Nucleic acid and amino acid sequences of gorilla adenovirus, vectors containing them, and their uses

Abstract

This invention provides a novel adenovirus strain that exhibits high immunogenicity and very low pre-existing immunity in the general human population. [Solution] An isolated polynucleotide is provided which encodes an adenovirus hexone protein comprising an HVR having a specific amino acid sequence, or a variant thereof containing up to two mutations, wherein the polynucleotide encoding the adenovirus hexone protein having a specific amino acid sequence further encodes an adenovirus fiber protein having a specific amino acid sequence, or a variant thereof containing up to two mutations.

Description

[Technical Field]

[0001] This invention relates to novel adenovirus strains that are highly immunogenic and have very low pre-existing immunity in the general human population. The absence of detectable neutralizing antibodies is due to a novel hypervariable region in the adenovirus capsid protein hexon. This invention provides the nucleotide and amino acid sequences of these novel adenovirus strains, as well as recombinant viruses, virus-like particles, and vectors based on these strains. Furthermore, it provides pharmaceutical compositions and pharmaceutical uses for the treatment or prevention of diseases, as well as methods for producing adenoviruses or virus-like particles using the novel sequences, recombinant viruses, virus-like particles, and vectors. [Background technology]

[0002] Adenoviruses (Ad) constitute a large family of double-stranded DNA viruses found in amphibians, birds, and mammals, and possess a non-enveloped icosahedral capsid structure (Non-Patent Literature 1; Non-Patent Literature 2; Non-Patent Literature 3; Non-Patent Literature 4). In contrast to retroviruses, adenoviruses can transduce numerous cell types in several mammalian species, including both dividing and non-dividing cells, without integrating into the host cell genome.

[0003] Generally, adenovirus DNA is usually very stable and remains in the episome (e.g., extrachromosomally) unless transformation or tumorigenesis occurs. Furthermore, adenovirus vectors can be grown in high yields in well-defined production systems that are readily adaptable to the pharmaceutical-scale production of clinical-grade compositions. These characteristics, along with well-characterized molecular genetics, make recombinant adenovirus vectors excellent candidates for use as vaccine carriers. The production of recombinant adenovirus vectors may depend on the use of packaging cell lines that can complement the function of adenovirus gene products that have been deleted or manipulated to be non-functional.

[0004] Currently, two well-characterized human subgroup C adenovirus serotypes (i.e., hAd2 and hAd5) are widely used as the source of the viral scaffold for most adenovirus vectors used in gene therapy. Replication-deficient human adenovirus vectors have also been tested as vaccine carriers for delivering various immunogens derived from various infectious pathogens. Studies conducted in experimental animals (e.g., rodents, canids, and non-human primates) have shown that recombinant replication-deficient human adenovirus vectors carrying transgenes encoding immunogens and other antigens induce both humoral and cellular immune responses to the transgene products. Generally, researchers have reported success using human adenovirus vectors as vaccine carriers in non-human experimental systems by using immunization protocols that utilize high doses of recombinant adenovirus vectors predicted to induce an immune response, or by employing immunization protocols that employ sequential administration as booster immunizations of adenovirus vectors derived from different serotypes but having the same transgene product (Non-Patent Literature 5).

