Adenovirus nanoparticles

Abstract

The present disclosure relates to modified recombinant adenoviruses comprising a chimeric hexon polypeptide comprising a first partner peptide capable of covalent coupling to a second partner peptide, wherein the chimeric hexon polypeptide comprises a portion derived from one or more adenoviruses. The modified recombinant adenoviruses of the present disclosure can be prepared as a vaccine platform to achieve display of a variety of target proteins on the surface of the viral particle.

Description

Technical Field

[0001] Adenovirus vectors are the most commonly used vectors in gene therapy and vaccine research, but the problem of anti-vector immune responses limits their widespread application. Another method for delivering antigens using adenoviruses is to display the target antigen on the surface of the adenovirus capsid protein. This method is achieved through multiple capsid proteins, including but not limited to hexagonal, pentaagonal, fibrin, peptide VII, and peptide IX.

[0002] However, the primary neutralizing epitope of adenovirus is located in the hypervariable region (HVR) of the hexagonal domain; therefore, the hexagonal domain is the main target for adenovirus-mediated neutralizing antibody production. Although the neutralizing epitope of the target antigen can be displayed on the hexagonal domain, the immune response may still be concentrated on the adenovirus particle itself. Therefore, vaccine preparation via epitope display may result in lower immunogenicity, and the immunogenicity may vary depending on the location of the epitope at different capsid sites. Furthermore, hexagonal domains typically only allow for the insertion of small linear epitopes; embedding longer peptides within the hexagonal domain may affect the infectivity and stability of the adenovirus itself. This relationship between the display vector and antigen size may limit their application in vaccine development. Some pathogens exhibit high variability, meaning that even if a vaccine is successfully developed against a specific strain, effective protection against other strains cannot be guaranteed. Therefore, multivalent vaccines are also needed to address infections from different strains.

[0003] Therefore, there is an urgent need to obtain further optimized adenovirus vectors for vaccines, in order to address the stability of adenovirus vector vaccines, improve the immunogenicity of vaccines, and achieve the development of multivalent vaccines. Summary of the Invention

[0004] This invention provides an adenovirus nanoparticle carrier that can efficiently display target proteins on a surface, while maintaining the infectivity and stability of the adenovirus.

[0005] In a first aspect, the present invention provides a modified recombinant adenovirus comprising a chimeric hexagonal polypeptide, the chimeric hexagonal polypeptide further comprising a first chaperone peptide capable of covalently coupling with a second chaperone peptide, wherein the chimeric hexagonal polypeptide comprises a portion derived from one or more adenoviruses.

[0006] In some implementations, the chimeric hexagonal includes one or more hexagonal HVRs.

[0007] In some implementations, the chimeric six-neighbor rings include one or more of the HVR1 ring, HVR2 ring, HVR3 ring, HVR4 ring, HVR5 ring, HVR6 ring, and HVR7 ring.

[0008] In some implementations, the one or more six-neighbor HVRs are each independently derived from Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57. In some implementations, the one or more six-neighbor HVRs are the same as or different from each other.

[0009] In some embodiments, the first chaperone peptide is displayed on the outer surface of the chimeric hexane.

[0010] In some embodiments, the first chaperone peptide is displayed on the HVR ring of the chimeric hexane.

[0011] In some embodiments, the recombinant adenovirus is derived from a first adenovirus. In some embodiments, the chimeric hexagon comprises one or more hexagonal HVRs of the first adenovirus.

[0012] In some implementations, the first adenovirus is derived from any one of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57.

[0013] In some implementations, the recombinant adenovirus further comprises one or more hexa-neighbor HVRs derived from one or more additional adenoviruses.

[0014] In some embodiments, the one or more additional adenoviruses are derived from any one or more of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57.

[0015] In some implementations, the first adenovirus is different from the one or more additional adenoviruses.

[0016] In some implementations, the first adenovirus is derived from Ad3, and the one or more additional adenoviruses are derived from Ad7.

[0017] In some implementations, the backbone of the recombinant adenovirus is derived from Ad3.

[0018] In some embodiments, the chimeric hexagonal polypeptide comprises one or more hexagonal HVR rings from Ad7, wherein at least one hexagonal HVR of Ad7 is different from the HVR of Ad3. In some embodiments, the HVR5 ring of the chimeric hexagonal polypeptide comprises a major neutralizing antigenic epitope from Ad7.

[0019] In some implementations, the recombinant adenovirus backbone is derived from Ad3, and the HVR5 of Ad3 is replaced with HVR5 from Ad7.

[0020] In some embodiments, the first chaperone peptide and the second chaperone peptide are derived from any one of the following chaperone peptide pairs:

[0021] (1) SpyCatcher and SpyTag;

[0022] (2)SdyCatcher and SdyTag / SnoopTag / SpyTag / RumTag / / RumTrunkTag / PhoTag / EntTag / BacTag;

[0023] (3) SnoopCatcher and SnoopTag / SnoopTagJr;

[0024] (4)MoonCake and PhoTag / RumTrunkTag / RumTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag;

[0025] (5)KatI and PhoTag / RumTrunkTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag;

[0026] (6)QueenCatcher and PhoTag / RumTrunkTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag;

[0027] (7)DogCatcher and RrgATag / RrgATag2 / DogTag;

[0028] (8) Pilin-C and Isopeptag;

[0029] (9) Pilin-N and Isopeptag-N;

[0030] (10) PsCsCatcher and PsCsTag;

[0031] (11)In and Jo;

[0032] (12)RrgACatcher and RrgATag / RrgATag2 / DogTag.

[0033] In some embodiments, the first chaperone peptide is a "tag" and the second chaperone peptide is a "capturer". In other embodiments, the first chaperone peptide is a "capturer" and the second chaperone peptide is a "tag".

[0034] In some embodiments, the first or second chaperone peptide is derived from SpyTag, SdyTag, SnoopTag, PhoTag, EntTag, KTag, BacTag, Bac2Tag, Bac3Tag, Bac4Tag, RumTrunkTag, Rum7Tag, RumTag, Rum2Tag, Rum3Tag, Rum4Tag, Rum5Tag, Rum6Tag, Bac5Tag, SnoopTagJr, RrgATag, R rgATag2, DogTag, Isopeptag, Isopeptag-N, PsCsTag or Jo, and their homologs having at least 60% homology with them, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99%.

[0035] In some embodiments, the second chaperone peptide or the first chaperone peptide is derived from SpyCatcher, SdyCatcher, SnoopCatcher, MoonCake, KatI, QueenCatcher, DogCatcher, Pilin-C, Pilin-N, PsCsCatcher, In, or RrgACatcher, and their homologs, which have at least 60% homology with SpyCatcher, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology.

[0036] In some implementations, the second chaperone peptide is linked to the target protein.

[0037] In some implementations, the second chaperone peptide forms a fusion protein with the target protein.

[0038] In some implementations, the second chaperone peptide is directly linked to the target protein or linked via a connector.

[0039] In some embodiments, the adapter is selected from glycine adapters, serine adapters, glycine-serine adapters, for example, GnS (n=1-4), SnG (n=1-4), (GSG)n (n=1-4).

[0040] In some implementations, each second chaperone peptide is linked to one or more target proteins.

[0041] In some embodiments, the target protein can be any protein known to those skilled in the art that is suitable for displaying viral particles or nanoparticles, including, but not limited to, one or more of antigens, antibodies or their antigen-binding fragments, therapeutic peptides, detectable tags, cell-penetrating peptides, targeting peptides or membrane fusion peptides.

[0042] In some implementations, the hexapods of the recombinant adenovirus display one or more target proteins on their surface.

[0043] In a preferred embodiment, the target protein is an antigen.

[0044] In some embodiments, the antigen may be an antigen fragment, such as an antigen fragment having a receptor-binding domain.

[0045] In some embodiments, the antigen is selected from viral antigens, bacterial antigens, parasitic antigens, or fungal antigens.

[0046] In some embodiments, the second chaperone peptide can be covalently coupled to the first chaperone peptide via an isopeptide bond or an ester bond.

[0047] In some embodiments, the first and second chaperone peptides are capable of forming covalent bonds, such as isopeptide or ester bonds, under suitable conditions. The first and second chaperone peptides are also referred to as a tag-and-capture pair. The formed covalent bond can be a spontaneous reaction or require the assistance of enzymes such as ligases.

[0048] In some embodiments, the first chaperone peptide is SpyTag and the second chaperone peptide is SpyCatcher, which can form a chaperone peptide pair.

[0049] Therefore, the target protein can be attached to the capsid protein of the adenovirus via covalent coupling of chaperone peptide pairs, specifically, the target protein and the fusion protein of the second chaperone peptide are covalently coupled to the first chaperone peptide contained in the modified capsid protein.

[0050] More specifically, the target protein and the fusion protein of the second chaperone peptide are covalently coupled to the first chaperone peptide contained in a modified hexagonal HVR ring.

[0051] In some embodiments, in the recombinant adenovirus, the HVR loops other than HVR5 are derived from Ad3, and the HVR5 of Ad3 contains a major neutralizing antigenic epitope from Ad7; and the first chaperone peptide is SpyTag, and the second chaperone peptide is SpyCatcher.

[0052] In some embodiments, the recombinant adenovirus is derived from Ad3, and the HVR5 of Ad3 contains a neutralizing epitope peptide from Ad7 (Ad7 hexagonal amino acids 258-276: DGREAADAFSPEIVLYTEN, SEQ ID NO:22); and the first chaperone peptide is SpyTag (GSGAHIVMVDAYKPTKGSG, SEQ ID NO:1), and the second chaperone peptide is SpyCatcher.

[0053] (GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTCNEDGQVTVDGEATEGDAHT, SEQ ID NO: 15).

[0054] In a second aspect, the present invention provides a composition comprising the recombinant adenovirus of the first aspect.

[0055] In some embodiments, the composition further includes a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient.

[0056] In some embodiments, the composition further includes a fusion protein with a second chaperone peptide and the target protein.

[0057] In some implementations, the second chaperone peptide is directly linked to the target protein or linked via a connector.

[0058] In some embodiments, the adapter is selected from glycine adapters, serine adapters, glycine-serine adapters, for example, GnS (n=1-4), SnG (n=1-4), (GSG)n (n=1-4).

[0059] In some embodiments, the target protein includes one or more of an antigen, antibody or antigen-binding fragment thereof, therapeutic peptide, detectable tag, cell-penetrating peptide, targeting peptide or membrane fusion peptide.

[0060] In some implementations, the recombinant adenovirus displays one or more target proteins.

[0061] In some implementations, the target protein is an antigen.

[0062] In some embodiments, the antigen is an antigen fragment, preferably an antigen fragment having a receptor-binding domain.

[0063] In some embodiments, the composition may be an immunogenic composition, preferably a vaccine.

[0064] In some embodiments, the antigen is selected from viral antigens, bacterial antigens, parasitic antigens, or fungal antigens.

[0065] In some embodiments, the second chaperone peptide can be covalently coupled to the first chaperone peptide via an isopeptide bond or an ester bond.

[0066] In some embodiments, the first and second chaperone peptides are capable of forming covalent bonds, such as isopeptide or ester bonds, under suitable conditions. The first and second chaperone peptides are also referred to as a tag-and-capture pair. The formed covalent bond can be a spontaneous reaction or require the assistance of enzymes such as ligases.

