A vaccine composition comprising an adenoviral vector and an adenoviral vector delivery enhancing agent

Abstract

The present invention is directed to a vaccine composition comprising i) an adenoviral vector delivery enhancing agent selected from the group consisting of SYN3, and SYN3 analogs, and ii) an adenoviral vector comprising, at an insertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism. The present invention is also directed to a method for delivering an adenoviral vector to a mammalian subject, said method comprising a step of administering onto the nasal and / or oral mucosa of the subject a dose of a delivery enhancing agent selected from the group consisting of SYN3 and SYN3 analogs and a dose of an adenoviral vector, wherein the first dose of said delivery enhancing agent and the first dose of said adenoviral vector are administered simultaneously or sequentially.

Description

A vaccine composition comprising an adenoviral vector and an adenoviral vector delivery enhancing agent FIELD

[0001] The present invention relates to vaccine compositions comprising an adenoviralvector and an adenoviral vector delivery enhancing agent as well as to methods for deliveringan adenoviral vector to a mammalian subject. In particular, the present invention relates to a vaccine composition against viral or bacterial infections and its combined mucosal delivery with SYN3 or SYN3 analogs. BACKGROUND

[0002] Adenoviruses have been widely studied as infectious agents, as a subject for basicresearch, and for their potential use in gene therapy and vaccines. Many human adenoviralserotypes have been identified and they are categorized into six subgenera (A through G) based on biological properties. For example, group A includes serotypes 12 and 31, group B includes serotypes 3 and 7, group C includes serotypes 2 and 5, group D includes serotypes 8 and 26, group E includes serotype 4, group F includes serotypes 40 and 41, and group G includesserotype 52. In addition, multiple gorilla and chimpanzee adenoviruses have been widelystudied for their potential use in gene therapy and vaccines.

[0003] Adenoviral vectors allow for the transmission of the transgene they carry into thehost nucleus but do not integrate viral DNA into the host chromosome. Additionally, adenoviralvectors have emerged as a promising vaccine delivery vehicle due to their ability to induce bothinnate and adaptive immune responses; having the capacity to induce potent antigen-specific B and T cell immune responses. Adenoviral vectors are highly immunogenic and are efficient in delivering antigens.

[0004] [N-(3-cholamidopropyl)-N-(3-lactobionamidopropyl)]-cholamide (SYN3) is asurfactant-like molecule that is known to enhance transduction of recombinant adenoviral genevectors for treatment of tumors, specifically bladder tumor (see EP1456377).SUMMARY OF THE INVENTION

[0005] The invention is defined by the features of the independent claims. Some specificembodiments are defined in the dependent claims.

[0006] According to a first aspect of the present invention, there is provided a vaccinecomposition comprising i) an adenoviral vector delivery enhancing agent selected from thegroup consisting of SYN3, and SYN3 analogs, and ii) an adenoviral vector comprising, at aninsertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism.

[0007] According to a second aspect of the present invention, there is provided a methodfor delivering an adenoviral vector to a mammalian subject, said method comprising a step of administering onto the nasal and / or oral mucosa of the subject a dose of a delivery enhancing agent selected from the group consisting of SYN3 and SYN3 analogs and a dose of anadenoviral vector, wherein the first dose of said delivery enhancing agent and the first dose ofsaid adenoviral vector are administered simultaneously or sequentially.

[0008] According to a third aspect of the present invention, there is provided a kitcomprising at least two containers, wherein a first container comprises a delivery enhancing agent selected from the group consisting of SYN3 and SYN3 analogs, and a second container comprises an adenoviral vector, wherein said adenoviral vector comprises, at an insertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGURE 1. Pre-treatment with Syn3 enhances intranasal adenovector SARS-CoV-2 vaccine immunogenicity. A) Pre-treatment with Syn3 enhances the immunogenicity of intranasally administered adenovector SARS-CoV-2 vaccine in a dose-dependent manner, with no clinical signs of toxicity. Spike-specific serum IgG levels were analyzed 21 days post- vaccination. B) Dodecyl Maltoside induces severe toxicity at higher concentrations (5mg / ml), but not at lower doses (1mg / ml), which result in minor enhancement of immunogenicity.

[0010] FIGURE 2. Syn3 enhances the immunogenicity of an intranasal adenovectorSARS-CoV-2 vaccine when formulated in a vaccine formulation buffer. The inclusion of Syn3 in the vaccine formulation enhances the immunogenicity of the intranasally administered adenovector SARS-CoV-2 vaccine in a dose-dependent manner, with no clinical signs oftoxicity. Spike-specific serum IgG levels were analysed 21 days post-vaccination. The controlgroup, which did not receive Syn3 in the vaccine formulation buffer, showed no signs of immunogenicity at the very low dose used in this experiment. Similarly, a Syn3 concentrationof 1 mg / mL was insufficient to induce immunogenicity when included in the vaccine formulation buffer and co-administered with the vaccine. EMBODIMENTS

[0011] DEFINITIONS

[0012] In the present context, the term “vector” is used to refer to a nucleic acid moleculecapable of mediating entry of, e.g., transferring, transporting, etc., another nucleic acid molecule into a cell. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids, cosmids, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses, adenoviruses, adeno-associated viruses, poxviruses and lentiviruses. As will be evident to one of ordinary skill in the art, viral vectors may include various viral components in addition to nucleic acid(s) that mediate entry of the transferred nucleic acid. Thus, the term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself.