[0005] Vectors derived from adenovirus type C (e.g., Ad5, Ad6, ChAd3, etc.) are the most immunogenic (Non-Patent Literature 6). In particular, viral vectors based on human adenovirus type 5 (Ad5) have been developed for gene therapy and vaccine applications. While Ad5-based vectors are highly efficient in animal models, clinical trials have demonstrated that the efficiency of gene delivery decreases in the presence of pre-existing neutralizing antibodies in humans against wild-type Ad5 virus (especially those directed to the capsid, as shown in Figure 1) (Non-Patent Literature 7). Such antibodies are primarily directed to the hypervariable region of the hexon protein. Immunity in the general population limits the widespread application of Ad5-based Ad-vectorized vaccines. On the other hand, rare human adenoviruses are less immunogenic than Ad5 (Non-Patent Literature 6). Vectors based on non-human adenoviruses are subject to very low pre-existing immunity in the general human population (Non-Patent Literature 8). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Straus, Adenovirus infections in humans; TheAdenoviruses, 451-498, 1984 [Non-Patent Document 2] Hierholzer et al.,J. Infect.Dis.,158:804-813,1988 [Non-Patent Document 3] Schnurr and Dondero, Intervirology., 36:79-83,1993 [Non-Patent Document 4] Jong et al., J. Clin. Microbiol., 37:3940-3945: 1999 [Non-Patent Document 5] Mastrangeli, et. al., Human Gene Therapy, 7:79-87 (1996) [Non-Patent Document 6] Colloca et al., Sci. Transl. Med. 4 (115),2012 [Non-Patent Document 7] Moore JP et al. Science. 2008 May 9;320(5877):753-5 [Non-Patent Document 8] Farina et al., J. Virol. 75 (23), 11603-11613, 2001 [Overview of the project] [Problems that the invention aims to solve]

[0007] Although several non-human adenovirus vectors are known, there is a possibility that immunity against these may develop in humans. Therefore, there is a continuing need for adenovirus vectors that are highly immunogenic and have few or no existing neutralizing antibodies in humans.

Means for Solving the Problems

[0008] In a first aspect, the present invention provides A) (i) HVR1 comprising the amino acid sequence at positions 136 to 168 of SEQ ID NO: 2, or a variant thereof containing up to two mutations, (ii) HVR2 comprising the amino acid sequence at positions 187 to 201 of SEQ ID NO: 2, or a variant thereof containing up to two mutations, (iii) HVR3 comprising the amino acid sequence at positions 219 to 225 of SEQ ID NO: 2, or a variant thereof containing up to two mutations, (iv) HVR4 comprising the amino acid sequence at positions 257 to 268 of SEQ ID NO: 2, or a variant thereof containing up to two mutations, (v) HVR5 comprising the amino acid sequence at positions 276 to 290 of SEQ ID NO: 2, or a variant thereof containing up to two mutations, (vi) HVR6 comprising the amino acid sequence at positions 314 to 322Y of SEQ ID NO: 2, or a variant thereof containing up to two mutations, and (vii) HVR7 comprising the amino acid sequence at positions 431 to 456 of SEQ ID NO: 2, or a variant thereof containing up to two mutations, or B) (i) HVR1 comprising the amino acid sequence at positions 136 to 168 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, [[ID=2(4]] (ii) HVR2 comprising the amino acid sequence at positions 187 to 201 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, (iii) HVR3 comprising the amino acid sequence at positions 219 to 225 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, (iv) HVR4 comprising the amino acid sequence at positions 257 to 268 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence from positions 276 to 290 of SEQ ID NO: 9, or a variant thereof containing up to two mutations. (vi) HVR6 containing the amino acid sequence at positions 314-322 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 431-456 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, C) (i) HVR1 containing the amino acid sequence from positions 136 to 163 of SEQ ID NO: 11, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence at positions 182-196 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 214-220 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, (iv) HVR4 containing the amino acid sequence at positions 252-262 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence at positions 270-278 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, (vi) HVR6 containing the amino acid sequence at positions 302-310 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 419-442 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, D) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 17, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219-225 of SEQ ID NO: 17, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257-267 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence at positions 275-289 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, (vi) HVR7 containing the amino acid sequence at positions 313-321 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 430-455 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, E) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 19, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 19, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219-225 of SEQ ID NO: 19, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257-268 of SEQ ID NO: 19, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence from positions 276 to 290 of SEQ ID NO: 19, or a variant thereof containing up to two mutations, (vi) HVR6 containing the amino acid sequence at positions 314-322 of SEQ ID NO: 19, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 431-456 of SEQ ID NO: 19, or a variant thereof containing up to two mutations, F) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219-225 of SEQ ID NO: 21, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257-267 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence at positions 275-289 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, (vi) HVR6 containing the amino acid sequence at positions 313-321 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 430-455 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, G) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 23, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 23, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219-225 of SEQ ID NO: 23, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257-268 of SEQ ID NO: 23, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence from positions 276 to 290 of SEQ ID NO: 23, or a variant thereof containing up to two mutations. (vi) HVR6 containing the amino acid sequence at positions 314-322 of SEQ ID NO: 23, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence from positions 431 to 456 of SEQ ID NO: 23, or a variant thereof containing up to two mutations. The present invention provides a polynucleotide encoding an adenovirus hexone protein, wherein the polynucleotide encoding the adenovirus hexone protein described in G) further encodes the adenovirus fiber protein of SEQ ID NO: 6, or a variant thereof containing up to two mutations.