[0067] In some embodiments, the first chaperone peptide is SpyTag and the second chaperone peptide is SpyCatcher, which together constitute a chaperone peptide pair.

[0068] Therefore, the target protein can be attached to the capsid protein of the adenovirus via covalent coupling of chaperone peptide pairs, specifically, the target protein and the fusion protein of the second chaperone peptide are covalently coupled to the first chaperone peptide contained in the modified capsid protein.

[0069] More specifically, the target protein and the fusion protein of the second chaperone peptide are covalently coupled to the first chaperone peptide contained in a modified hexagonal HVR ring.

[0070] In some embodiments, the fusion protein of the target protein and the second chaperone peptide is spontaneously covalently coupled to the first chaperone peptide contained in a modified hexagonal HVR ring, thereby forming recombinant adenovirus nanoparticles displaying the target protein on the hexagonal surface. More specifically, recombinant adenovirus nanoparticles displaying the target protein are formed on the HVR ring of the hexagonal surface.

[0071] In some embodiments, the recombinant adenovirus is derived from Ad3, and the HVR5 of Ad3 contains a major neutralizing antigenic epitope from Ad7; and the first chaperone peptide is SpyTag, and the second chaperone peptide is SpyCatcher.

[0072] In some embodiments, the recombinant adenovirus is derived from Ad3, and the HVR5 of Ad3 contains a neutralizing epitope peptide from Ad7 (Ad7 hexagonal amino acids 258-276: DGREAADAFSPEIVLYTEN, SEQ ID NO:22); and the first chaperone peptide is SpyTag (GSGAHIVMVDAYKPTKGSG, SEQ ID NO:1), and the second chaperone peptide is SpyCatcher (GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTCNEDGQVTVDGEATEGDAHT, SEQ ID NO:15).

[0073] In some embodiments, the immunogenic composition is a vaccine.

[0074] Thirdly, the present invention provides an immunogenic material comprising a modified recombinant adenovirus, the recombinant adenovirus comprising a chimeric hexagonal polypeptide, the chimeric hexagonal polypeptide further comprising a first chaperone peptide, the first chaperone peptide being covalently coupled to a second chaperone peptide, the second chaperone peptide being linked to a target protein.

[0075] The chimeric hexagonal polypeptide comprises a portion derived from a first adenovirus and a portion derived from one or more other adenoviruses.

[0076] In some embodiments, the first chaperone peptide is displayed on the outer surface of the chimeric hexane.

[0077] In some embodiments, the first chaperone peptide is displayed on the HVR ring of the chimeric hexane.

[0078] In some implementations, the recombinant adenovirus is derived from a first adenovirus and contains one or more hexa-neighbor HVRs of a second adenovirus.

[0079] In some implementations, the recombinant adenovirus is derived from a first adenovirus and contains a hexa-neighbor HVR of a second adenovirus.

[0080] In some implementations, the first adenovirus is derived from any one of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57.

[0081] In some implementations, the second adenovirus is derived from any one of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57.

[0082] In some implementations, the first adenovirus is derived from Ad3, and the second adenovirus is derived from Ad7.

[0083] In some embodiments, the recombinant adenovirus is derived from Ad3 and contains one or more hexa-neighbor HVRs from Ad7, wherein at least one hexa-neighbor HVR of Ad7 is different from the HVR of Ad3.

[0084] In some embodiments, the recombinant adenovirus is derived from Ad3, and the HVR5 of Ad3 contains a major neutralizing antigenic epitope from Ad7.

[0085] In some embodiments, the recombinant adenovirus is derived from Ad3, and the HVR5 of Ad3 contains a neutralizing epitope peptide from Ad7 (Ad7 hexagonal amino acids 258-276: DGREAADAFSPEIVLYTEN, SEQ ID NO:22).

[0086] In some embodiments, the first chaperone peptide is derived from SpyTag, SdyTag, SnoopTag, PhoTag, EntTag, KTag, BacTag, Bac2Tag, Bac3Tag, Bac4Tag, RumTrunkTag, Rum7Tag, RumTag, Rum2Tag, Rum3Tag, Rum4Tag, Rum5Tag, Rum6Tag, and Bac5Tag, and their homologs, which have at least 60% homology with them, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology.

[0087] In some embodiments, the second chaperone peptide is derived from SpyCatcher, SdyCatcher, or SnoopCatcher, and its homologs, which have at least 60% homology with it, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology.

[0088] In some implementations, the second chaperone peptide forms a fusion protein with the target protein.

[0089] In some implementations, the second chaperone peptide is directly linked to the target protein or linked via a connector.

[0090] In some embodiments, the adapter is selected from glycine adapters, serine adapters, glycine-serine adapters, for example, GnS (n=1-4), SnG (n=1-4), (GSG)n (n=1-4).

[0091] In some implementations, the target protein is an antigen.

[0092] In some embodiments, the antigen is an antigen fragment, preferably an antigen fragment having a receptor-binding domain.

[0093] In some embodiments, the antigen is selected from viral antigens, bacterial antigens, parasitic antigens, or fungal antigens.

[0094] In some embodiments, the second chaperone peptide is covalently coupled to the first chaperone peptide via an isopeptide bond or an ester bond.

[0095] In some embodiments, the first and second chaperone peptides are capable of forming covalent bonds, such as isopeptide or ester bonds, under suitable conditions. The first and second chaperone peptides are also referred to as a tag-and-capture pair. The formed covalent bond can be a spontaneous reaction or require the assistance of enzymes such as ligases.

[0096] In some embodiments, the first chaperone peptide is SpyTag and the second chaperone peptide is SpyCatcher, which together constitute a chaperone peptide pair.

[0097] Therefore, the target protein is attached to the capsid protein of the adenovirus via covalent coupling of chaperone peptide pairs, specifically, the target protein and the fusion protein of the second chaperone peptide are covalently coupled to the first chaperone peptide contained in the modified capsid protein.

[0098] More specifically, the target protein and the fusion protein of the second chaperone peptide are covalently coupled to the first chaperone peptide contained in a modified hexagonal HVR ring.

[0099] In some embodiments, the fusion protein of the target protein and the second chaperone peptide is spontaneously covalently coupled to the first chaperone peptide contained in a modified hexagonal HVR ring, thereby forming recombinant adenovirus nanoparticles displaying the target protein on the hexagonal surface. More specifically, recombinant adenovirus nanoparticles displaying the target protein are formed on the HVR ring of the hexagonal surface.

[0100] In some embodiments, the recombinant adenovirus is derived from Ad3, and the HVR5 of Ad3 contains a major neutralizing antigenic epitope from Ad7; and the first chaperone peptide is SpyTag, and the second chaperone peptide is SpyCatcher.

[0101] In some embodiments, the recombinant adenovirus is derived from Ad3, and the HVR5 of Ad3 contains a neutralizing epitope peptide from Ad7 (Ad7 hexagonal amino acids 258-276: DGREAADAFSPEIVLYTEN, SEQ ID NO:22); and the first chaperone peptide is SpyTag (GSGAHIVMVDAYKPTKGSG, SEQ ID NO:1), and the second chaperone peptide is SpyCatcher (GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTCNEDGQVTVDGEATEGDAHT, SEQ ID NO:15).

[0102] Fourthly, the present invention provides isolated nucleic acids that encode a recombinant adenovirus of the first aspect, an immunogenic composition of the second aspect, or an immunogenic substance of the third aspect.

[0103] In some implementations, the isolated nucleic acid comprises a first nucleic acid that encodes a recombinant adenovirus of the first aspect.

[0104] In some embodiments, the isolated nucleic acid comprises a first nucleic acid and a second nucleic acid, the first nucleic acid encoding a recombinant adenovirus of a first aspect, and the second nucleic acid encoding a fusion protein of a second chaperone peptide and a target protein.

[0105] Fifthly, the present invention provides an expression vector comprising the nucleic acid described in the fourth aspect.

[0106] In a sixth aspect, the present invention provides a host cell comprising the nucleic acid of the fourth aspect or the expression vector of the fifth aspect.

[0107] In a seventh aspect, the present invention provides the use of a recombinant adenovirus of the first aspect, an immunogenic composition of the second aspect, an immunogenic substance of the third aspect, a nucleic acid of the fourth aspect, an expression vector of the fifth aspect, or a host cell of the sixth aspect in the preparation of a medicament for treating or preventing a disease in a subject.

[0108] In some implementations, the drug is a vaccine.

[0109] Eighthly, the present invention provides a method for preventing or treating a disease, the method comprising administering to a subject in need a recombinant adenovirus of the first aspect, an immunogenic composition of the second aspect, an immunogenic substance of the third aspect, a nucleic acid of the fourth aspect, an expression vector of the fifth aspect, or a host cell of the sixth aspect.

[0110] In a ninth aspect, the present invention provides the use of the recombinant adenovirus of the first aspect, the nucleic acid of the fourth aspect, the expression vector of the fifth aspect, or the host cell of the sixth aspect as a vector (e.g., a delivery vector).

[0111] In some embodiments, the recombinant adenovirus may also load one or more additional peptides, proteins, drugs, or nucleic acid molecules. Attached Figure Description

[0112] Figure 1 A schematic diagram of Spytag tags embedded in HVR2, 4, and 7 of recombinant adenovirus rAdMH5 is shown.

[0113] Figure 2 The results of PCR identification of recombinant adenovirus plasmid rAdMH5-HVR2 / 4 / 7-Spytag colonies using specific primers are shown.

[0114] Figure 3 The sequencing results of the recombinant adenovirus plasmid rAdMH5-HVR2 / 4 / 7-Spytag with specific primers are shown.

[0115] Figure 4 The image shows rAdMH5-HVR4-Spytag after multiple consecutive transfers (fluorescence represents the rescued live virus).

[0116] Figure 5 The growth kinetics analysis of recombinant adenovirus rAdMH5 is shown.

[0117] Figure 6 The image shows that the recombinant adenovirus rAdMH5-Spytag was purified by cesium chloride and separated into two viral bands by ultracentrifugation (indicated by red arrows).

[0118] Figure 7 The results of SDS-PAGE electrophoresis of Spycatcher-VP1 purified are shown.

[0119] Figure 8 The Western Blot analysis shows the in vitro binding effect of rAdMH5-Spytag and Spycatcher-VP1 (M: protein marker, 1: MH5-SpyVP1, 2: Spycatcher-VP1, 3: rAdMH5-Spytag).

[0120] Figure 9 The in vitro binding efficacy of rAdMH5-Spytag and Spycatcher-VP1 was demonstrated using ELISA analysis.

[0121] Figure 10 The in vitro binding efficiency of rAdMH5-Spytag and Spycatcher-VP1 was shown using nanoparticle size and Zeta potential analysis (particle size is expressed as Z-mean (nm), rAdMH5-Spytag is marked with a blue line, and rAdMH5-SpyVP1 is marked with an orange line).

[0122] Figure 11 The kinetics of antibody production against EV71 VP1 following vaccination are shown.

[0123] Figure 12 The results of ELISA analysis of the titer of the two vaccines against EV71 VP1 after two immunizations are shown.

[0124] Figure 13 The results of ELISPOT detection of the cytokine IFN-γ are shown.

[0125] Figure 14 The results of ELISPOT detection of the cytokine IL-4 are shown.

[0126] Figure 15 The titers of neutralizing antibodies against EV71 in mouse serum after three immunizations are shown.

[0127] Figure 16 The analysis of neutralizing antibody titers of rAdMH5-SpyVP1 and rAdMH5-Spytag against Ad3E and Ad7E is shown.