[0013] The term “signal sequence” means herein a signal peptide that is a part of the N-terminus of a secretory protein that is secreted outside a cell and thus passes through the cell membrane. The signal peptide is usually composed of approximately 10 to 30 amino acids, andis subsequently cleaved and removed by a protease specific for the cell membrane, and only thesecretory protein is transferred outside the cell. Signal peptides serve as targeting signals, enabling cellular transport machinery to direct proteins to specific intracellular or extracellular locations. To date, more than 4000 signal peptides present in eukaryotic cells are known. DNA libraries encoding signal peptides are disclosed, e.g., in WO2021045541A1 and KR20210028116A.

[0014] The term “transmembrane domain” refers herein to a hydrophobic alpha helixstructure that transverses the host cell membrane. The transmembrane domain may be directly fused to the C-terminal part of the fusion protein encoded by a vector. For example, thetransmembrane domain may be derived from an integral membrane protein (e.g., receptor,cluster of differentiation molecule, enzyme, transporter, cell adhesion molecule, or the like). Aparticular example is the transmembrane domain derived from Type 1 transmembrane proteinssuch as human VCAM-1 protein (vascular cell adhesion molecule 1). Type I transmembraneproteins are anchored to the lipid membrane with a stop-transfer anchor sequence and have their N-terminal domains targeted to the extracellular space, when a mature form of the protein is located on the cell membrane.

[0015] [N-(3-cholamidopropyl)-N-(3(lactobionamidopropyl)]-cholamide is the genericname of the compound this is also referred to by the name "SYN3". The terms are usedinterchangeably throughout the description.

[0016] SYN3 has the CAS Registry No. 2127407-44-5. The compound has a secondgeneric name NODA: (N-[3-[(4-O-β-D-galactopyranosyl-D-gluconoyl)amino]propyl]-3,7,12-trihydroxy-N[3-[[(3α,5β,7α,12α)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino]propyl]-, (3α,5β,7α,12α)-cholan-24-amide). As will be apparent to those of skill in the art, SYN3 exists in various optical, tautomeric, stereoisomeric and isomeric forms. Buffered and lyophilized aqueous formulations as well as nonaqueous solution formulations of SYN3 are known.

[0017] SYN3 is a surfactant-like polyamide molecule that is known to enhancetransduction of recombinant adenoviral gene vectors for treatment of tumors, more specificallyin bladder tumors. SYN3 can be present in a concentration of from about 0.001 mg / ml to about 150 mg / ml.

[0018] Examples of SYN3 analogs, such as Syn3-Mel and Syn3-Suc, are disclosed inConnor et al., 2001, Gene Therapy 8:41-48. Syn3-Mel as described by Connor et al., is one ofthe preferred SYN3 analogs in the present invention.

[0019] A "delivery-enhancing agent" refers herein to any agent which enhances deliveryof an adenoviral vector. Such enhanced delivery may be achieved by various mechanisms. Onesuch mechanism may involve the disruption of the protective glycosaminoglycan layer on theepithelial / mucosal surface. SYN3 and its analogs are examples of preferred delivery-enhancingagents in the present invention.

[0020] In an embodiment, the delivery-enhancing agent is included in the buffer in whichthe recombinant adenoviral vector delivery system is formulated. The delivery-enhancing agentmay be administered prior to the recombinant virus or concomitant with the virus. In someembodiments, the delivery-enhancing agent is provided with the virus by mixing a virus preparation with a delivery-enhancing agent formulation just prior to administration to the patient. In other embodiments, the delivery-enhancing agent and virus are provided in a single vial for administration.

[0021] In the case of a vaccine composition comprising a recombinant adenoviral vectordelivery system formulated in a buffer which further comprises a delivery-enhancing agent, the delivery-enhancing agent may be administered prior to administration of the recombinantadenoviral vector delivery system. The prior administration of the delivery-enhancing agentmay be in the range of about 30 seconds to 2 hours, preferably about 15 minutes to 90 minutes,and preferably about 30 minutes to 60 minutes prior to administration of the adenoviral vectordelivery system.

[0022] Accordingly, the present invention is providing a vaccine composition comprisingi) an adenoviral vector delivery enhancing agent selected from the group consisting of SYN3, and SYN3 analogs, and ii) an adenoviral vector comprising, at an insertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism.

[0023] In a preferred embodiment, said immunogenic protein is a polypeptide derivedfrom, or expressed by a pathogenic microbe or a modified polypeptide thereof.

[0024] In another preferred embodiment, said pathogenic microbe is selected from thegroup consisting of: influenza virus, respiratory syncytial virus, coronavirus andMycobacterium tuberculosis.

[0025] In another preferred embodiment, said adenoviral vector comprises humanadenovirus serotype 26 or serotype 5 backbone.

[0026] In another preferred embodiment, said adenoviral vector is a non-replicatinghuman adenovirus serotype 5 vector.

[0027] In another preferred embodiment, said adenoviral vector comprises simian orgorilla adenovirus backbone.

[0028] In another preferred embodiment, said pathogenic microbe is from the genusCoronaviridae such as SARS-CoV-2 coronavirus.

[0029] In another preferred embodiment, said adenoviral vector comprises, at aninsertion site in the viral genome, a nucleic acid sequence encoding a modified extracellular domain of the SARS-CoV-2 spike protein, wherein said modified extracellular domaincomprises an N-terminal signal peptide causing the spike protein to enter a secretory system ina host cell, wherein said modified extracellular domain comprises a C-terminal deletion of atleast of heptad repeat 2 (HR2), transmembrane segment (TM) and cytoplasmic tail (CT) of the spike protein.

[0030] In another preferred embodiment, the deletion site of said C-terminal deletion islocated at the region corresponding to positions 1030-1157 of the omicron BA.5 strain spike protein of SEQ ID NO:1.