[0009] In a second embodiment, a hexone polypeptide encoded by a polynucleotide as defined in A), B), C), D), E), or F) of the first embodiment is provided.

[0010] In a third embodiment, the present invention provides an adenovirus capsid comprising a hexone, a fiber, and a penton protein, wherein, with respect to A) to F), the hexone is a hexone encoded by a polynucleotide of the first embodiment, and with respect to G), the hexone and fiber are a hexone and fiber encoded by a polynucleotide of the first embodiment.

[0011] In a fourth embodiment, the present invention provides (i) an adenovirus encoded by a polynucleotide according to the first embodiment, (ii) an adenovirus comprising a polynucleotide according to the first embodiment, and / or (iii) an adenovirus comprising a hexone polypeptide according to the second embodiment or a capsid according to the third embodiment.

[0012] In a fifth embodiment, the present invention provides (i) a virus-like particle encoded by a polynucleotide of the first embodiment, and / or (ii) a virus-like particle comprising a hexone polypeptide of the second embodiment or a capsid of the third embodiment.

[0013] In a sixth embodiment, the present invention provides a vector comprising a polynucleotide according to the first embodiment.

[0014] In a seventh embodiment, the present invention provides a composition comprising (i) an adjuvant, (ii) a polynucleotide according to the first embodiment, a hexone polypeptide according to the second embodiment, an adenovirus capsid polypeptide according to the third embodiment, an adenovirus according to the fourth embodiment, a virus-like particle according to the fifth embodiment, or a vector according to the sixth embodiment, and optionally (iii) a pharmaceutically acceptable excipient.

[0015] In an eighth aspect, the present invention provides a cell comprising a polynucleotide of the first aspect, a hexone polypeptide of the second aspect, an adenovirus capsid polypeptide of the third aspect, an adenovirus of the fourth aspect, a virus-like particle of the fifth aspect, or a vector of the sixth aspect.

[0016] In a ninth aspect, the present invention provides a polynucleotide of a first aspect, a hexone polypeptide of a second aspect, an adenovirus capsid polypeptide of a third aspect, an adenovirus of a fourth aspect, a virus-like particle of a fifth aspect, a vector of a sixth aspect, a composition of a seventh aspect, and / or a cell of an eighth aspect for use in the treatment or prevention of a disease.

[0017] In a tenth embodiment, the present invention relates to an in vitro method for producing adenovirus or adenovirus-like particles, (i) A step of expressing a polynucleotide of the first aspect in a cell so that adenoviruses or adenovirus-like particles aggregate within the cell, (ii) relating to an in vitro method comprising the step of isolating adenoviruses or adenovirus-like particles from cells or a culture medium surrounding cells. [Brief explanation of the drawing]