[0128] Figure 17 The results of SDS-PAGE electrophoresis after Spycatcher-RBD purification are shown.

[0129] Figure 18 The Western Blot analysis shows the in vitro binding effect of rAdMH5-Spytag and Spycatcher-RBD (1: rAdMH5-Spytag, 2: Spycatcher-RBD, 3: MH5-SpyRBD, M: protein marker).

[0130] Figure 19 The in vitro binding efficacy of rAdMH5-Spytag and Spycatcher-RBD was demonstrated using ELISA analysis.

[0131] Figure 20 The in vitro binding efficiency of rAdMH5-Spytag and Spycatcher-RBD was shown using nanoparticle size and Zeta potential analysis (particle size is expressed as Z-mean (nm), rAdMH5-Spytag is marked with a blue line, and rAdMH5-SpyRBD is marked with an orange line).

[0132] Figure 21 The kinetics of antibody production against SARS-CoV-2RBD following vaccination are shown.

[0133] Figure 22 The results show the titer analysis of antibodies against SARS-CoV-2RBD in the two vaccine groups after two immunizations using ELISA.

[0134] Figure 23 The results of ELISPOT detection of the cytokine IFN-γ are shown.

[0135] Figure 24 The results of ELISPOT detection of the cytokine IL-4 are shown.

[0136] Figure 25The titers of neutralizing antibodies against live SARS-CoV-2 BA5.2, EG.8 and WT in mouse serum after three immunizations are shown.

[0137] Figure 26 The neutralizing antibody titers of rAdMH5-SpyRBD and rAdMH5-Spytag against Ad3E and Ad7E are shown. Detailed Implementation

[0138] The recombinant adenovirus (Ad) of the present invention is a recombinant adenovirus (Ad) comprising a chimeric hexagonal polypeptide. In some embodiments, the chimeric hexagonal polypeptide comprises a portion derived from a first Ad and a portion derived from a second Ad. In some embodiments, the first portion may be referred to as the main chain, and a portion of the second Ad is grafted onto the main chain. In some embodiments, the hexagonal polypeptide sequence of the second Ad is used to replace a portion of the hexagonal polypeptide sequence of the first Ad. In some embodiments, the hexagonal polypeptide sequence of the second Ad is inserted into a region of the hexagonal polypeptide sequence of the first Ad. For example, the hexagonal polypeptide from the first Ad (such as Ad3) can be modified with one or more HVRs from the second Ad (such as Ad7).

[0139] The first and second chaperone peptides of this invention are selected from peptide pairs capable of forming isopeptide or ester bonds, thereby possessing an intrinsic ability to bind to each other. The first and second chaperone peptides can be derived from proteins or peptides that naturally form intramolecular isopeptide bonds. For example, the first chaperone peptide is typically a shorter peptide, also referred to as a "tag," and the second chaperone peptide is typically a longer peptide, also referred to as a "catcher." For example, the SpyTag / SpyCatcher system consists of a first chaperone peptide (SpyTag) and a second chaperone peptide (SpyCatcher).

[0140] The SpyTag and SpyCatcher may be derived from the fibronectin-binding protein FbaB of Streptococcus pyogenes.

[0141] In some embodiments, the length of the tag can be between 5 and 50 amino acids, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0142] In some embodiments, the length of the "capturer" is at least 20 amino acids. Preferably, the length of the "capturer" is at least 25 amino acids, for example, 25,400 amino acids, more preferably, 30,350 amino acids, and more preferably, 50,150 amino acids. For example, the length of the "capturer" is 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 amino acids or more.

[0143] In some implementations, the tag may also be derived from SdyTag, SnoopTag, PhoTag, EntTag, KTag, BacTag, Bac2Tag, Bac3Tag, Bac4Tag, RumTrunkTag, Rum7Tag, RumTag, Rum2Tag, Rum3Tag, Rum4Tag, Rum5Tag, Rum6Tag, and Bac5Tag, and their homologs, which have at least 60% homology with it, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology.

[0144] In some embodiments, the catcher may also be derived from SdyCatcher or SnoopCatcher, and its homologs, which have at least 60% homology with it, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology.

[0145] In some embodiments, the protein encoded by the first polynucleotide spontaneously forms particles, such as nanoparticles, virus-like particles (VLPs), or capsid particles. Upon expression in a cell, the protein expressed by the first polynucleotide forms said particles; specifically, said particles are formed from a protein fused with a first chaperone peptide, and a second polynucleotide expresses an antigen, which, together with the second chaperone peptide, constitutes a fusion protein. The particles may be very similar to viruses or other pathogenic organisms recognized by the immune system, but are non-infectious because they do not contain pathogenic genetic material. Assembled particles are formed by the spontaneous formation of isopeptide or ester bonds between the first and second chaperone peptides, thereby displaying said antigen on the surface of said assembled particles, preferably on their outer surface.

[0146] In some implementations, the assembled particles are extracted from the expressed cells.

[0147] Therefore, in this invention, the active ingredient in the vaccine can be a nucleic acid vaccine or an assembled particle.

[0148] In some embodiments, the protein may be a viral capsid protein or a viral envelope protein, such as a glycoprotein.

[0149] In some embodiments, the protein is derived from mammalian viruses, such as human viruses. For example, the protein may be derived from hepatitis viruses, noroviruses, papillomaviruses such as human papillomavirus (HPV), polyomaviruses, caliciviruses, porcine circoviruses, neuronecrosis viruses, parvoviruses, or enteroviruses.

[0150] In some implementations, the protein is a particle-forming protein.

[0151] In some embodiments, the particle-forming protein is a carrier protein capable of forming nanoparticles.

[0152] For example, the particle-forming protein is an adenovirus capsid protein, preferably a hexagonal protein.

[0153] In some embodiments, the capsid protein is a modified capsid protein.

[0154] In some embodiments, the modified capsid protein is a hexagonal protein with HVR ring modification.

[0155] In some embodiments, the hexagonal protein with HVR ring modification expresses a first chaperone peptide (SpyTag or a portion thereof) on the HVR ring.

[0156] In some embodiments, the antigen is an antigen fragment, preferably an antigen fragment having a receptor-binding domain.

[0157] In some implementations, the antigen is selected from viral, bacterial, parasitic, or fungal antigens.

[0158] In some embodiments, the virus is selected from human papillomavirus (HPV), enteroviruses (primarily Coxsackie A virus and enterovirus 71 (EV71), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV2 / HSV1), influenza virus (types A, B, and C), Ebola virus (EBOV), poliovirus, dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4), cytomegalovirus (CMV), respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus group, mumps virus, astrovirus, measles virus, parainfluenza virus, mumps virus, varicella-zoster virus, human herpesvirus (HHV), Japanese encephalitis virus, and rabies virus. Hepatitis viruses, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), adenovirus, rubella virus, human T-cell lymphoma virus type I (HTLV-I), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), poxvirus, flavivirus, human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), Marburg virus, and coronavirus SARS-CoV-2.

[0159] In some embodiments, the bacteria are selected from Acinetobacter baumannii, Cryptobacterium hemolyticus, Bacillus anthracis, Bacillus cereus, Blastomyces dermatitidis, Burkholderia cepacia, Burkholderia glanderi, Burkholderia pseudomeliformis, Campylobacter genus, Candida albicans, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, certain species of Clostridium spp, Clostridium tetani, certain species of Coccidioides spp, Listeria monocytogenes, Corynebacterium diphtheriae, and Cryptococcus neoformans. * *Neoflavian*, *Enterococcus genus*, *Escherichia coli*, *Francisella tularensis*, *Fusobacterium genus*, *Malassezia spp.*, *Haemophilus ducreyi*, *Haemophilus influenzae*, *Helicobacter pylori*, *Histoplasma capsulatum*, *Kingella kingae*, *Klebsiella granulomatis*, *Legionella pneumophila*, *Mycobacterium leprae*, *Mycobacterium lepromatosis*, *Mycobacterium tuberculosis*, *Mycobacterium ulcerans* ulcerans), Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp.*S. spp.*, *Paracoccidioides brasiliensis*, *Pasteurella genus*, *Salmonella genus*, *Shigella genus*, *Sporothrix schenckii*, *Staphylococcus genus*, *Streptococcus agalactiae*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Yersinia enterocolitica*, *Yersinia pestis*, and *Yersinia pseudotuberculosis*.

[0160] In some embodiments, the parasite is selected from *Ancylostomabraziliense*, *Ancylostoma duodenale*, *Arcanobacterium haemolyticum*, *Ascaris lumbricoides*, *Brugia malayi*, *Strongyloides stercoralis*, *Taeniagenus*, *Taenia solium*, *Toxocara canis*, *Toxocara cati*, *Toxoplasma gondii*, *Trichuris trichiura*, *Trypanosoma brucei*, *Trypanosoma cruzi*, *Trichinella spiralis*, and *Trichomonas vaginalis*. The following are listed: vaginalis, Cryptosporidium genus, Echinococcus genus, Fasciola hepatica, Fasciola gigantica, Giardia intestinalis, Gnathostoma spp, Metagonimus yokagawai, Microsporidiaphylum, Necator americanus, Onchocercavolvulus, and Pneumocystis jirovecii.

[0161] In some embodiments, the fungus is selected from Candida albicans, Microsporum gypseum, Epidermophyton floccosum, Aspergillus fumigatus, Candida auris, Trichophyton verrucosum, Trichophyton mentagrophytes, Trichophyton equinum, Trichophyton rubrum, and Microsporum canis.

[0162] In some embodiments, the adenovirus vector is a recombinant adenovirus constructed by inserting the hexagonal major neutralizing antigen epitope of human adenovirus type 7 (rAd7) into the hypervariable 5 region (HVR5) of the hexagonal protein of the human adenovirus type 3 (rAd3) vector.

[0163] In some embodiments, the adenovirus vector is constructed by inserting the hexonal major neutralizing antigenic epitope of human adenovirus type 7 (rAd3) into the hypervariable 5 region (rAd3-HVR5) of the hexonal protein of human adenovirus type 3 (rAd3-eGFP) vector, thus constructing the rAdMH5 recombinant (also known as rAdMHE3, see: H.Qiu, X.Li, X.Tian, ​​Z.Zhou, K.Xing, H.Li, N.Tang, W.Liu, P.Bai and R.Zhou. 2012. Serotype-specific neutralizing antibody epitopesof human adenovirus type 3 (HAdV-3) and HAdV-7reside in multiple hexonhypervariable regions. J Virol 86(15). 7964-75. 10.1128 / JVI.07076-11.).

[0164] Definitions:

[0165] adenovirus

[0166] Adenoviruses (Ad) are non-enveloped, double-stranded DNA viruses with a genome of approximately 36 kilobases (kb). There are over 60 human adenovirus serotypes, grouped as A, B, and C. Ad5 is the most widely studied serotype and the most widely used platform in oncolytic virus development. In oncolytic virus development, the goal is to target specific tissues and thus alter tropism. A major problem in using some adenovirus serotypes (including Ad5) in clinical settings is pre-existing human immunity. Adenoviruses are typically 70–90 nm in size and have an icosahedral capsid shape. The capsid structure (also known as “capsid proteins”) comprises three main protein types: hexagonal, fibrils, and penton bases. Additional minor proteins are present in the capsid, including VI, VIII, IX, IIIa, and IVa2. Hexagonal proteins are the major component of the adenovirus capsid, accounting for over 83% of the capsid proteins, and are approximately 100 kDa in size, with 720 monomers per viral particle. The hexagonal monomers organize into trimers, resulting in 12 trimers on each of the 20 faces, leading to 240 trimers per viral particle. The hexagonal sequence contains loop hypervariable regions (HVRs) corresponding to the outer surface of the virus, thus covering almost the entire surface of the virus. Each monomer has 7 HVRs, identified as HVR1-HVR7, which are serotype-specific. Because the loops are located on the outer surface of the virus, the hexagonal loops are the primary antigen recognition sites, i.e., targets of the host immune response.

