32). The alternative construct of RBD-ETSD could serve as an alternative vaccine.

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

[0106] Example 4: Immunogenicity Testing of Candidate COVID-19 Vaccine Constructs Two adenovirus-based COVID-19 vaccine constructs are tested in preclinical experiments including in vitro expression; murine immunogenicity, and non-human primate immunogenicity and efficacy.

[0107] Description of constructs: Two second-generation hAd5-based COVID-19 vaccine constructs were evaluated. The first is an hAd5 vector with SARS-CoV-2 having a spike protein insert (see Figure 26). The second is an E1-, E2b-, E3 hAd5 vector having a SARS-CoV-2 wild-type spike protein (S) insert and a nucleocapsid protein (N) insert containing an endosome targeting domain sequence (ETSD) in the same vector backbone.

[0108] Immunogenicity studies: Homologous prime-boost immunogenicity in mice was tested by treating the mice with 1, 2, or 3 doses of the adenovirus vaccine candidates listed in Figure 26, and serum and splenocyte samples were tested for SARS-CoV-2 antigen-specific immune responses. Serum has been tested for anti-spike and anti-nucleocapsid antibody responses by ELISA. Splenocytes were 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 studies: Serum from mice immunized during the course of the immunogenicity studies described above is used and sent to a test laboratory for SARS-CoV-2 neutralization studies conducted in their ABSL-3 facilities. Serum dilutions are mixed with the COVID 19 virus, the mixture is incubated, and then the mixture is exposed to Vero cells to detect the cytopathic effect (CPE) to test the serum for COVID 19 virus neutralizing activity. The last dilution that prevents CPE is considered the end-point neutralizing titer.

[0110] Immunogenicity and Efficacy Assessment in Non-Human Primates: Rhesus macaques are treated with 3 doses of the adenovirus vaccine candidates listed in Figure 26. The SARS-CoV-2 antigen-specific immune response is monitored in serum and PBMC by ELISA, ELISPOT, and ICS throughout the course of treatment. Four weeks after the final vaccination, the animals are challenged with SARS-CoV-2, and the disease characteristics and viral excretion are monitored.

[0111] Example 5: Phase Ib Clinical Trial of the hAd5[E1-, E2b-, E3-]CoV-2 Vaccine. Study Design: This is a Phase 1b open-label trial 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 hAd5[E1-, E2b-, E3-] vector-based targeted vaccines that encode the SARS-CoV-2 spike (S) protein alone or together with the SARS-CoV-2 nucleocapsid (N) protein. The hAd5[E1-, E2b-, E3-] vector is a platform technology for targeted vaccines that has demonstrated safety at high doses of virus particles in over 125 cancer patients. Three different hAd5[E1-, E2b-, E3-] vector-based vaccines are co-administered on the same day at 5×10 11 virus particles per dose (1.5×10 11 total virus particles), and it has also been demonstrated to be safe. 12

[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. Therefore, there is a possibility that hAd5-COVID-19-S and hAd5-COVID-19-S / N can 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 are divided into four dosing cohorts (Cohort 1A, 1B, 2A, 2B; n = 10 for each cohort): · Cohort 1A - hAd5-COVID-19-S 5×10 10 virus 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 receives a subcutaneous (SC) injection of hAd5-COVID-19-S or hAd5-COVID-19-S / N on Day 1 and Day 22 (i.e., 2 doses). This dosing schedule is consistent with the hAd5[E1-, E2b-, E3-] vector-based vaccines currently in clinical trials. Cohorts 1-2 are registered in parallel and can be opened simultaneously or with a time lag depending on the availability of the investigational drug. Subjects in Cohorts 1A and 2A first complete a 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 assessment period up to Day 8 of the trial, the sponsor and an independent Safety Review Committee (SRC) and at least one qualified infectious disease physician confirm that there are no safety concerns, and enrollment proceeds. Subjects are then enrolled in the high-dose Cohorts 1B and 2B and vaccinated. For all subjects, follow-up study visits are conducted on Days 8, 22, 29, 52 after the final vaccination, and at 3, 6, and 12 months. Additional follow-up for safety information is conducted by telephone as described in the event schedule. The primary objective of this 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. The secondary objective is 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 Based on the review of safety data from the Phase 1b safety assessment, if the SRC determines that it is safe to do so, the expansion of Phase 1b proceeds. In the expansion of Phase 1b, a total of 60 healthy subjects are divided into 4 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 / dosage (n = 15) · Cohort 2B - hAd5-COVID-19-S / N 1×10 11 VP / dosage (n = 15)

[0116] Each subject receives an SC injection of hAd5-COVID-19-S or hAd5-COVID-19-S / N on Day 1 and Day 22 (i.e., 2 dosages). For all subjects, follow-up study visits are conducted on Day 8, 22, 29, 52 after the final vaccination, as well as at 3, 6, and 12 months. Additional follow-up of safety information is conducted by telephone contact as described in the event schedule. The primary objective of the expanded Phase 1b study is to select the most immunogenic construct between hAd5-COVID-19-S and hAd5-COVID-19-S / N and the dosage level determined by the changes in humoral and cellular immunogenicity indices. The secondary objective is 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. The examples are merely illustrative and do not limit the scope of the claimed invention in any way.

[0118] Example 7: hAd5[E1-, E2b-, E3-] Platform and Construct For the examples shown here, a viral vaccine candidate construct was generated using a next-generation hAd5[E1-, E2b-, E3-] vector (Figure 33, panel C). As shown in Figure 33, panels D–H, diverse 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 an enhanced T cell-stimulating domain (ETSD); Figure 33, panel F: S fusion: S optimized to enhance surface expression and display of the RBD; Figure 33, panel G: N-ETSD: nucleocapsid (N) sequence with an ETSD; and Figure 33, panel H: bivalent S fusion + N-ETSD; S-WT + N-ETSD and S RBD-ETSD + N-ETSD constructs were also generated but not shown.

[0119] Example 8: Enhancement of HEK 293T cell surface expression of RBD after transfection with Ad5 S fusion + N-ETSD As shown in Figure 34, anti-RBD specific antibodies did not detect RBD on the surface of HEK 293T cells transfected with the 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 with RBD, hAd5 RBD-ETSD, and RBD-ETSD + N-ETSD showed high binding of anti-RBD antibodies (Figure 34, panels C and D). In particular, 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 containing multiple 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: Enhancement of S expression and immunoblot correlation with hAd5 S-fusion + N-ETSD Immunoblot analysis of S expression correlated with enhanced S expression (Figure 35), again showing that the bivalent hAd5 S-fusion + N-ETSD construct enhances S expression compared to the S-fusion alone. Figure 35 shows the immunoblot analysis of S expression. Cell surface RBD expression using (a) hAd5 S-WT, S-fusion, and (c) S-fusion + N-ETSD in HEK 293T cells showed high correlation with S expression (d) in immunoblots of HEK 293T cell lysates probed with an anti-full length (S2) antibody. The Y-axis scale is normalized to the mode (NM).

[0121] Example 10: Confirmation of native folding of enhanced surface RBD after hAd5 S-fusion + N-ETSD transfection Determination of the binding of recombinant ACE2-Fc was performed to confirm the native physiologically relevant folding of S RBD after expression from the hAd5 S-fusion + N-ETSD vaccine candidate. 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 when produced in response to S presented in the correct conformation. In addition to enhanced cell surface expression, the optimized S allows for proper protein folding. It was found that ACE2-Fc binding to the S RBD expressed from hAd5 S-fusion + N-ETSD was significantly enhanced compared to either hAd5 S-WT or hAd5 S-fusion (Figure 36, panels A and B, respectively) (Figure 36, panel C). Anti-RBD antibody binding tests performed in the same experiment (Figure 36, panels F-J) confirmed the enhanced surface expression findings shown by ACE2-Fc binding. The hAd5 S-fusion + N-ETSD vaccine candidate was selected for clinical trials based on these findings of conformationally accurate and enhanced S RBD expression that is important for the production of neutralizing antibodies.

[0122] Example 11: hAd5 N-ETSD successfully guides N to the endosome / lysosome compartment The ETSD design successfully translocated N into the intracellular compartment of endosomes. After infection of HeLa cells with N-ETSD, N co-localized with the endosome marker transferrin receptor (CD71), as shown in Panel C of Figure 37, and also co-localized with the lysosome marker Lamp1 (Figure 37, Panel D), demonstrating that N-ETSD translocates to lysosomes through the endosomal pathway and enables processing for MHC II presentation. Compared with N-ETSD, N-wild type (N-WT) showed a diffuse cytoplasmic distribution and did not co-localize with the lysosome marker (Figure 37, Panel E). These findings confirm the role of ETSD in guiding N to the endosome / lysosome compartment, which results in increased MHC II presentation and CD4+ activation by N.

[0123] Example 12: In vivo hAd5 S-fusion + N-ETSD vaccine immunogenicity test Based on evidence that S-fusion + N-ETSD resulted in enhanced expression of the physiologically relevant RBD and that N-ETSD successfully translocated to the endosome / lysosome compartment, the 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 result in the generation of both CD8+ and CD4+ T cell responses and neutralizing antibodies. As described in the method, mice received an initial injection on Day 0 and a second injection on Day 21. Serum was collected at the end of the tests on Day 0 and Day 28 for antibody and neutralization analysis. Spleen cells were also collected on Day 28 for intracellular cytokine staining (ICS) and ELISpot analysis. All age- and sex-matched animals assigned to the study were free of site reactions, showed no weight loss throughout the administration, and appeared normal, consistent with previous observations with the hAd5[E1-, E2b-, E3-] platform.

[0124] Example 13: hAd5 S-fusion + N-ETSD elicits 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. Evaluation of simultaneous IFN-γ / TNF-α expression from CD8β+ splenocytes (Figure 38, panel C) reflected 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 the 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 the N peptide pool stimulated CD4+ splenocytes from hAd5 S-fusion + N-ETSD vaccinated mice to express IFN-γ (Figure 38, panel B) or IFN-γ / TNF-α (Figure 38, panel D) at substantially higher levels than the hAd5 null control. The contribution of N to the CD4+ T cell response is essential for an effective immune response to the candidate vaccine.

[0126] Example 14: hAd5 S-fusion + N-ETSD elicits antibody responses to both S and N antigens The main goal of currently developed coronavirus vaccines is neutralizing antibodies against the spike. In CD-1 mice vaccinated with the bivalent vaccine, significant production of both anti-S (Figure 39, panel A) and anti-N (Figure 39, panel C) antibodies was observed in sera from mice vaccinated with hAd5 S-fusion + N-ETSD on day 28 of the study. Anti-N antibodies were higher in serum compared to anti-S antibodies, with a serum dilution factor of 1:90 for anti-N antibody analysis and 1:30 for anti-S antibody analysis.

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

[0128] Example 15: The hAd5 S-fusion + N-ETSD vaccine generates potent neutralizing antibodies as evaluated by both the cPass and live virus neutralization assays Neutralizing antibody activity was evaluated 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 at both 1:20 and 1:60 dilutions, and approximately 100% inhibition for two mice. The results of the Vero E6 neutralization assay 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 interesting possibility that the generation of bivalent multi-antigen multi-epitopes by the hAd5 S-fusion + N-ETSD vaccine may result in a synergistic effect on the neutralizing immune response by epitopes in addition to those related to RBD-ACE2 binding (Figure 40, panel B). As seen in Figure 40, panel B, the 50% neutralization (IC50) value was present at a 1:10,000 serum dilution for the G4 pool of sera from mice that showed S-specific antibodies, 10-fold higher than the convalescent sera at a 1:1,000 dilution. The potent neutralization confirmed by both assays supports the predicted efficacy of the hAd5 S-fusion + ETSD vaccine candidate and its progression to clinical trials.