[0031] The vaccine composition of the present invention has been developed to suite asa booster vaccine for those who have received full vaccination cycle (1 or more doses) of forexample Comirnaty, Spikevax, or ADZ1222 as well as a primary vaccine for those who havenot yet received any vaccine against SARS-CoV-2 infection. The present viral vector in saidvaccine produces a secreted version of a C-terminally truncated SARS-CoV-2 spike protein. In a preferred embodiment, the C-terminally truncated spike protein comprises mutated furin cleavage site with single-amino acid modifications R677G, R678S and R680S (correspondingto positions 685, 686 and 688 in SEQ ID NO:2, respectively). In an embodiment, the C-terminally truncated spike protein has been modified by proline substitutions at residues 981and 982 (corresponding to positions 989 and 990 in SEQ ID NO:2, respectively).

[0032] The spike protein mediates the entry of SARS-CoV-2 into the host cell and is themain target of neutralizing antibodies arising in infected individuals, and a predominant target of antibody-mediated immunity (Chaudhary et al., 2021). Therefore, apart from inactivated and attenuated whole virus approaches, virtually all ongoing COVID-19 vaccine projects use the spike protein as the immunogen. All COVID-19 vaccines authorized in EU and in the United States are based on the spike protein of SARS-CoV-2.

[0033] The omicron BA.5 strain spike protein has the following amino acid sequence(SEQ ID NO:1): MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVT 60 WFHAISGTNGIKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATN 120 VVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN 180 FKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALH 240 RSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS 300 FTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADY 360 SVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKL 420 PDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAG 480 VNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG 540LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS 600 NQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIP 660 IGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTE 720 ILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQV 780 KQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIA 840 ARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYR 900 FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQ 960 LSSKFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLA 1020 ATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG 1080 KAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPEL 1140 DSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK 1200 YEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL 1260 KGVKLHYT 1268

[0034] In the above sequence (SEQ ID NO:1), amino acid positions 1-13 correspond toa signal sequence, amino acids 14-1203 correspond to the extracellular domain (ectodomain) of the spike protein, amino acids 1209-1229 correspond to the transmembrane domain of the spike protein and amino acids 1230-1268 correspond to cytoplasmic region of the spike protein.

[0035] In a preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein has the following amino acid sequence (SEQ ID NO:2): MSRLPVLLLLQLLVRPGLQAPQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLF 60 LPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSL 120 LIVNNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLM 180 DLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITR 240 FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLS 300 ETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKR 360 ISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGN 420 IADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGN 480 KPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 540 CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSV 600 ITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVN 660 NSYECDIPIGAGICASYQTQTKSHGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTN 720 FTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKN 780 TQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY 840GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP 900 FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQ 960 ALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAE 1020 IRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTT 1080 APAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTV 1140 YD 1142

[0036] In the above sequence (SEQ ID NO:2), amino acid positions 1-19 correspond toa signal sequence, and amino acids 22-1142 correspond to the extracellular domain (ectodomain) of the spike protein with a deletion at the C-terminal end. In another embodiment, the C-terminal deletion site is between the connector domain, CD, (amino acids 1030-1062 of SEQ ID NO:1) and the heptad repeat 2, HR2 (amino acids 1158-1197 of SEQ ID NO:1). In another embodiment, the C-terminal deletion site is in the connector domain, CD, (amino acids1030-1062 of SEQ ID NO:1). In another preferred embodiment, the deletion site of said C-terminal deletion is located at the region corresponding to positions 1030-1157 of the omicronBA.5 strain spike protein of SEQ ID NO:1.

[0037] In another preferred embodiment, said C-terminal deletion comprises part but notall of connector domain (CD) of the spike protein.

[0038] In another preferred embodiment, said N-terminal signal peptide is the humaninterleukin-3 secretion signal peptide or the natural signal peptide of the SARS-CoV-2 spike protein.

[0039] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises a mutated furin cleavage site with single amino acid modifications in positions R677G, R678S and R680S, wherein said positions corresponds tothe respectively numbered amino acid positions of the omicron BA.5 strain spike protein ofSEQ ID NO:1.

[0040] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2, amino acids 22-1142 of SEQ ID NO:2, or a sequence which has at least 80 % sequence identity with the sequence of SEQ ID NO:2, or with amino acids 22-1142 of SEQ ID NO:2.

[0041] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises at least N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), and central helix (CH).

[0042] In another preferred embodiment, said nucleic acid sequence encoding saidmodified extracellular domain is as set forth in SEQ ID NO:3, or a nucleotide sequence having at least 70%, 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence as set forth in SEQ ID NO:3.

[0043] The term “sequence identity,” in the context of two or more amino acid sequences,refers to two or more sequences or subsequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues that are the same (i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0044] Two examples of algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915- 10919] alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of bothstrands. For short amino acid sequences, PAM30 scoring matrix can be applied.

[0045] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises in the C-terminal end one or more further receptor binding domains (RBD) from SARS-CoV-2 spike proteins which are immunologically distinctfrom said modified extracellular domain of the SARS-CoV-2 spike protein.

[0046] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises in the C-terminal end a trimerization domain, preferably T4 fibritin trimerization domain, human collagen trimerization domain, leuzine zipper domainor any other additional C-terminal amino acid sequence promoting trimerization of said spike protein.

[0047] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises in the C-terminal end a transmembrane domain, or any other additional C-terminal amino acid sequence promoting transmembrane anchoring of said spike protein.

[0048] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises in the C-terminal region in any order i) a trimerization domain, preferably T4 fibritin trimerization domain, human collagen trimerization domain, leuzine zipper domain or any other additional C-terminal amino acid sequence promoting trimerization of said spike protein, and ii) a transmembrane domain, or any other additional C- terminal amino acid sequence promoting transmembrane anchoring of said spike protein.

