Replication-Competent Adenovirus Type 4 SARS-COV-2 Vaccines and Their Use

Abstract

A replication-competent adenovirus type 4 (Ad4) modified to express the SARS-CoV-2 spike protein is described. The genome of the recombinant Ad4 is modified to have a deletion of at least a portion of the adenovirus E3 region to accommodate the insertion of the spike protein coding sequence. Administration of the recombinant Ad4 to the upper respiratory tract induces mucosal immunity, which is important for protection against SARS-CoV-2 infection and for preventing the spread of the virus.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 138,221, filed January 15, 2021, which is incorporated by reference in its entirety herein.

[0002] Field The present disclosure relates to a recombinant replication-competent adenovirus type 4 (Ad4) expressing the SARS-CoV-2 spike protein and its use as an immunogenic composition for inhibiting SARS-CoV-2 infection and transmission. [Background technology]

[0003] background Coronaviruses are a large family of viruses that typically cause mild to moderate upper respiratory tract illness; however, some members of this family can cause severe disease and death in humans. In the past two decades, there have been three major coronavirus outbreaks in humans resulting from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, the latter of which first emerged in December 2019 in Wuhan, China. As of January 2021, SARS-CoV-2 had infected over 84 million people worldwide and caused approximately 2 million deaths. Although several SARS-CoV-2 vaccines have been approved for use in the United States and other countries, there remains a need for an effective SARS-CoV-2 vaccine that induces mucosal immunity and can be rapidly produced in large quantities. Summary of the Invention [Means for solving the problem]

[0004] Abstract Disclosed herein is an immunogenic composition comprised of a replication-competent adenovirus type 4 (Ad4) expressing a SARS-CoV-2 spike (S) protein ("Ad4-Spike"), such as a wild-type or modified version of the S protein derived from the original Wuhan strain or from a SARS-CoV-2 variant (e.g., beta (B.1.351), delta (B.1.617.2), gamma (P.1), delta plus, or omicron (B.1.1.529) variant). In the Ad4 vector of the present disclosure, the gene encoding the SARS-CoV-2 S protein is cloned into the E3 region of the Ad4 vaccine strain. At least a portion of the E3 region is deleted to accommodate the insertion of the S protein. The Ad4-Spike vaccine of the present disclosure has several important advantages over other recommended and approved SARS-CoV-2 vaccine platforms. In particular, as a replicating vector, Ad4-Spike can induce long-lasting immune responses, including mucosal immunity, which is an important factor for inhibiting both viral infection and transmission. Furthermore, Ad4-Spike vaccines can be rapidly produced to high titers at a relatively low cost.

[0005] Provided herein is a recombinant replication-competent Ad4 expressing a SARS-CoV-2 S protein. The genome of the recombinant Ad4 comprises a deletion in the adenovirus E3 region and an insertion of the coding sequence of the SARS-CoV-2 S protein. The SARS-CoV-2 S protein can be a native S protein or a modified S protein (e.g., a stabilized or truncated S protein). Additionally, the S protein can be derived from the Wuhan strain of SARS-CoV-2 or a variant thereof (e.g., a variant of concern (VOC)).

[0006] Also provided is a recombinant replicable Ad4 vector having a deletion in the adenovirus E3 region and an insertion of the coding sequence of the SARS-CoV-2 S protein, which can be the native S protein or a modified S protein (e.g., a stabilized or truncated S protein) from either the Wuhan strain or a SARS-CoV-2 mutant strain (e.g., VOC).

[0007] Further provided is an immunogenic composition comprising a recombinant Ad4 or recombinant Ad4 vector disclosed herein and a pharma- ceutically acceptable carrier.

[0008] Also provided are methods of inducing an immune response against SARS-CoV-2 in a subject and methods of immunizing a subject against SARS-CoV-2 infection by administering to the subject a therapeutically effective amount of a recombinant Ad4, recombinant Ad4 vector, or immunogenic composition disclosed herein. In some embodiments, the recombinant Ad4, recombinant Ad4 vector, or immunogenic composition is administered to the upper respiratory tract (e.g., intranasally).

[0009] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0010] [Figure 1]Figure 1: SARS-CoV-2 spike expression of stabilized and truncated designs in transfected A549 cells. A549 cells were transfected with a shuttle vector plasmid containing the gene for the SARS-CoV-2 spike protein from the Wuhan strain (nCoV). Four spike protein constructs were generated: wild type (WT), stabilized (PP), tail-truncated (TT), and endocytosis motif-truncated (noEndo). Controls included untransfected (unTF) cells and cells transfected with a plasmid expressing the HIV-1 envelope (Env) protein (FDE3). Spike and Env expression were measured by flow cytometry using SARS-CoV-2 spike protein-specific and HIV-1 Env-specific antibodies (VRC01), respectively. SARS-CoV-2 spike protein expression in transfected A549 cells was decreased with stabilizing mutations, shortened tails, and truncated endocytic motifs compared to the wild-type spike protein. [Figure 2A]Figures 2A-2B: SARS-CoV-2 spike expression of stabilized and truncated designs in infected A549 cells. Replicating adenoviruses carrying SARS-CoV-2 protein genes were used to infect A549 cells. Three spike protein designs based on the Wuhan strain were tested for expression on the surface of A549 cells: wild-type (nCoV-WT), PP-stabilized (nCoV-PP), and tail-truncated (nCoV-TT) spike proteins. A replicating adenovirus expressing the HIV-1 Env protein (FDE3) was used as a positive control for infection, and uninfected (unIF) cells were used as a negative control. Spike protein expression was measured by flow cytometry using a SARS-CoV-2 spike protein-specific antibody. Antibody VRC01 was used to detect HIV Env expression. Spike expression by nCoV-WT is shown in Figure 2A; spike expression by FDE3, nCoV-PP and nCoV-TT is shown in Figure 2B. As shown in Figures 2A-2B, spike protein expression was high in both nCoV-WT and nCoV-PP constructs. [Figure 2B] Same as above. [Diagram 3] Figure 3: Immunization with replicating Ad4 containing the SARS-CoV-2 spike protein gene induces neutralization in rabbits. New Zealand White rabbits were immunized with 1.29 × 109 infectious units (IFU) of purified replicating Ad4 nCoV-WT on days 0 and 28 (indicated by arrows). Using a luciferase assay, serum neutralization against the Wuhan SARS-CoV-2 pseudovirus was detected starting 4 weeks after immunization (before the second dose) and continued to increase up to 12 weeks after immunization. [Figure 4]Figure 4: Amino acid alignment of nCoV-PP, nCoV-WT, nCoV-tail-truncated, and nCoV-No-Endo spike proteins. The alignment shows the positions of the three mutations introduced into the SARS-Cov-2 wild-type (Wuhan) spike protein. nCoV-PP contains a double proline stabilizing substitution at amino acid positions 986 and 987; nCoV-tail-truncated contains a deletion of the terminal 24 amino acids in the cytoplasmic tail; nCoV-No-Endo contains a deletion of the terminal endocytosis signaling motif (terminal 5 residues). Amino acid numbering refers to the wild-type spike protein shown herein as SEQ ID NO:2. [Diagram 5] Figures 5A-5B: Serum neutralization against Wuhan pseudovirus at escalating doses of intranasal Ad4-SARS-CoV-2WuPP in hamsters. Syrian golden hamsters were intranasally administered 102-107 infection-forming units (IFU) of Ad4-SARS-CoV-2 Wuhan spike with PP stabilization (Ad4-SARS-CoV-2WuPP). Serum neutralization against Wuhan pseudovirus was measured at weeks 4 (Figure 5A) and 8 (Figure 5B). Robust neutralization was observed at both time points for the highest dose of Ad4-SARS-CoV-2WuPP. [Figure 6A-B]Figures 6A-6E: Seroneutralization of intranasal Ad4-SARS-CoV-2 expressing the indicated VOC spikes in hamsters. Syrian golden hamsters were immunized with intranasal Ad4 expressing stabilized spike proteins from either the Wuhan strain (Ad4-CoV2-Wuhan), beta mutant (Ad4-CoV2-SA), delta mutant (Ad4-CoV2-India) or gamma mutant (Ad4-CoV2-Brazil), or stabilized chimeric spike protein bearing the beta mutant RBD (Ad-CoV2-Wu / RBD-SA). Ad4 expressing influenza virus H5 hemagglutinin (Ad4-H5) and mock inoculation were included as negative controls. Seroneutralization against Wuhan pseudovirus (Figure 6A) or delta pseudovirus (Figure 6B) was determined 28 days after intranasal administration. Furthermore, serum neutralization against Wuhan pseudovirus (Fig. 6C), Delta pseudovirus (Fig. 6D), and Omicron pseudovirus (Fig. 6E) was determined 56 days after intranasal administration. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Sequence Listing The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as specified in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. The sequence listing is submitted as an ASCII text file (created Jan. 14, 2022, 199 KB), which is incorporated herein by reference. The attached sequence listing is as follows:

[0012] SEQ ID NO:1 is the nucleotide sequence of the Ad4-SARS-CoV-2 spike vector. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0013] SEQ ID NO:2 is the amino acid sequence of the wild-type SARS-CoV-2 (Wuhan strain) spike protein deposited under GenBank Accession No. YP_009724390.1. [ka] [ka]

[0014] SEQ ID NO:3 is the amino acid sequence of a stabilized SARS-CoV-2 spike protein (nCoV-PP) with double proline substitutions. [ka]

[0015] SEQ ID NO:4 is the amino acid sequence of the tail-truncated SARS-CoV-2 spike protein (nCoV-TT). [ka]