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

[0130] T Cells Responding to N and S, Th1 Bias: IFN-γ production correlates with CTL activity47 (Th1 bias), while IL-4 causes delayed virus clearance48 (Th2 bias). The ratio of IFN-γ to IL-4 is balanced, and a ratio greater than 1 indicates Th1 bias. ELISpots from animals immunized with the bivalent S+N vaccine showed that IFN-γ secretion was significantly higher for hAd5 S-fusion + N-ETSD compared to hAd5 null splenocytes in response to both the S peptide pool 1 and the N peptide pool (Figure 42, panel A), while IL-4 was secreted at significantly higher levels for hAd5 S-fusion + N-ETSD in response to the N peptide pool only (Figure 42, panel B).

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

[0132] This Th1 bias profile of the hAd5 S-fusion + N-ETSD vaccine candidate provides further justification for hAd5 S-fusion + N-ETSD to be the lead candidate in clinical trials.

[0133] The hAd5 S-fusion + N-ETSD vaccine is 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 the generation of long-lasting antibody responses and the direct killing of infected cells. Both CD4+ and CD8+ T cells are multifunctional, and the induction of such multifunctional T cells by the vaccine has been correlated with better defense against infection. The expression of the S antigen optimized for surface display and the N antigen optimized for endosome / lysosome intracellular compartment localization, and thus the enhanced CD4+ T cell response and Th1 dominance resulting from 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 in pooled sera from mice vaccinated with the hAd5 S-fusion + N-ETSD vaccine, combined with the Th1 dominance of the antibodies generated in response to both the S and N antigens, supports the objectives of this vaccine design.

[0134] The simultaneous MHC I and MHC II presentation of antigens by antigen-presenting cells activates both CD4+ and CD8+ T cells simultaneously and is optimal for the generation of memory B and T cells. An important finding of the construct is that N-ETSD is directed to the endosome / lysosome compartment. N-ETSD induces the CD4+ response, which is required for the induction of memory T cells and helper cells for B cell antibody production. The importance of lysosomal localization for inducing the strongest T cell IFN-γ and CTL responses has also been reported compared to native N.50, 51

[0135] The T cell responses against the S and N antigens expressed by hAd5 S-fusion + N-ETSD were polycytokines including IFN-γ and TNF-α, which were consistent with the success of antibacterial immunity in bacterial and viral infections. The polycytokine T cell responses after vaccination have been shown to correlate with vaccine efficacy, including those by viral vectors. Of high relevance here is that the polycytokine T cell responses against the SARS-CoV-2 N protein are consistent with recovered COVID-19 patients, suggesting that the bivalent hAd5 S-fusion + N-ETSD vaccine provides greater protection against SARS-CoV-2 to the vaccinated subjects.

[0136] In contrast to N, the S protein, expressed here as S-fusion, was confirmed to have enhanced RBD cell surface expression and conformational integrity, as demonstrated by high ACE2-Fc binding, and mainly generated CD8+ T cells. Our results confirmed the vaccine design goal and showed that the S-fusion induced an increase in the level of antigen-specific T cell responses against 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 produce this coordinated response.

[0137] The neutralization data with live SARS-CoV-2 virus demonstrated the efficacy 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 the pooled sera was evident, with more potent neutralization than the control convalescent sera at ≧1:1,000 dilution.

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

[0139] Example 17: Methods and Constructs hAd5[E1-, E2b-, E3-] Platform and Constructs For the tests herein, a second-generation hAd5[E1-, E2b-, E3-] vector was used (Figure 44, panel A) to generate viral vaccine candidate constructs. The hAd5[E1-, E2b-, E3-] backbone containing the SARS-CoV-2 antigen expression insert and viral particles was generated as previously described. Briefly, 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) from ViraQuest Inc. (North Liberty, IA) was used to generate high-titer adenovirus stocks. Virus particle numbers were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280 nm, and virus titers were determined using the Adeno-X™ Rapid Titer Kit (Takara Bio). The constructs generated contained the following:

[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-stimulating domain (ETSD); S-RBD-ETSD + N-ETSD; S-fusion: S optimized to enhance surface expression and display of RBD; and bivalent S-fusion + N-ETSD;

[0141] Transfection of HEK 293T cells with the hAd5 construct To determine the surface presentation of the RBD epitope by the vaccine candidate constructs, the inventors transfected HEK 293T cells with the hAd5 construct DNA and quantified the surface RBD by flow cytometry detection using an anti-RBD antibody. There were 7 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 a 24-well plate 5Cells / well) were grown at 37°C in DMEM (Gibco Cat# 11995-065) containing 10% FBS and 1X PSA (100 units / mL penicillin, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B). 0.5 μg of hAd5 plasmid DNA was transfected into the cells using JetPrime transfection reagent (Polyplus catalog# 89129-924) according to the manufacturer's instructions. At 1, 2, 3, and 7 days post-transfection, cells were harvested by gently pipetting them into the medium and labeled with anti-RBD monoclonal antibody (clone D003 Sino Biological Catalog # 40150-D003), and F(ab’)2-goat anti-human IgG-Fc secondary antibody conjugated to R-phycoerythrin (ThermoFisher Catalog # 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 intracellular localization of N after HeLa cells were infected or transfected with hAd5 N-wild type (WT) or hAd5 N-ETSD (each having a flag tag enabling labeling), 48 hours after infection or transfection, the 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, the cells were then incubated overnight at 4°C at a 1:1000 dilution with an anti-flag monoclonal antibody (anti-Flag M2 produced in mouse, Sigma cat# F1804) in phosphate-buffered saline containing 3% BSA, followed by washing in PBS and incubation for 1 hour at a 1:500 dilution with a goat anti-mouse IgG(H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 555 (Life Technologies, CAt# A32727). For the co-localization assay, the cells were also incubated overnight at 4°C at a 1:10 dilution with a sheep anti-Lamp1 Alexa Fluor 488-conjugated (lysosome marker) antibody (R&D systems, Cat# IC7985G) or at a 1:200 dilution with a rabbit anti-CD71 (transferrin receptor, endosome marker) antibody (ThermoFisher Cat# PA5-83022). After removing the primary antibody and washing twice in PBS and three times in PBS containing 3% BSA, when applicable, the cells were incubated for 1 hour at room temperature at a 1:500 dilution with a fluorescent-conjugated secondary antibody (goat anti-rabbit IgG(H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488, Life technologies, A-11034). After a short wash, the cells were mounted with Vectashield Antifade mounting medium containing DAPI (Fisher Scientific, catalog number 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 herein and harvested 3 days post-transfection into 150 ml of RIPA lysis buffer containing 1X final Protease Inhibitor cocktail (Roche). After protein assay, equal amounts of total protein were loaded onto a 4–12% gradient polyacrylamide gel (type), run, 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 using 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 generated 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] The purified ACE2-IgG1Fc was dialyzed against 10 mM HEPES, pH 7.4, 150 mM NaCl and concentrated to 2.6 mg / mL. For the binding assay, ACE2-IgG1Fc was used at a concentration of 1 μg / mL for binding. Cells were incubated with ACE2-Fc for 20 minutes, and after the washing step, labeled with a PE-conjugated F(ab’)2-goat anti-human IgG Fc secondary antibody at a 1:100 dilution, incubated for 20 minutes, washed, and acquired on a flow cytometer. The histogram was based on the number of positive cells - in the normalized mode (NM)-PE channel of the cell number - signal.

[0146] Vaccination of CD-1 mice with hAd5 S-fusion + N-ETSD vaccine candidates Seven-week-old female CD-1 mice (Charles River Laboratories) were used in the immunological tests conducted at the vivarium facility of Omeros Inc. (Seattle, WA). After the first blood collection, on day 0, mice were injected with hAd5 null (negative control) or the vaccine candidate hAd5 S-fusion + N-ETSD at a dose of 1×10 10 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 then the mice were euthanized for collection of the spleen and other tissues.

[0147] Spleen cell collection and intracellular cytokine staining (ICS) Spleens were removed from each mouse and placed in 5 mL of sterile medium of RPMI (Gibco Cat# 22400105), HEPES (Hyclone Cat# SH30237.01), 1X Pen / Strep (Gibco Cat# 15140122), and 10% FBS (Gibco Cat# 16140-089). Spleen cells were isolated within 2 hours of collection. ICS for flow cytometric detection of CD8β+ and CD4+ T cell-related 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 antibody detection For the detection of antibodies in sera 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 either 100 ng of purified recombinant SARS-CoV-S-S-FTD (full-length S with fibrin 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 protein, and the plates were blocked with 250 μL of PBST for 60 minutes at room temperature. After blocking, the wells were washed with PBST, and 100 μL of diluted serum sample was added to the wells, and the samples were incubated for 60 minutes at room temperature. After incubation, the wells were washed with PBST, and 100 μL of 1 / 5000-diluted anti-mouse IgG HRP (GE Health Care; Cat# NA9310V), or anti-mouse IgG1 HRP (Sigma; Cat# SAB3701171), or anti-mouse IgG2a HRP (Sigma; Cat# SAB3701178), or anti-mouse IgG2b HRP (Sigma; Cat# SAB3701185), or anti-mouse IgG3 HRP-conjugated antibody (Sigma; Cat# SAB3701192) was added to the wells. For the positive control, 100 μL of 1 / 5000-diluted rabbit anti-N IgG Ab or 100 μL of 1 / 25-diluted mouse anti-S serum (derived from mice immunized with purified S antigen in adjuvant) was added to the appropriate wells.After incubating at room temperature for 1 hour, 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) to water, and the absorbance at 490 nm was determined using a microplate reader (SoftMax® Pro, Molecular Devices).

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

[0152] cPass™ neutralizing antibody detection GenScript cPass™ for the detection of neutralizing antibodies was used according to the manufacturer's instructions. The kit detects circulating neutralizing antibodies against SARS-CoV-2 that block the interaction between the S RBD and the ACE2 cell surface receptor. This is suitable for all antibody isotypes and for use in animal models without modification.

[0153] Vero E6 cell neutralization assay All aspects of the assays using the virus were conducted in a BSL3 containment facility in accordance with the ISMMS Conventional Biocontainment Facility SOP for SARS-CoV-2 cell culture testing. Vero E6 kidney epithelial cells, derived from African green monkey (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 antibody or heat-inactivated serum pre-diluted in 3-fold steps in DMEM containing 2% FBS, 1% NEAA, and 1% Pen-Strep; the diluted samples were mixed 1:1 with SARS-CoV-2 in DMEM containing 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 contain cells to generate neutralizing activity prior to infection. Samples for testing included sera from 4 mice that showed >20% inhibition of ACE2 binding in cPass, pooled sera from these 4 mice, sera from COVID-19 convalescent patients, and media only. For detection of neutralization, 120 μL of the virus / sample mixture was transferred to Vero E6 cells, incubated for 48 hours, and then fixed with 4% PFA. Each well received 60 μL of virus or an infectious dose of 600 TCID50. For virus-free and virus-only controls, control wells containing 6 wells each were used on each plate. Percent neutralization was calculated as 100 - ((mean of the sample of interest - [mean of "virus-free"]) / [mean of "virus-only"] * 100) using staining of CoV-2 Np imaged on a Celigo Imaging Cytometer (Nexcelom Bioscience).