[0167] Adrenal vectors (AdVs) are commonly used in gene therapy, particularly as gene delivery vectors, because of their ability to contain other gene sequences. Adenovirus vectors can deliver the transgenes they carry into the host cell nucleus without integrating viral DNA into the host chromosome. When used as gene therapy vectors, AdVs can have long insert sequences, with a usable capacity of 8–36 kb. Due to their ability to induce both innate and adaptive immune responses, as well as strong antigen-specific B-cell and T-cell immune responses, AdVs have become a promising vaccine delivery vector.

[0168] An isopeptide bond is an amide bond formed between a carboxyl / carboxamide group and an amino group, where at least one of the carboxyl or amino groups is not derived from or considered part of the protein backbone. Isopeptide bonds can form within a single protein, between two peptides, or between a peptide and a protein. Therefore, isopeptide bonds can form intramolecularly within a single protein molecule or intermolecularly (i.e., between two peptide / protein molecules). Isopeptide bonds are chemically irreversible under typical biological conditions and are resistant to most proteases. Because isopeptide bonds are inherently covalent, they are stable under conditions of rapid dissociation (e.g., long periods of time, high temperatures (up to at least 95°C), high forces, or harsh chemical treatments (e.g., pH 2–11, organic solvents, detergents, or denaturants)).

[0169] Spontaneous formation of isopeptide bonds refers to the formation of isopeptide bonds within proteins, within peptides, between proteins, between peptides, or between peptides and proteins (e.g., between a first chaperone peptide and a second chaperone peptide) without the presence of any other reagents (e.g., enzyme catalysts) and / or without chemical modification of the protein or peptide, such as without natural chemical linkages or chemical couplings. Therefore, isopeptide bonds can form spontaneously in the absence of enzymes or other exogenous substances or without chemical modification.

[0170] The term "subject" refers to any animal classified as a mammal, such as humans and non-human mammals. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably herein. Preferably, the subject is a human.

[0171] The terms “treatment” or “relief” include administering a compound or agent to a subject to prevent or delay the onset of symptoms, complications, or biochemical markers of a disease (such as coronavirus infection), to alleviate symptoms, or to stop or suppress the further development of the disease, condition, or disorder. Subjects requiring treatment include those who already have the disease or condition and those at risk of developing it. Treatment can be preventative (preventing or delaying the onset of the disease, or preventing the manifestation of its clinical or subclinical symptoms) or therapeutic suppression or symptom relief following the manifestation of the disease.

[0172] A vaccine is a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is protective. Typically, a vaccine elicits an antigen-specific immune response against an antigen of a pathogen, such as a viral pathogen, or against a cellular composition associated with a pathological condition. Vaccines may comprise polynucleotides (e.g., nucleic acids encoding known antigens), peptides or polypeptides (e.g., publicly disclosed antigens), viruses, cells, or one or more cellular compositions. In some embodiments of the invention, the vaccine, vaccine antigen, or vaccine composition is expressed from a fusion construct and self-assembles into nanoparticles displaying antigenic peptides or proteins on their surface.

[0173] Currently, existing technologies primarily utilize adenovirus capsid proteins to display neutralizing epitopes of pathogens. This approach may induce a low level of immune response, and the immunogenicity may vary depending on the epitope's embedding site on the capsid. Further optimization of the epitope embedding site and method is needed. Since hexagonal proteins are the main capsid proteins of adenoviruses and contain the main neutralizing sites, embedding long peptides into the hexagonal proteins may affect the infectivity and stability of the adenovirus itself. This relationship between display vector and antigen size may limit their application in vaccine development.

[0174] This invention improves the efficiency of vaccine development by using spytag / spycatcher technology to prepare adenovirus into universal nanoparticles, which can quickly and efficiently display target proteins on the surface of adenovirus particles, greatly shortening the vaccine development cycle.

[0175] Maintaining the stability of viral particles is a critical issue in vaccine preparation. This invention addresses this by embedding a spytag into the recombinant adenovirus rAdMH5-spytag, which does not affect the infectivity and stability of the adenovirus itself, thus ensuring the stability of the subsequently displayed antigen.

[0176] Current vaccines typically target only one pathogen, but this invention, by preserving the main neutralizing epitopes of adenovirus, can be used as both a nanoparticle vaccine against EV71 or SARS-CoV-2 and an adenovirus vaccine, thus enabling the development of multivalent vaccines.

[0177] The complex procedures and high costs involved in traditional vaccine preparation are significant factors hindering widespread vaccine adoption. This invention utilizes in vitro incubation to display the target protein on the surface of adenovirus particles, avoiding the complex procedures and high costs of traditional vaccine preparation and thus reducing vaccine production costs.

[0178] Improving the immunogenicity and efficacy of vaccines is a key issue in vaccine development. This invention displays the target protein on the surface of adenovirus particles, which helps to improve the immunogenicity and efficacy of the vaccine, thereby achieving better prevention and treatment effects.

[0179] Current vaccines are typically only applicable to specific pathogens or diseases. The technology of this invention can be applied not only to the development of nanoparticle vaccines against EV71 or SARS-CoV-2, but also to the development of vaccines against other pathogens, thus expanding the scope of vaccine applications.

[0180] This invention provides an innovative and effective technological platform for the development of novel vaccines. Utilizing Spytag / Spycatcher technology, multiple target proteins can be displayed on the surface of viral particles, offering greater possibilities and flexibility for vaccine design.

[0181] The technical solution of this invention is simple to operate, relatively low in cost, and applicable to a variety of different target proteins and viral vectors. This makes the technical solution have broad application prospects, not only for vaccine development but also for other biotechnology fields.

[0182] In summary, the technical solution of this invention solves several technical problems in traditional vaccine development and provides an innovative and effective technical platform for the development of new vaccines.

[0183] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0184] Unless otherwise specified, the experimental methods in the embodiments of this invention are all conventional methods.

[0185] Unless otherwise specified, the experimental materials used in the embodiments of this invention are all commercially available conventional biochemical reagents.

[0186] Example 1. Preparation of adenovirus nanoparticles

[0187] Based on the human adenovirus type 3 and 7 bivalent vaccine candidate strain rAdMH5 (also known as rAdMHE3, detailed preparation method can be found in the literature H. Qiu, X. Li, X. Tian, ​​Z. Zhou, K. Xing, H. Li, N. Tang, W. Liu, P. Bai and R. Zhou. 2012. Serotype-specific neutralizing antibody epitopes of human adenovirus type 3 (HAdV-3) and HAdV-7 reside in multiple hexon hypervariable regions. J Virol 86(15).7964-75.10.1128 / JVI.07076-11.), while retaining the major neutralizing epitope HVR1 of human type 3 and the embedded epitope of adenovirus type 7 HVR5, appropriate restriction enzyme sites AvrII and PacI were selected at both ends of the hexapod sequence of the pBR322-rAdMH5 plasmid to delete some amino acids in hexapods HVR2 (DITTTEGEEKPI, SEQ ID NO:16), HVR4 (VKPTTEGGVET, SEQ ID NO:17) and HVR7 (IKVKTDDANGWEKDANVDTAN, SEQ ID NO:18), respectively, and a Spytag tag (GSGAHIVMVDAYKPTKGSG, SEQ ID NO:18) was inserted at the deleted amino acid positions. NO:1), by PCR, the Spytag sequence was modified on primers, and recombinant adenovirus plasmids pAdMH5-HVR2-Spytag, pAdMH5-HVR4-Spytag, and pAdMH5-HVR7-Spytag were constructed using a three-fragment homologous recombination method. Figure 1 (A structural diagram is shown). The specific construction process is as follows:

[0188] The primers for constructing the clone and the primers for colony identification are shown in the table below:

[0189]

[0190]

[0191] 1.1.1 Construction of recombinant plasmid pClone007-AP

[0192] Select appropriate restriction enzyme sites, AvrII and PacI, at both ends of the hexaplex sequence of the pBR322-rAdMH5 plasmid, and use AP primer pairs Pac18303F and Avr23600R, diluted 10... -4Using pBR322-rAdMH5 plasmid as a template, the amplification product size was approximately 5300 bp. The amplification system is as follows:

[0193]

[0194] PCR reaction procedure: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min to obtain the 007-AP fragment.

[0195] After being recovered by gel electrophoresis, the product was ligated to the pClone007 Versatile Simple Vector (Beijing Qingke Biotechnology Co., Ltd., Guangzhou Branch) with blunt ends. The ligation system is as follows:

[0196]

[0197] The blunt-end ligation reaction procedure was as follows: 25℃, 5 min, PCR instrument temperature control. The recombinant was named pClone007-AP. After the ligation reaction, DH5α was transformed using the following method: 100 μL / tube of DH5α competent cells were thawed on ice, 10 μL of pClone007 ligation product was added, and the mixture was gently pipetted and incubated on ice for 30 min. After heat shock at 42℃ for 60 s, the mixture was quickly transferred to an ice bath and incubated for 3 min. LB medium without antibiotics was added to a 1.5 ml centrifuge tube, and the mixture was incubated at 37℃, 200 rpm for 1 h on a shaker. The precipitate was collected by centrifugation at 5000 rpm for 5 min, and approximately 100 μL of medium was used to resuspend the precipitate. The precipitate was then evenly spread onto LB agar plates containing Amp antibiotics and incubated upside down at 37℃ overnight. The next day, single clones were picked and sent for sequencing. Correct single clones were selected for subsequent experiments.

[0198] 1.1.2 Construction of the mutant shuttle vector pClone007-HVR2 / 4 / 7-spytag

[0199] A three-fragment homologous recombination method was used to delete amino acids 2, 4, and 7 of the hexagonal HVR sequence, and Spytag tags were inserted at the deleted amino acid positions. The Spytag sequences were then modified onto primers by PCR. The specific method is as follows:

[0200] Using primer pairs Pac18303F / HVR2-R, HVR4-R, or HVR7-R, PacI was amplified by PCR to the sequences of hexagonal HVR2, 4, and 7, approximately 680-1440 bp. Using primer pairs HVR2-F, HVR4-F, or HVR7-F / Avr23600R, the sequences of hexagonal HVR2, 4, and 7 were amplified by PCR to AvrII, approximately 3800-4600 bp. The plasmid pClone007-AP was double-digested with AvrII and PacI to obtain three fragments. Homologous recombination yielded the vectors pClone007-HVR2-spytag, pClone007-HVR4-spytag, and pClone007-HVR7-spytag.

[0201] The PCR amplification system is as follows:

[0202]

[0203] PCR reaction procedure: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 50 s, 30 cycles; 72℃ extension for 10 min, the product was recovered by gel electrophoresis and then used for the next experiment.

[0204] Double enzyme digestion system:

[0205]

[0206] Double enzyme digestion conditions: 37℃, 4 hours or more. After the enzyme digestion product is recovered by gel electrophoresis, the vector of about 2000 bp is used for the next experiment.