[0016] SEQ ID NO:5 is the amino acid sequence of the SARS-CoV-2 spike protein lacking the C-terminal endocytosis motif (nCoV-noEndo). [ka] [ka]

[0017] SEQ ID NO:6 is a nucleic acid sequence encoding the SARS-CoV-2 spike protein. [ka]

[0018] SEQ ID NO:7 is the amino acid sequence of a stabilized SARS-CoV-2 beta mutant spike protein with a double proline substitution. [ka] [ka]

[0019] SEQ ID NO:8 is the amino acid sequence of a stabilized double proline substituted chimeric SARS-CoV-2 spike protein containing the RBD of the beta variant and the remaining sequence derived from the Wuhan strain. [ka]

[0020] SEQ ID NO:9 is the amino acid sequence of a stabilized SARS-CoV-2 delta mutant spike protein with a double proline substitution. [ka]

[0021] SEQ ID NO: 10 is the amino acid sequence of a stabilized SARS-CoV-2 gamma mutant spike protein with a double proline substitution. [ka]

[0022] SEQ ID NO:11 is the amino acid sequence of a stabilized SARS-CoV-2 delta plus mutant spike protein with a double proline substitution. [ka]

[0023] SEQ ID NO: 12 is the amino acid sequence of a stabilized SARS-CoV-2 omicron mutant spike protein with a double proline substitution. [ka]

[0024] SEQ ID NO: 13 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-CoV-2 beta mutant spike protein with a double proline substitution. [ka]

[0025] SEQ ID NO:14 is a codon-optimized nucleic acid sequence encoding a stabilized double proline-substituted chimeric SARS-CoV-2 spike protein containing the RBD of the beta variant and the remaining sequence derived from the Wuhan strain. [ka] [ka]

[0026] SEQ ID NO: 15 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-CoV-2 delta mutant spike protein with a double proline substitution. [ka] [ka]

[0027] SEQ ID NO: 16 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-CoV-2 gamma mutant spike protein with a double proline substitution. [ka] [ka]

[0028] SEQ ID NO:17 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-CoV-2 delta plus mutant spike protein with a double proline substitution. [ka] [ka]

[0029] SEQ ID NO:18 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-CoV-2 omicron mutant spike protein with a double proline substitution. [ka] [ka]

[0030] SEQ ID NO:19 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-CoV-2 Wuhan strain spike protein with double proline substitutions. [ka]

[0031] Detailed Description I. Abbreviations Ad Adenovirus CoV Coronavirus COVID-19 Coronavirus Disease 2019 Env Envelope GI HIV Human Immunodeficiency Virus IFU infection forming unit IM Intramuscular IN Nasal OPV Oral poliovirus PP double protein replacement S spike protein SARS Severe Acute Respiratory Syndrome TT Tail shortening URT Upper Airway VOC Variants of Concern Wu Wuhan Stock

[0032] II. Terminology Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in: Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers in 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, 16 volumes, published by Wiley-VCH in 2008; and other similar references.

[0033] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." It should be further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate unless otherwise indicated and are provided for illustrative purposes. Although many methods and materials similar or equivalent to those described herein can be used, certain suitable methods and materials are described herein. In case of conflict, the present specification will control, including the terminology. Additionally, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. In order to facilitate review of the various embodiments, the following explanations of terms are provided:

[0034] Adenovirus: A non-enveloped virus with a linear double-stranded DNA genome and an icosahedral capsid. There are at least 68 known serotypes of human adenovirus, which are divided into seven species (species A, B, C, D, E, F and G). Different serotypes of adenovirus are associated with different types of disease, with some serotypes causing respiratory disease (mainly species B and C), conjunctivitis (species B and D), and / or gastroenteritis (species F and G). Adenovirus type 4 (Ad4) is a virus of species E that can cause acute respiratory and eye disease. Adenovirus-based vectors are commonly used for various therapeutic applications, including vaccines and gene therapy vectors. In some embodiments herein, the adenovirus vector is a human replication-competent Ad4 with a complete or partial deletion in the E3 region.

[0035] Adjuvant: A component of an immunogenic composition used to enhance antigenicity. In some embodiments, adjuvants include suspensions of minerals (alum, aluminum hydroxide, or phosphates) to which antigens are adsorbed; or water-in-oil emulsions (e.g., where an antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), sometimes including killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibit antigen degradation and / or induce macrophage influx). In some embodiments, adjuvants used in the immunogenic compositions of the present disclosure include lecithin and carbomer homopolymer (e.g., ADJUPLEX, commercially available from Advanced BioAdjuvants, LLC). TM Adjuvants are combinations of adjuvants (see also Wegmann, Clin Vaccine Immunol 22(9): 1004-1012, 2015). Additional adjuvants for use in the immunogenic compositions of the present disclosure include QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides (e.g., those containing CpG motifs) can also be used as adjuvants. Adjuvants include biological molecules ("biological adjuvants") such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, and toll-like receptor (TLR) agonists (e.g., TLR-9 agonists). Those of skill in the art are familiar with adjuvants (see, for example, Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).

[0036] Administration: The introduction of a composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intravenous, the composition is administered by introducing the composition into the subject's vein. Exemplary administration routes include, but are not limited to, intranasal, inhalation, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical) and vaginal routes.

[0037] Codon-optimized: A nucleic acid sequence in which the codons have been changed to be optimal for expression in a particular system (e.g., a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or in a particular mammalian species (e.g., human cells). Codon optimization does not change the amino acid sequence of the encoded protein.

[0038] Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or reduce the function of a protein (e.g., a coronavirus spike protein). "Conservative" amino acid substitutions are those substitutions that do not substantially affect or reduce the function of a protein (e.g., the ability of the protein to elicit an immune response when administered to a subject). The term conservative variation also includes the use of a substituted amino acid in place of the unsubstituted parent amino acid. Furthermore, individual substitutions, deletions, or additions that change, add, or delete one amino acid or a small percentage of amino acids (e.g., less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations if the change results in the replacement of an amino acid with a chemically similar amino acid.

[0039] The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0040] A non-conservative substitution reduces the activity or function of a protein, such as a recombinant Env protein (e.g., its ability to induce an immune response when administered to a subject). For example, if an amino acid residue is essential for the function of the protein, an otherwise conservative substitution may interfere with its activity. Thus, a conservative substitution does not change the basic function of the protein of interest.

[0041] Coronavirus: A large family of positive-sense, single-stranded RNA viruses that can infect humans and non-human animals. Coronaviruses get their name from the crown-like spikes on their surface. The viral envelope is composed of a lipid bilayer that contains viral membrane (M), envelope (E), and spike (S) proteins. Most coronaviruses cause mild to moderate upper respiratory tract illness (e.g., the common cold). However, three coronaviruses have emerged that can cause more severe illness and death: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Other coronaviruses that infect humans include human coronavirus HKU1 (HKU1-CoV), human coronavirus OC43 (OC43-CoV), human coronavirus 229E (229E-CoV), and human coronavirus NL63 (NL63-CoV).

[0042] COVID-19: The disease caused by the coronavirus SARS-CoV-2.

[0043] Degenerate variant: A polynucleotide encoding a polypeptide that contains a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

[0044] E3 region: refers to the adenovirus early region 3 (E3) gene. It contains multiple open reading frames (ORFs). The E3 region of human adenovirus type 4 (Ad4) contains the following ORFs: 12.1K, 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K. In some embodiments herein, the deletion in the E3 region includes the deletion of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K ORFs. In other embodiments, the deletion in the E3 region is the deletion of only the 24.8K, 6.3K and 29.7K ORFs.

[0045] Heterologous: Derived from a distinct genetic source or species. For example, a heterologous polypeptide or polynucleotide refers to a polypeptide or polynucleotide that is derived from a different source or species.

[0046] Immune response: A response of a cell of the immune system (e.g., a B cell, T cell, or monocyte) to a stimulus. In some embodiments, the response is specific for a particular antigen, such as the SARS-CoV-2 spike protein (an "antigen-specific response"). In some embodiments, the immune response is a T cell response (e.g., a CD4+ or CD8+ response). In other embodiments, the response is a B cell response and results in the production of specific antibodies. "Priming an immune response" refers to the treatment of a subject with a "prime" immunogen / immunogenic composition to induce an immune response. That immune response is then "boosted" with a boost immunogen / immunogenic composition. Taken together, priming and boosting immunization generate the desired immune response in a subject.

[0047] Immunogenic composition: A composition that includes an immunogen or a nucleic acid molecule or vector that encodes an immunogen (e.g., SARS-CoV-2 spike protein) and that, when administered to a subject, induces a measurable CTL response to the immunogen and / or induces a measurable B cell response (e.g., production of antibodies) to the immunogen. It further refers to an isolated nucleic acid that encodes an immunogen (e.g., a nucleic acid that can be used to express an immunogen (and thus can be used to induce an immune response to the immunogen)). For in vivo use, the immunogenic composition can include the protein or nucleic acid molecule in a pharma- ceutically acceptable carrier and can also include other agents, such as adjuvants.

[0048] Immunize: To render a subject protected from infection by a particular infectious agent (e.g., SARS-CoV-2). Immunity does not require 100% protection. In some instances, immunization provides at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% protection against infection compared to infection in the absence of immunity.

[0049] Isolated: An "isolated" biological component is one that has been substantially separated or purified from other biological components (e.g., other chromosomal and extrachromosomal DNA, RNA, and proteins) in which it naturally occurs. "Isolated" proteins, peptides, nucleic acids, and viruses include those purified by standard purification methods. Isolated does not require absolute purity and can include protein, peptide, nucleic acid, or viral molecules that are at least 50% isolated (e.g., at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated).