[0018] [Figure 1] Structure of adenovirus capsid [Figure 2] Schematic diagram of a shuttle plasmid [Figure 3] pGRAd23 DE1 GAG BAC schematic diagram [Figure 4] pGRAd23 DE1 GAG DE3 BAC schematic diagram [Figure 5] pGRAd23 DE1 GAG DE3 DE4 hAd5 E4orf6 Schematic diagram of BAC [Figure 6] T cell response to mouse splenocytes. Each point represents the response of one mouse, and the line corresponds to the average for each dose group. The injected dose, expressed as the number of viral particles, is shown on the x-axis. [Figure 7] Humoral response to GRAd23 DE1, which encodes the Gag antigen. Each point represents the response of one mouse, and the lines correspond to the mean for each dose group. [Figure 8]Serum prevalence of GRAd23 vectors in a panel of human serum. Each dot represents a single serum sample (neutralizing titer on the y-axis). The table shows the percentage of serum with negative (<18), moderate neutralizing titer (<200), or high titer (>200). [Figure 9] Serum prevalence of GRAd32 vector in a panel of human serum. Each point represents a single serum sample, and the vertical axis represents the neutralizing titer. [Figure 10] Humoral response to GRAd21 DE1, which encodes the Gag antigen. Each point represents the response of one mouse, and the lines correspond to the mean for each dose group. [Figure 11] Spike antigen expression using GRAd32 DE1, which encodes the SARS-CoV-2 spike antigen. [Figure 12] Spike antigen immunogenicity (ELIspot) using GRAd32 DE1, which encodes the SARS-CoV-2 spike antigen. [Figure 13] Post-immunization with GRAd32 DE1 encoding the SARS-CoV-2 spike antigen, followed by anti-spike serum antibody response. [Figure 14] Spike 2P expression. HeLa cells were infected with a specified vector at 50 MOI. 48 hours after infection, cell lysates were collected and analyzed by SDS-PAGE Western blot. In the upper panel, membranes were blotted with an anti-HA antibody that recognizes the spike 2P protein (HA tag). In the lower panel, GAPDH is used as a loading control. 1: Mock, 2: GRAd23b-S2P, 3: GRAd33b-S2P, 4: GRAd34b-S2P, 2: GRAd39b-S2P. [Figure 15] Immunogenicity is measured by antibody endpoint titers of the GRAd vector 2 weeks (w2) or 5 weeks (w5) after immunization of BALB / c mice with a specified dose (viral particles). The numbers below each group of data points represent the geometric mean. [Figure 16-1]SARS-CoV-2 specific binding and neutralizing antibody responses in GRAd-COV2 vaccinated volunteers. Antibody responses to SARS-CoV-2 induced by low-dose (LD-5x10^10 vp-circle), medium-dose (ID-1x10^11 vp-upright triangle), and high-dose (HD-2x10^11 vp-inverted triangle) GRAd-COV2 vaccination. Ages 18–55 and 65–85 distinguish the younger and older age cohorts, respectively. Horizontal black lines within each group of data points are set to the median of all panels. Human convalescent sera (HCS) (diamond) and NIBSC 20 / 130 standard plasma (black circle) obtained from previously hospitalized (hosp-dark gray) or non-hosp-light gray COVID-19 patients are shown for reference. (A) IgG binding to S1-S2 measured by CLIA on vaccination day (d0) and 1, 2, or 4 weeks after vaccination. Data are expressed in arbitrary units (AU) / ml. Filled and dashed lines are set at 12 and 15 AU / ml. According to the manufacturer, results above 15 are clearly positive, between 12 and 15 are ambiguous, and below 12 may indicate a negative result or a low level of IgG antibody against the pathogen. (BC) SARS-CoV-2 specific IgG titers in serum collected on d0 and w4 after vaccination were measured by ELISA with recombinant full-length spike (B) or RBD (C). Data are expressed as endpoint titers, and for negative serum where titer cannot be calculated, an arbitrary value of 50 (or half of the first serum dilution tested) is assigned. (DE) SARS-CoV-2 neutralizing antibodies four weeks after vaccination were detected by SARS-CoV-2 microneutralization assay (D) or plaque reduction neutralization test (E). SARS-CoV-2 neutralizing titers are expressed as MNA90 and PRNT50, or the reciprocal of the serum dilution that achieves 90% or 50% neutralization, respectively. The dashed line indicates LOD, and negative serum is assigned a value of half the LOD. [Figure 16-2] Same as above [Figure 16-3] Same as above [Figure 17-1]T cell responses to spike peptides induced by low-dose (LD-5x10^10 vp-round), medium-dose (ID-1x10^11 vp-right triangle), and high-dose (HD-2x10^11 vp-inverted triangle) GRAd-COV2 vaccination. The labels 18–55 years and 65–85 years distinguish the younger and older age cohorts, respectively. The horizontal black line is set to the median of all panels. (AB) IFNγELISpot on newly isolated PBMCs in w2. Data are represented as IFN-γ spot-forming cells (SFC) / 106 PBMCs. (A) Individual data points represent the cumulative spike T cell response, calculated by summing the responses to S1a, S1b, S2a, and S2b peptide pool stimulation for each volunteer and correcting for background (DMSO stimulation). (B) Newly isolated human convalescent PBMCs (HCPs) obtained from subjects who recovered from symptomatic SARS-CoV-2 infection. Distribution of IFNγ ELISpot responses to individual spike peptide pools. The dashed line indicates the assay-positive cutoff (48 SFCs / 1 million PBMCs). (CDEF) Intracellular staining of IFNγ / IL2 / IL4 / IL17 and FACS analysis in w2 of fresh PBMCs from young (CD) and aged (EF) volunteers. Data are expressed as the percentage of spike-specific CD4 (CE) or CD8 (DF) secreting each cytokine (or the sum of any Th1 cytokine combination, i.e., IFNγ alone, IL2 alone, and CD4 secreting both IFNγ and IL2), obtained by summing the responses to each of the four spike peptide pools and corrected for background (DMSO stimulation). The table below CE (CD4 graph) shows the p-values ​​obtained by the Kruskal-Wallis test, which compares arbitrary Th1, IFN-g, and IL-2 profiles within each dose group with IL-4 and IL-17 profiles. [Figure 17-2] Same as above [Figure 17-3] Same as above [Modes for carrying out the invention]