[0207] Three-segment homologous recombination system:

[0208]

[0209] Homologous recombination conditions: 50℃, 15 min, PCR instrument temperature control. The recombinant was named pClone007-HVR2 / 4 / 7-spytag. After the ligation reaction, it was transformed into DH5α. The next day, single colonies were picked and resuspended in 10 μL of liquid LB medium containing Amp resistance for colony identification. Colony identification system:

[0210]

[0211] PCR reaction procedure: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 10 s, 30 cycles; 72℃ extension for 10 min, the size was identified as approximately 750 bp. After the PCR product was verified to be correct by gel electrophoresis, it was sent for sequencing. The correct clone was used for the next experiment.

[0212] 1.1.3 Construction of recombinant adenovirus plasmid rAdMH5-Spytag:

[0213] The shuttle plasmid carrying the Spytag from the previous step was cloned into the rAdMH5 plasmid via PCR, enzyme digestion, and homologous recombination.

[0214] PCR amplification of AvrII to PacI sequences tagged with spytags:

[0215]

[0216]

[0217] PCR reaction program: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min, PCR product size approximately 5300 bp.

[0218] rAdMH5 plasmid was used as a vector by double enzyme digestion. The enzyme digestion system is as follows:

[0219]

[0220] Double digestion conditions: 37℃, 4 hours or more, digestion vector approximately 32kbp.

[0221] The PCR products and enzyme digestion products were recovered by agarose gel electrophoresis and then subjected to homologous recombination of the two fragments:

[0222]

[0223] Homologous recombination conditions: 50℃, 15 min, PCR instrument temperature control. The recombinant was named rAdMH5-HVR2 / 4 / 7-spytag. After the ligation reaction, it was transformed into DH5α. The next day, single colonies were picked and resuspended in 10 μL of liquid LB medium containing Amp resistance for colony identification. Colony identification system:

[0224]

[0225] PCR reaction procedure: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 10 s, 30 cycles; 72℃ extension for 10 min, identification size approximately 750 bp. After verification by gel electrophoresis, the PCR products were sent for sequencing identification and named pAdMH5-HVR2-Spytag, pAdMH5-HVR4-Spytag, and pAdMH5-HVR7-Spytag, respectively. The sequences of HVR2-Spytag (DITGSGAHIVMVDAYKPTKGSGKPI, SEQ ID NO:19), HVR4-Spytag (VKPTGSGAHIVMVDAYKPTKGSGVET, SEQ ID NO:20), and HVR7-Spytag (IKVKGSGAHIVMVDAYKPTKGSGWEKDANVDTAN, SEQ ID NO:21) with inserted spytags were correctly identified. The gel electrophoresis and sequencing identification results of the PCR products are as follows: Figure 2 and Figure 3 As shown.

[0226] 1.2 Rescue of Recombinant Adenovirus

[0227] Using a recombinant adenovirus plasmid with a correctly identified sequence, a novel recombinant virus was rescued in Ad293 cells. Finally, the recombinant adenovirus rAdMH5-HVR4-Spytag (rAdMH5-Spytag) with Spytag inserted in HVR4 was successfully rescued, as shown in the results. Figure 4 As shown, the fluorescent light represents the rescued live virus.

[0228] 1.3 Analysis of the growth characteristics of recombinant adenovirus

[0229] Analysis of the growth characteristics of rAdMH5-Spytag showed that the prepared recombinant adenovirus rAdMH5-Spytag had replication ability, similar to that of Ad3E (e.g., Figure 5 (As shown). Furthermore, compared to other expression systems, it is easy to culture and can be scaled up in various cell lines to obtain high-titer recombinant adenovirus. rAdMH5-Spytag was cultured in large quantities in A549 cells and then purified by CsCl density gradient centrifugation (e.g., ...). Figure 6 (As shown), it is used to prepare nanoparticle vaccines.

[0230] Example 2. Preparation of adenovirus-EV71 trivalent vaccine

[0231] Using rAdMH5-Spytag viral particles as a platform for antigen display, the VP1 protein of enterovirus 71 was fused with Spycather for expression. After in vitro incubation, VP1 was displayed on the surface of rAdMH5-Spytag particles, yielding a novel anti-EV71 nanoparticle vaccine candidate, rAdMH5-SpyVP1. Because rAdMH5-SpyVP1 retains the major neutralizing antigenic epitopes of human adenovirus types 3 and 7, it can serve as a candidate for a trivalent vaccine against EV71, human adenovirus type 3, and human adenovirus type 7.

[0232] 1. Expression and purification of Spycatcher-VP1

[0233] VP1 and Spycatcher were linked via a 3GSG linker to form the Spycatcher-VP1 fusion protein (amino acid sequence shown in SEQ ID NO:14). The gene encoding Spycatcher-VP1 was cloned into the pFast-dual vector and expressed and purified using an insect expression system. SDS-PAGE results are shown below. Figure 7 As shown, from Figure 7 It can be seen that the Spycatcher-VP1 protein was successfully expressed and purified, yielding a protein band of approximately 50 kDa.

[0234] 2. In vitro binding of rAdMH5-Spytag and Spycatcher-VP1

[0235] We used Western blotting, ELISA, and particle size analysis to verify whether rAdMH5-Spytag and Spycatcher-VP1 could bind in vitro.

[0236] (1) Western Blot

[0237] Sample preparation: Take 10 μL of rAdMH5-Spytag (2×10⁻⁶) 12 VPs) and 1 μL Spycatcher-VP1 (1 μg / μL) were mixed and incubated overnight at 4 °C. 10 μL rAdMH5-Spytag and 1 μL Spycatcher-VP1 were used as controls. All samples were treated with 4×SDS Loading Buffer, heated at 98 °C for 5 min, and then rapidly cooled on ice.

[0238] SDS-PAGE gel: Prepare an 8% discontinuous gel according to the Molecular Cloning Laboratory Guide.

[0239] Electrophoresis: Set the voltage to a constant 80V, adjust it to 120V after 30 minutes, and continue electrophoresis until the dye reaches the bottom of the separating gel, then disconnect the power.

[0240] Transfer: After SDS-PAGE electrophoresis, wet transfer of PVDF membrane was performed at 260mA for 90min. After transfer, the membrane was blocked overnight in PBST containing 5% skim milk powder, 0.5% tryptone, and 0.05% Tween 20.

[0241] Antibody incubation: Wash the membrane once with PBST. The primary antibody is His monoclonal antibody (catalog number: 40592-MM117). Dilute with blocking buffer according to the instructions. Incubate at 37°C for 1 hour. Wash with PBST 4 times for 10 minutes each time. Then add HRP-labeled goat anti-mouse IgG secondary antibody diluted according to the instructions. Incubate at 37°C for about 45 minutes. Wash with PBST 4 times for 10 minutes each time.

[0242] Imaging: After membrane washing, images were captured and saved using a high-sensitivity ECL chemiluminescence kit on a fully automated chemiluminescence imaging system (iBright FL1500). Imaging results are shown below. Figure 8 As shown, from Figure 8 It can be seen that His monoclonal antibody can specifically recognize the approximately 230 kDa band of rAdMH5-SpyVP1, and at the same time specifically recognize the approximately 60 kDa band of Spycatcher-VP1 (lane 1), indicating that Spycatcher-VP1 can bind to rAdMH5-Spytag.

[0243] (2) ELISA

[0244] Coating: The ELISA plates were coated with a concentration of 2×10⁻⁶. 10 The rAdMH5-Spytag strain of VPs and the wild-type Ad7 GZ08 strain (shown as Ad7W in the attached figure, GenBank No. GQ478341.1, see the literature Xingui Tian, ​​et al. Seroprevalence of Neutralizing Antibodies against Six Human Adenovirus Types Indicates the Low Level of Herd Immunity in Young Children from Guangzhou, China, Virol Sin. 2021 Jun; 36(3):373-381) were coated overnight at 4℃.

[0245] Blocking: Wash once with 250 μL PBST and blot dry on paper. Add 200 μL of PBST containing 3% BSA to each well of a 96-well microplate and incubate at 37°C for 2 hours for blocking.

[0246] In vitro binding of recombinant adenovirus rAdMH5-Spytag to Spycatcher-VP1 protein: Wash once with PBST and blot dry on paper. Add 2 μg / mL Spycatcher-VP1 protein to ensure complete binding of the protein to rAdMH5-Spytag, and incubate at room temperature for 4 h.

[0247] Antibody incubation: Wash once with PBST. The primary antibody is His monoclonal antibody (catalog number: 40592-MM117), diluted according to the manufacturer's instructions with PBST containing 1% BSA. Use 3% BSA blocking buffer as a blank control. Incubate at 37°C for 1 hour, wash four times with PBST, and pat dry. Dilute the HRP-labeled goat anti-mouse IgG secondary antibody according to the manufacturer's instructions and incubate at 37°C for 1 hour.

[0248] Color development: After incubation with the secondary antibody, wash four times with PBST, pat dry, add 100 μL of TMB substrate, and incubate at room temperature in the dark for 5-10 minutes for color development. After color development, add 100 μL of 2M H2SO4 to stop the reaction, and measure the OD value at 450 nm using a microplate reader.

[0249] 96-well microplates were simultaneously coated with recombinant adenovirus strains rAdMH5-Spytag and Ad7 GZ08. After blocking with 3% BSA, 2 μg / mL Spycatcher-VP1 protein was added to each plate, followed by the addition of His monoclonal antibody and secondary antibody. The experimental results are as follows: Figure 9 As shown, from Figure 9 It can be seen that only rAdMH5-Spytag coated with Spycatcher-VP1 protein can be recognized by His monoclonal antibody.

[0250] (3) Particle size

[0251] Instrument: Nanoparticle size and Zeta potential analyzer (Malvin)

[0252] After the samples were incubated overnight at 4°C, they were centrifuged at 12,000 rpm for 5 min at low temperature. The supernatant was then collected, and the particle size was measured using a nanoparticle size and Zeta potential analyzer. Among all the measured data, the Z-mean value was the average particle size obtained through the intensity distribution. Therefore, the Z-mean value (nm) was used to represent the particle size.

[0253] The results are as follows Figure 10 As shown. From Figure 10It can be seen that the particle size of rAdMH5-Spytag is 128.7 nm, and the particle size of rAdMH5-SpyVP1 is 145.3 nm. The particle size of rAdMH5-SpyVP1 is larger than that of rAdMH5-Spytag, indicating that the VP1 protein has been bound to rAdMH5-Spytag.

[0254] Results from combined Western blotting, ELISA, and particle size analysis indicate that the Spycatcher-VP1 protein can bind to and be displayed on the surface of rAdMH5-Spytag particles.

[0255] Example 3. Immunogenicity analysis of adenovirus-EV71 (rAdMH5-SpyVP1) trivalent vaccine

[0256] 1. Animal immunization

[0257] Recombinant adenovirus rAdMH5-Spytag and Spycatcher-VP1 (SEQ ID NO:14) were conjugated in vitro and then used to immunize mice. An experimental group and a control group were designed to be immunized simultaneously.

[0258] Group 1 (rAdMH5-SpyVP1): Hexagonal and VP1 protein molar ratio 1:1, rAdMH5-Spytag, 2×10 12 VPs+spycatcher-VP1, 1μg+Addavax;

[0259] Group 2 (Spycatcher-VP1): Spycatcher-VP1: 1μg + Addavax;

[0260] Group 3 (blank group): PBS + Addavax.