[0050] Neutralizing antibody: An antibody that reduces the infectious titer of an infectious agent (e.g., a virus (e.g., a coronavirus) by binding to a specific antigen on the infectious agent. In some embodiments, an antibody specific for the SARS-CoV-2 spike protein neutralizes the infectious titer of SARS-CoV-2. For example, an antibody that neutralizes SARS-CoV-2 may interfere with the virus by directly binding the virus and restricting its entry into cells. Alternatively, a neutralizing antibody may interfere with one or more post-binding interactions between the pathogen and a receptor, for example, by interfering with virus entry using the receptor. In some embodiments, a SARS-CoV-2 neutralizing antibody inhibits SARS-CoV-2 infection of cells by, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to a control antibody.

[0051] Pharmaceutically acceptable carriers: Useful pharma- ceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19th ed., 1995, describes compositions and formulations suitable for pharmaceutical delivery of the immunogens (e.g., recombinant Ad4 expressing SARS-CoV-2 S protein) and immunogenic compositions of the present disclosure.

[0052] Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain injectable fluids that contain pharma- ceutically and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle). For solid compositions (e.g., in powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances (e.g., wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate). In certain embodiments suitable for administration to a subject, the carrier may be sterile and / or may be suspended or otherwise included in a unit dosage form containing one or more measured doses of a composition suitable for eliciting the desired anti-SARS-CoV-2 immune response. It may also be accompanied by a drug therapy for its use for treatment purposes. The unit dosage form may be, for example, a sealed vial containing sterile contents, a syringe for injection into a subject, or a lyophilized form for subsequent solubilization and administration, or a solid or controlled release dose.

[0053] Preventing, Treating, or Ameliorating a Disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after the disease or pathological condition has begun to develop (e.g., reducing viral load). "Ameliorating" refers to a reduction in the number or severity of signs or symptoms of a disease (e.g., coronavirus infection).

[0054] Recombinant: A recombinant nucleic acid, vector, or virus is one that has a sequence that is not naturally occurring or that is made by the artificial combination of two otherwise separated segments of sequences, which can be accomplished, for example, by the artificial manipulation of isolated segments of nucleic acid using genetic engineering techniques.

[0055] Replication-competent virus: A virus that can undergo genome replication and protein synthesis to produce progeny virus.

[0056] Sequence identity: The similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide or polynucleotide have a relatively high degree of sequence identity when aligned using standard methods.

[0057] Methods of alignment of sequences for comparison are well known. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents detailed considerations of sequence alignment methods and homology calculations.

[0058] Variants of a polypeptide or nucleic acid sequence are typically characterized by having at least about 75%, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, counted over the full length alignment with the amino acid or nucleotide sequence of interest. Sequences with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity). When less than the entire sequence is being compared for sequence identity, homologs and variants will typically have at least 80% sequence identity over a short window of 10-20 amino acids (or 30-60 nucleotides), and may have at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short stretches are available on the internet at the NCBI website.

[0059] As used herein, reference to "at least 90% identity" (or similar language) refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a particular reference sequence.

[0060] SARS-CoV-2: A coronavirus of the genus Betacoronavirus that first emerged in humans in 2019. The virus is also known as Wuhancoronavirus, 2019-nCoV, or 2019 novel coronavirus. The term "SARS-CoV-2" includes variants thereof, including, but not limited to, alpha (B.1.1.7 and Q lineages); beta (B.1.351 and descendant lineages); delta (B.1.617.2 and AY lineages); gamma (P.1 and descendant lineages); epsilon (B.1.427 and B.1.429); eta (B.1.525); iota (B.1.526); kappa (B.1.617.1); 1.617.3; mu (B.1.621, B.1.621.1), zeta (P.2) and omicron (B.1.1.529 and BA lineages). Symptoms of SARS-CoV-2 infection include fever, chills, dry cough, shortness of breath, fatigue, muscle / body aches, headache, new loss of taste or smell, sore throat, nausea or vomiting, and diarrhea. Patients with severe disease may develop pneumonia, multiple organ failure, and death. The time from exposure to onset of symptoms is approximately 2-14 days. SARS-CoV-2 virions contain a viral envelope with a large spike glycoprotein. The SARS-CoV-2 genome, like most coronaviridae, has a common genomic organization with replicase genes in the 5' 2 / 3 of the genome and structural genes in the 3' 1 / 3 of the genome. The SARS-CoV-2 genome encodes a standard set of structural protein genes, in order: 5'-spike (S)-envelope (E)-membrane (M) and nucleocapsid (N)-3'.

[0061] SARS spike (S) protein: a class I fusion glycoprotein that is initially synthesized as a precursor protein of approximately 1256 amino acids for SARS-CoV and 1273 amino acids for SARS-CoV-2. The individual precursor S polypeptides form homotrimers, undergo glycosylation in the Golgi apparatus, and processing to remove the signal peptide and cleavage by cellular proteases between approximately positions 679 / 680 for SARS-CoV and 685 / 686 for SARS-CoV-2 to generate separate S1 and S2 polypeptide chains. These remain associated as S1 / S2 protomers within the homotrimer, thereby forming a trimer of heterodimers. The S1 subunit is distal to the viral membrane and contains a receptor binding domain (RBD) that is believed to mediate viral binding to its host receptor. The S2 subunit is believed to contain the fusion protein apparatus (e.g., fusion peptide). S2 also contains two heptad repeat sequences (HR1 and HR2) and a central helix, typical of a fusion glycoprotein, a transmembrane domain, and a cytosolic tail domain. An exemplary wild-type (Wuhan strain) SARS-CoV-2 spike protein sequence is set forth herein as SEQ ID NO:2. Exemplary modified Wuhan SARS-CoV-2 spike protein sequences are set forth herein as SEQ ID NOs:3-5. Additionally, exemplary SARS-CoV-2 mutant spike protein sequences are set forth herein as SEQ ID NOs:7-12.

[0062] Subject: A living multicellular vertebrate, a category that includes humans and non-human mammals. In some embodiments, the subject is a human. In some examples, a subject is selected that is in need of inhibiting or preventing SARS-CoV-2 infection. For example, the subject may be uninfected and at risk of SARS-CoV-2 infection.

[0063] Therapeutically effective amount: A quantity of a particular substance (e.g., an immunogen of the present disclosure (e.g., a recombinant Ad4 expressing a SARS-CoV-2 S protein) or immunogenic composition sufficient to achieve a desired effect (e.g., a protective immune response) in a subject being treated. A "therapeutically effective amount" can be the amount necessary to inhibit SARS-CoV-2 replication or to treat COVID-19 in a subject with an existing SARS-CoV-2 infection. A "prophylactically effective amount" can be the amount necessary to inhibit SARS-CoV-2 replication or to treat COVID-19 in a subject with an existing SARS-CoV-2 infection. "An effective amount" refers to the administration of an agent or composition that inhibits or prevents the establishment of an infection (e.g., infection by SARS-CoV-2). It is understood that multiple administrations of the immunogen / immunogenic composition of the present disclosure may be required to obtain a protective immune response to the antigen of interest, and / or administration of the composition of the present disclosure as a "priming" in a prime-boost protocol, where the boost immunogen may be different from the priming immunogenic composition. Thus, an effective amount of the immunogen / immunogenic composition of the present disclosure may be an amount of the immunogen or immunogenic composition sufficient to induce a priming immune response in a subject. The subject may be subsequently boosted with the same or a different immunogen to induce a protective immune response.

[0064] In one example, the desired response is to induce an immune response that inhibits or prevents SARS-CoV-2 infection. SARS-CoV-2 infected cells do not need to be completely eliminated or prevented for the composition to be effective. For example, administration of an effective amount of an immunogen or immunogenic composition may induce an immune response that reduces the number of SARS-CoV-2 infected cells (or prevents infection of cells) by the desired amount, for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 infected cells) compared to the number of SARS-CoV-2 infected cells in the absence of immunity.

[0065] Unit Dosage Form: A physically discrete unit (e.g., capsule, tablet, or liquid) suitable as a unitary dosage for a human patient, each unit containing a predetermined quantity of one or more active ingredients calculated to produce a therapeutic effect, in association with at least one pharma- ceutically acceptable diluent or carrier, or a combination thereof.

[0066] Vaccine: A pharmaceutical composition that induces a preventative or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response against an antigen of a pathogen (e.g., a viral pathogen) or against a cellular component that correlates with a pathological condition. A vaccine may include a polynucleotide (e.g., a nucleic acid encoding an antigen of the present disclosure), a peptide or polypeptide (e.g., an antigen of the present disclosure), a virus, a cell, or one or more cellular components. In one specific non-limiting example, the vaccine reduces the severity of symptoms associated with SARS-CoV-2 infection and / or reduces the viral load compared to a control. In another non-limiting example, the vaccine reduces SARS-CoV-2 infection and / or transmission compared to a control.

[0067] Vector: An entity that contains a DNA or RNA molecule that has a promoter(s) operably linked to a coding sequence of a protein of interest (e.g., an immunogenic protein) and can express the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, a component of a virus or bacteria or other microorganism that may be replication incompetent, or a virus or bacteria or other microorganism that may be replication competent. A vector is sometimes referred to as a construct. A recombinant DNA vector is a vector that contains recombinant DNA. A vector may contain a nucleic acid sequence (e.g., an origin of replication) that allows it to replicate in a host cell. A vector may also contain one or more selectable marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector that has at least some nucleic acid sequence derived from one or more viruses. Non-limiting examples of viral vectors include adenovirus vectors, adeno-associated virus (AAV) vectors, and poxvirus vectors (e.g., vaccinia, fowlpox).