[0154] Characterization of the Vaccine Most of the current prophylactic anti-SARS-CoV-2 vaccines under development are designed to prevent further disease-related deaths and morbidity by targeting the viral spike (S) protein, with the aim of generating a neutralizing antibody response in recipients prior to virus exposure. However, recent characterization of the COVID-19 patient immune response to SARS-CoV-2 has shown that other immune cells, such as T cells, are important for clearing infection and generating long-term immunity against coronavirus infection. Both CD4+ T cells and CD8+ T cells support a durable humoral response because, although CD4+ T cells are not effector cells like CD8+ T cells, they are important for the generation of robust and persistent immunity brought about by antibody-secreting plasma cells and 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 fight SARS-CoV-2 by including a modified viral nucleocapsid (N) antigen, a potent CD4+ and CD8+ T cell target, along with an optimized S protein (S-fusion) to stimulate the humoral response.

[0155] The human adenovirus serotype 5 (hAd5) E1-, E2b-, and E3-deleted [E1−, E2b−, E3−] vaccine platform (Figure 44, panel A) is effective in the presence of pre-existing adenovirus immunity, is less likely to generate vector-targeted host immune responses, and can thus be used as both a prime and a boost, making it superior to the adenovirus platforms currently used in other COVID-19 vaccines in clinical trials. Using this platform, the vaccine contains an optimized S surface protein, S-fusion, for increasing cell surface display and humoral responses; and a highly conserved antigenic N protein found within the viral particle, which here has an intracellular compartmentalization targeting sequence for enhanced antigen presentation. This strategy is safe and robust in inducing humoral and T cell responses against SARS-CoV-2. The addition of N addresses the risk of loss of vaccine efficacy against a monovalent S-only vaccine due to the emergence of S mutations over time in the population. In contrast, N is highly conserved, with a lower risk of mutation, while being highly immunogenic. It is a known target antigen of innate immunity, and antibodies and T cells against N are found in the majority of people recovered from SARS-CoV-2 and similar virus SARS-CoV infections. In this vaccine, an enhanced T cell stimulation domain (ETSD) directs the N protein to the endosome-lysosome intracellular compartment after translation, supporting MHC class II presentation for T helper cell activation and promoting 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, displayed as a trimer on the virus surface (Figure 44, panel B), has a receptor-binding domain (RBD) that interacts with the host angiotensin-converting enzyme 2 (ACE2) to facilitate entry and replication in host cells, and thus antibodies against S are key to neutralizing infection. Antibodies against the S RBD are commonly found in patients recovered from COVID19 51, as are antibodies against other S epitopes.In the bivalent vaccine construct, the S-fusion optimizes S by addition of a fusion linker and displays the S RBD in a physiologically relevant conformation on the cell surface, with the aim of improving the generation of anti-S RBD antibodies that are virus neutralizing.

[0156] The bivalent hAd5 S-fusion + N-ETSD vaccine of the present disclosure elicits an excellent T cell response. Vaccines currently in clinical trials focus on generating a humoral response as a means of neutralizing infection. However, considering that the antibody may decline over time even if well generated and fully neutralizing, the T cell response becomes important. In the absence of a T cell response due to viral-induced lymphopenia, infected individuals are at risk of developing acute symptoms of the disease even in the presence of a large amount of neutralizing antibody. Although it cannot be excluded that S (and other viral proteins) can induce a T cell response, the evidence in the literature supports an important role for N. T cell responses against N have been found not only in the majority of patients recovered from COVID-19, but these responses against N in patients exposed to the highly similar virus SARS-CoV are remarkably durable. Strong evidence for the importance of N in natural T cells can be found in a recent report by Ferretti et al., who used unbiased genome-wide screening for the exact peptide sequences recognized by memory CD8+ T cells in COVID-19 patients and found that only 3 out of 29 shared epitopes are from the spike protein, whereas the highest density of epitopes is located in the nucleocapsid protein. Thus, in the hAd5 S-fusion + N-ETSD vaccine, N is expected to not only induce a humoral response but also induce a T cell response that better recapitulates the innate immunity that limits the disease.

[0157] In the initial report, the hAd5 S-fusion + N-ETSD vaccine provided enhanced cell surface expression of the S RBD that is readily recognized by ACE2, reflecting its conformational integrity. N-ETSD, which has endosome / lysosome localization for enhanced antigen presentation, generated both neutralizing antibodies and a Th1-dominant CD4+ / CD8+ T cell-mediated response in vaccinated mice. The present disclosure expands on these findings by confirming native S antigen expression using plasma and monocyte-derived dendritic cells (MoDCs) from previously SARS-CoV-2-infected patients. The inventors elucidated N-ETSD localization in antigen-presenting MoDCs and showed that it localizes to endosomes, lysosomes, and autophagosomes. Protein processing via this intracellular pathway enhances MHC class II presentation, increases peptide recycling, and enables MHC class I presentation as well. By localizing the nucleocapsid protein to the late lysosome-autophagosome compartment, both CD4+ and CD8+ SARS-CoV-2-specific memory T cells are recalled from patients previously infected with SARS-CoV-2. Furthermore, in these immune response recall assays, in vitro, hAd5-infected MoDCs presenting S-fusion and N-ETSD induce a dominant Th1 response from autologous memory T cells of previously SARS-CoV-2-infected patients. N in particular drives the CD8+ T cell response in in vitro recall assays. The recapitulation of natural infection and immunity to the extent achievable by vaccination with the hAd5 S-fusion + N-ETSD vaccine makes it a leading candidate for clinical trials of its ability to protect individuals from SARS-CoV-2 infection and COVID-19, and this second-generation vaccine construct is currently in a Phase I clinical trial.

[0158] Example 18: hAd5[E1-, E2b-, E3-] Platform and Construct For the tests here, virus vaccine candidate constructs were generated using the next-generation hAd5[E1-, E2b-, E3-] vector (Figure 44, panel A). A variety of 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 fusion: S optimized to enhance surface expression and presentation of the RBD; Figure 44, panel E: N (N without ETSD): nucleocapsid (wild-type) sequence with a tag for immunodetection but without ETSD modification and with mainly cytoplasmic localization. Figure 44, panel F. N with enhanced T cell stimulating domain (N-ETSD): nucleocapsid (wild-type) with ETSD to direct lysosome / endosome localization and a tag for immunodetection; and Figure 44, panel G: bivalent hAd5 S-fusion + N-ETSD vaccine.

[0159] Nucleocapsid antigens engineered with an enhanced T cell stimulating domain (ETSD) direct N to endosomes, lysosomes, and autophagosomes in MoDCs and drive enhanced CD4+ T cell activation:

[0160] The hAd5 bivalent vaccine construct contains sequences designed to target N to the MHC class II antigen-loading compartment. To further investigate factors affecting 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 that somewhat overlapped 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 co-localization with autophagosome markers (Figure 45, panels M–O). Protein processing in autophagosomes plays an important role in MHC-mediated antigen presentation in DCs and provides a potential mechanism for enhanced CD4+ T cells induced by N-ETSD in vaccine constructs. Evidence of this T cell interaction with MoDCs infected with N-ETSD translocated to autophagosomes (and endosomes and lysosomes are also assumed) 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 distinguishable 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] Verification of SARS-CoV-2 antibody and cell-mediated immune responses from previously SARS-CoV-2-infected and virus-naïve patients for memory T cell recall assays:

[0162] For the tests described below, plasma samples were collected from four individuals who had recovered from SARS-CoV-2 infection, as confirmed by antibody assays and the patient's medical history as described below. The presence of anti-spike IgG, as well as neutralizing antibodies, by both cPass 66 and live virus assays, was confirmed in all patient samples. Samples were also collected from four virus-naïve individuals and used as controls. In additional tests to verify the immune response to SARS-CoV-2 antigens, the binding of plasma from previously SARS-CoV-2-infected patients and virus-naïve control individuals to human embryonic kidney (HEK) 293T cells transfected with either the hAd5 S-fusion alone or the hAd5 S-fusion + N-ETSD was evaluated. This binding reflects the presence of antibodies in the plasma that recognize the antigen expressed by the hAd5-vectored vaccine. Quantification of the histograms showed little or no binding of virus-naïve plasma antibodies to cells expressing either construct, and the highest binding of plasma antibodies from previously SARS-CoV-2-infected patients to cells expressing the bivalent S-fusion + N-ETSD construct. This is due to either enhanced surface expression of S found in hAd5 S-fusion + N-ETSD-infected HEK 293T cells compared to hAd5 S-fusion alone, or the expression of both S and N antigens.

[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 tests here focus on the response of T cells from patients previously infected with SARS-CoV-2 to autologous MoDCs infected with the hAd5 vaccine construct. DCs are powerful antigen-presenting cells for processing and presenting complex antigens acquired through infection or phagocytosis to induce T cell responses. Thus, in addition to evaluating the binding of patient plasma antibodies to the hAd5 vaccine expressed by 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 after 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 the in vitro system of human MoDCs and plasma is not only an important confirmation of the results of tests using HEK 293T cells and commercially available anti-RBD antibodies, but also indicates a potential method for screening plasma for SARS-CoV-2 antigen 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 the S1, S2, and N CoRSSAN-2 peptide pools, compared to T cells from virus-naive controls. To demonstrate the reactivity of T cells from four previously infected SARS-CoV-2 patients and 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 sequences of the N and S proteins. T cells from previously infected SARS-CoV-2 patients secreted IFNg in response to the SARS-CoV-2 antigen, while 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 SARS-CoV-2 patients recognize S and N peptide pool antigens, while CD8+ T cells show greater recognition of the N peptide antigen. CD4+ T cells from two patient samples tested responded to both the S and N peptide pools and had a higher response to N by Pt3 (Figure 48, panel B). In contrast, CD8+ T cells from both patients responded highly significantly to N but not to the 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: Endo / Lysosome-Targeted Nucleocapsid - Autologous MoDCs infected with ETSD induce higher levels of IFN-γ secretion from CD4+ and CD8+ T cells from previously infected SARS-CoV-2 patients compared to the cytoplasmic nucleocapsid protein (hAd5 N). To evaluate the immunogenicity of endo / lysosome-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 comparing N-ETSD and N (Figure 49, panels C and D). For all patients, interleukin-4 (IL-4) secreted by CD3+ T cells was relatively low (Figure 49, panels E–G). Both CD4+ and CD8+ selected T cell populations showed significantly greater IFN-γ responses to N-ETSD than null (Figure 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 dominant Th1 cytokine response to N / N-ETSD. The cell surface expression of NETSD and N was equivalent in 293T HEK cells, suggesting that the reason for the improved T cell response to N-ETSD is likely epitope processing and MHC loading.

[0167] Example 23: Th1-dominant SARS-CoV-2-specific CD4+ and CD8+ memory T cell recall to 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 inducing the patient's T cell cytokine response. For total T cells (CD3+), the IFN-γ response was similar for S-fusion + N-ETSD and N-ETSD, and the response to S-fusion was 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-fusion, the increased T cell response can be explained by either an increase in S or T cells that recognize the presence of N. Importantly, these T cell responses were characterized by the 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). In some individuals, the response to a particular construct was high, but 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 significantly higher levels 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 show that T cells from previously infected SARS-CoV-2 patients are reactive and have immune memory recall to both vaccine antigens (S and N) in the vaccine vector.

[0168] One feature of the S-fusion is the high expression of the S RBD compared to S-WT. This was the goal of vaccine design based on findings from initial studies of the S cryo-electron microscopy structure suggesting that the RBD epitope is largely inaccessible for immunodetection. A further advantage results from combining S with N in a bivalent vaccine, through the ability of N to enhance the immunodetection of S, a phenomenon generally observed by others with respect to gene expression. The N protein expressed by the vaccine is transported to the endosome / lysosome intracellular compartment, an important antigen presentation pathway for stimulating CD4+ T cells, and as a result, they can license dendritic cells to activate naive CD8+ CTLs. N-ETSD localizes to endosomes and lysosomes, as well as autophagosomes, in MoDCs. Targeting both endosomes and lysosomes is desirable for enhancing antigen presentation and CD4+ T cell activation. Lysosomes can fuse with 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 recognized N-ETSD more readily than N. The data presented herein strongly support the potential for enhanced efficacy of vaccine constructs that specifically express modified N-ETSD.