[0261] Both rAdMH5-Spytag and Spycatcher-VP1 were diluted with PBS and incubated overnight at 4°C. Four 6-8 week old female BALB / c mice were immunized intramuscularly with 50 μL of the solution. Immunization was repeated every two weeks for a total of three immunizations. 10 μL of serum was collected before each immunization to analyze the enhanced antibody response. Two weeks after the third immunization, mice were euthanized by enucleation and cervical dislocation. Lymphocytes were isolated for ELISPOT assay. Serum was incubated overnight at 4°C and centrifuged at 5000 rpm for 5 min to collect antiserum. Tail vein blood was collected from mice at W0, W2, W4, and W6 after vaccination.

[0262] 2. ELISA detection of mouse VP1 antiserum

[0263] Coating: 1 μg / mL of VP1 protein was coated onto a 96-well ELISA plate and incubated overnight at 4°C.

[0264] Blocking: After washing once with PBST, block with 3% BSA at 37°C for 2 hours.

[0265] The remaining steps are performed according to the ELISA operation in Example 2 (2).

[0266] ELISA was used to detect and analyze the kinetics of serum antibody production against EV71 VP1, and the results are as follows: Figure 11 As shown, from Figure 11 It can be seen that, with the use of Addavax adjuvant, after a booster immunization, mice in group 1 (rAdMH5-SpyVP1 trivalent vaccine group) and group 2 (Spycatcher-VP1 monotherapy group) began to produce antibodies against EV71-VP1. Moreover, the production rate of anti-VP1 antibodies by rAdMH5-SpyVP1 was faster than that of Spycatcher-VP1 monotherapy, with a significant difference (P<0.05), indicating that granulated VP1 can rapidly induce the production of VP1 antibodies.

[0267] In addition, serum dilutions of 2×10⁻⁶ were used to detect VP1 antibody kinetics. 4 The dilution of VP1 antibody titer in serum after three immunizations was 10. -3 ~10 -7 ELISA results are as follows: Figure 12 As shown, from Figure 12 It can be seen that in the continuous gradient dilution, the titer of the antibody against VP1 obtained by the rAdMH5-SpyVP1 group was consistent with that of the Spycatcher-VP1 group, which may be because the effect of multiple immunizations and adjuvant treatments masked the role of particulate formation.

[0268] 3. ELISPOT

[0269] To evaluate whether two vaccine groups, Spycatcher-VP1 and granulated rAdMH5-SpyVP1, can induce T-cell immune responses.

[0270] Mouse lymphocyte isolation: Mice were euthanized by dislocation and soaked in medical alcohol for about 30 seconds. The spleen was dissected in a clean bench, and the fascia and adipose tissue were removed. The spleen was placed in a 6-well plate, washed with serum-free medium, and then minced with scissors. The spleen was ground on a 70-mesh screen and washed again with 1 mL of serum-free medium. Cells were collected, centrifuged at 500g for 10 min, the supernatant was discarded, 6 mL of erythrocyte lysis buffer was added, and the mixture was incubated at room temperature for 2 min. The cells were then centrifuged at 500g for 10 min, the supernatant was discarded, and the cells were resuspended in 10 mL of PBS. The cells were then centrifuged at 250g for 10 min, the supernatant was discarded, and the mixture was resuspended in 5 mL of PBS. The cells were then centrifuged at 250g for 10 min, the supernatant was discarded, and the mixture was centrifuged again at 250g for 10 min. Finally, the cells were resuspended in 1 mL of 10% FBS in RPMI-1640 medium and counted using a cell counter. After the mouse lymphocytes were isolated, the IFN-γ and IL4 cytokines (catalog numbers: 2210002 and 2210402) were detected according to the ELISPOT kit instructions.

[0271] Spot counting: Use an ELISPOT counter to read the color development of the spots on the board and count them according to the size of the spots.

[0272] The detection results of cytokine IFN-γ are as follows: Figure 13 As shown, the detection results of cytokine IL-4 are as follows: Figure 14 As shown. From Figure 13-14 It can be seen that, compared with the experimental control group, the mice that were only vaccinated with rAdMH5-SpyVP1 vaccine could not detect the cytokine IFN-γ, and only a low level of cytokine IL-4 was detected.

[0273] 4. Cell micro-neutralization experiment

[0274] The EV71 virus neutralization experiment was conducted using a microcell neutralization assay to verify whether the serum of mice vaccinated with two VP1 vaccines had different neutralizing activities against the EV71 virus.

[0275] Neutralization assay for EV71: Prior to the neutralization assay for infectious virus, mouse serum collected after three immunizations was inactivated at 56°C for complement and other potential neutralizing agents. Serum samples were then serially diluted 2-fold from 1:20 to 1:1280 and compared with an equal volume of 200 TCID50. 50 The EV-71 virus was incubated at 37°C for 1 hour. The serum-virus mixture was then added to Vero cells, and the cells were cultured for another 3 days in fresh serum containing 1% FBS. Results were as follows... Figure 15 As shown. From Figure 15 It can be seen that the mice vaccinated with VP1 have similar effective neutralizing activity, and there is no significant difference in the neutralizing antibody titers between the two groups.

[0276] Neutralization assays for human adenovirus types 3 and 7: Before the neutralization assay, the TCID50 of adenovirus Ad3E and Ad7E was measured. All mouse antiserum was inactivated for complement at 56°C for 30 min. Cells were seeded at a density of 2 × 10⁶ cells per well in 96-well plates. 4 A549 cells were cultured until the cell density reached 60%–80% for subsequent experiments. Both the immune antiserum and control serum were diluted with serum-free DMEM medium at dilution gradients of 1:100 to 1:102400. Ad3E and Ad7E viruses were diluted with serum-free DMEM medium to a final concentration of 200 TCID50 / 100 μL. An equal volume of virus and antiserum was mixed in each well. After incubation at 37°C for 1 hour, the cells were discarded, washed once with serum-free DMEM medium, and the virus-serum mixture was added. The cells were incubated at 37°C for 2 hours, then the medium was changed, and 200 μL of serum-free DMEM medium was added. The cells were then cultured at 37°C for 2–3 days. The cells were observed under a fluorescence microscope, and the results were interpreted based on the fluorescence count. Results are shown below. Figure 16 As shown. From Figure 16 It can be seen that, compared with rAdMH5-Spytag, rAdMH5-SpyVP1 does not affect the neutralizing antibody titer of Ad3E, but it reduces the neutralizing antibody titer of Ad7E.

[0277] Example 4. Preparation of a trivalent adenovirus-SARS-CoV-2 vaccine

[0278] Using rAdMH5-Spytag viral particles as a platform for antigen display, the RBD protein (GenBank: OP603965) of SARS-CoV-2 BA.5 strain was fused with Spycather for expression. After in vitro incubation, the RBD was displayed on the surface of rAdMH5-Spytag particles, yielding a novel anti-SARS-CoV-2 nanoparticle vaccine candidate, rAdMH5-SpyRBD. Because rAdMH5-SpyRBD retains the major neutralizing antigenic epitopes of human adenovirus types 3 and 7, it can serve as a candidate for a trivalent vaccine against SARS-CoV-2 and human adenovirus types 3 and 7.

[0279] 1. Expression and purification of Spycatcher-RBD

[0280] The RBD and Spycatcher sequences were linked using a 3GSG linker. The nucleic acid sequence encoding Spycatcher-RBD was optimized and synthesized by the Guangzhou branch of Beijing Qingke Biotechnology Co., Ltd., and constructed into the pcDNA3.1 vector. The Spycatcher-RBD protein was expressed and purified by Nanjing GenScript Biotech Co., Ltd. using the CHO cell line. SDS-PAGE results are shown below. Figure 17 As shown, from Figure 17It can be seen that the Spycatcher-RBD protein was successfully expressed and purified, yielding a protein band of approximately 50 kDa.

[0281] 2. In vitro binding of rAdMH5-Spytag and Spycatcher-RBD

[0282] We used Western blotting, ELISA, and particle size analysis to verify whether rAdMH5-Spytag and Spycatcher-RBD can bind in vitro.

[0283] (1) Western Blot

[0284] Sample preparation: Mix 10 μL of rAdMH5-Spytag and 1 μL of Spycatcher-RBD (MH5-SpyRBD), and incubate overnight at 4°C. Separately, use 10 μL of rAdMH5-Spytag and 1 μL of Spycatcher-RBD as controls. All samples were treated with 4×SDS Loading Buffer, heated at 98°C for 5 min, and then rapidly cooled on ice.

[0285] SDS-PAGE gel: Prepare an 8% discontinuous gel according to the Molecular Cloning Laboratory Guide.

[0286] Electrophoresis: Set the voltage to a constant 80V, adjust it to 120V after 30 minutes, and continue electrophoresis until the dye reaches the bottom of the separating gel, then disconnect the power.

[0287] Transfer: After SDS-PAGE electrophoresis, wet transfer of PVDF membrane was performed at 260mA for 90min. After transfer, the membrane was blocked overnight in PBST containing 5% skim milk powder, 0.5% tryptone, and 0.05% Tween 20.

[0288] Antibody incubation: Wash the membrane once with PBST. The primary antibody was spike monoclonal antibody (Sino Biological, Inc., catalog number: 40592-MM117), diluted according to the instructions using blocking buffer. Incubate at 37°C for 1 hour, wash four times with PBST for 10 minutes each time, then add HRP-labeled goat anti-mouse IgG secondary antibody diluted according to the instructions, incubate at 37°C for approximately 45 minutes, and wash four times with PBST for 10 minutes each time.

[0289] Imaging: After membrane washing, images were captured and saved using a high-sensitivity ECL chemiluminescence kit on a fully automated chemiluminescence imaging system (iBright FL1500). Imaging results are shown below. Figure 18 As shown, from Figure 18It can be seen that Spike monoclonal antibody can specifically recognize the band of rAdMH5-SpyRBD located at approximately 210 kDa, and can also specifically recognize the band of Spycatcher-RBD located at approximately 50 kDa (lane 3), indicating that Spycatcher-RBD can bind to rAdMH5-Spytag.

[0290] (2) ELISA

[0291] Coating: The ELISA plates were coated with a concentration of 2×10⁻⁶. 10 VPs rAdMH5-Spytag and Ad7 GZ08 strains were encapsulated overnight at 4°C.

[0292] Blocking: Wash once with 250 μL PBST and blot dry on paper. Add 200 μL of PBST containing 3% BSA to each well of a 96-well microplate and incubate at 37°C for 2 hours for blocking.

[0293] In vitro binding of recombinant adenovirus rAdMH5-Spytag to Spycatcher-RBD protein: Wash once with PBST and blot dry on paper. Add 2 μg / mL Spycatcher-RBD protein to ensure complete binding of the protein to rAdMH5-Spytag, and incubate at room temperature for 4 h.

[0294] Antibody incubation: Wash once with PBST. The primary antibody was Spike monoclonal antibody (Sino Biological, Inc., catalog number: 40592-MM117), diluted according to the manufacturer's instructions with PBST containing 1% BSA. A 3% BSA blocking buffer was used as a blank control. Incubate at 37°C for 1 hour, wash four times with PBST, and blot dry. Dilute the HRP-labeled goat anti-mouse IgG secondary antibody according to the manufacturer's instructions and incubate at 37°C for 1 hour.