[0068] III. Introduction Among the available vaccine platforms for presenting viral glycoproteins to the immune system, replicating vectors have several important advantages over most non-replicating vectors (Robert-Guroff, Curr Opin Biotechnol 18(6):546-556, 2007). Replication-competent vectors can express viral surface proteins, so that the total dose of antigen far exceeds that of non-replicating vectors. Replicating mucosal vaccines induce mucosal immunity (including IgA and IgG antibodies) and balanced T cell responses (including resident memory T cells). In addition, replicating vectors (e.g., replication-competent adenovirus (Ad) vectors) express viral glycoproteins for extended periods of time, similar to live virus infection. This feature is thought to be important for loading dendritic cells in lymph nodes and for the induction of durable antibody responses (Cirelli et al., Cell 177(5): 1153-1171, 2019; Tam et al., Proc Natl Acad Sci USA 113(43): E6639-E6648, 2016; Mueller et al., Mol Pharm 12(5): 1356-1365, 2015). Each of these features contributes to the magnitude and persistence of the immune responses observed following replicating virus vaccination.

[0069] The vaccine construct disclosed herein is a replication-competent Ad4 encoding the SARS-CoV-2 spike (S) protein. In the Ad4 vector of the present disclosure, derived from the Ad4 vaccine strain, the gene encoding the SARS-CoV-2 spike protein is cloned into the E3 region with multiple E3 ORF deletions. The parent Ad4 vaccine vector has been given to over 10 million people with an excellent safety record. Ad4 recombinants have been developed for both influenza virus H5 and human immunodeficiency virus (HIV) envelope (Env) and Gag proteins. These Ad4-based vaccines have undergone preclinical testing in rabbits for immunogenicity and human testing in Phase I clinical trials.

[0070] The replicable Ad4-based vaccine platform has several distinct advantages over other proposed and approved SARS-CoV-2 vaccines. For example, the efficacy of the Ad4 vaccine has already been established as it is routinely administered as a single-dose enteric capsule in the US military and has been found to prevent respiratory disease with greater than 95% efficacy. Furthermore, when administered intranasally or into the tonsils, the replicable Ad4-based vaccine induces a neutralizing antibody response in human subjects. Upper respiratory tract administration also circumvents pre-existing Ad4 immunity in most people. By inducing mucosal immunity, the Ad4-based vaccine platform not only provides protection to vaccinated subjects, but also has the potential to impede the transmission of SARS-CoV-2 to others. In contrast to non-replicating viral vaccines, the replicable Ad4-based system generates a durable immune response. Moreover, unlike mRNA-based SARS-CoV-2 vaccines, the Ad4 vaccine can be stored for extended periods at 4-8°C. Moreover, the vaccine platform of the present disclosure is unparalleled in terms of scalability and cost. It is expected that the SARS-CoV-2 vaccine of the present disclosure can be produced for less than 1 cent per dose.

[0071] IV. Overview of the Embodiments Disclosed herein are recombinant adenovirus type 4 (Ad4) expressing the SARS-CoV-2 spike (S) protein (referred to in some embodiments as "Ad4-SARS-CoV-2-spike" or "Ad4-Spike"), recombinant Ad4 nucleic acid vectors encoding the recombinant Ad4-Spike, and immunogenic compositions thereof.

[0072] In one aspect, provided herein is a recombinant Ad4 expressing a SARS-CoV-2 S protein. The recombinant Ad4 is replication competent, and the genome of the Ad4 comprises a deletion in the adenovirus E3 region and an insertion of the coding sequence of the SARS-CoV-2 S protein. In some embodiments, the amino acid sequence of the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of a native S protein (e.g., the S protein of the Wuhan SARS-CoV-2 strain shown herein as SEQ ID NO:2). In a specific example, the amino acid sequence of the S protein comprises or consists of SEQ ID NO:2.

[0073] Amino acid numbering as used herein with respect to residues of the SARS-CoV-2 S protein is done with reference to the wild type Wuhan strain SARS-CoV-2 S sequence provided as SEQ ID NO:2. With reference to the SARS-CoV-2 S protein sequence provided as SEQ ID NO:2, the ectodomain of the SARS-CoV-2 S protein includes approximately residues 16-1208. Residues 1-15 are a signal peptide, which is removed during cellular processing. The S1 / S2 cleavage site is located at positions 685 / 686. HR1 is located at approximately residues 915-983. The central helix is ​​located at approximately residues 988-1029. HR2 is located at approximately residues 1162-1194. The C-terminus of the S2 ectodomain is located at approximately residue 1208. Although the numbering of positions in the S protein may vary between SARS-CoV-2 strains, the sequences may be aligned to determine the associated structural domains and cleavage sites (see, e.g., FIG. 4).

[0074] In some embodiments, the recombinant Ad4 comprises a coding sequence for a SARS-CoV-2 S protein that includes one or more (e.g., two, e.g., two consecutive) proline substitutions at or near the interface between the HR1 domain and the central helix domain that stabilize the S protein in the prefusion conformation. In some such embodiments, the one or more (e.g., two, e.g., two consecutive) proline substitutions that stabilize the S protein in the prefusion conformation are located between the amino acid at position 15, which is the N-terminal of the C-terminal residue of HR1, and the amino acid at position 5, which is the C-terminal of the N-terminal residue of the central helix. In some embodiments, the one or more (e.g., two, e.g., two consecutive) proline substitutions that stabilize the SARS-CoV-2 S protein in the prefusion conformation are located between residues 975-995 (e.g., 981-992). In some embodiments, the SARS-CoV-2 S protein is stabilized in a prefusion conformation by K986P and V987P substitutions ("PP" or "2P"). In some embodiments, the SARS-CoV-2 S protein is stabilized in a prefusion conformation by one or two proline substitutions at positions D985, K986, or V987 of the S ectodomain promoter in the trimer. In some examples, the SARS-CoV-2 S protein stabilized in a prefusion conformation by one or more proline substitutions (e.g., K986P and V987P substitutions) includes one or more additional modifications for stabilization in a prefusion conformation.

[0075] In some embodiments, the SARS-CoV-2 S protein encoded by the recombinant Ad4 genome comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:3 (Wuhan-PP), where the SARS-CoV-2 S protein is stabilized in a prefusion conformation with one or more of the modifications provided herein (e.g., K986P and V987P substitutions). In other embodiments, the stabilized proline-substituted S protein is derived from a SARS-CoV-2 mutant strain. In some examples, the stabilized S protein from a SARS-CoV-2 mutant comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:7 (beta-PP), SEQ ID NO:8 (Wuhan / RDB-beta-PP), SEQ ID NO:9 (delta-PP), SEQ ID NO:10 (gamma-PP), SEQ ID NO:11 (deltaplus-PP), or SEQ ID NO:12 (omicron-PP). In certain examples, the amino acid sequence of the stabilized SARS-CoV-2 S protein comprises or consists of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0076] In other embodiments, the SARS-CoV-2 S protein encoded by the recombinant Ad4 genome comprises a C-terminal truncation (e.g., a truncated cytoplasmic tail or a truncated endocytosis motif). In specific examples, the truncated SARS-CoV-2 S protein comprises or consists of the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0077] An exemplary nucleic acid sequence encoding a SARS-CoV-2 S protein is provided as SEQ ID NO: 6. In some examples, the nucleic acid sequence encoding the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In specific, non-limiting examples, the nucleic acid sequence encoding the S protein comprises or consists of SEQ ID NO: 6.

[0078] The exemplary DNA sequence of the SARS-CoV-2 S protein provided above can be modified to introduce the amino acid substitutions and deletions disclosed herein for prefusion stabilization. In some embodiments, this DNA sequence (with or without modification to introduce amino acid substitutions) can be included in the recombinant Ad4 vector as a sequence encoding the SARS-CoV-2 S protein. In some embodiments, the S protein is encoded by a codon-optimized nucleic acid sequence. In some examples, the nucleic acid sequence encoding the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:13 (beta-PP), SEQ ID NO:14 (Wuhan / RBD beta-PP), SEQ ID NO:15 (delta-PP), SEQ ID NO:16 (gamma-PP), SEQ ID NO:17 (delta plus-PP), SEQ ID NO:18 (omicron-PP) or SEQ ID NO:19 (Wuhan-PP). In a specific example, the nucleic acid sequence encoding the S protein comprises or consists of any one of SEQ ID NOs:13-19.

[0079] In some embodiments, the deletion in the E3 region is a deletion of at least two, at least three, at least four, at least five, at least six, or at least seven E3 open reading frames (ORFs). In some examples, the deletion includes at least two, at least three, at least four, at least five, at least six, or at least seven of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K, and 14.7K ORFs. In a specific, non-limiting example, the deletion in the E3 region includes a deletion of each of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K, and 14.7K ORFs.

[0080] In some embodiments, the coding sequence for the SARS-CoV-2 S protein is inserted in place of the deleted portion of the E3 region.

[0081] In some embodiments, the nucleotide sequence of the recombinant Ad4 genome is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some examples, the nucleotide sequence of the recombinant Ad4 genome comprises or consists of SEQ ID NO: 1.

[0082] Recombinant replicable Ad4 nucleic acid vectors are also provided herein. In some embodiments, the recombinant Ad4 vector comprises a deletion in the adenovirus E3 region and an insertion of the coding sequence of the SARS-CoV-2 S protein. In some embodiments, the amino acid sequence of the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of a native S protein (e.g., the S protein of the Wuhan SARS-CoV-2 strain shown herein as SEQ ID NO:2). In specific examples, the amino acid sequence of the S protein comprises or consists of SEQ ID NO:2.