[0049] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises in the C-terminal end in any order i) one or more receptor binding domains (RBD) from SARS-CoV-2 spike proteins which are immunologically distinctfrom said modified extracellular domain of the SARS-CoV-2 spike protein, and ii) atrimerization domain, preferably T4 fibritin trimerization domain, human collagen trimerization domain, leuzine zipper domain or any other additional C-terminal amino acid sequence promoting trimerization of said spike protein.

[0050] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises in the C-terminal end in any order i) one or more receptor binding domains (RBD) from SARS-CoV-2 spike proteins which are immunologically distinctfrom said modified extracellular domain of the SARS-CoV-2 spike protein, and ii) atransmembrane domain, or any other additional C-terminal amino acid sequence promoting transmembrane anchoring of said spike protein.

[0051] In another preferred embodiment, said modified extracellular domain of theSARS-CoV-2 spike protein comprises in the C-terminal end in any order i) one or more receptor binding domains (RBD) from SARS-CoV-2 spike proteins which are immunologically distinctfrom said modified extracellular domain of the SARS-CoV-2 spike protein, ii) a trimerizationdomain, preferably T4 fibritin trimerization domain, human collagen trimerization domain, leuzine zipper domain or any other additional C-terminal amino acid sequence promotingtrimerization of said spike protein, and iii) a transmembrane domain or any other additional C- terminal amino acid sequence promoting transmembrane anchoring of said spike protein.

[0052] In another preferred embodiment, said adenoviral vector delivery enhancing agentis SYN3 or a SYN3 analog.

[0053] In another preferred embodiment, said vaccine is in a mucosal administrationformulation.

[0054] In another preferred embodiment, said mucosal administration formulation isselected from the group consisting of nasal drops, aerosols, sprays, powder sprays, gels, microspheres, liposomes, membranes, and suspensions.

[0055] In another preferred embodiment, said vaccine composition is a spray or sprayablecomposition in a liquid dosage form.

[0056] In another preferred embodiment, the vaccine composition further comprises apharmaceutically-acceptable adjuvant, carrier, diluent or excipient.

[0057] In another preferred embodiment, the vaccine composition comprises a buffercontaining Tris, sodium chloride, magnesium chloride, histidine, sucrose, polysorbate-80,EDTA, and ethanol.

[0058] In another preferred embodiment, said buffer comprises 5-15 mM Tris-HCl, 50-100 mM NaCl, 0.5-2 mM MgCl2, 5-15 mM histidine, 2%-8% (wt / vol) sucrose, 0.02%-1% (wt / vol) polysorbate-80, 0.05-05 mM EDTA, and 0.25%-1% (vol / vol) ethanol. Preferably, thebuffer contains 0.05%-0.5% (wt / vol) polysorbate-80, more preferably 0.2%-0.5% (wt / vol). Theeffect of higher concentration (i.e. > 0.02%) of polysorbate-80 is improved solubility of SYN3 in the buffer.

[0059] In another preferred embodiment, said buffer contains 10 mM Tris-HCl, 75 mMNaCl, 1 mM MgCl2, 10 mM histidine, 5% (wt / vol) sucrose, 0.2%-0.5% (wt / vol) polysorbate- 80, 0.1 mM EDTA, and 0.5% (vol / vol) ethanol.

[0060] In another preferred embodiment, said buffer is the A195 buffer containing 10mM Tris-HCl at a pH of 7.4, 75 mM NaCl, 1 mM MgCl2, 10 mM histidine, 5% (wt / vol) sucrose,0.02% polysorbate-80 (wt / vol), 0.1 mM EDTA, and 0.5% (vol / vol) ethanol.

[0061] In another preferred embodiment, said composition is to be administeredintranasally.

[0062] In an embodiment, the present invention is also providing a method for deliveringan adenoviral vector to a mammalian subject, said method comprising a step of administering onto the nasal and / or oral mucosa of the subject a dose of a delivery enhancing agent selected from the group consisting of SYN3 and SYN3 analogs and a dose of an adenoviral vector, wherein the first dose of said delivery enhancing agent and the first dose of said adenoviral vector are administered simultaneously or sequentially.

[0063] In a preferred embodiment, said adenoviral vector comprises, at an insertion sitein the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism.

[0064] In another preferred embodiment, said immunogenic protein is a polypeptidederived from, or expressed by a pathogenic microbe or a modified polypeptide thereof.

[0065] In another preferred embodiment, said pathogenic microbe is selected from thegroup consisting of: influenza virus, respiratory syncytial virus, coronavirus andMycobacterium tuberculosis.

[0066] In another preferred embodiment, said adenoviral vector comprises humanadenovirus serotype 26 or serotype 5 backbone.

[0067] In another preferred embodiment, said adenoviral vector is a non-replicatinghuman adenovirus serotype 5 vector.

[0068] In another preferred embodiment, said adenoviral vector comprises, at aninsertion site in the viral genome, a nucleic acid sequence encoding a modified extracellulardomain of the coronavirus spike protein such as the SARS-CoV-2 spike protein, wherein saidmodified extracellular domain comprises an N-terminal signal peptide causing the spike proteinto enter a secretory system in a host cell, wherein said modified extracellular domain comprisesa C-terminal deletion of at least of heptad repeat 2 (HR2), transmembrane segment (TM) and cytoplasmic tail (CT) of the spike protein.

[0069] In another preferred embodiment, the first dose of said delivery enhancing agentand the first dose of said adenoviral vector are administered simultaneously.

[0070] In another preferred embodiment, a vaccine composition of the present inventionis administered to said subject.

[0071] In another preferred embodiment, the first dose of said delivery enhancing agentand the first dose of said adenoviral vector are administered sequentially within 24 hours, preferably within 1-4 hours from the first dose of either said delivery enhancing agent or said adenoviral vector.