[0169] T cells are important for the elimination of SARS-CoV infection. 36,74-78 Here, hAd5 expressed S and N and induced strong antigen-specific IFN-γ, but substantially did not induce IL-4 secretion from T cells of previously infected SARS-CoV-2 patients, indicating a Th1 predominance. The antiviral Th1 cytokine response eliminates diverse viruses, including viruses closely related to SARS-CoV-2 and SARS-CoV, from infected hosts. 79,80 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 assist both antibody production from B cell and CD8+ T cell memory, and that these function together 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 not only protects non-infected patients, but also has the potential to be used as a therapeutic for already infected patients by activating T cells to kill virus-infected cells, thereby inducing rapid clearance of the virus and reducing virus replication and lateral spread. Importantly, T cell recall of N-ETSD was shown to be Th1-predominant, as indicated by a vigorous interferon g response and a low IL-4 response.

[0170] The methods and constructs used above Example 25: hAd5[E1-, E2b-, E3-] platform and constructs For the tests described herein, virus vaccine candidate constructs were generated using a second-generation hAd5[E1-, E2b-, E3-] vector (Figure 44, panel A). The hAd5[E1-, E2b-, E3-] backbone containing the SARSCoV-2 antigen expression insert and virus particles was generated as previously described. Briefly, high-titer adenovirus stocks were generated by serial propagation in the E1- and E2b-expressing E.C7 packaging cell line, followed by CsCl2 purification and dialysis into storage buffer (2.5% glycerol, 20 mM Tris pH 8, 25 mM NaCl) from ViraQuest Inc. (North Liberty, IA). Virus particle numbers were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280 nm. Virus titers were determined using the Adeno-X (trademark) 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-fusion: S optimized to enhance surface expression and presentation of the RBD; iii. N: nucleocapsid (N) wild-type sequence protein containing a tag for immunodetection; iv. N-ETSD: N with an enhanced T cell stimulation domain (ETSD) together with a tag for immunodetection; and v. bivalent S-fusion + N-ETSD;

[0171] Collection of plasma and peripheral blood mononuclear cells from patients with a previous confirmed SARS-CoV-2 infection and naïve volunteers: Blood was collected via venipuncture with informed consent from volunteers who had not been exposed to SARS-CoV-2 (UNEX) as confirmed by ELISA, and volunteers confirmed by multiple negative SARS-CoV-2 tests, or volunteers who had recovered from COVID-19 as indicated by recent medical history and positive SARS-CoV-2 antibody tests (patients, Pt). A third source of whole blood was apheresis 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 86, GM-CSF (200 U / ml), and IL-4 (100 U / ml) as described above. Briefly, monocytes were enriched by adhesion on plastic while non-adherent cells were saved and frozen as a source of lymphocytes, particularly T cells. Adherent cells were differentiated into dendritic cells (3 - 5 days in RPMI containing 10% FBS) and then frozen in liquid nitrogen for later use. T cells were enriched from the non-adherent fraction of 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 evaluated 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 4Plated using cells / wells and transduced at an MOI of 5000 1 hour after plating with hAd5 N-ETSD or hAd5 N. The slides were incubated overnight at 37 °C, fixed in 4% paraformaldehyde for 15 minutes, and then permeabilized in 1% Triton X100 (in PBS) for 15 minutes at room temperature. To label N, the cells were then incubated at 1:1000 in phosphate-buffered saline (PBS) containing 3% BSA, 0.5% Triton X100 and 0.01% saponin overnight at 4 °C with an anti-Flag monoclonal (anti-Flag M2 produced in mouse) antibody, followed by washing 3 times in PBS and incubating at 1:500 for 1 hour with a goat anti-mouse IgG(H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 555 (Life Technologies). For the co-localization assay, the cells were also incubated overnight at 4 °C with a rabbit anti-CD71 (transferrin receptor, recycling / sorting endosome marker) antibody (ThermoFisher) at 1:200, a sheep anti-Lamp1 Alexa Fluor 488 conjugated (lysosome marker) antibody (R&D systems) at 1:10, or a 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, the cells were washed 2 times in PBS and 3 times in PBS containing 3% BSA and, where applicable, incubated at 1:500 (goat anti-rabbit IgG(H+L) secondary antibody, Alexa Fluor 488; 1:500 dilution) for 1 hour at room temperature with a fluorescent-conjugated secondary antibody. After a short wash, the cells were mounted with Vectashield Antifade mounting medium containing DAPI (Fisher Scientific) and imaged immediately 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 differentiation of MoDCs from peripheral blood mononuclear cells (PBMCs) to DCs, infection of MoDCs, incubation with plasma from previously infected patients, and detection of binding to infected and non-infected MoDCs by flow cytometry.

[0175] MoDCs were infected at an MOI of 5000 (0.5 x 106 / well in a 12-well plate) with hAd5 S-WT, S-fusion, S-fusion + N-ETSD, or a "null" construct expressing green fluorescent protein (GFP). One day after infection, MoDCs were detached using EDTA (0.5 mM), gently pipetted, and transferred for incubation (4°C) for 30 minutes with plasma from a previously infected patient diluted 1:100 from a single patient (Pt4). Plasma antibodies were detected on the MoDC surface with goat anti-human IgG (phycoerythrin-conjugated). For flow cytometry analysis, cells were acquired as described above. Data were graphed as DMFI, i.e., the difference in binding between infected and non-infected MoDCs.

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

[0177] The ability of T cells from previously infected patients used in these tests to recognize SARS-CoV-2 antigens in vitro was verified, and then a similar analysis was performed for selected CD4+ and CD8+ T cells. Briefly, MoDCs (2 x 10 4) were pulsed with SARS-CoV-2 peptide antigens (1 μg / ml, PepMix S containing S1 and S2 pools PM-WCPV-S-1; and N PM-WCPVNCAP-1, both from JPT Peptide Technologies), and then autologous T cells (1×10 5 ) concentrated from the non-adherent fraction of PBMCs using MojoSort (BioLegend CD3 enrichment) were added to enriched 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 with a secondary antibody against the cytokine conjugated to biotin. The biotin-conjugated secondary antibody was detected using streptavidin / horseradish peroxidase conjugate. The number of spots per well (1×10 5 cells) was 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 the same as those for IFN-γ, but with alkaline phosphatase detection instead of peroxidase.

[0178] Determination of T cell reactivity from previously SARS-CoV-2-infected patients against autologous hAd5-vaccinated 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 formulations In addition to viral constructs that express N-ETSD and / or S-fusion (or proteins having similarity to N-ETSD and / or S-fusion), N-ETSD and / or S-fusion (or proteins having similarity to N-ETSD and / or S-fusion) can also be expressed in yeast, either on yeast cells presenting an 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 conceptual diagram of an ideal vaccine that is durable across multiple pathways and induces effective immunity. As can be readily understood, the vaccine composition preferably (but not necessarily) consists of a yeast vaccine composition and a viral vaccine composition, and optionally, is composed in combination with a macrophage polarizing agent such as RP182 and an immunostimulatory cytokine or cytokine analog (e.g., N-803).

[0181] Regarding the yeast component, the yeast is most typically preferably a recombinant yeast that expresses one or more recombinant nucleic acid target antigens, and perhaps additional DAMPs or PAMPs or STING pathway signals. The recombinant yeast so produced is typically heat-inactivated, and after heat-inactivation, the yeast is lysed. Most preferably, the yeast is lysed using pressure homogenization, and then the homogenate is preferably clarified by filtration. However, it should be understood that other lysis methods, including enzymatic or chemical lysis of the cell wall, sonication, flash freezing / thawing, etc., are also considered appropriate. Similarly, clarification of the lysate can also include centrifugation, sedimentation, aggregation, etc.

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

[0183] Using a mouse model, lysed tarmogen (yeast having a recombinant expressed antigen) was up to 150-fold more immunogenic than intact tarmogen with respect to induction of a T cell response (based on multiple antigen-specific T cell activation assays in hundreds of mice). Advantageously, the lysed yeast has a favorable safety profile and can be prepared with a single small-scale pressure homogenizer at a dose of 10,000 10 YU in 2 hours. Intact yeast induces Th1-Th17 and elicits IFNγ, IL2, IL12, IL6, TNFα, GM-CSF, whereas yeast lysates clearly induced a strong Th1 effect and reduced Th17. Thus, intact or lysed yeast induces coronavirus-specific antibodies (see, for example, doi.org / 10.1016 / j.jaut.2005.01.008, doi.org / 10.1155 / 2016 / 4131324, or sfamjournals.onlinelibrary.wiley.com / doi / pdf / 10.1111 / lam.12188).

[0184] Formulations for oral / mucosal administration In further embodiments, compositions of a vaccine composition and methods for manufacturing a vaccine composition are disclosed herein that use aragonite to form a solid dosage form (e.g., powder, tablet, or capsule) that is stable during storage, is easily administered (e.g., orally), and dissolves (e.g., releases 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 from a plurality of aragonite particles loaded with a vaccine active ingredient that provides a solid dosage vaccine in the form of a powder, tablet, or capsule. More specifically, the vaccine composition can include a recombinant expression construct in powder form (e.g., lyophilized) that is blended with the plurality of aragonite particles and thereby loaded onto its surface to express an antigen corresponding to the relevant infection / disease. In an exemplary embodiment, the vaccine composition immunizes against coronaviruses. Preferably, the recombinant expression construct is an adenovirus construct that expresses at least one antigenic coronavirus protein or protein fragment.

[0186] In particular, the use of aragonite in currently contemplated solid dosage forms enables cost - effective manufacture and easy administration of a stable vaccine composition. Thus, currently contemplated vaccine tablets or capsules can be mass - produced and easily transported. Further, the solid dosage form enables oral administration that can be self - administered by most people without the need for medical personnel. The tablet form can also be made using additional excipients and / or additives (e.g., flavors and gelatin) to form lozenges.

[0187] Aragonite (e.g., oolith aragonite) is one of the purest forms of naturally precipitated calcium carbonate. Referring to FIG. 1, aragonite has an orthorhombic, dipyramidal, characteristic acicular crystal form and is thus distinguishable from calcite and vaterite. Aragonite can be processed to recrystallize and / or reform into various shapes so that it can be used for various purposes utilizing the mechanical and chemical properties of calcium carbonate minerals. Aragonite particles as disclosed herein are solid substances having regular (e.g., spherical or oval) or irregular shapes. As used herein, aragonite particles have an average particle size of 100 nm to 1 mm. A method for grinding aragonite particles is described in U.S. Patent Application Publication No. 2020 / 0308015, the entire content of which is incorporated herein by reference. For example, a method for grinding aragonite particles to a size of 2.0 to 3.5 microns with a clean top size is disclosed. A clean top size means that when manufactured using the disclosed grinding method with a classifier set to a size range of 2.0 to 3.5 microns or 2.5 to 3.5 microns, very few particles are larger than 3.5 microns. Thus, aragonite particles as disclosed herein using the method of U.S. Patent Application Publication No. 2020 / 0308015 have a cleaner 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 onto the surface of aragonite particles under given conditions. In particular, the zeta potential of aragonite increases the stability of surfactants such as glycerol and sorbitol.