[0083] In some embodiments, the SARS-CoV-2 S protein is stabilized in the prefusion conformation by K986P and V987P substitutions ("PP" or "2P"). In some embodiments, the SARS-CoV-2 S protein is stabilized in the prefusion conformation by one or two proline substitutions at positions D985, K986, or V987 of the S ectodomain promoter in the trimer. In some examples, a SARS-CoV-2 S protein stabilized in the prefusion conformation by one or more proline substitutions (e.g., K986P and V987P substitutions) includes one or more additional modifications for stabilization in the prefusion conformation.

[0084] In some embodiments, the SARS-CoV-2 S protein encoded by the recombinant Ad4 nucleic acid vector comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:3 (Wuhan-PP), where the SARS-CoV-2 S protein is stabilized in a pre-fusion conformation with one or more of the modifications provided herein (e.g., K986P and V987P substitutions). In other embodiments, the stabilized proline-substituted S protein is derived from a SARS-CoV-2 mutant strain. In some embodiments, the S protein is encoded by a codon-optimized nucleic acid sequence. In some examples, the stabilized S protein from a SARS-CoV-2 mutant comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:7 (beta-PP), SEQ ID NO:8 (Wuhan / RDB-beta-PP), SEQ ID NO:9 (delta-PP), SEQ ID NO:10 (gamma-PP), SEQ ID NO:11 (deltaplus-PP), or SEQ ID NO:12 (omicron-PP). In certain examples, the amino acid sequence of the stabilized SARS-CoV-2 S protein comprises or consists of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0085] In other embodiments, the SARS-CoV-2 S protein encoded by the recombinant Ad4 nucleic acid vector comprises a C-terminal truncation (e.g., a truncated cytoplasmic tail or a truncated endocytosis motif). In specific examples, the truncated SARS-CoV-2 S protein comprises or consists of the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0086] In some embodiments of the Ad4 vectors of the present disclosure, the deletion in the E3 region is a deletion of at least two, at least three, at least four, at least five, at least six, or at least seven E3 ORFs. In some examples, the deletion includes at least two, at least three, at least four, at least five, at least six, or at least seven of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K, and 14.7K ORFs. In certain non-limiting examples, the deletion in the E3 region includes a deletion of each of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K, and 14.7K ORFs.

[0087] In some embodiments of the Ad4 vectors of the present disclosure, the coding sequence of the SARS-CoV-2 S protein is inserted in place of the deleted portion of the E3 region. In some examples, the coding sequence of the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 2-5 and 7-12. In specific non-limiting examples, the coding sequence of the S protein comprises or consists of any one of SEQ ID NOs: 2-5 and 7-12.

[0088] In some embodiments, the nucleotide sequence of the Ad4 vector is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some examples, the nucleotide sequence of the Ad4 vector comprises or consists of SEQ ID NO:1.

[0089] Further provided herein is an immunogenic composition comprising recombinant Ad4 or recombinant Ad4 vector and a pharma- ceutically acceptable carrier. In some embodiments, the immunogenic composition further comprises an adjuvant. In other embodiments, the immunogenic composition does not comprise an adjuvant.

[0090] Methods of inducing an immune response against SARS-CoV-2 in a subject are also provided. In some embodiments, the methods include administering to the subject a therapeutically effective amount of a recombinant Ad4, a recombinant Ad4 (nucleic acid) vector, or an immunogenic composition disclosed herein. Methods of immunizing a subject against SARS-CoV-2 infection are also provided. In some embodiments, the methods include administering to the subject a therapeutically effective amount of a recombinant Ad4, a recombinant Ad4 vector, or an immunogenic composition disclosed herein.

[0091] In some embodiments of the disclosed methods, the recombinant Ad4, recombinant Ad4 vector, or immunogenic composition is administered intranasally or to the tonsils. In some examples, intranasal administration includes administration of an aerosol. The particle size of the aerosol should allow delivery to the upper respiratory tract, but not the lower respiratory tract. In specific examples, the aerosol includes particles greater than 10 microns in diameter (e.g., greater than 20 microns, greater than 30 microns, greater than 40 microns, or greater than 50 microns). In particular examples, the aerosol includes particles from about 10 to about 150 microns (e.g., from about 20 to about 125 microns or from about 30 to about 100 microns). One of skill in the art can select an appropriate device for intranasal delivery of the disclosed recombinant Ad4, recombinant Ad4 vector, or immunogenic composition to the upper respiratory tract. Non-limiting examples of devices include the Accuspray TM (Becton-Dickinson) and MAD Nasal TM (Teleflex®) atomizer.

[0092] In some embodiments, the method comprises the step of: 4 ~about 10 6 Recombinant Ad4 particles (e.g., about 5×10 4 ~Approx. 5×10 5 Viral particles or approximately 1 x 10 5In some examples, the dose is about 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , or 1 × 10 6 Recombinant Ad4 particles.

[0093] In some embodiments, the recombinant Ad4, recombinant Ad4 vector, or immunogenic composition is administered in a single dose.

[0094] In some embodiments, the recombinant Ad4, recombinant Ad4 vector, or immunogenic composition is administered as part of a priming-boost immunization protocol. In some examples, the recombinant Ad4, recombinant Ad4 vector, or immunogenic composition is a priming dose. In other examples, the recombinant Ad4, recombinant Ad4 vector, or immunogenic composition is a boost dose.

[0095] V. Preclinical and Clinical Trials Related to COVID-19 Vaccine Development By testing vaccine-induced mucosal neutralizing antibody responses in a series of oral live poliovirus (OPV) challenge studies, investigators have convincingly demonstrated a striking separation of systemic and mucosal antibody systems (Brickley et al., Clin Infect Dis. 2018;67(suppl_1):S42-S50). This study clearly shows that, despite inducing high levels of serum antibodies and providing individual protection from paralytic polio, the inactivated Salk vaccine fails to induce the intestinal IgA response that is crucial to inhibit enteric poliovirus replication and prevent fecal-oral transmission. In contrast, initial vaccination with live attenuated Sabin OPV induces strong mucosal IgA responses and sterilizing immunity upon challenge with live OPV. This observation highlights the crucial nature of inducing mucosal immunity to prevent COVID-19 infection and transmission. It is believed that the lack of mucosal immunogenicity observed with OPV is recapitulated by subunit or replication-incompetent systemically administered SARS-CoV-2 vaccines.

[0096] A similar benefit of mucosal immunization in blocking infection was observed in preclinical trials of SARS-CoV-2 vaccines: in ferrets, IM or mucosal immunization with a replication-deficient Ad5-spike recombinant induced similar levels of spike-specific antibodies in serum, but only mucosal immunization induced bactericidal protection in the upper respiratory tract (URT) (Wu et al., Nat Commun 11(1): 4081, 2020). Similar advantages of intranasal over intramuscular administration in inducing mucosal immunity and bactericidal defenses in URT were observed in mouse models permissive to SARS-CoV-2 infection using lentivirus or chimpanzee adenovirus spike recombinants (Ku et al., Cell Host Microbe S1931-3128(20)30672-7, 2020; Hassan et al., Cell 183(1): 169-184, 2020; King et al., King et al., bioRxiv 2020.10.10.331348, 2020). Local specific IgA was observed to be highly associated with cessation of viral shedding in humans following challenge with coronavirus 229E (Callow et al., J Hyg 95(1): 173-189, 1985).

[0097] Previous attempts to protect against naive host viral mucosal infections using parenterally administered non-replicating vaccines have failed or resulted in enhanced disease. Examples include respiratory syncytial virus (RSV), parainfluenza virus (PIV)-3, Ad4, rotavirus, and measles virus. The reasons for these failures are, in part, the difficulty in protecting mucosal surfaces coated on their apical surfaces with viral receptors, 100-1000-fold lower antibodies at these surfaces compared to serum, and the distorted and short-lived immune responses generated by non-replicating vectors. Clinical trials of the Ad4-SARS-CoV-2-spike vaccine of the present disclosure will evaluate in detail humoral and mucosal responses to the SARS-CoV-2 spike protein and adenoviral vectors. The Ad4-SARS-CoV-2-spike vaccine of the present disclosure is predicted to generate mucosal antibodies in the respiratory tract, most closely mimicking the immune profile observed after natural SARS-CoV-2 infection. Furthermore, it is believed that the vaccine of the present disclosure offers the best chance of permanently interrupting transmission during the COVID-19 pandemic.

[0098] Among the recombinant viral vectors available for human use, replicating adenoviruses offer several important advantages. Replicating Ad4 has been given to over 10 million people in the military as a vaccine against Ad4 respiratory disease, with an exceptional safety and efficacy record (Gaydos and Gaydos, Mil Med. 1995;160(6):300-304). This recombinant Ad4 is attenuated by administration to the digestive tract in the form of an enteric-coated tablet and does not cause respiratory disease (Choudhry et al., Vaccine 2016:34(38) 4558-4564). Using enteric capsule delivery, a phase 3 trial was conducted in 4,000 volunteers undergoing military basic training. The results demonstrated a vaccine efficacy of 99.3% and a seroconversion of 94.5% against respiratory disease caused by Ad4 (Kuschner et al., Vaccine 2013:31 2963-2971).

[0099] In one human trial, enterally delivered replicative recombinant adenovirus vectors expressing influenza virus H5 were only moderately immunogenic. This is most likely related to the attenuation of replication upon administration to the gastrointestinal tract in conjunction with E3 deletion (Gurwith et al., Lancet Infect Dis. 2013;13(3):238-50). Introduction of large genes (e.g., genes encoding coronavirus spike proteins) into adenovirus vectors, including removal of the immediate early (in this case, E3) genes, results in at least 10-fold attenuation relative to the parental adenovirus in tissue culture, chimpanzees, and humans (Lubeck et al., Nat Med. 1997;3(6):651-8).