[0072] In another preferred embodiment, said adenoviral vector or said vaccinecomposition is administered by spraying a liquid dosage form of said adenoviral vector or vaccine composition onto the nasal and / or oral mucosa of the subject.

[0073] In an embodiment, the present invention further provides a kit comprising at leasttwo containers, wherein a first container comprises a delivery enhancing agent selected from the group consisting of SYN3 and SYN3 analogs, and a second container comprises an adenoviral vector, wherein said adenoviral vector comprises, at an insertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism.

[0074] It is to be understood that the embodiments of the invention disclosed are notlimited to the particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0075] Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

[0076] As used herein, a plurality of items, structural elements, compositional elements,and / or materials may be presented in a common list for convenience. However, these listsshould be construed as though each member of the list is individually identified as a separateand unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example ofthe present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another, but are to be considered as separate and autonomous representations of the present invention.

[0077] Furthermore, the described features, structures, or characteristics may becombined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of lengths, widths, shapes, etc., to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well- known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

[0078] While the forgoing examples are illustrative of the principles of the presentinvention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below.

[0079] The verbs “to comprise” and “to include” are used in this document as openlimitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality. EXPERIMENTAL SECTION EXAMPLE 1 Reagents Two chemical compounds, Syn3 and Dodecyl Maltoside (DM), was evaluated astransduction-enhancing pretreatment excipient for FCV2.1 vaccination, which is based on anon-replicating Ad5 vector expressing a modified extracellular domain (ectodomain) of theSARS-CoV-2 spike protein from the omicron BA.5 strain (Fig. 1). FINCoVac 2.1 is designedfor intranasal administration (see WO2024223994), and when administered into the nasalcavity, it was anticipated that the encoded modified spike ectodomain will generate a hostimmune reaction leading to immunity to SARS-CoV-2. Cloning of truncated SARS-CoV-2 spike ectodomain into pRLF1 for obtaining non- replicating adenoviral vaccine vector plasmid pFCV2.1The truncated spike ectodomain gene was synthesized as eBlocks™ gene fragments(Integrated DNA Technologies) and then assembled together creating longer fragmentcontaining CMV promoter and enhancer region, the truncated spike ectodomain, WPREelement, and a polyadenylation signal sequence by using Gibson assembly master mix (NEB,E2611) followed by PCR amplification of the assembled fragment. To assemble the newlymade truncated spike ectodomain fragment into an adenoviral vector, the adenoviral vector genomic backbone plasmid pRLF1 was first linearized using BstZ17I restriction enzymefollowed by ethanol precipitation. pRLF1 is an adenoviral vector genomic backbone plasmiddeveloped by Rokote Laboratories Finland to suit Gibson assembly recombination of transgenes into the deleted E1 region. To obtain pFCV2.1, the truncated spike ectodomain fragment was assembled into the digested viral backbone pRLF1 using Gibson assembly master mix (NEB, E2611) according to the manufacturer’s instructions. The Gibson assembly reaction was then transformed into NEB® 5-alpha Competent E. coli (NEB, C2987H) according to manufacturer’s instructions. Positive colonies were screened by PCR and the correct recombination events were further confirmed by sequencing the construct. Final viral vaccine Drug Product will be stored in below -60°C. A long term stability study will be conducted to provide data for assigning the batch expiration date. Based on preliminary data available on adenoviruses stored in the proposed final formulation buffer, at least 3 years stability is expected. Virus productionpFCV2.1 adenoviral vaccine vector plasmid was digested with PacI (New England Biolabs)to release the adenoviral genome. Digested viral genome was purified by ethanol precipitation and dissolved in TE buffer.2 µg of the linearized viral genome was then transfected into CAPAd cells using Trans-IT (MirusBio) transfection reagent according to manufacturer’s instructions. Recovered virus from the transfection was then further amplified in CAP Ad cells and purified using CsCl2 purification. Animals A total of 25 Syrian golden hamsters (Mesocricetus auratus) was used for excipient testing. Method Briefly, animals received pre-treatment with transduction-enhancing excipients by intranasally administering Syn3 or DM (diluted in the A195 formulation buffer: 10 mM Tris-HCl, 10 mM histidine, 75 mM NaCl, 5% sucrose (w / v), 1 mM MgCl2, 0.02% (w / v) PS-80 (polysorbate-80), 0.1 mM EDTA, 0.5% Ethanol (v / v), pH 7.4) prior to FCV2.1 vaccination. Each nostril received 25µL of Syn3 or DM 15-30 minutes before FCV2.1 vaccination (25µL of FCV2.1 per nostril). Daily monitoring for signs of stress was conducted, and blood was collected for immunological analyses 21 days post-vaccination. The experimental setup is outlined in Table 1. Table 1: Experimental Setup Group Syn3 conc. 1 Syn3 conc. 2 DM conc. 1 DM conc. 2 Controln 5 5 5 5 5Day 0 PretreatmentPretreatment Pretreatment Pretreatment Pretreatment with Syn3 conc.1 with Syn3 conc.2 with DM conc.1 with DM conc.2 with A195 buffer 107VP dose of 107VP dose of 107VP dose of107 VP dose of107VP dose of FCV2.1 FCV2.1 FCV2.1 FCV2.1 FCV2.1 Day 21 End of study (blood collection for immunolocigal analyses)Results Syn3 enhances vaccine immunogenicity in dose dependent manner, DM induce toxicity at higher concentration. Pretreatment with Syn3 at a concentration of 5mg / ml induced approximately a 2-fold enhancement of spike-specific serum IgG levels 21 days post- vaccination. At the lower concentration (1mg / ml) of Syn3, the enhancement was significantly lower, albeit still visible (see Figure 1A). In contrast to Syn3, pretreatment with DM at a concentration of 5mg / ml induced severe toxicity, leading to the euthanasia of all animals in the high concentration group due to toxicity. However, at the lower concentration (1mg / ml) of DM, there were no clinical signs of toxicity, allowing the experiment to proceed according to theplanned setup. DM at the lower concentration was able to induce a similar enhancement of spike-specific serum IgG levels as the 1mg / ml Syn3 pretreatment (see Figure 1B). EXAMPLE 2 Reagents Syn3 was included in the vaccine formulation buffer and evaluated as a transduction-enhancing excipient for FCV2.1 vaccination. Animals A total of 20 Syrian golden hamsters (Mesocricetus auratus) was used for excipient testing. Method Briefly, animals received FCV2.1 vaccination with transduction-enhancing excipient via intranasal administration of Syn3 (formulated in A195 formulation buffer: 10 mM Tris-HCl, 10 mM histidine, 75 mM NaCl, 5% sucrose (w / v), 1 mM MgCl₂, 0.02% (w / v) PS-80 (polysorbate-80), 0.1 mM EDTA, and 0.5% ethanol (v / v), pH 7.4). Each animal received 50 µL of FCV2.1 formulated with varying concentrations of Syn3 (25 µL of FCV2.1 per nostril). Daily monitoring for signs of stress was conducted, and blood was collected for immunological analyses 21 days post-vaccination. The experimental setup is outlined in Table 2. Table 2: Experimental Setup Group Control Syn3 conc. 1 Syn3 conc. 2 Syn3 conc. 2 Syn3 conc. 2Day 0 FCV2.1FCV2.1 FCV2.1 FCV2.1 FCV2.1 vaccination with vaccination with vaccination with vaccination with vaccination with no Syn3 in 1mg / ml Syn3 2mg / ml Syn3 4mg / ml Syn3 5mg / ml Syn3 vaccine formulated in the formulated in the formulated in the formulated in the formulation buffer vaccine vaccine vaccine vaccine formulation buffer formulation buffer formulation buffer formulation buffer 5x104VP dose of 5x104VP dose of 5x104VP dose of 5x104VP dose of 5x104VP dose of FCV2.1 FCV2.1 FCV2.1 FCV2.1 FCV2.1 Day 21 End of study (blood collection for immunolocigal analyses)Results Syn3 enhances vaccine immunogenicity in a dose-dependent manner. The inclusion of Syn3 in the vaccine formulation significantly enhances the immunogenicity of the intranasally administered adenovector vaccine. The control group, which did not receive Syn3 in the vaccine formulation buffer, showed no signs of immunogenicity at the very low dose used in this experiment. Similarly, a Syn3 concentration of 1 mg / mL was insufficient to induce immunogenicity when included in the vaccine formulation buffer and co-administered with the vaccine. However, concentrations of 2 mg / mL up to the highest tested dose (5 mg / mL) markedly enhanced vaccine immunogenicity, as shown in Figure 2. CITATION LIST Patent Literature EP1456377 WO2021045541 KR20210028116 WO2024223994 Non-Patent Literature Connor et al., 2001, Identification of polyamides that enhance adenovirus-mediated gene expression in the urothelium, Gene Therapy 8:41-48.