[0295] Color development: After incubation with the secondary antibody, wash four times with PBST, pat dry, add 100 μL of TMB substrate, and incubate at room temperature in the dark for 5-10 minutes for color development. After color development, add 100 μL of 2M H2SO4 to stop the reaction, and measure the OD value at 450 nm using a microplate reader.

[0296] The results of the ELISA reader test are as follows Figure 19 As shown. From Figure 19It can be seen that only rAdMH5-Spytag coated with Spycatcher-RBD protein can be recognized by Spike antibody; the Ad7 GZ08 strain cannot be recognized by Spike antibody when Spycatcher-RBD protein or BSA is added. Simultaneously, ELISA experiments also show that the Spytag tag embedded in HVR4 is exposed on the surface of adenovirus particles.

[0297] (3) Particle size

[0298] Instrument: Nanoparticle size and Zeta potential analyzer (Malvin)

[0299] After the samples were incubated overnight at 4°C, they were centrifuged at 12,000 rpm for 5 min at low temperature. The supernatant was then collected, and the particle size was measured using a nanoparticle size and Zeta potential analyzer. Among all the measured data, the Z-mean value was the average particle size obtained through the intensity distribution. Therefore, the Z-mean value (nm) was used to represent the particle size.

[0300] The results are as follows Figure 20 As shown. From Figure 20 It can be seen that the particle size of rAdMH5-Spytag is 128.7 nm, and the particle size of rAdMH5-SpyRBD is 143.3 nm. The particle size of rAdMH5-SpyRBD is larger than that of rAdMH5-Spytag, indicating that the Spycatcher-RBD protein can bind to the rAdMH5-Spytag particles.

[0301] The results of Western blotting, ELISA, and particle size analysis all indicate that the Spycatcher-RBD protein can bind to and be displayed on the surface of rAdMH5-Spytag particles.

[0302] Example 5. Immunogenicity analysis of adenovirus-SARS-CoV-2 (rAdMH5-SpyRBD) trivalent vaccine

[0303] To verify the immunogenicity of granulated rAdMH5-SpyRBD and standalone Spycatcher-RBD in animals.

[0304] 1. Animal immunization

[0305] Recombinant adenovirus rAdMH5-Spytag and Spycatcher-RBD were conjugated in vitro and then used to immunize mice. An experimental group and a control group were designed to be immunized simultaneously.

[0306] Group 1 (Recombinant Adenovirus Control Group): rAdMH5-Spytag: 2×10 12 VPs;

[0307] Group 2 (rAdMH5-SpyRBD adjuvant experimental group): hexagonal protein to RBD protein molar ratio 1:1, rAdMH5-Spytag, 2×10 12 VPs+spycatcher-RBD, 1μg+Addavax;

[0308] Group 3 (rAdMH5-SpyRBD adjuvant-free experimental group): hexagonal protein to RBD protein molar ratio 1:1, rAdMH5-Spytag, 2×10 12 VPs+Spycatcher-RBD, 1μg;

[0309] Group 4 (Spycatcher-RBD experimental group): Spycatcher-RBD: 1μg + Addavax;

[0310] Group 5 (Spycatcher-RBD adjuvant-free experimental group): Spycatcher-RBD: 1 μg;

[0311] Group 6 (control group): PBS + Addavax.

[0312] Both rAdMH5-Spytag and Spycatcher-RBD dilutions were performed using PBS and incubated overnight at 4°C. Four 6-8 week old female BALB / c mice were immunized intramuscularly with 50 μL of the solution. Immunization was repeated every two weeks for a total of three immunizations. Before each immunization, 10 μL of serum was collected to analyze the enhanced antibody response. Two weeks after the third immunization, mice were euthanized by enucleation and cervical dislocation. Lymphocytes were isolated for ELISPOT assay. Serum was incubated overnight at 4°C, centrifuged at 5000 rpm for 5 min, and antiserum was collected.

[0313] After vaccination, tail vein blood was collected from mice at W0, W2, W4 and W6.

[0314] 2. ELISA detection of mouse RBD antiserum

[0315] Coating: 96-well ELISA plates were coated with 1 μg / mL of SARS-CoV-2 (BA.4 / BA.5 / BA.5.2) SpikeRBD and incubated overnight at 4°C.

[0316] Blocking: After washing once with PBST, block with 3% BSA at 37°C for 2 hours. The remaining steps are performed according to the ELISA procedure in Example 4, 2(2).

[0317] The kinetics of antibody production against SARS-CoV-2 RBD in serum were detected and analyzed by ELISA, and the results are as follows: Figure 21 As shown, from Figure 21 It can be seen that, with the use of Addavax adjuvant, after a booster immunization, mice in group 2 (rAdMH5-SpyVP1 trivalent vaccine group) and group 4 (Spycatcher-VP1 monotherapy group) began to produce antibodies against BA5.2-RBD. Moreover, the production rate of anti-RBD IgG antibodies by rAdMH5-SpyRBD was faster than that by Spycatcher-RBD monotherapy, with a significant difference (P<0.05). This indicates that the granulated RBD can rapidly induce the production of RBD antibodies under the action of adjuvant.

[0318] In addition, serum dilutions of 2×10⁻⁶ were used to detect the kinetics of RBD antibodies. 4 The dilution of RBD antibody titer in serum after three immunizations was 10. -3 -10 -7 ELISA results are as follows: Figure 22 As shown, from Figure 22 It can be seen that in the serial dilution, the rAdMH5-SpyRBD group obtained a higher titer of anti-RBD antibody than the Spycatcher-RBD group, indicating that the granulated RBD protein can induce a higher antibody titer after three immunizations.

[0319] (3) ELISPOT

[0320] Activating T-cell immune responses is crucial for vaccine development. To test whether Spycatcher-RBD and granulated rAdMH5-SpyRBD vaccines could induce T-cell immunity, ELISPOT was used to measure the IFN-γ and IL-4 factors secreted by T lymphocytes that specifically responded to the BA5-RBD antigen.

[0321] Mouse lymphocyte isolation: Mice were euthanized by dislocation and soaked in medical alcohol for about 30 seconds. The spleen was dissected in a clean bench, and the fascia and adipose tissue were removed. The spleen was placed in a 6-well plate, washed with serum-free medium, and then minced with scissors. The spleen was ground on a 70-mesh screen and washed again with 1 mL of serum-free medium. Cells were collected, centrifuged at 500g for 10 min, the supernatant was discarded, 6 mL of erythrocyte lysis buffer was added, and the mixture was incubated at room temperature for 2 min. The cells were then centrifuged at 500g for 10 min, the supernatant was discarded, and the cells were resuspended in 10 mL of PBS. The cells were then centrifuged at 250g for 10 min, the supernatant was discarded, and the mixture was resuspended in 5 mL of PBS. The cells were then centrifuged at 250g for 10 min, the supernatant was discarded, and the mixture was centrifuged again at 250g for 10 min. Finally, the cells were resuspended in 1 mL of 10% FBS in RPMI-1640 medium and counted using a cell counter. After the mouse lymphocytes were isolated, the IFN-γ and IL4 cytokines (Daiyou, catalog numbers: 2210002 and 2210402) were detected according to the ELISPOT kit instructions.

[0322] Spot counting: Use an ELISPOT counter to read the color development of the spots on the board and count them according to the size of the spots.

[0323] The detection results of cytokine IFN-γ are as follows: Figure 23 As shown, the detection results of cytokine IL-4 are as follows: Figure 24 As shown. From Figure 23-24 It can be seen that, compared with the experimental control group, the production of cytokines IFN-γ and IL-4 can be detected in mice vaccinated with both groups of vaccines, indicating that both groups of vaccines induced T cell immune responses.

[0324] (4) Cell micro-neutralization experiment

[0325] A SARS-CoV-2BA5.2 virus neutralization experiment was conducted using a cellular micro-neutralization assay to verify whether the serum of mice vaccinated with two RBD vaccines had different neutralizing activities against the SARS-CoV-2BA5.2 virus.

[0326] Neutralization assay for BA5.2: Prior to the neutralization assay for infectious viruses, mouse serum collected after three immunizations was inactivated with complement and other potential neutralizing agents at 56°C. Serum samples were then serially diluted 2-fold, from 1:32 to 1:4096, and mixed with 150 FFU of SARS-CoV-2 Omicron BA5.2, EG.5, and the original WT strain. The serum and virus were incubated together at 37°C for 1 hour. The serum-virus mixture was then added to Vero-E6 cells and incubated for another 1 hour at 37°C. After removing the inoculum, the cells were incubated with 1.2% methylcellulose for 24 h, fixed in 4% paraformaldehyde, and then permeabilized with 0.2% Triton X-100. The cells were then treated with a primary antibody against SARS-CoV / SARS-CoV-2N protein (Sinochem, 40143-T62) and a secondary antibody against HRP-labeled goat anti-rabbit IgG (H+L) (Jackson, 111-035-144). Finally, viral foci were observed using TrueBlue Peroxidase Substrate (KPL, Gaithersburg, MD) and counted using an ELISPOT Reader. Results are as follows: Figure 25 As shown. From Figure 25 It can be seen that both groups of mice vaccinated with RBD vaccines exhibited similar effective neutralizing activity, with no significant difference in neutralizing antibody titers. Furthermore, in the cross-reaction experiment against the SARS-CoV-2 variant EG.5 and the original strain WT, only the antiserum induced by the rAdMH5-SpyRBD vaccine showed low neutralizing activity against the variant EG.5, while the antiserum induced by both RBD vaccines showed no neutralizing activity against the original strain WT.

[0327] Neutralization assays for human adenovirus types 3 and 7: Before the neutralization assay, the TCID50 of adenovirus Ad3E and Ad7E was measured. All mouse antiserum was inactivated for complement at 56°C for 30 min. Cells were seeded at a density of 2 × 10⁶ cells per well in 96-well plates. 4 A549 cells were cultured until the cell density reached 60%–80% for subsequent experiments. Both the immune antiserum and control serum were diluted with serum-free DMEM medium at dilution gradients of 1:100 to 1:102400. Ad3E and Ad7E viruses were diluted with serum-free DMEM medium to a final concentration of 200 TCID50 / 100 μL. An equal volume of virus and antiserum was mixed in each well. After incubation at 37°C for 1 hour, the cells were discarded, washed once with serum-free DMEM medium, and the virus-serum mixture was added. The cells were incubated at 37°C for 2 hours, then the medium was changed, and 200 μL of serum-free DMEM medium was added. The cells were then cultured at 37°C for 2–3 days. The cells were observed under a fluorescence microscope, and the results were interpreted based on the fluorescence count. Results are shown below. Figure 26 As shown. From Figure 26 It can be seen that, compared with rAdMH5-Spytag, rAdMH5-SpyRBD does not affect the neutralizing antibody titer of Ad3E, but it does reduce the neutralizing antibody titer of Ad7E.

[0328] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A modified recombinant adenovirus, said recombinant adenovirus comprising a chimeric hexagonal polypeptide, said chimeric hexagonal polypeptide comprising a first chaperone peptide, the first chaperone peptide being covalently coupled to a second chaperone peptide. wherein The chimeric hexagonal polypeptide contains a portion derived from one or more adenoviruses.