[0100] In another clinical trial, higher and significantly more durable levels of influenza-specific neutralizing antibodies were observed when replication-competent Ad4 expressing influenza virus hemagglutinin type 5 Vietnam (Ad4-H5-Vtn) was administered to the URT compared to the gastrointestinal tract (GI) (Matsuda et al., Sci Immunol. 2019;4(34):eaau2710; Matsuda et al., J Clin Invest 131(5):e140794, 2021). Vaccine delivered to the URT increased 10 8 Up to doses of 100 mg / kg / day were very safe (nasal congestion or throat discomfort in 25% of participants, none above grade 2). This level of reactivity is roughly the same as that seen with placebo, several parenterally administered non-replicating platforms are currently being tested against SARS-CoV-2, and is below that of the currently licensed Varicella Zoster (Shingrix) vaccine. URT administration of adenovirus to Ad4 seropositive humans did not result in reinfection. URT administration advantageously uses the difficulty in protecting the upper respiratory tract to overcome vector-specific immunity. One example is the ability of adenovirus expressing the Ebola glycoprotein to induce protective immunity upon Ebola challenge by the intranasal route in adeno-immune primates, whereas no protection was observed following IM administration of Ebola constructs in previously adeno-immune animals.

[0101] Previous results with Ad4-H5-Vtn and Ad4-HIV recombinants showed that nearly all human participants developed a response to the transgene. Increases in H5-specific B cells, H5-specific antibody somatic hypermutation, and potency were observed after a single intranasal or tonsillar administration of the vaccine. The vaccine also induced highly durable responses. Responses to licensed split influenza vaccines typically fade by 5-10 fold within 2-6 months after immunization. However, when Ad4-H5-Vtn participants were asked to return for a boost 3-5 years later, neutralizing antibodies were still at the levels observed at peak levels after immunization with the licensed vaccine. The Ad4-SARS-CoV-2-spike vaccine constructs disclosed herein can be used to generate mucosal immunity after systemic vaccination. Alternatively, a subunit vaccine can be administered after immunization with the vaccine of the present disclosure to boost mucosal and systemic antibodies, which has been shown to occur with the H5-Vtn vaccine construct.

[0102] VI. Immunogenic composition Immunogenic compositions comprising an immunogen of the present disclosure (e.g., a recombinant Ad expressing a SARS-CoV-2 S protein, or a recombinant Ad4 nucleic acid vector comprising a SARS-CoV-2 S protein coding sequence) and a pharma- ceutically acceptable carrier are also provided. Such compositions can be administered to a subject by a variety of modes of administration (e.g., intranasal, supratonsillar, inhalation, oral, intramuscular, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, or parenteral routes). Methods for preparing administrable compositions are described in more detail in publications such as Remingtons Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, Easton, Pennsylvania, 1995).

[0103] Thus, the immunogens described herein may be formulated with pharma- ceutically acceptable carriers to help retain biological activity while also promoting increased stability during storage within acceptable temperature ranges. Potential carriers include, but are not limited to, physiologically balanced culture media, phosphate buffer saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotectant additives or stabilizers (e.g., proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., monosodium glutamate), or other protective agents). The resulting aqueous solutions may be packaged for immediate use or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration, either for single or multiple doses.

[0104] Formulated compositions, particularly liquid compositions, may contain bacteriostatic agents to prevent or minimize degradation during storage, including but not limited to benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben at effective concentrations, usually ≦1% w / v. Bacteriostatic agents may be contraindicated for some patients; thus, lyophilized formulations may be reconstituted in solutions with or without such ingredients.

[0105] The immunogenic compositions of the disclosure may include pharma- ceutically acceptable vehicle substances as necessary to approximate physiological conditions, such as, for example, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like (e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate).

[0106] The pharmaceutical composition may optionally contain an adjuvant to enhance the immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-A and derivatives or modifications thereof, oil-emulsions, saponins, neutral liposomes, liposomes including vaccines and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block copolymers including polyoxyethylene (POE) and polyoxypropylene (POP) (e.g., POE-POP-POE block copolymers, MPL, etc.) are also suitable. TM (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA) can be used as adjuvants (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage that they help stimulate the immune system in a non-specific manner, thus enhancing the immune response to the pharmaceutical product. In some embodiments, adjuvants are not required and therefore are not administered with the Ad4-Spike vaccine.

[0107] In some embodiments, the compositions may be provided as sterile compositions. The pharmaceutical compositions typically contain an effective amount of the immunogen of the present disclosure and may be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected as an amount that induces an immune response without significant adverse side effects. In some examples, the dose is about 1×10 4 ~about 10 6 Viral particles (e.g., about 5 × 10 4 ~Approx. 5×10 5 Viral particles or approximately 1 x 10 5 virus particles).

[0108] In some embodiments, the compositions may be provided in a unit dosage form for use in eliciting an immune response in a subject, e.g., for preventing SARS-CoV-2 infection in a subject. The unit dosage form includes a single preselected dose suitable for administration to a subject, or a suitably marked or measured multiple of two or more preselected unit doses, and / or a metering mechanism for administering the unit dose or multiples thereof. In some examples, the unit dosage is about 1×10 4 ~about 10 6 Viral particles (e.g., about 5 × 10 4 ~Approx. 5×10 5 In a specific example, the unit dose is about 1×10 5 It is a virus particle.

[0109] VII. Methods of Inducing an Immune Response The immunogens of the disclosure (e.g., recombinant replication-competent adenoviruses expressing the SARS-CoV-2 spike protein), polynucleotides and vectors encoding the immunogens of the disclosure, and compositions comprising same may be used in methods of inducing an immune response against SARS-CoV-2 to prevent, inhibit (including inhibiting transmission), and / or treat SARS-CoV-2 infection.

[0110] Provided herein is a method for inducing an immune response against SARS-CoV-2 in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a recombinant adenovirus, adenovirus vector or immunogenic composition disclosed herein. In some examples, the recombinant adenovirus, vector or immunogenic composition is administered intranasally (e.g., in a spray) or orally (e.g., by using an enteric-coated tablet).

[0111] When inhibiting, treating, or preventing a SARS-CoV-2 infection, the methods can be used to either avoid infection in a SARS-CoV-2 seronegative subject (e.g., by inducing an immune response that protects against SARS-CoV-2 infection) or to treat an existing infection in a SARS-CoV-2 seropositive subject.

[0112] To identify subjects for prevention or treatment according to the methods of the present disclosure, accepted screening methods are used to determine risk factors associated with the targeted or suspected disease or condition, or to determine the status of the disease or condition present in the subject. These screening methods include, for example, conventional post-treatment to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods (e.g., various ELISA and other immunoassay methods for detecting and / or characterizing SARS-CoV-2 infection). These and other conventional methods allow clinicians to select patients in need of treatment using the methods and immunogenic compositions of the present disclosure. In accordance with these methods and principles, the compositions can be administered according to the teachings herein, or other conventional methods, as an independent prevention or treatment program, or as a follow-up, adjunctive, or coordinate treatment regimen to other treatments.

[0113] The immunogens of the present disclosure may be used in coordinate (or prime-boost) immunization protocols or combination formulations. In certain embodiments, the novel combination immunogenic compositions and coordinate immunization protocols use separate immunogens or formulations, each directed to eliciting an anti-SARS-CoV-2 immune response (e.g., an immune response against the SARS-CoV-2 spike protein). The separate immunogenic compositions that elicit an anti-SARS-CoV-2 immune response may be combined into a multivalent immunogenic composition administered to a subject in a single immunization step, or they may be administered separately (in a monovalent immunogenic composition) in a coordinate immunization protocol.

[0114] In one embodiment, a suitable immunization regimen includes at least two separate inoculations with one or more immunogenic compositions comprising the Ad4-Spike of the present disclosure (the second inoculation is administered more than about 2 weeks, more than about 3-8 weeks, or more than about 4 weeks after the first inoculation). The third inoculation may be administered more than several months after the second inoculation, and in specific embodiments, more than about 5 months after the first inoculation, about 6 months to more than about 2 years after the first inoculation, or about 8 months to more than about 1 year after the first inoculation. A third or subsequent regular inoculation is also desirable to enhance the "immunological memory" of the subject. The appropriateness of the selected vaccination parameters, e.g., formulation, dose, regimen, etc., may be determined by taking aliquots of serum from the subject and assaying for antibody titers during the course of the immunization program. Alternatively, T cell populations may be monitored by conventional methods. In addition, the subject's clinical condition can be monitored for the desired effect, such as prevention of SARS-CoV-2 infection, amelioration of disease state (e.g., reduction of viral load), or reduction of transmission frequency. If such monitoring indicates that vaccination is suboptimal, the subject can be boosted with additional doses of the immunogenic composition, and the vaccination parameters can be modified in a manner predicted to enhance the immune response. Thus, for example, the dose of the immunogen of the present disclosure can be increased or the route of administration can be changed.

[0115] It is contemplated that there may be several boosts, and that each boost may be a different immunogen. It is also contemplated that in some instances the boost may be the same immunogen as another boost, or as the priming.

[0116] The priming and the boost can be administered as a single dose or multiple doses. For example, two, three, four, five, six or more doses can be administered to the subject over the course of several days, weeks or months. Multiple boosts can also be given (e.g., one to five or more). Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose for the first inoculation, followed by a relatively small dose for the boost. An immune response against the selected antigenic surface can be elicited by one or more inoculations of the subject.