Claims

CLAIMS1. A vaccine composition comprising i) an adenoviral vector delivery enhancing agentselected from the group consisting of SYN3, and SYN3 analogs, and ii) an adenoviral vectorcomprising, at an insertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism.

2. The vaccine composition according to claim 1, wherein said immunogenic protein is apolypeptide derived of, or expressed by a pathogenic microbe or a modified polypeptidethereof.

3. The vaccine composition according to claim 2, wherein said pathogenic microbe is selected from the group consisting of: influenza virus, respiratory syncytial virus, coronavirus and Mycobacterium tuberculosis.

4. The vaccine composition according to any one of claims 1-3, wherein said adenoviralvector comprises human adenovirus serotype 26 or serotype 5 backbone.

5. The vaccine composition according to claim 4, wherein said adenoviral vector is a non-replicating human adenovirus serotype 5 vector.

6. The vaccine composition according to any one of claims 2-5, wherein said pathogenic microbe is a coronavirus.

7. The vaccine composition according to claim 6, wherein said adenoviral vector comprises, at an insertion site in the viral genome, a nucleic acid sequence encoding one or more immunogenic proteins that generate an adaptive immune response against one or more coronaviruses.

8. The vaccine composition according to claim 6, wherein said adenoviral vector comprises,at an insertion site in the viral genome, a nucleic acid sequence encoding a modifiedextracellular domain of a coronavirus spike protein.

9. The vaccine composition according to claim 8, wherein said adenoviral vector comprises,at an insertion site in the viral genome, a nucleic acid sequence encoding a modifiedextracellular domain of the SARS-CoV-2 spike protein,wherein said modified extracellular domain comprises an N-terminal signal peptide causingthe spike protein to enter a secretory system in a host cell,wherein said modified extracellular domain comprises a C-terminal deletion of at least ofheptad repeat 2 (HR2), transmembrane segment (TM) and cytoplasmic tail (CT) of the spike protein.

10. The vaccine composition according to claim 9, wherein the deletion site of said C-terminal deletion is located at the region corresponding to positions 1030-1157 of the omicronBA.5 strain spike protein of SEQ ID NO:1.

11. The vaccine composition according to claim 9 or 10, wherein said C-terminal deletioncomprises part but not all of connector domain (CD) of the spike protein.

12. The vaccine composition according to any one of claims 9-11, wherein said N-terminal signal peptide is the human interleukin-3 secretion signal peptide or the natural signal peptide of the SARS-CoV-2 spike protein.