2. The recombinant adenovirus according to claim 1, wherein the chimeric hexagon comprises one or more hexagonal HVRs, preferably including one or more of HVR1 loop, HVR2 loop, HVR3 loop, HVR4 loop, HVR5 loop, HVR6 loop, and HVR7 loop. Preferably, the one or more six-neighbor HVRs are each independently derived from Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57. Preferably, the recombinant adenovirus is derived from a first adenovirus, and the chimeric hexagon includes one or more hexagonal HVR variants of the first adenovirus. Preferably, the first adenovirus originates from any one of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57, and is more preferably derived from Ad3. Preferably, the recombinant adenovirus further comprises one or more hexagonal HVRs derived from one or more additional adenoviruses. Preferably, the one or more additional adenoviruses are derived from any one or more of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57. Preferably, the first adenovirus is different from the one or more additional adenoviruses. Preferably, the one or more additional adenoviruses are derived from or include Ad7.

3. The recombinant adenovirus of claim 1, wherein the first chaperone peptide is disposed on the outer surface of the chimeric hexagon, preferably, the first chaperone peptide is disposed on one or more HVR rings of the chimeric hexagon, more preferably on the HVR4 ring of the chimeric hexagon. Preferably, the first chaperone peptide and the second chaperone peptide are derived from any one of the following chaperone peptide pairs: (1) SpyCatcher and SpyTag; (2)SdyCatcher and SdyTag / SnoopTag / SpyTag / RumTag / / RumTrunkTag / PhoTag / EntTag / BacTag; (3) SnoopCatcher and SnoopTag / SnoopTagJr; (4) MoonCake and PhoTag / RumTrunkTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag; (5) KatI and PhoTag / RumTrunkTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag; (6) QueenCatcher and PhoTag / RumTrunkTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag; (7) DogCatcher and RrgATag / RrgATag2 / DogTag; (8) Pilin-C and Isopeptag; (9) Pilin-N and Isopeptag-N; (10) PsCsCatcher and PsCsTag; (11) In and Jo; (12) RrgACatcher and RrgATag / RrgATag2 / DogTag, Preferably, the first or second chaperone peptide is derived from SpyTag, SdyTag, SnoopTag, PhoTag, EntTag, KTag, BacTag, Bac2Tag, Bac3Tag, Bac4Tag, RumTrunkTag, Rum7Tag, RumTag, Rum2Tag, Rum3Tag, Rum4Tag, Rum5Tag, Rum6Tag, Bac5Tag, SnoopTagJr, RrgATag, or RrgA. Tag2, DogTag, Isopeptag, Isopeptag-N, PsCsTag, or Jo, or their homologs, wherein the homologs have at least 60% homology with it, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology. Preferably, the second chaperone peptide or the first chaperone peptide is derived from SpyCatcher, SdyCatcher, SnoopCatcher, MoonCake, KatI, QueenCatcher, DogCatcher, Pilin-C, Pilin-N, PsCsCatcher, In, or RrgACatcher, or a homolog thereof, wherein the homolog has at least 60% homology with it, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology.

4. The recombinant adenovirus according to claim 1, wherein the second chaperone peptide and the target protein constitute a fusion protein. Preferably, the second chaperone peptide and the first chaperone peptide can be covalently coupled via an isopeptide bond or an ester bond. Preferably, the target protein includes one or more of the following: antigen, antibody or antigen-binding fragment thereof, therapeutic peptide, detectable tag, cell-penetrating peptide, targeting peptide or membrane fusion peptide. Preferably, the hexapods of the recombinant adenovirus display one or more target proteins on their surface. Preferably, the antigen is selected from viral antigens, bacterial antigens, parasitic antigens, or fungal antigens.

5. The recombinant adenovirus according to claim 1, wherein the backbone of the recombinant adenovirus is derived from Ad3. Preferably, the chimeric hexagonal polypeptide includes an HVR5 ring derived from Ad7. Preferably, the chimeric hexagonal polypeptide further includes HVR1, HVR2, HVR3, HVR4 and / or HVR7 rings derived from Ad3. Preferably, the first chaperone peptide is inserted into or replaces the HVR4 ring. Preferably, the first and second chaperone peptides are derived from the SpyCatcher and SpyTag chaperone peptide pair. Preferably, the first chaperone peptide is derived from SpyTag and the second chaperone peptide is derived from SpyCatcher, or the second chaperone peptide is derived from SpyTag and the first chaperone peptide is derived from SpyCatcher.

6. A composition comprising the recombinant adenovirus of any one of claims 1-5, and a pharmaceutically acceptable vector or pharmaceutically acceptable excipient.

7. The composition of claim 6, further comprising a fusion protein of a second chaperone peptide and the target protein. Preferably, the second chaperone peptide is directly linked to the target protein or linked via a linker. Preferably, the adapter is selected from glycine adapters, serine adapters, glycine-serine adapters, and more preferably from GnS, SnG, or (GSG)n, wherein n is selected from 1, 2, 3, or 4. Preferably, the target protein includes one or more of the following: antigen, antibody or antigen-binding fragment thereof, therapeutic peptide, detectable tag, cell-penetrating peptide, targeting peptide or membrane fusion peptide. Preferably, the recombinant adenovirus displays one or more target proteins. Preferably, the antigen is selected from viral antigens, bacterial antigens, parasitic antigens, or fungal antigens. Preferably, the second chaperone peptide is covalently coupled to the first chaperone peptide via an isopeptide bond or an ester bond. Preferably, the target protein is an antigen, and more preferably, the composition is a vaccine.

8. An immunogenic agent comprising a modified recombinant adenovirus, the recombinant adenovirus comprising a chimeric hexagonal polypeptide, the chimeric hexagonal polypeptide further comprising a first chaperone peptide, the first chaperone peptide being covalently coupled to a second chaperone peptide, the second chaperone peptide being linked to a target protein. wherein The chimeric hexagonal polypeptide contains a portion derived from one or more adenoviruses.

9. The immunogenicity of claim 8, wherein the chimeric hexagon comprises one or more hexagonal HVRs, preferably including one or more of HVR1, HVR2, HVR3, HVR4, HVR5, HVR6, and HVR7 rings. Preferably, the one or more six-neighbor HVRs are each independently derived from Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57. Preferably, the recombinant adenovirus is derived from a first adenovirus, and the chimeric hexagon includes one or more hexagonal HVR variants of the first adenovirus. Preferably, the first adenovirus originates from any one of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57, more preferably from Ad3. Preferably, the recombinant adenovirus further comprises one or more hexagonal HVRs derived from one or more additional adenoviruses. Preferably, the one or more additional adenoviruses are derived from any one or more of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad8, Ad9, Ad10, Ad11, Ad12, Ad13, Ad14, Ad15, Ad16, Ad55, and Ad57, more preferably from Ad7; Preferably, the first adenovirus is different from the one or more additional adenoviruses. Preferably, the one or more additional adenoviruses are derived from Ad7.

10. The immunogenicity of claim 8, wherein the first chaperone peptide is disposed on the outer surface of the chimeric hexagon, preferably, the first chaperone peptide is disposed on one or more HVR rings of the chimeric hexagon, more preferably on the HVR4 ring of the chimeric hexagon. Preferably, the first chaperone peptide and the second chaperone peptide are derived from any one of the following chaperone peptide pairs: (1) SpyCatcher and SpyTag; (2)SdyCatcher and SdyTag / SnoopTag / SpyTag / RumTag / / RumTrunkTag / PhoTag / EntTag / BacTag; (3) SnoopCatcher and SnoopTag / SnoopTagJr; (4)MoonCake and PhoTag / RumTrunkTag / RumTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag; (5)KatI and PhoTag / RumTrunkTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag; (6)QueenCatcher and PhoTag / RumTrunkTag / RumTrunkD9NTag / RumTag / Rum2Tag / Rum3Tag / Rum4Tag / Rum5Tag / Rum6Tag / Rum7Tag / BacTag / Bac2Tag / Bac3Tag / Bac4Tag / Bac5Tag / SpyTag / SdyTag; (7)DogCatcher and RrgATag / RrgATag2 / DogTag; (8) Pilin-C and Isopeptag; (9) Pilin-N and Isopeptag-N; (10) PsCsCatcher and PsCsTag; (11)In and Jo; (12)RrgACatcher and RrgATag / RrgATag2 / DogTag, Preferably, the first chaperone peptide is derived from SpyTag, SdyTag, SnoopTag, PhoTag, EntTag, KTag, BacTag, Bac2Tag, Bac3Tag, Bac4Tag, RumTrunkTag, Rum7Tag, RumTag, Rum2Tag, Rum3Tag, Rum4Tag, Rum5Tag, Rum6Tag, Bac5Tag, SnoopTagJr, RrgATag, RrgATag2 DogTag, Isopeptag, Isopeptag-N, PsCsTag, or Jo, or their homologs, wherein the homologs have at least 60% homology with it, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology. Preferably, the second chaperone peptide is derived from SpyCatcher, SdyCatcher, SnoopCatcher, MoonCake, KatI, QueenCatcher, DogCatcher, Pilin-C, Pilin-N, PsCsCatcher, In, or RrgACatcher, or a homolog thereof, wherein the homolog has at least 60% homology with it, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% homology.

11. The immunogenicity of claim 8, wherein the second chaperone peptide constitutes a fusion protein with the target protein. Preferably, the second chaperone peptide is covalently coupled to the first chaperone peptide via an isopeptide bond or an ester bond. Preferably, the target protein is an antigen or a fragment thereof. Preferably, the recombinant adenovirus displays one or more target proteins. Preferably, the antigen is selected from viral antigens, bacterial antigens, parasitic antigens, or fungal antigens.

12. The immunogenicity of claim 8, wherein the target protein and the second chaperone peptide are covalently coupled to a first chaperone peptide contained in a modified hexagonal HVR ring, thereby forming recombinant adenovirus nanoparticles displaying the target protein on the hexagonal surface.

13. An isolated nucleic acid, said nucleic acid encoding a recombinant adenovirus of any one of claims 1-5, an immunogenic composition of claim 6 or 7, or an immunogenic substance of any one of claims 8-12.

14. The nucleic acid of claim 13, comprising a first nucleic acid, wherein the first nucleic acid encodes a recombinant adenovirus of any one of claims 1-5. Preferably, the nucleic acid further comprises a second nucleic acid, the second nucleic acid encoding a fusion protein of a second chaperone peptide and the target protein.

15. An expression vector comprising the nucleic acid of claim 13 or 14.

16. A host cell comprising the nucleic acid of claim 13 or 14 or the expression vector of claim 15.

17. The use of the recombinant adenovirus of any one of claims 1-5, the composition of claim 6 or 7, the immunogenicity of any one of claims 8-12, the nucleic acid of any one of claims 13 or 14, the expression vector of claim 15, or the host cell of claim 16 in the preparation of a medicament for treating or preventing a disease in a subject.

18. In the application according to claim 17, the drug is a vaccine, preferably a multivalent vaccine. Preferably, the disease includes diseases related to the target protein, and more preferably, diseases related to the antigen.

19. The recombinant adenovirus of any one of claims 1-5, the composition of claim 6 or 7, the nucleic acid of any one of claims 13 or 14, the expression vector of claim 15, or the host cell of claim 16 as a vector.

20. A method for preventing or treating a disease, the method comprising administering to a subject in need a recombinant adenovirus of any one of claims 1-5, a composition of claim 6 or 7, an immunogenic agent of any one of claims 8-12, a nucleic acid of any one of claims 13 or 14, an expression vector of claim 15, or a host cell of claim 16.

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