[0117] In some embodiments, the immunogen of the present disclosure may be administered to a subject simultaneously with administration of an adjuvant. In other embodiments, the immunogen may be administered to a subject after administration of an adjuvant and within a sufficient amount of time to elicit the immune response. In other embodiments, no adjuvant is administered.

[0118] The above method does not need to completely inhibit SARS-CoV-2 infection to be effective. For example, eliciting an immune response against SARS-CoV-2 can reduce or inhibit SARS-CoV-2 infection to a desired amount, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 infected cells) compared to SARS-CoV-2 infection in the absence of immunity. In a further example, SARS-CoV-2 replication can be reduced or inhibited by the method of the present disclosure. SARS-CoV-2 replication does not need to be completely eliminated to be effective. For example, an immune response elicited using one or more of the immunogens of the present disclosure may reduce SARS-CoV-2 replication by a desired amount, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (eliminating or preventing detectable SARS-CoV-2 replication) compared to SARS-CoV-2 replication in the absence of an immune response.

[0119] After immunization of the subject, serum can be collected from the subject at the appropriate time, frozen and stored for neutralization testing.Methods for assaying for neutralization activity include, but are not limited to, plaque reduction neutralization (PRNT) assay, microneutralization assay, flow cytometry-based assay, single cycle infection assay, and pseudovirus neutralization assay.

[0120] In some embodiments, immunization is achieved by administration of recombinant Ad4 vector DNA. Immunization with nucleic acid constructs is taught, for example, in U.S. Patent Nos. 5,643,578 (which describes a method of immunizing a vertebrate by introducing DNA encoding a desired antigen to elicit a cell-mediated or humoral response), 5,593,972 and 5,817,637 (which describes operably linking a nucleic acid sequence encoding an antigen to a regulatory sequence to allow expression), and is described generally in Janeway & Travers, Immunobiology: The Immune System In Health and Disease, page 13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari, N. Engl. J. Med. 334:42-45, 1996.

[0121] The following examples are provided to illustrate certain particular features and / or embodiments and should not be construed as limiting the disclosure to the particular features or embodiments described. EXAMPLES

[0122] Working Example Example 1: Expression of wild-type and modified SARS-CoV-2 spike proteins The following study evaluated cell surface expression of wild-type Wuhan strain SARS-CoV-2 spike protein (SEQ ID NO:2) and three modified versions of the Wuhan strain spike protein: stabilized (PP), tail-truncated (TT), and endocytosis motif-truncated (no-Endo). PP contains a double proline stabilizing substitution at amino acid positions 986 and 987 (SEQ ID NO:3); TT contains a deletion of the terminal 24 amino acids of the cytoplasmic tail (SEQ ID NO:4); no-Endo contains a deletion of the C-terminal endocytosis signaling motif (SEQ ID NO:5) (see FIG. 4).

[0123] The expression of SARS-CoV-2 WT, PP, TT and no-Endo spike proteins was evaluated in A549 cells. Cells were transfected with shuttle vector plasmids containing genes for WT or modified SARS-CoV-2 spike proteins. Untransfected cells served as negative controls, and cells transfected with a plasmid expressing HIV-1 Env protein were used as positive controls for transfection. Spike and Env expression was measured by flow cytometry using SARS-CoV-2 spike protein-specific and HIV Env-specific antibodies (VRC01), respectively. As shown in Figure 1, SARS-CoV-2 spike protein expression in transfected A549 cells was reduced with shortening of the tail and shortening of the endocytosis motif compared to the wild-type spike protein.

[0124] Nucleic acid sequences encoding WT, PP or TT SARS-CoV-2 spike proteins were inserted into the E3 region of a replicative Ad4 vector with deletions of the E3 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K ORFs described above. The nucleotide sequence of recombinant Ad4 containing the WT spike protein coding sequence is shown herein as SEQ ID NO:1. Expression of WT, stabilized and truncated spike proteins in recombinant Ad4-infected A549 cells was evaluated. Replicating Ad4 with the WT spike nucleic acid sequence (nCoV-WT), PP stabilized spike nucleic acid sequence (nCov-PP) or tail truncated spike nucleic acid sequence (nCov-TT) was used to infect A549 cells. Replicating adenovirus expressing HIV-1 Env protein (FDE3) was used as a positive control for infection, and uninfected (unIF) cells were used as a negative control. Spike protein expression was measured by flow cytometry using a SARS-CoV-2 spike protein specific antibody. Antibody VRC01 was used to detect HIV-1 Env expression. Spike protein expression from Ad4-Spike 2 days after infection is shown in Figure 2A. In Figure 2B, expression of PP stabilized and truncated spike proteins is shown. As shown in Figures 2A-2B, spike protein expression from both nCoV-WT and nCoV-PP constructs was high.

[0125] Example 2: Immunogenicity of Ad4-Spike (WT) in rabbits The immunogenicity of Ad4-Spike (expressing the WT spike protein sequence of SEQ ID NO:2) was tested in New Zealand White rabbits. Although rabbits and other laboratory animals do not replicate Ad4 virus, intramuscular administration (IM) is commonly used as a screen for immunogenicity. Rabbits were inoculated with 1.29 x 10 of purified replicating Ad4-Spike. 9Infectious units (IFU) were administered IM on days 0 and 28. Using a luciferase assay, serum neutralization against the Wuhan SARS-CoV-2 pseudovirus was detected at 4 weeks (before the second immunization) and continued to increase throughout the 12-week study period.

[0126] Example 3: Immunogenicity study in hamsters Human adenoviruses are capable of infecting Syrian golden hamsters (van der Lubbe et al., NPJ Vaccines 6(1):39, 2021). Therefore, immunogenicity studies were performed in these animals. WuPP ) of 10 2 ~10 7 Dose-escalation of infection-forming units (IFU) was performed. Strong serum neutralization was observed in the Ad4-SARS-CoV-2 WuPP At the highest dose, increased expression of IgG1 was observed at 4 weeks (Figure 5A) and 8 weeks (Figure 5B) in the lentiviral pseudotype assay.

[0127] These results suggested that hamsters were semi-permissive for Ad4 but replicated the virus sufficiently to induce serum neutralizing antibodies. Spike-specific IgA and IgG were also observed in nasal washes at day 60.

[0128] Hamsters were then immunized with intranasal Ad4 expressing a stabilized (double proline substituted -PP) spike protein derived from a variant of concern (VOC). Ad4-CoV2-Wuhan, Ad4-CoV2-SA (beta), Ad-CoV2-Wu / RBD-SA, Ad4-CoV2-India (delta) and Ad4-CoV2-Brazil (gamma) were included in the study. Ad4 expressing influenza virus H5 hemagglutinin (Ad4-H5) and mock inoculation were included as negative controls.

[0129] Serum neutralization against Wuhan, Delta and Omicron pseudoviruses was determined 28 and 56 days after intranasal challenge. The results are shown in Figures 6A-6E. Ad4 expressing Wuhan-PP (SEQ ID NO: 3) or Delta-PP (SEQ ID NO: 9) were the most immunogenic.

[0130] Example 4: Challenge study in hamsters This example describes a study testing a candidate vaccine in the Syrian golden hamster model.

[0131] In this study, Syrian golden hamsters were administered 10 mL of the immunogenic candidates identified in Example 3 (candidate 1 or candidate 2). 7 The animals are administered intranasally at a dose of 100 mg / kg / day (IFU) and then challenged with SARS-CoV-2 by cohabiting with SARS-CoV-2 delta- or omicron-infected animals (van Doremalen et al., Sci Transl Med 13(607):eabh0755, 2021). Table 1 shows the groups of animals used. Animals in group A are challenged 60 days after immunization, while animals in group B are challenged 6 months after immunization. Hamsters receiving intranasal administration of Ad4-H5-Vtn are included as negative controls. Pfizer mRNA or Ad26-Spike are administered intramuscularly as comparisons. [Table 1]

[0132] The intranasal Ad4-Spike vaccine is predicted to confer systemic neutralizing antibodies of the same order of magnitude as mRNA or Ad26, but that are more durable. It is also predicted that Ad4-Spike will cause greater restriction of the challenge virus compared to parenterally administered vaccines.

[0133] Example 4: Human Clinical Trials A phase 1 / 2 open-label study of a single dose of intranasally administered Ad4-Spike in healthy volunteers will be conducted. Enrollment will begin with volunteers who may or may not have had coronavirus disease 2019 (COVID-19) or have been vaccinated. The selected international setting is one where COVID-19 vaccine supply is limited and SARS-CoV-2-free volunteers may be more easily enrolled. All SARS-CoV-2-free participants will be offered the emergency use authorization (EUA) vaccine upon study completion or if their neutralization titers fall below approximately 40 at the lower interquartile range of the Moderna mRNA 1272 vaccine after 6 months. Each study participant will receive a single dose of intranasal Ad4-SARS-CoV-2 vaccine or intramuscular (IM) immunization with a licensed or approved booster. Study participants will be monitored for adverse events (AEs) and blood and respiratory secretions will be collected periodically throughout the study for immunogenicity and safety testing. Nasal swabs will be collected to monitor adenovirus shedding and nasal washes will be collected to monitor mucosal immune responses. Household and close contacts who wish to participate will also be enrolled and monitored for transmission of vaccine virus by serology.