13. The vaccine composition according to any one of claims 9-12, wherein said modifiedextracellular domain of the SARS-CoV-2 spike protein comprises a mutated furin cleavagesite with single amino acid modifications in positions R677G, R678S and R680S, whereinsaid positions corresponds to the respectively numbered amino acid positions of the omicronBA.5 strain spike protein of SEQ ID NO:1.

14. The vaccine composition according to claim 13, wherein said modified extracellulardomain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ IDNO:2, amino acids 22-1142 of SEQ ID NO:2, or a sequence which has at least 80 % sequenceidentity with the sequence of SEQ ID NO:2, or with amino acids 22-1142 of SEQ ID NO:2.

15. The vaccine composition according to any one of claims 9-14, wherein said modifiedextracellular domain of the SARS-CoV-2 spike protein comprises at least N-terminal domain(NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), andcentral helix (CH).

16. The vaccine composition according to any one of claims 9-15, wherein said nucleic acidsequence encoding said modified extracellular domain is as set forth in SEQ ID NO:3, or anucleotide sequence having at least 70%, 80%, 85%, 90%, or 95% sequence identity with thenucleotide sequence as set forth in SEQ ID NO:3.

17. The vaccine composition according to claim 16, wherein said modified extracellulardomain of the SARS-CoV-2 spike protein comprises in the C-terminal end one or morefurther receptor binding domains (RBD) from SARS-CoV-2 spike proteins which areimmunologically distinct from said modified extracellular domain of the SARS-CoV-2 spikeprotein.

18. The vaccine composition according to any one of claims 9-17, wherein said modifiedextracellular domain of the SARS-CoV-2 spike protein comprises in the C-terminal end atrimerization domain, preferably T4 fibritin trimerization domain, human collagen trimerization domain, leuzine zipper domain or any other additional C-terminal amino acid sequence promoting trimerization of said spike protein.

19. The vaccine composition according to any one of claims 9-18, wherein said modifiedextracellular domain of the SARS-CoV-2 spike protein comprises in the C-terminal end atransmembrane domain, or any other additional C-terminal amino acid sequence promoting transmembrane anchoring of said spike protein.

20. The vaccine composition according to claim 18 or 19, wherein said modified extracellulardomain of the SARS-CoV-2 spike protein comprises in the C-terminal region in any order i) atrimerization domain, preferably T4 fibritin trimerization domain, human collagen trimerization domain, leuzine zipper domain or any other additional C-terminal amino acid sequence promoting trimerization of said spike protein, and ii) a transmembrane domain, or any other additional C-terminal amino acid sequence promoting transmembrane anchoring of said spike protein.

21. The vaccine composition according to claim 18 or 19, wherein said modified extracellulardomain of the SARS-CoV-2 spike protein comprises in the C-terminal end in any order i) oneor more receptor binding domains (RBD) from SARS-CoV-2 spike proteins which areimmunologically distinct from said modified extracellular domain of the SARS-CoV-2 spikeprotein, and ii) a trimerization domain, preferably T4 fibritin trimerization domain, humancollagen trimerization domain, leuzine zipper domain or any other additional C-terminalamino acid sequence promoting trimerization of said spike protein.

22. The vaccine composition according to claim 19 or 21, wherein said modified extracellulardomain of the SARS-CoV-2 spike protein comprises in the C-terminal end in any order i) oneor more receptor binding domains (RBD) from SARS-CoV-2 spike proteins which areimmunologically distinct from said modified extracellular domain of the SARS-CoV-2 spikeprotein, and ii) a transmembrane domain, or any other additional C-terminal amino acidsequence promoting transmembrane anchoring of said spike protein.

23. The vaccine composition according to any one of claims 19-21, wherein said modifiedextracellular domain of the SARS-CoV-2 spike protein comprises in the C-terminal end inany order i) one or more receptor binding domains (RBD) from SARS-CoV-2 spike proteinswhich are immunologically distinct from said modified extracellular domain of the SARS-CoV-2 spike protein, ii) a trimerization domain, preferably T4 fibritin trimerization domain,human collagen trimerization domain, leuzine zipper domain or any other additional C- terminal amino acid sequence promoting trimerization of said spike protein, and iii) a transmembrane domain or any other additional C-terminal amino acid sequence promoting transmembrane anchoring of said spike protein.

24. The vaccine composition according to any one of claim 9-23, wherein said adenoviral vector delivery enhancing agent is SYN3.

25. The vaccine composition according to any one of claims 9-24 in a mucosal administrationformulation.

26. The vaccine composition according to claim 25, wherein said mucosal administration formulation is selected from the group consisting of nasal drops, aerosols, sprays, powder sprays, gels, microspheres, liposomes, membranes, and suspensions.

27. The vaccine composition according to claim 26, wherein said vaccine composition is a spray or sprayable composition in a liquid dosage form.

28. The vaccine composition according to any one of claims 25-27 further comprising apharmaceutically-acceptable adjuvant, buffer, carrier, diluent or excipient.

29. The vaccine composition according to any one of claims 1-28 comprising a buffer containing Tris, sodium chloride, magnesium chloride, histidine, sucrose, polysorbate-80, EDTA, and ethanol.

30. The vaccine composition according to claim 29, wherein the buffer comprises 5-15 mM Tris-HCl, 50-100 mM NaCl, 0.5-2 mM MgCl2, 5-15 mM histidine, 2%-8% (wt / vol) sucrose, 0.02%-1% (wt / vol) polysorbate-80, 0.05-05 mM EDTA, and 0.25%-1% (vol / vol) ethanol, preferably the buffer comprises 0.05%-0.5% (wt / vol) polysorbate-80, more preferably 0.2%- 0.5% (wt / vol) polysorbate-80.