[0019] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0020] Preferably, terms used herein are defined as those found in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," Leuenberger, HGW, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland), as well as in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals," edited by Susan Budavari et al., CRC Press, 1996; and the United States Pharmacopeia-25 / National Formulary-20. It is defined as described in the States Pharmacopeia-25 / National Formulary-20), published by the United States Pharmaceutical Convention, Inc., Rockville Md., 2001.

[0021] Throughout this specification and the subsequent claims, unless otherwise required by context, the word “comprise,” and variations such as “comprises” and “comprising,” are understood to mean that they include the described feature, integer or process or group of features, integer or process, but not that they exclude other features, integer or process or group of integers or processes. Different aspects of the invention are defined in more detail in the following sections. Each of these defined aspects can be combined with any other aspect unless it is expressly shown to be contrary thereto. In particular, any feature indicated as preferred or advantageous can be combined with any other feature indicated as preferred or advantageous.

[0022] Throughout this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference, whether above or below.

[0023] Nucleotide and amino acid sequences Tables 1a and 1b below provide an overview of GRAd and its sequences as referred to herein (GRAd + number: isolated adenovirus strain; *: corresponding nucleotide sequence of the GRAd genome encoding the amino acid sequence). GRAd (Gorilla Adenovirus) is the inventor's strain name. The range of hexon, penton, and fiber genomic coordinates shown below does not include the final stop codon, but is optionally included / added to this disclosure when referring to polynucleotides encoding hexons, pentons, or fibers using these coordinates.