[0134] The primary endpoint is safety, measured by the frequency and grade of solicited and unsolicited adverse events during the first 28 days after vaccination. Safety will be assessed by evaluating the incidence, severity, and type of adverse events separately in the candidate vaccine arm of the trial over the duration of follow-up. It is expected that 21% (N=10 / 48) of vaccine recipients may experience vaccine-related signs and symptoms (e.g., headache fatigue, myalgia, rhinorrhea, nausea, diarrhea). Vaccine virus shedding will be assessed by documenting the presence, amount, and duration of shed virus in serially collected nasal wash samples.

[0135] A secondary endpoint is immunogenicity. Immunogenicity will be assessed in serially collected serum, nasal and fecal samples. Immunogenicity will be determined by lentivirus-based pseudovirus neutralization assays, including functional antibodies as measured by B cell clones, complement and antibody-dependent enhancement, characterization of mucosal and T cell immunity. Respiratory mucosal responses are emerging following COVID-19 infection and are therefore predicted to be a distinguishing feature of Ad4-Spike vaccines. If an Ad4-vectored SARS-CoV-2 vaccine is "taken" in 95% of recipients and immunogenic to adenovirus 4 and SARS-CoV-2 spike proteins in 90% of these recipients, systemic immune responses are predicted to be induced in 85% (N=44 / 52) of vaccine recipients and mucosal responses in 90-100% of volunteers.

[0136] In the rare case that there is no evidence of vaccination at 30 days, a second dose at 60 days will be administered. However, the primary analysis will be after one dose, because this vaccine is expected to be a single-dose regimen. Most participants in previous Ad4-based vaccine trials did not develop a higher response after the second immunization. The second dose only induces a response in the rare case that the participant is not infected with the first dose.

[0137] Volunteers were not prescreened for serum antibodies, and a subset of them were seropositive at baseline for Ad4 (approximately 30%, N=20 / 60) as a result of exposure to circulating wild-type adenovirus. Responses in individuals with pre-existing Ad4 immunity in previous vectored vaccine trials suggest that Ad4 immunity may modulate responses to the vector, limiting viral shedding, although vector-specific immunity may still be induced.

[0138] Participants will be monitored for safety and immunogenicity over a one-year period. The Phase 1 trial will include a parallel exploratory arm designed to allow for the use of Ad4-Spike with other SARS-CoV-2 Spike immunogens (e.g., DNA, mRNA, or protein vaccines) as needed. Ad4-Spike is predicted to confer greater persistence and mucosal T and B cell responses compared to non-replicating parenterally administered protein or nucleic acid vaccines.

[0139] The target study population excludes only those who may be adversely affected by respiratory viral infections (e.g., pregnant women or those with severe immunodeficiency). Symptoms of recombinant Ad4 vaccination, if they occur, tend to be mild and self-limited. Those people who do not have difficulty dealing with upper respiratory tract infections should not experience severe symptoms with the Ad4-Spike vaccine. Pre-existing immunity to Ad4 is not uncommon (30%), but it is largely overcome by intranasal vaccination. The extent to which vector-specific immunity is overcome will be assessed and is expected to be a function of vaccine virus replication and the immunogenicity of the spike protein. Prevalence of Ad4 antibodies in individuals under 16 years of age is extremely low, making this vaccine a very attractive modality for inducing durable immunity in school aged children. The primary endpoints are safety and immunogenicity. Safety will be ultimately addressed in phase 2 of the trial if the primary endpoint is achieved.

[0140] When previous Ad4 recombinant virus vaccines were given intranasally, the virus replicated at low levels for 2–4 weeks. However, viral shedding detected by viral culture was very low, with a median of 1 day. Participants are advised to avoid close contact for 14 days after vaccination. For these reasons, transmission of vaccine virus to household and close contacts has not been observed. The majority of vaccines are asymptomatic. However, the most common adverse events (AEs) were throat discomfort and nasal congestion in 25% of participants, none above grade 2. Recombinant Ad4 containing the SARS-CoV-2 spike protein is predicted to produce similar outcomes to previous Ad4-based intranasally administered vaccines.

[0141] Phase 3 trials and / or challenge trials will follow Phase 2.

[0142] In view of the many possible embodiments to which the principles of the subject matter of this disclosure may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the disclosure and should not be understood as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.

Claims

1. 1. A recombinant adenovirus type 4 (Ad4) expressing SARS-CoV-2 spike (S) protein, comprising: The amino acid sequence of the S protein is at least 95% identical to SEQ ID NO:2; the recombinant Ad4 is replication competent; and The genome of said recombinant Ad4 comprises a deletion in the adenovirus E3 region and an insertion of the coding sequence for the SARS-CoV-2 S protein.

2. The recombinant Ad4 of claim 1, wherein the amino acid sequence of the S protein is at least 99% identical to SEQ ID NO:

2.

3. 3. The recombinant Ad4 of claim 1 or claim 2, wherein the amino acid sequence of the S protein comprises or consists of SEQ ID NO:

2.

4. 2. The recombinant Ad4 of claim 1, wherein the amino acid sequence of the S protein contains at least one modification to stabilize the protein in a prefusion conformation.

5. The recombinant Ad4 of claim 4, wherein the at least one modification comprises a K986P and a V987P substitution.

6. 6. The recombinant Ad4 of claim 4 or claim 5, wherein the amino acid sequence of the S protein comprises or consists of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:

12.

7. The recombinant Ad4 of any one of claims 1 to 6, wherein the deletions in the E3 region include deletions of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K open reading frames (ORFs).

8. The recombinant Ad4 of any one of claims 1 to 7, wherein the coding sequence for the SARS-CoV-2 S protein is inserted in place of the deleted E3 region.

9. The recombinant Ad4 of any one of claims 1 to 8, wherein the S protein is encoded by a codon-optimized nucleic acid sequence.

10. 10. The recombinant Ad4 of claim 9, wherein the codon-optimized nucleic acid sequence comprises or consists of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:

19.

11. The recombinant Ad4 according to any one of claims 1 to 3, wherein the nucleotide sequence of said genome is at least 95% identical to SEQ ID NO:

1.

12. The recombinant Ad4 according to any one of claims 1 to 3, wherein the nucleotide sequence of said genome is at least 99% identical to SEQ ID NO:

1.

13. The recombinant Ad4 according to any one of claims 1 to 3, wherein the genomic nucleotide sequence comprises or consists of SEQ ID NO:

1.

14. A recombinant adenovirus type 4 (Ad4) vector comprising a deletion in the adenovirus E3 region and an insertion of a coding sequence for the SARS-CoV-2 S protein, wherein the amino acid sequence of the S protein is at least 95% identical to SEQ ID NO:

2.

15. 15. The recombinant Ad4 vector of claim 14, wherein the amino acid sequence of the S protein is at least 99% identical to SEQ ID NO:

2.

16. 16. The recombinant Ad4 vector of claim 14 or claim 15, wherein the amino acid sequence of the S protein comprises or consists of SEQ ID NO:

2.

17. 15. The recombinant Ad4 vector of claim 14, wherein the amino acid sequence of the S protein comprises at least one modification to stabilize the protein in a prefusion conformation.

18. 18. The recombinant Ad4 of claim 17, wherein the at least one modification comprises a K986P and a V987P substitution.

19. 19. The recombinant Ad4 of claim 17 or claim 18, wherein the amino acid sequence of the S protein comprises or consists of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:

12.

20. The recombinant Ad4 vector of any one of claims 14 to 19, wherein the deletions in the E3 region include deletions of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K open reading frames (ORFs).

21. The recombinant Ad4 vector of any one of claims 14 to 20, wherein the coding sequence for the SARS-CoV-2 S protein is inserted in place of the deleted E3 region.

22. The recombinant Ad4 vector of any one of claims 14 to 21, wherein the S protein is encoded by a codon-optimized nucleic acid sequence.

23. 23. The recombinant Ad4 vector of claim 22, wherein the codon-optimized nucleic acid sequence comprises or consists of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:

19.

24. The recombinant Ad4 vector of any one of claims 14 to 16, wherein the nucleotide sequence of the vector is at least 95% identical to SEQ ID NO:

1.

25. The recombinant Ad4 vector of any one of claims 14 to 16, wherein the nucleotide sequence of the vector is at least 99% identical to SEQ ID NO:

1.

26. The recombinant Ad4 vector of any one of claims 14 to 16, wherein the nucleotide sequence of said vector comprises or consists of SEQ ID NO:

1.

27. An immunogenic composition comprising the recombinant Ad4 of any one of claims 1 to 13 or the recombinant Ad4 vector of any one of claims 14 to 26 and a pharma- ceutically acceptable carrier.

28. A composition comprising a recombinant Ad4 according to any one of claims 1 to 13, a recombinant replicable Ad4 vector according to any one of claims 14 to 26, or an immunogenic composition according to claim 27, for inducing an immune response against SARS-CoV-2 in a subject.

29. A composition comprising a recombinant Ad4 according to any one of claims 1 to 13, a recombinant replicable Ad4 vector according to any one of claims 14 to 26, or an immunogenic composition according to claim 27, for immunizing a subject against SARS-CoV-2 infection.

30. A composition or immunogenic composition described in claim 28 or 29, characterized in that administration of the composition or immunogenic composition includes intranasal administration.

31. 31. The composition or immunogenic composition of claim 30, wherein intranasal administration comprises administration of an aerosol comprising particles greater than 10 microns in diameter.

32. about 10 ４ ~about 10 ６ A composition or immunogenic composition according to any one of claims 28 to 31, characterized in that it is administered in a dose of recombinant Ad4 particles.

33. about 10 ５ 33. The composition or immunogenic composition of claim 32, administered in a dose of recombinant Ad4 particles.

34. A composition or immunogenic composition according to any one of claims 28 to 33, characterized in that it is administered in a single dose.

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