[0189] Furthermore, aragonite has a large number of measurable pores in nature in particles having a diameter of less than 2 nm (i.e., high "microporosity"). See, for example, European Patent No. 2719373. Thus, the aragonite platform enables the active ingredient particles to be strongly held together and the filled 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, and an electronic charge (zeta potential), and has already created a nitrogenous pairing with amino acids and proteins. Without being bound by any one theory, these advantageous properties of aragonite make aragonite metastable under ambient conditions. More specifically, aragonite particles contain a natural amino acid content of about 2 - 3%, most of which is aspartic acid (about 25 - 30%) and glutamic acid (about 8 - 10%) that make the aragonite surface hydrophilic. See, for example, Mitterer, 1972, Geochimic et Cosmochimica Acta, 36:1407 - 1422. Thus, in some embodiments, the vaccine composition (e.g., recombinant adenovirus) is directly bound to the natural untreated surface of the aragonite particles.

[0191] Currently, calcium carbonate used in the market is processed from, or as, ground calcium carbonate (GCC), precipitated calcium carbonate (PCC) (synthetic), and / or limestone products. The products produced are of commercial grades with different attributes. To obtain a clean particle size distribution (PSD) top size and low hold-up, most companies utilize a wet grinding process with either high solid or low solid. As used herein, aragonite refers to natural aragonite, which is distinct from GCC, PCC, and limestone, and has a crystal form of orthorhombic, dipyramid, and characteristic acicular crystals. For example, aragonite ball milled using the systems and methods disclosed in U.S. Patent Application Publication No. 2020 / 0308015 can produce aragonite particles with a clean top size in the range of 2.0 to 3.5 microns. A clean top size means that when manufactured using this system and method with a classifier set in the range of 2. to 3.5 microns or 2.0 to 3.5 microns, very few particles are larger than 3.5 microns. For example, for aragonite produced in this set range using the disclosed system, compared to a GCC product with the same median (D50) particle size distribution (PSD), only <0.0005% is retained on 325 mesh, and only <0.0007% is retained on 500 mesh. Therefore, aragonite produced using the contemplated system and method has a cleaner top size than conventional GCC.

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

[0193] In an exemplary embodiment, the aragonite vaccine composition comprises: i) an inner core made from 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 contacts only the outer core and the surface of the inner core is not exposed; and iii) a coating that covers the entire outer surface of the outer core. Preferably, the inner core is made from aragonite particles having a diameter of at least 2 μm or more, thereby providing a surface area for capturing and loading the specific antigenic molecule thereon. Preferably, the outer core contains no antigenic molecules and is made from 90% to 100% aragonite. More preferably, the outer core contains only 100% aragonite. The outer coating of the solid composition can be any suitable coating (e.g., enteric coating) that is stable in the highly acidic, low pH environment of the stomach (e.g., pH of about 3) and dissolves at the higher pH of the small intestine (e.g., pH of about 7 - 9). Suitable examples of enteric coatings include bio-polymer dispersions such as methacrylic acid, ethyl acrylate, and / or a plasticizer / stabilizer (e.g., triethyl citrate (TEC)). Additionally, an anti-tacking agent can also be combined with the enteric coating (e.g., glycerol monostearate).

[0194] In certain embodiments, the inner core is made from aragonite particles having a diameter of at least 2 μm or more (e.g., 2 - 3.5 μm) blended with a lyophilized powder of the antigenic molecule. In some embodiments, additional excipients are blended with the aragonite and the antigenic molecule. For example, dimethylglycine and / or methylsulfonylmethane (MSM) can be combined with the lyophilized powder of the antigenic molecule and 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 an optimized protein or a fragment thereof of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or CoV2). For example, the contemplated solid dosage form vaccine composition disclosed herein may encode at least one antigen of the nucleocapsid (N) protein and the spike (S) protein of the 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 of CoV2 or a fragment thereof, and / or the recombinant encoding the spike protein of CoV2 or a fragment thereof. The vaccine formulation may be useful for treating diseases such as coronavirus-mediated diseases or infectious diseases. Thus, in another embodiment, a method for treating coronavirus disease in a patient in need thereof is disclosed, the method comprising administering to the subject a solid dosage form vaccine composition comprising a recombinant comprising a nucleic acid encoding at least the CoV2 N protein or a fragment thereof, preferably encoding both the CoV2 N protein or a fragment thereof and the CoV2 S protein or a fragment thereof. The coronaviruses contemplated herein can be Coronavirus Disease 2019 (COVID-19) and / or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0196] In a further exemplary embodiment, the contemplated solid dosage form vaccine composition disclosed herein comprises an aragonite blended with a lyophilized recombinant produced with a bivalent human adenovirus serotype 5 (hAd5) expression vector. hAd5 can induce immunity in patients with existing adenovirus immunity and expresses an antigen for producing antibodies that target the coronavirus 2 spike (S) protein and / or nucleocapsid (N) protein. For example, hAd5 CoV2 can encode a modified nucleocapsid protein having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In a more specific embodiment, hAd5 comprises both an S sequence (S-fusion) optimized for cell surface expression and a conserved nucleocapsid (N) antigen designed to be transported to the endosomal intracellular compartment, which may result in a durable immune defense against CoV2, as disclosed in U.S. Patent Application No. 16 / 883,263, the entire content of which is incorporated herein by reference. For example, hAd5 CoV2 can encode an S-fusion or S-HA protein having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4 or SEQ ID NO: 3, respectively.

[0197] Advantageously, the bivalent hAD5 vaccine provides the following: (i) an optimized S-fusion having improved S-receptor binding domain (RBD) cell surface expression compared to S-WT (wild type) with little 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 stimulating domain (ETSD) was localized to the endosome / lysosome intracellular compartment for MHC I / II presentation; and (iv) these optimizations to S and N (S-fusion and N-ETSD) resulted in 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 having E1, E2b, and E3 gene region deletions, together with a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid (N) protein fused to an endosome targeting sequence (N-ETSD), and a nucleic acid encoding a CoV2 spike (S) protein sequence optimized for cell surface expression (S-fusion). Typically, the nucleic acid encoding the CoV2 N-ETSD protein in the hAd5 adenovirus 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 acids encoding the CoV2 S-HA or S-fusion proteins in the hAd5 adenovirus each have 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 proteins encoded in the hAd5 adenovirus have 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, respectively.

[0199] In a further embodiment, the lyophilized bivalent hAd5 vaccine may also contain a nucleic acid encoding a trafficking sequence, a co-stimulatory molecule, and / or an immunostimulatory cytokine. Examples of co-stimulatory molecules encoded 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 containing a recombinant adenovirus genome, optionally containing a deleted or non-functional E2b gene) made from aragonite particles blended with a virus vector in lyophilized form, which contains (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 virus vector may further contain a co-stimulatory molecule. Typically, the nucleic acid encodes 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 contain a sequence portion that enhances the intracellular turnover of the peptide product.

[0201] As discussed, since the aragonite enables direct binding of the lyophilized vaccine active ingredient, it is possible to manufacture and prepare a solid dosage vaccine composition. Thus, the contemplated method for making the disclosed solid dosage form vaccine involves forming an inner core of aragonite and the vaccine active ingredient by loading the vaccine active ingredient (e.g., by mixing) onto the ground aragonite particles, as disclosed herein and in U.S. Patent Application Publication No. 2020 / 0308015, the entire content of which is incorporated herein by reference. Any suitable method for blending the lyophilized vaccine active ingredient with the aragonite particles can be used. Loading or mixing of the ground aragonite particles (e.g., having a D50 PSD of 2 um to 3.5 um) with the lyophilized vaccine active ingredient can be done by any conventional methodology. Mixing of the aragonite and the active ingredient involves mixing the dry weights of both the aragonite and the lyophilized active ingredient in a sealed container suitable for rotation / reversal. For example, the reversal or rotation of the aragonite and the active ingredient can be done for about 5 minutes to 30 minutes. For example, the vaccine composition can be loaded onto the solid dosage form in a mixer (e.g., a tumbling mixer) or blender. The amount of aragonite can vary depending on the amount of the lyophilized active ingredient and the determined titer of the active ingredient for inducing an immune response. For example, the effective dose of the lyophilized bivalent hAd5 vaccine disclosed herein can be about 1×10 9 IU per capsule (or tablet). Thus, a lyophilized bivalent hAd5 vaccine composition having a titer of 2.21×10 7 IU / mg requires about 45.25 mg of the lyophilized bivalent hAd5 powder per capsule. In some embodiments, the weight of the solid single capsule can be about 300 mg to 600 mg. In a particular embodiment, for a 550 mg capsule, about 40 - 60 mg of the 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 7In the case of an active ingredient having a titer of IU / mg, 45.25 mg of the lyophilized active ingredient can be blended with about 504.75 mg of aragonite. Tablet or caplet formation of the mixture can be carried out using any suitable encapsulation method and / or kit known in the art. For example, Capsule Machine Filler (Item# CPM1001 / 2081677).

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

[0203] To deliver an antigenic molecule into the bloodstream in a tablet or capsule for oral administration, the antigenic molecule must remain within the inner core encompassed by the outer core until it passes through the stomach and becomes available for absorption in the intestine (e.g., the 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 an enteric coating are well known in the art. For example, a coating device or apparatus can be used. A specific example of a coating device is 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 photographs (left to right: 1, 2, 3) of the 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), and as shown, having wrinkles, swelling, or holes observed in the capsules: 1) after 2 minutes of HCL acid exposure; 2) after 2 hours of HCL acid exposure; and 3) after 2 hours of HCL acid exposure and drying. Figure 3 shows three photographs (left to right: 1, 2, 3) of hAD5-COVID-S / N in uncoated aragonite capsules (sample #7 or #8) in 0.1 M HCL, and as shown, having swelling, twisting, or holes observed in the capsules: 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 (left to right: 1, 2) of hAD5-COVID-S / N in uncoated lactose capsules (sample #3 or #4) in 0.1 M HCL, and as shown, having swelling of the observed capsules: 1) sample #3 after 2 hours of HCL acid exposure; 2) sample #4 after 2 hours of HCL acid exposure. Figure 5 shows two photographs (left to right: 1, 2) of hAD5-COVID-S / N in coated aragonite capsules (sample #1 or #5) in 0.1 M HCL, and as shown, having swelling of the observed capsules: 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] As shown in FIGS. 6 to 11, the sample capsules were further assayed for infectious units per gram (IFU / gram) and percent virus recovery. More specifically, FIG. 6 shows the infectious units per gram (IFU / gram) (y-axis) for the indicated hAD5-COVID-S / N capsule type as shown. FIG. 7 shows the percent virus recovery (%) (y-axis) for each hAD5-COVID-S / N capsule type as shown. FIG. 8 shows the IFU / gram and the corresponding pH for each hAD5-COVID-S / N capsule type as shown. FIG. 9 shows the percent virus recovery (%) for each hAD5-COVID-S / N capsule type as shown. FIG. 10 shows the percent of virus recovered for each hAD5-COVID-S / N capsule type as shown, with acid treatment shown for those with shading. FIG. 11 shows the infectious units / gram for each hAD5-COVID-S / N capsule type as shown, with acid treatment shown for those with shading. The results are summarized in Table 2 below.

[0206]

Table 1

[0207] In alternative embodiments, the contemplated dosage form includes an inner core of aragonite that has been impregnated (i.e., bound) with carbon dioxide (CO2) prior to addition of the vaccine composition. See, e.g., European Patent No. 2719373 and U.S. Patent Application Publication No. 2020 / 0155458. In further embodiments, the contemplated dosage form includes aragonite having a biocompatible polymer and / or a disintegrant that has been mixed and processed with the aragonite prior to addition of the vaccine composition. Typically, prior to adding the vaccine composition, the aragonite is impregnated with CO2 and mixed with both the biocompatible polymer and the disintegrant. More typically, prior to adding the vaccine composition, the aragonite is impregnated with CO2, mixed with the biocompatible polymer and the disintegrant, and formed (e.g., compressed) into a solid form. See, e.g., European Patent No. 2719373 and U.S. Patent Application Publication No. 2020 / 0155458.