[0024] Table 1a: GRAd having sequence numbers referred to in this application [Table 1a]

[0025] Table 1b: Sequence IDs referenced in this application [Table 1b] JPEG2026082958000003.jpg159137

[0026] Tables 2a, 2b, 2c, 2d, and 2e provide genomic boundaries / coordinates of CDS, RNA, and ITR in the genome. These apply to references to genomic elements herein that are listed in these tables and incorporated as preferred in each embodiment.

[0027] Table 2a: Genomic boundaries of CDS, RNA, and ITR for GRAd32 and GRAd23. E3_CR1-alpha represents the estimated open reading frame with GTG as the start codon. rc represents the reverse complement. The splicing product is shown in multiple coordinate pairs. [Table 2a]

[0028] Table 2b: Genomic boundaries of CDS, RNA, and ITR in GRAd21 and GRAd37. E3_CR1-alpha represents the estimated open reading frame with GTG as the start codon. rc represents the reverse complement. The splicing product is shown in multiple coordinate pairs. [Table 2b]

[0029] Table 2c: Genomic boundaries of CDS, RNA, and ITR for GRAd33 and GRAd34. E3_CR1-alpha represents the estimated open reading frame with GTG as the start codon. rc represents the reverse complement. The splicing product is shown in multiple coordinate pairs. [Table 2c]

[0030] Table 2d: Genomic boundaries of CDS, RNA, and ITR in GRAd35 and GRAd36. E3_CR1-alpha represents the estimated open reading frame with GTG as the start codon. rc represents the reverse complement. The splicing product is shown in multiple coordinate pairs. [Table 2d]

[0031] Table 2e: Genomic boundaries of CDS, RNA, and ITR in GRAd37 and GRAd38. E3_CR1-alpha represents the estimated open reading frame with GTG as the start codon. rc represents the reverse complement. The splicing product is shown in multiple coordinate pairs. [Table 2e]

[0032] Aspects of the present invention and specific embodiments thereof The present invention relates to some of the embodiments described in the summary of the invention. These embodiments include alternative and preferred embodiments described below.

[0033] First aspectIn this invention, the present invention provides polynucleotides as outlined in the abstract of the invention, where “the variant” refers to the cited amino acid fragment, not the entire cited sequence number. In one preferred embodiment, the HVR variant comprises one mutation. The polynucleotide is preferably an isolated polynucleotide. In the art, as is known, for example, by Bradley et al. (J Virol., 2012 Jan;86(2):1267-72), adenovirus neutralizing antibodies often target the hexon hypervariable region, and by replacing the HVR region of the adenovirus with serumprevalence, the adenovirus can evade the immune system of the immune host. Thus, the above HVRs can be used with each of the hexon proteins defined below, but are useful independently of these hexon proteins, and the penton and fiber proteins below, i.e., they exert such usefulness by replacing the hexon HVR in other adenoviruses having other hexon, penton, and / or fiber proteins.

[0034] Preferably, A) The hexone protein described herein includes the amino acid sequence shown in Sequence ID No. 2, or a variant thereof. The hexone protein described in B) includes the amino acid sequence shown in SEQ ID NO: 9, or a variant thereof. The hexone protein described in C) includes the amino acid sequence shown in SEQ ID NO: 11, or a variant thereof. The hexone protein described in D) includes the amino acid sequence shown in SEQ ID NO: 17, or a variant thereof. The hexone protein described in E) includes the amino acid sequence shown in SEQ ID NO: 19, or a variant thereof. The hexone protein described in F) includes the amino acid sequence shown in SEQ ID NO: 21, or a variant thereof, and / or The hexone protein described in G) includes the amino acid sequence shown in SEQ ID NO: 23, or a variant thereof.

[0035] In one preferred embodiment, the polynucleotide further encodes an adenovirus fiber protein and / or an adenovirus penton protein. Here, the adenovirus fiber protein is Regarding A), the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6, or a variant thereof, With respect to B), D), E), and / or F), the amino acid sequence shown in SEQ ID NO: 6, or its variants, and / or Regarding C), this includes the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 15, or a variant thereof.