31. The vaccine composition according to claim 29 comprising10 mM Tris at a pH of 7.4, 75mM NaCl, 1 mM MgCl2, 10 mM histidine, 5% (wt / vol) sucrose, 0.02% polysorbate-80 (wt / vol), 0.1 mM EDTA, and 0.5% (vol / vol) ethanol).

32. The vaccine composition according to any one of claims 25-30, wherein said composition is formulated to be administered intranasally.

33. A method for delivering an adenoviral vector to a mammalian subject, said methodcomprising a step of administering onto the nasal and / or oral mucosa of the subject a dose of adelivery enhancing agent selected from the group consisting of SYN3 and SYN3 analogs and a dose of an adenoviral vector,wherein the first dose of said delivery enhancing agent and the first dose of said adenoviralvector are administered simultaneously or sequentially.

34. The method according to claim 33, wherein said adenoviral vector comprises, at an insertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism.

35. The method according to claim 34, wherein said immunogenic protein is a polypeptidederived of, or expressed by a pathogenic microbe or a modified polypeptide thereof.

36. The method according to claim 35, wherein said pathogenic microbe is selected from thegroup consisting of: influenza virus, respiratory syncytial virus, coronavirus and Mycobacterium tuberculosis.

37. The method according to any one of claims 34-36, wherein said adenoviral vectorcomprises human adenovirus serotype 26 or serotype 5 backbone.

38. The method according to claim 37, wherein said adenoviral vector is a non-replicatinghuman adenovirus serotype 5 vector.

39. The method according to claim 37 or 38, wherein said adenoviral vector comprises, at aninsertion site in the viral genome, a nucleic acid sequence encoding one or more immunogenic proteins that generate an adaptive immune response against one or more coronaviruses.

40. The method according to claim 37 or 38, wherein said adenoviral vector comprises, at aninsertion site in the viral genome, a nucleic acid sequence encoding a modified extracellular domain of a coronavirus spike protein.

41. The method according to claim 37 or 38, wherein said adenoviral vector comprises, at aninsertion site in the viral genome, a nucleic acid sequence encoding a modified extracellulardomain of the SARS-CoV-2 spike protein,wherein said modified extracellular domain comprises an N-terminal signal peptide causingthe spike protein to enter a secretory system in a host cell,wherein said modified extracellular domain comprises a C-terminal deletion of at least ofheptad repeat 2 (HR2), transmembrane segment (TM) and cytoplasmic tail (CT) of the spike protein.

42. The method according to any one of claims 33-41, wherein the first dose of said deliveryenhancing agent and the first dose of said adenoviral vector are administered simultaneously.

43. The method according to claim 42, wherein a vaccine composition according to any oneof claims 1-31 is administered to said subject.

44. The method according to any one of claims 33-42, wherein the first dose of said deliveryenhancing agent and the first dose of said adenoviral vector are administered sequentiallywithin 24 hours, preferably within 1-4 hours from the first dose of either said deliveryenhancing agent or said adenoviral vector.

45. The method according to any one of claims 33-44, wherein said adenoviral vector or saidvaccine composition is administered by spraying a liquid dosage form of said adenoviralvector or vaccine composition onto the nasal and / or oral mucosa of the subject.

46. A kit comprising at least two containers, wherein a first container comprises a deliveryenhancing agent selected from the group consisting of SYN3 and SYN3 analogs, and a second container comprises an adenoviral vector, wherein said adenoviral vector comprises, at an insertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism.

47. The kit according to claim 46, wherein said immunogenic protein is a polypeptide derivedof, or expressed by a pathogenic microbe or a modified polypeptide thereof.

48. The kit according to claim 47, wherein said pathogenic microbe is selected from the group consisting of: influenza virus, respiratory syncytial virus, coronavirus and Mycobacterium tuberculosis.

49. The kit according to any one of claims 46-48, wherein said adenoviral vector compriseshuman adenovirus serotype 26 or serotype 5 backbone.

50. The kit according to claim 49, wherein said adenoviral vector is a non-replicating humanadenovirus serotype 5 vector.

51. The kit according to any one of claims 47-50, wherein said pathogenic microbe is coronavirus.

52. The kit according to claim 51, wherein said adenoviral vector comprises, at an insertion site in the viral genome, a nucleic acid sequence encoding one or more immunogenic proteins that generate an adaptive immune response against one or more coronaviruses.

53. The kit according to claim 51, wherein said adenoviral vector comprises, at an insertionsite in the viral genome, a nucleic acid sequence encoding a modified extracellular domain ofa coronavirus spike protein.

54. The kit according to claim 51. wherein said adenoviral vector comprises, at an insertionsite in the viral genome, a nucleic acid sequence encoding a modified extracellular domain ofthe SARS-CoV-2 spike protein,wherein said modified extracellular domain comprises an N-terminal signal peptide causingthe spike protein to enter a secretory system in a host cell,wherein said modified extracellular domain comprises a C-terminal deletion of at least ofheptad repeat 2 (HR2), transmembrane segment (TM) and cytoplasmic tail (CT) of the spike protein.

55. An adenoviral vector together with a separate delivery enhancing agent selected from thegroup consisting of SYN3 and SYN3 analogs administered onto the nasal and / or oral mucosaof a mammalian subject, for use in the prevention or treatment of an infectious disease causedby a pathogenic microbe,wherein the first dose of said delivery enhancing agent and the first dose of said adenoviralvector are administered simultaneously or sequentially, andwherein said adenoviral vector preferably comprises, at an insertion site in the viral genome, a nucleic acid sequence encoding an immunogenic protein generating adaptive immune response upon exposure to a host organism.

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