[0208] In further embodiments, as described herein, the aragonite may be combined with carbon dioxide (CO2) and mixed with at least one biocompatible polymer. Typically, the weight ratio of the CO2-bonded aragonite to the biocompatible polymer is from about 95:5 to 5:95. In further embodiments, the biocompatible polymer is a hot melt extrusion 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 copolymer, 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 core and / or outer core of the solid dosage form, as determined by manufacturing and packaging requirements. Examples of additional excipients include ion exchange resins, rubbers, chitin, chitosan, clays, gellan gum, crosslinked polyacrilin copolymers, agar, gelatin, dextrin, acrylic acid polymers, sodium / calcium carboxymethyl cellulose, hydroxypropyl methylcellulose phthalate, shellac or mixtures thereof, lubricants, internal phase lubricants, external phase lubricants, impact modifiers, plasticizers, waxes, stabilizers, pigments, colorants, flavoring agents, taste modifiers, fragrances, sweeteners, mouthfeel improvers, binders, diluents, film formers, adhesives, buffers, adsorbents, odor masking agents and mixtures thereof.

[0210] In alternative embodiments, the surface of the aragonite particles 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 U.S. Patent Application No. 16 / 858,548 and PCT / US20 / 29949. Treatment of aragonite with phosphoric acid forms a lamellar structure for protein loading. Further conjugation techniques for attaching 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 Vaccine Based at least in part on the formulations described above, the present disclosure also provides methods and compositions for administering, monitoring, and assaying vaccines. The contemplated methods include administering a vaccine composition to a patient by inducing immunity against a virus in the patient and by delivering the vaccine composition to the patient by delivery to the nasal mucosa, oral mucosa, and / or alimentary mucosa of the patient. Preferably, the vaccine targets Severe Acute Respiratory Syndrome-like Coronavirus 2 (SARS-CoV-2). The oral vaccine compositions described herein can serve as a booster vaccination for any initial prime vaccination against the SARS-CoV-2 S or N protein.

[0212] The oral vaccine compositions described herein can be used as a booster vaccine for any anti-SARS-CoV-2 vaccine against the SARS-CoV-2 spike (S) and / or nucleocapsid (N) protein. This booster can function even 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 the 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, such as at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 28 days, at least 35 days, or at least 42 days after. The 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 a 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 certain embodiments, the adenovirus comprises a sequence (e.g., SEQ ID NO: 12) 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. In certain embodiments, the adenovirus comprises a sequence (e.g., SEQ ID NO: 13) 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. In certain embodiments, the adenovirus comprises a sequence encoding a soluble ACE2 protein that binds to the immunoglobulin Fc portion to form an ACE2-Fc hybrid construct, which 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), which mutant has at least one mutant amino acid residue (e.g., by substitution) that confers an increased binding affinity of the ACE2 protein to the RBD protein domain of the SARS-CoV-2 spike protein, as disclosed in U.S. Patent No. 63 / 022,146, the entire contents of which are incorporated herein by reference.In another exemplary embodiment, a SARS-CoV-2 vaccine (e.g., an adenovirus 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, a SARS-CoV-2 vaccine comprises a modified yeast cell (e.g., Saccharomyces cerevisiae) genetically engineered to express a coronavirus spike protein on the surface of the yeast cell, as disclosed in U.S. Patent No. 63 / 010,010, whereby the yeast presenting cell stimulates 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 cynomolgus monkeys (5 monkeys per group) were subcutaneously immunized on day 0 with the adenovirus anti-SARS-CoV-2 vaccine described above. Blood was collected from each cynomolgus monkey prior to immunization. On day 14, one group of cynomolgus monkeys (Group 1) received another subcutaneous booster injection of the same vaccine, while another group (Group 2) received the oral vaccine (E1- / E2b-Ad5 having SEQ ID NO: 12 or SEQ ID NO: 13) described herein. On day 28, both groups received an oral vaccine booster dose. Two cynomolgus monkeys (controls) were vaccinated with sham at the indicated time points. Blood was collected on days 14, 21, 28, 35, and 42.

[0215] Next, serum samples collected from these rhesus monkeys at the indicated time points were evaluated by ELISA for anti-spike protein IgG and IgM serum reactivity. 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 by washing each well three times with 150 μL of TPBS solution (PBS + 0.05% Tween 20) for each immunoglobulin type (IgG or IgM). Next, 100 μL / well of blocking solution (2% non-fat milk in TPBS) was added and incubated for 1 hour 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. The plates were washed as described above, and 50 μL / well of each serial dilution was added to the plates and incubated for 1 hour at room temperature. The plates were washed three 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 was added to separate reactions / plates for each immunoglobulin type (IgG or IgM) and incubated for 1 hour at RT. The plates were washed three 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, and then the reaction was stopped by adding 50 μL / well of 1 N sulfuric acid (H2SO4). The optical density at 450 nm was measured using a Synergy 2 plate reader (BioTek Instruments, Inc).Data were analyzed using Prism 8 (GraphPad Software, LLC), and shown in Figure 14 which presents ELISA results detecting IgG serum reactivity against SARS-CoV-2 spike in serum samples collected from immunized rhesus monkeys.

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

[0217] Example 28: Serum Reactivity Serum samples were collected from various volunteers who received various experimental anti-SARS-CoV-2 vaccines and assayed by ELISA for IgG and IgM serum reactivity against the SARS-CoV-2 S protein as described above. The results are shown in Figure 18, and the ELISA results detect IgG and IgM serum reactivity against 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) was immunized by subcutaneous injection of 5×10 10 virus particles (E1− / E2b-Ad5 containing SEQ ID NO: 12 or SEQ ID NO: 13) as described herein. Cohort 2 (10 individuals) was immunized by subcutaneous injection of 10 11 virus particles as described herein. Cohort 3 (15 individuals) was immunized by subcutaneous injection of 10 11 virus particles (or 5×10 10 virus particles if a lower dose with less safety concern was indicated) as described herein. Blood was collected from each volunteer on the same day as the day the first prime vaccination was administered. Blood was collected again on days 8, 15, and 22. A booster injection of the same vaccine was administered on day 22.

[0219] The ELISpot assay was performed on blood samples collected on day 1 and day 15 to evaluate cell-mediated immunity against SARS-CoV-2. 400,000 viable PBMCs (Cellometer K2w / AO / PI viability staining) from each blood collection well were stimulated with empty 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 the ELISpot assay from Th1 N-responsive patients 3, 6, and 11. Figure 20 shows the results from patient 4 (N-non-responsive) and patient 10 (weak Th1 N-responsiveness). None of these patients showed a Th2 response to N.

[0220] Airway protection In further experiments, the intended vaccine formulations and methods of use provided protection of the nasal and lung airways against SARS-CoV-2 challenge in non-human primates. As shown in more detail below, the dual antigen COVID-19 vaccine incorporating the genes for a modified SARS-CoV-2 spike (S-fusion) protein and a viral nucleocapsid (N) protein with an enhanced T cell stimulating domain (N-ETSD) increased 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. Evaluation of the hAd5 S-fusion + N-ETSD vaccine in rhesus monkeys showed that upon oral challenge after subcutaneous priming, humoral and Th1-predominant T cell responses to both S and N were induced and the upper and lower airways were protected from a high-titer (1x10 6 TCID50) SARS-CoV-2 challenge. In particular, viral replication was inhibited within 24 hours of challenge in both the lung and nasal cavity and became undetectable within 7 days after challenge. Figure 25 shows the hAd5 platform and the hAd5 S-fusion + N-ETSD construct. (A) Deletion of the E1, E2b, and E3 regions ([[]] *)Shows the 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 and has a C-terminal SV40 polyA sequence supplied by the hAd5[E1-, E2B-, E3-] platform.

[0221] The dual antigen hAd5 S-fusion + N-ETSD vaccine of Figure 25 expresses the viral spike (S) protein (S-fusion) fused to a signal sequence, which enhances the 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 for similar sequences, and antigens used in most other vaccines have been developed. This vaccine also expresses the viral nucleocapsid (N) protein with an enhanced T cell stimulation domain (N-ETSD) that directs N to the endo / lysosomal intracellular compartment predicted to enhance MHC class II responses.

[0222] The SARS-CoV-2 vaccine antigen is delivered by a recombinant human adenovirus serotype 5 (hAd5)[E1-, E2b-, E3-] vector platform (Figure 25), enabling the rapid generation of vaccines against multiple agents and allowing 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 under 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 an hAd5[E1-, E2b-, E3-] vector expressing the H1N1 hemagglutinin and neuraminidase genes induced both cell-mediated immunity and humoral responses that protected the animals from lethal virus challenge.

[0223] The overwhelming majority of other SARS-CoV-2 vaccines under development target only the wild-type S antigen and are expected to induce a SARS-CoV-2 neutralizing antibody response. Vaccine development prioritizes activated T cells to enhance the duration of respiratory and protective immune responses; in particular, the addition of N was predicted to provide a greater opportunity for T cell responses. T cells can provide immune protection that is at least as important as antibody production. In tests of SARS-CoV-2 convalescent patients, virus-specific T cells were seen in most patients, including asymptomatic individuals, even in patients with undetectable antibody responses.

[0224] In a preliminary test of the hAd5 S-fusion + N-ETSD vaccine in a mouse model, the vaccine not only induces a T helper cell 1 (Th1)-biased antibody response against both S and N, but also activates T cells. MoDCs from SARS CoV-2 convalescent individuals were transduced with the dual antigen vaccine and the S-fusion and N ETSD-expressing MoDCs were incubated with T cells from the same individuals. The vaccine antigen induces the secretion of interferon-γ (IFN-γ) by both CD4+ T cells 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 the vaccine antigen, in turn, suggests that vaccination with the hAd5 S-fusion + N-ETSD vaccine generates T cells that recognize SARS-CoV-2 antigens during viral infection and protect vaccinated individuals from disease.

[0225] The generation of T cell responses can be an important feature for vaccines that are effective against many variants, where their emergence may be at least partially an escape response to antibodies generated by either the first wave of virus (28) or antibody-based vaccines. As reported elsewhere, neutralization by 14 out of 17 of the most potent mRNA vaccine-induced monoclonal antibodies (mAbs) was either reduced or lost for the variants E484K, N501Y or the K417N:E484K:N501Y combination. Also, these variants were found to be 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, when effectively established by vaccination, can provide protection against existing viral strains and avoid mutants.

[0226] In the next step in the development of the 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 test design was to evaluate the efficacy of a subcutaneous (SC) prime followed by a thermally stable oral boost. Oral boosting offers several advantages in SARS-CoV-2 vaccination, including the greater potential to generate mucosal immunity, particularly in the gastrointestinal tract, which is one of the major sites of infection. SARS-CoV-2 is a mucosal virus and is rarely detected in the blood, thus vaccines that specifically target mucosal immunity are of interest. A further compelling advantage of a thermally stable oral boost is its potential to transform the global distribution of the vaccine, particularly in developing countries, where patients may be able to self-administer the boost at home. The hAd5 S-fusion + N-ETSD construct may also function as a "universal" heterologous booster vaccine for a number of SARS-CoV-2 vaccines in development, as it induces both humoral and CMI responses to both antigens.