[0036] Adenovirus penton protein is Regarding A), the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 7, or a variant thereof, With respect to B), D), E), F), and / or G), the amino acid sequence shown in SEQ ID NO: 7, or its variants, and / or Regarding C), this includes the amino acid sequence shown in SEQ ID NO: 13, or its variants.

[0037] The above-mentioned variants of the hexon, fiber, and penton proteins of the adenovirus can be incorporated into the adenovirus capsid in place of the adenovirus hexon, fiber, and penton proteins according to their respective sequence numbers, and each variant independently possesses sequence identity of at least 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% relative to the amino acid sequence defined by each sequence number (i.e., each variant can have a different sequence identity), with each higher value taking precedence over any of the preceding lower values. Instead of defining sequence identity by percentage levels, hexon, fiber, and penton variants can be defined as having a certain number of amino acid mutations independently within their respective sequence numbers (i.e., each variant can have a different number). The number of mutations is as follows: up to 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation, with each lower value taking precedence over any preceding higher value.

[0038] Each of the three capsid proteins, hexon, fiber, and penton (see also Figure 1), has an independent utility because it can replace the corresponding capsid protein of different adenoviruses having other hexon, penton, and / or fiber proteins. Thus, in another embodiment of the present invention, the polynucleotide encodes one of the adenovirus hexon, fiber, and penton proteins. In one embodiment, the polynucleotide encodes two of the adenovirus hexon, fiber, and penton proteins, for example, (i) adenovirus hexon protein and adenovirus fiber protein, (ii) adenovirus hexon protein and adenovirus penton protein, and / or (iii) adenovirus fiber protein and adenovirus penton protein. However, in one preferred embodiment, the polynucleotide encodes all of the adenovirus hexon, fiber, and penton proteins.

[0039] The polynucleotide of the first embodiment preferably further comprises other adenovirus genes and nucleotide segments adjacent to the hexon, penton, and / or fiber genes in the adenovirus genome, using Sequence IDs 1, 5, 8, 10, 14, 16, 18, 20, and / or 22 as reference. These are shown in Table 2. The polynucleotide also preferably comprises sequences necessary for packaging the polynucleotide into adenovirus particles.

[0040] In general, the polynucleotide of the first embodiment preferably comprises at least one of the following: (a) The 5' end of the adenovirus, preferably the 5' inverted terminal repeat of the adenovirus; (b) the adenovirus E1a region, or a fragment thereof selected from the 13S, 12S, and 9S regions; (c) The adenovirus E1b region, or a fragment thereof selected from the group consisting of E1b 19k, E1b 55k, and IX regions; (d) an adenovirus VA RNA region, or a fragment thereof selected from the group consisting of VA RNA I and VA RNA II regions; (e) The adenovirus E2b region, or a fragment thereof selected from the group consisting of pTP, polymerase, and IVa2 region; (f) The adenovirus L1 region, or a fragment thereof encoding an adenovirus protein selected from the group consisting of 28.1 kD protein, polymerase, agunoprotein, 52 / 55 kDa protein, and IIIa protein; (g) The adenovirus L2 region, or a fragment thereof that encodes an adenovirus protein selected from the group consisting of the Penton proteins VII, V, and X as defined above; (h) The adenovirus L3 region, or a fragment thereof that encodes an adenovirus protein selected from the group consisting of the VI protein, the hexone protein defined above, and the endoprotease; (i) the adenovirus E2a region, or a fragment thereof that encodes an adenovirus protein consisting of the DBP protein; (j) The adenovirus L4 region, or a fragment thereof that encodes an adenovirus protein selected from the group consisting of 100 kD protein, 22 kD homolog, 33 kD homolog, and VIII protein; (k) The adenovirus E3 region, or a fragment thereof selected from the group consisting of E3 ORF1, E3 ORF2, E3 ORF3, E3 ORF4, E3 ORF5, E3 ORF6, E3 ORF7, E3 ORF8 and E3 ORF9; (l) The adenovirus L5 region, or a fragment thereof that encodes the fiber protein as defined above; (m) Adenovirus E4 region, or a fragment thereof selected from the group consisting of E4 ORF6 / 7, E4 ORF6, E4 ORF5, E4 ORF4, E4 ORF3, E4 ORF2, and E4 ORF1; and / or (n) Adenovirus 3' terminal, preferably an inverted terminal repeat of adenovirus 3'.