[0227] In one study, the efficacy of the hAd5 S-fusion + N-ETSD vaccine in rhesus macaques was investigated as either an SC prime and SC and oral boosts (SC-SC-oral; n = 5) or an SC prime and two oral boosts (SC-oral-oral; n = 5) using a regimen of a prime on day 0 and boosts on days 14 and 28, both of which were found to maximize T cell responses. The aim 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. The cell-mediated T cell response after challenge, as well as protection of the nasal cavity and lungs from SARS-CoV-2 infection and the rate of virus 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 nasal cavity and lung after SARS CoV-2 challenge: RT-qPCR analysis of genomic RNA (gRNA) was performed on nasal swab 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 starting on day 57, the first day after challenge (Figure 22, panels A and B). Viral gRNA in this group continued to decline to very low or low levels below the limit of detection (LOD) in all vaccinated animals by day 63, 7 days after challenge. The placebo controls had moderate to high levels of SARS-CoV-2 (range 2E+09 - 8.4E+03 gene copies / mL) present in nasal swab samples throughout the study period.

[0231] In the lungs (bronchoalveolar lavage, BAL) of SC-oral-oral NHPs, gRNA also rapidly declined, with the geometric mean showing a ~2 log reduction in vaccinated NHPs compared to placebo NHPs on day 57, just 1 day after challenge (Figure 22, panels C and D). In the group that received SC and oral boost (SC-SC-oral), SARS-CoV-2 gRNA in nasal swab samples also declined as seen in SC-oral vaccinated primates, and viral gRNA declined to very low or low levels below the LOD in all vaccinated animals by day 63 (Figure 22, panels E and F). In the lungs of SC-SC-oral NHPs, gRNA also showed a ~2 log reduction on day 57 (Figure 22, panels G and H).

[0232] SC prime, oral boost vaccination immediately inhibited viral replication 188 in the nasal cavity and lungs after SARS-CoV-2 challenge: The presence of replicating virus in nasal swab samples was determined by RT qPCR of subgenomic RNA (sgRNA). From 4 days after challenge until day 60, sgRNA was below the limit of detection (LOD) for two SC-oral-oral primates and, starting on day 61, for all primates that received only the oral boost (Figure 23, panels A and B). In the lungs of SC-oral-oral NHPs, sgRNA also decreased compared to placebo starting on day 57 and was below the LOD for all by day 63 (Figure 23, panels C and D). Evidence of replicating virus in the nasal cavity also decreased rapidly in SC-SC-oral NHPs, was below the LOD in two primates by day 59, and below the LOD in all primates by day 63 (Figure 23, panels E and F); in the lungs of this group, sgRNA decreased by approximately 2 logs compared to the placebo control on day 57, 1 day after challenge, and was below the LOD in 4 of 5 primates on day 63 and just above the LOD on day 5 (Figure 23, panels G and H). Not only was there a rapid decrease in both viral load and replicating virus in the nasal cavity and lungs, but it was noted that there was no viral growth after challenge. This implies the presence of existing humoral and cellular immunity that results in rapid clearance of the virus upon infection.

[0233] Immediate protection of NHPs from SARS-Cov-2 challenge may be due to the presence of T cells that respond to both S and N, and rapid viral clearance against the activation of memory B cell peripheral blood mononuclear cell (PBMC)-derived T cell responses to antigens delivered by the hAd5 S fusion + N+ETSD vaccine, spike, and nucleocapsid was determined by ELISpot on day 0, day 14 (before boost), and 1 week after the second boost on day 35. 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 Th1 dominant as reflected by the IFN-γ / IL-4 ratio (Figure 24, panel C).

[0234] For sera collected during the post-challenge period, the SARS-CoV-2 neutralizing capacity reflected by "MN50", i.e., the serum dilution correlated with a 50% reduction in viral infectivity compared to the serum-free control, was evaluated using a micro-neutralization assay (see methods). A rapid increase in the neutralizing capacity of sera from NHPs that received only the oral boost was seen over the 2 weeks post-challenge, which reflected the decrease in nasal gRNA and sgRNA (Figure 24, panels E and F, respectively) and lung gRNA and sgRNA (Figure 24, panels G and H, respectively) (Figure 24, panel D). In particular, sera from placebo group primates did not show an increase in neutralizing capacity post-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 unvaccinated placebo group. Further studies are needed to confirm this hypothesis.

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

[0236] The presence of cytotoxic T cells due to vaccination (Figure 24, Panels A-C and I-K) led to a near immediate reduction of viral replication within the first 24 hours after challenge (Figure 24, Panels F, H, N, and P), and the continuous reduction over two weeks reflecting the increased neutralization capacity of sera from vaccinated but not placebo NHPs reflects 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 cavity and lung airways against SARS-CoV-2 challenge in the rhesus macaque NHP model. The inhibition of viral replication in the nasal cavity as demonstrated by the reduction of sgRNA on the first day after viral challenge was significant, with continuous clearance of virus to levels below detection within 7 days of challenge in all (10 / 10) animals (Figures 22 and 23); and although rhesus macaques are not a model for evaluation of transmission, these rapid reductions in nasal virus replication encourage and support studies of the ability of this vaccine to prevent transmission in future studies.

[0238] The ability of hAd5 S-fusion + N-ETSD vaccination to induce virus-neutralizing anti-S antibodies (Figure 21) and the ability to induce T cells responsive to both S and N (Figure 23), particularly when seen with a rapid increase in the neutralization capacity of post-challenge sera, which is likely to indicate the presence of memory B cells, suggest 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 considering that T cells play a crucial role in protecting COVID-19 convalescent patients from infection, where SARS-CoV-2-specific T cells were identified even in the absence of an antibody response. The apparent almost immediate reduction in viral replication by hAd5 S-fusion + N-ETSD vaccination is in contrast to 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 post-challenge. Even when challenged at a relatively high titer of 1×10 6 281 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 by SC and oral boost administration is particularly resistant to antibodies and convalescent plasma, and highlights the potential for this vaccine to be developed for distribution worldwide, especially in light of escape mutants that are rapidly spreading around the world. The oral hAd5 S-fusion + N-ETSD formulation does not require ultra-low temperature freezing like many of the COVID vaccines currently under development. Dependence on a cold chain for distribution to geographically remote or underdeveloped regions causes shipping and storage challenges and is likely to reduce access to RNA-based COVID-19 vaccines.

[0241] The thermally stable oral hAd5 S-fusion + N-ETSD vaccine may also act as a "universal" boost to other previously administered vaccines that deliver only the S antigen, due to the expression of S and N. This use would 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. The contemplated methods include administering a vaccine composition to a patient by inducing immunity against a virus in the patient and delivering the vaccine composition to the patient by delivery to the nasal mucosa, oral mucosa, and / or gastrointestinal mucosa of the patient. Preferably, the vaccine targets Severe Acute Respiratory Syndrome-like Coronavirus (SARS-CoV-2).

[0243] In particular, the disclosed methods also include obtaining a saliva sample from the patient for a period of time after administering the vaccine. Typically, the saliva sample is stored in a stabilization solution containing glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, or any combination thereof. More typically, the stabilization solution contains 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 that target a virus or a virus-specific protein, and in the absence of antibodies in the sample saliva, the method further includes administering a booster of the vaccine to the patient.

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

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

[0246] In certain embodiments, the plurality of aragonite particle beads are functionalized with a moiety capable of binding to anti-SARS-CoV-2 comprising recombinant ACE2 protein. For example, the moiety capable of binding to anti-SARS-CoV-2 can be selected from recombinant ACE2 protein having at least 85% sequence identity to SEQ ID NO: 9, recombinant α-helix ACE2 protein of SEQ ID NO: 10, or recombinant α-helix 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 / D355L.

[0247] The contemplated subject matter includes methods for administering a vaccine to a patient by two or more routes of administration to induce both local and systemic immune responses to the vaccine. The 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's 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 affect whether the patient's immune response is strong enough to establish protection.

[0249] For emerging viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (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 not yet been tested across diverse populations. Given the current SARS-CoV-2 pandemic and the high transmission rate of the SARS-CoV-2 virus, effective tests for robust vaccination protocols and immunity to the virus or the virus (e.g., the presence of anti-SARS-CoV-2 antibodies) are needed.

[0250] The contemplated methods disclosed herein for inducing immunity in a patient include administering the vaccine by at least oral administration, preferably by oral administration and by injection into the blood supply. Many vaccines are given via the intramuscular (IM) route, using direct delivery of the vaccine to the intramuscular blood supply to optimize immunogenicity and induce systemic immunity. 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, induction of mucosal immunity has been reported to be essential to prevent human-to-human transmission of pathogenic microorganisms and limit their growth within mucosal tissues. Furthermore, for protective immunity against mucosal pathogens (e.g., SARS coronavirus), instead of a more general approach of tolerance to maintain mucosal homeostasis, immune activation in mucosal tissues enables enhanced mucosal immune responses and better local defense. For example, nasal vaccination (delivery of vaccine by nasal administration) induces both mucosal and systemic immunity (see, e.g., Fujkuyama 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 a vaccine to the patient, at least by administration to the nasal mucosa, oral mucosa, and / or gastrointestinal mucosa of the patient. In some embodiments, the route of administration includes administering the vaccine to the nasal mucosa, oral mucosa, and / or gastrointestinal mucosa of the patient, 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 administering 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 tissue of a patient is a SARS-CoV-2 vaccine. In an exemplary embodiment, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a soluble ACE2 protein bound to an immunoglobulin Fc portion that forms an ACE2-Fc hybrid construct that may also include a J chain portion, as disclosed in U.S. Patent 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) includes a mutant of a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10), the mutant having at least one mutant amino acid residue (e.g., by substitution) that confers an increased binding affinity of the ACE2 protein for the RBD protein domain of the SARS-CoV-2 spike protein, 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. In another exemplary embodiment, the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a CoV2 nucleocapsid protein or a CoV2 spike protein fused to an endosome targeting sequence (N-ETSD), as disclosed in U.S. Patent 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 includes a modified yeast cell (e.g., Saccharomyces cerevisiae) genetically engineered to express a coronavirus spike protein on the surface of the yeast cell, such that the yeast presenting cell stimulates B cells (e.g., humoral immunity), as disclosed in U.S. Patent No. 63 / 010,010.

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

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

[0256] To assay samples from patients who have received a vaccine against a pathogenic infection (e.g., targeting SARS-CoV-2) and / or who 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 enables the detection of antibodies in the presence of an antigen. Diagnostic tests using a cell viability assay for the detection of anti-SARS-CoV-2 antibodies are disclosed in U.S. Patent No. 62 / 053,691, the entire contents of which are incorporated herein by reference. The cell diagnostic assay relies 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 derivatives thereof, etc.) 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 (human receptor targeted by the SARS-CoV-2 spike protein) are disclosed in U.S. Patent Application No. 16 / 880,804, the entire content of which is incorporated herein by reference.

[0258] To more easily monitor patients for the presence of anti-pathogen antibodies, assaying saliva samples from patients enables rapid sample collection, increased patient participation, and may enable 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 against SARS-CoV-2, it may be necessary to stabilize proteins in saliva against degradation during transport and storage after pre-test sample collection.

[0259] Once a saliva sample is collected, the saliva is placed in a preservation solution to stabilize 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,” U.S. Department of Justice grant # 2010-DN-BX-K193) and U.S. Patent No. 6,133,036. For example, a stabilizing preservation solution for a patient's saliva sample may contain 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 0.1% (w / v) to 2.0% (w / v), such as about 0.2% (w / v), about 0.3% (w / v), about 0.4% (w / v), about 0.5% (w / v), about 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 1.0% (w / v), about 1.1% (w / v), about 1.2% (w / v), about 1.3% (w / v), about 1.4% (w / v), about 1.5% (w / v), about 1.6% (w / v), 1.7% (w / v), about 1.8% (w / v), or about 1.9% (w / v).

[0261] In additional or alternative embodiments, the saliva sample can be mixed with a stabilizing preservative solution of about 0.10% to about 1.00% sodium benzoate (weight / volume of the sample) and / or about 0.025% to about 0.20% citric acid (weight / volume of the sample). For example, the saliva sample can be mixed with 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, or 1.00% w / v sodium benzoate. In further embodiments, the saliva sample is mixed with a stabilizing preservative solution of at least 0.5 mg / mL (e.g., at least 0.6 mg / mL, at least 0.7 mg / mL, at least 0.8 mg / mL, at least 0.9 mg / mL, at least 1 mg / mL, at least 1.5 mg / mL, at least 2 mg / mL, at least 2.5 mg / mL, at least 3 mg / mL, at least 3.5 mg / mL, at least 4 mg / mL, at least 4.5 mg / mL, or even 5 mg / mL) benzoic acid and / or at least 0.2 mg / mL (e.g., at least 0.2 mg / mL, at least 0.25 mg / mL, at least 0.3 mg / mL, at least 0.35 mg / mL, at least 0.40 mg / mL, 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 (e.g., sodium benzoate), and "citric acid" is interchangeable with citrate (e.g., sodium citrate).

[0262] A saliva sample containing the preservative described above is stable for storage at a temperature of 15°C to 40°C 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). Thus, a method for storing a saliva sample for a neutralizing antibody test, the method 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 is disclosed herein. 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 contain citric acid and / or benzoic acid at the concentrations disclosed herein.

[0263] Aragonite: In some embodiments, any antibody protein or any specific antibody protein can be captured from a saliva sample using roe-shaped aragonite particles. For example, the saliva preservation solutions disclosed herein, such as glutaraldehyde, sodium benzoate and citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, and any combination thereof, can also contain roe-shaped aragonite (calcium carbonate, CaCO3) particles. The use of aragonite particles for binding to proteins is disclosed, for example, in U.S. Patent Application No. 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 antibody present in a saliva sample or to those modified to specifically capture antibodies against a particular antigen. For example, aragonite can be functionalized with moieties that can bind to immunoglobulin (Ig) proteins. Preferably, the Ig protein is an immunoglobulin A (IgA), immunoglobulin G (IgG), or immunoglobulin E (IgE) protein. More preferably, aragonite is functionalized to bind to IgA protein. Most preferably, the aragonite particles are functionalized with moieties that can bind to specific antibodies. For example, the aragonite particles can be bound to a moiety specific for an anti-SARS-CoV-2 antibody. Preferably, the aragonite particles are bound to, for example, the recombinant ACE2 protein disclosed in the aforementioned U.S. Patent Application No. 16 / 880,804. In a typical embodiment, the aragonite particles are bound 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) with a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10). To more efficiently capture or bind anti-SARS-CoV-2 antibodies or the spike protein of SARS CoV-2, the recombinant soluble ACE2 can be mutated to form an ACE2 variant having a higher binding affinity for the SARS-CoV-2 spike protein (e.g., the RBD domain of the spike protein). These ACE2 variants of the recombinant soluble ACE2 protein include T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and / or D355L.

[0265] As used herein, the term “functionalized” refers to the coupling or binding of a moiety to the aragonite particles, thereby imparting any function of the attached moiety to the aragonite particles. For example, the aragonite particles can 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 have an average particle size of 100 nm to 1 mm.

[0266] In some embodiments, the protein moiety is directly bound to the native untreated surface of the aragonite particles. The aragonite particles have 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 can be directly bound to the surface of the aragonite particles.

[0267] In alternative embodiments, the aragonite particle surface can be treated to modify the binding surface. For example, as disclosed in U.S. 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 attaching 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] Oral dosages of each vaccine (e.g., the SARS-CoV-2 vaccines disclosed herein) can be sent to patients who do not show a sufficient titer (e.g., presence) of neutralizing antibodies in saliva. The patients inhale or ingest these vaccine dosages and then, two weeks later, send another saliva sample prepared and stored in the same manner as above to a test facility to confirm that the oral vaccine dosage has restored the titer of its anti-SARS-CoV-2 antibodies (e.g., IgA).

[0269] Accordingly, in further embodiments, a kit for collecting a saliva sample from a patient includes a collection container having the saliva preservative solution disclosed herein. For example, the kit includes a collection container containing 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 for securing the collection container having the saliva sample for transport or mailing. In some embodiments, the kit may also include at least one dosage of a vaccine for oral administration.

[0270] Immunostimulation In addition to vaccination, or even as an alternative treatment for subjects at high risk of or actually diagnosed with coronavirus infection, particularly SARS-CoV-2, immune stimulation with one or more immunostimulatory cytokines may prevent or alleviate lymphopenia, which is frequently associated with COVID-19. Among other immunostimulatory cytokines, N-803 is particularly contemplated.

[0271] COVID-19 infection causes lymphopenia, particularly suppression of NK and CD8 T cells, and severe cases and subsequent deaths are associated with this significant decrease in lymphocytes. Evidence also suggests that COVID-19 infection results in macrophage death and a decrease in natural killer (NK) cells and CD8+ T cells. Analysis of data from 1,099 COVID-19 patients confirmed in the laboratories of 552 hospitals across China revealed that lymphopenia was observed in 83.2% of patients at admission. On average, patients with severe disease had more pronounced clinical test abnormalities, such as lower lymphocyte counts, compared to patients with non-severe disease. Similarly, another research group showed that the median lymphocyte count of patients admitted to the ICU was significantly lower compared to patients who did not require ICU care.

[0272] At the cellular level, COVID-19 appears to induce immune evasion by decreasing NK, CD4+ and CD8+ T cells. The mechanism of lymphopenia remains unclear, but the rapid decrease of both CD4 and CD8 T cells may be associated with adverse outcomes. Lymphopenia and increased viral load in the first 10 days of SARS suggest immune evasion by SARS coronavirus. The lack of interferon (IFN)-γ response in SARS-infected cells has been reported in vitro using human primary bone marrow-derived dendritic cells and epithelial 293 cell lines. Other researchers have proposed a mechanism of immune evasion by SARS-CoV in dendritic cells (DC) based on the discovery 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 depletion, lack of T cell reactivity through insufficient T cell diversity may contribute to an insufficient immune response. This occurs particularly in the elderly population.

[0273] N-803 has demonstrated significant ability to induce increased broad T cell reactivity in both preclinical (mouse) and clinical (breast cancer, lung cancer, and pancreatic cancer) settings. By activating T cell diversity, cross-reactivity to past coronavirus infections may provide immunity against COVID-19 after N-803 enhancement of T cell reactivity, making it possible to overcome immune evasion. N-803 significantly promotes the proliferation of NK cells and CD8-T cells and the 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. There is additional preclinical evidence regarding the antiviral effect of N-803, including in mouse and non-human primate (NHP) models.

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

[0275] NK cell and CD8+ T cell activation has been demonstrated in healthy volunteers. In studies of healthy volunteers administered N-803 at SC doses of 10 μg / kg and 20 μg / kg, N-803-treated subjects showed more than 20-fold increases in Ki-67 stained NK and CD8+ T cells. The clinical efficacy of N-803 in stimulating lymphopenia responses has been demonstrated in patients with acute myeloid leukemia (AML), breast cancer, lung cancer, and pancreatic cancer.

[0276] Therefore, preclinical in vitro and in vivo studies, together with clinical data, have demonstrated that N-803 binds to IL-15 receptor-presenting cells with higher affinity, enhances lymphocyte distribution, extends half-life, causes the proliferation and activation of effector NK cells and CD8+ memory T cells, and results in anti-tumor activity. Thus, N-803 can be used as a treatment option to counter COVID-19-related lymphopenia and rescue lymphopenia in both normal healthy subjects and patients with cancer in a similar manner to N-803 that stimulates both NK and CD8 T cells. Therefore, N-803 can be used for the recovery or alleviation of lymphopenia in patients infected with COVID-19 and to improve disease outcome. For example, infected subjects can be treated by receiving a subcutaneous (SC) injection of N-803 in the abdomen on day 1 and, if necessary, on day 15. Most typically, N-803 will be provided in a liquid injectable form at a dose of 10 mcg / kg.

[0277] In some embodiments, the numbers representing characteristics such as amounts of ingredients, concentrations, reaction conditions, etc., which are used to describe and claim particular embodiments of the present invention, are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and the appended claims are approximations that may vary depending upon the desired characteristics sought to be obtained by a particular embodiment. The recitation of a range of values herein is merely intended to serve as a concise method of referring individually 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 the pharmaceutical composition or drug, and direct administration of the pharmaceutical composition or drug is typically performed by a medical professional (e.g., a physician, nurse, etc.), and indirect administration includes the step of providing or making available to a medical professional a pharmaceutical composition or drug for direct administration (e.g., by injection, infusion, oral delivery, topical delivery, etc.). It should be further noted that the term "predicting" or "prediction" of a condition, susceptibility to the onset of a disease, or response to an intended treatment covers the act or prediction (not a treatment or diagnosis) of predicting a condition, susceptibility, and / or response, including the rate of progression, improvement, and / or persistence of the condition in a subject.

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

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

[0281] It will be apparent to those skilled in the art that many more changes other than those already described can be made without departing from the inventive concept of this specification. Accordingly, the subject matter of this invention should not be limited except as by the appended claims. Further, when 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 "comprising" and "comprises" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that other elements, components, or steps not explicitly referenced may be present, utilized, or combined. When the specification or claims refer to at least one selected from the group consisting of A, B, C... and N, the passage should be interpreted as requiring only one element from the group, not A+N, or B+N, etc.

Claims

1. Deletion of the E1 gene, deletion of the E2b gene, and a. A first part encoding a SARS-CoV-2 coronavirus nucleocapsid protein (N)-ETS-D fused to an endosomal target sequence, where N has the amino acid sequence of SEQ ID NO: 7, ETS-D has the amino acid sequence of SEQ ID NO: 8, and is functionally linked to one or more regulatory elements enabling N-ETS-D expression, the first part; b. A second part encoding a SARS-CoV-2 spike protein (S), where S has the amino acid sequence of SEQ ID NO: 4 and is each functionally linked to one or more regulatory elements enabling S expression, the second part; A recombinant replication-deficient adenovirus comprising a recombinant nucleic acid comprising the above.

2. The adenovirus according to claim 1, wherein the endosomal target sequence of the N-ETS-D is encoded at the 5' end of the first part.

3. The adenovirus according to claim 1, wherein the endosomal target sequence of the N-ETS-D is encoded at the 3' end of the first part.

4. The adenovirus according to claim 3, wherein the first and second parts are arranged in a bicistronic sequence.

5. The adenovirus according to claim 3, wherein the N-ETS-D has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

6. The adenovirus according to claim 5, wherein the N-ETS-D has an amino acid sequence having the amino acid sequence of SEQ ID NO:

1.

7. The adenovirus according to claim 5, wherein the first part has the nucleotide sequence of SEQ ID NO:

2.

8. The adenovirus according to claim 5, wherein the second part has the nucleotide sequence of SEQ ID NO:

6.

9. An adenovirus according to any one of claims 1 to 8, further comprising a third portion encoding a co-stimulatory molecule or an immunostimulatory cytokine.

10. The adenovirus according to any one of claims 1 to 9, wherein the recombinant adenovirus has a deletion in the E3 gene region.

11. A vaccine composition comprising aragonite particles mixed with a recombinant replication-deficient adenovirus according to any one of claims 1 to 10, wherein the recombinant replication-deficient adenovirus is lyophilized.

12. The vaccine composition according to claim 11, wherein the aragonite particles have an average particle size of 100 nm to 1 mm.

13. A recombinant replication-deficient adenovirus according to any one of claims 1 to 10 for use in inducing immunity against SARS virus.

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