[0041] These elements may originate from adenoviruses or other adenoviruses represented by sequence numbers 1, 5, 8, 10, 14, 16, 18, 20, or 22 (i.e., as shown in Table 2), in particular from one of the other species, such as human adenoviruses that form chimeric adenoviruses.

[0042] In some embodiments of the polynucleotides described above, it may be desirable that the polynucleotides include adenovirus genes that do not contain one or more genomic regions (e.g., E3 and / or E4 regions, as described in (a) to (m)) and / or have deletions and / or mutations that render at least one gene nonfunctional, as outlined above. In these preferred embodiments, suitable adenovirus regions are modified to not contain the aforementioned regions / genes, or to render selected regions / genes nonfunctional. One possibility for rendering them nonfunctional is to introduce one or more stop codons (e.g., TAA) into the open reading frames of these genes. Methods for rendering viruses non-replicating are well known in the art (see, for example, Brody et al, 1994 Ann NY Acad Sci., 716: 90-101). The deletions can preferably create space for inserting the transgene into an expression cassette, such as the minigene cassette described herein. Furthermore, the deletions can be used to generate adenovirus vectors that lack replication ability without the use of a packaging cell line or helper virus, as is well known in the art. Thus, the final recombinant adenoviruses containing one or more deletions or loss-of-function mutations of specific genes / regions, as outlined above, can provide safer recombinant adenoviruses for applications such as gene therapy and vaccination.

[0043] A polynucleotide is (i) at least one genomic region / gene (e.g., E3 and / or E4 region) as outlined herein, in particular E1A, E1B, E2A, E2B, E3 ORF1, E3 ORF2, E3 ORF3, E3ORF4, E3 ORF5, E3 ORF6, E3 ORF7, E3 ORF8, E3ORF9, E4 ORF6 / 7, E4 ORF6, E4 ORF5, E4 ORF4, E4ORF3, E4 ORF2 and / or E4 ORF1, preferably E1A, E1B, E2A, E2B, E3 and / or E4 may not be included, and / or (ii) may include an adenovirus genomic region / gene (e.g., as identified in (i) above) that contains a deletion and / or mutation that renders at least one genomic region / gene nonfunctional, but intact E1A and / or E1B regions may be optionally retained. Such intact E1 regions may be located in their original positions in the adenovirus genome, or may be located at a deletion site in the native adenovirus genome (e.g., E3 region).

[0044] In one preferred embodiment, the polynucleotide of the first embodiment further encodes one or more, preferably all, of the following adenovirus proteins: protein VI, protein VIII, protein IX, protein IIIa and / or protein IVa2.

[0045] A typical person skilled in the art of adenoviruses is well familiar with how to determine the open reading frames encoding the particular adenovirus proteins described above. Those skilled in the art are also familiar with the structure of adenovirus genomes and can, without undue burden, map the individual adenovirus regions and ORFs outlined herein to any adenovirus genome.

[0046] In another embodiment, the polynucleotide of the first embodiment further encodes one or more heterologous proteins or fragments thereof. The one or more heterologous proteins or fragments thereof are preferably non-adenovirus proteins or fragments thereof. In one preferred embodiment, the one or more non-adenovirus proteins or fragments thereof are one or more antigenic proteins or antigenic fragments thereof. Preferably, the one or more heterologous proteins or fragments thereof are encoded by a gene that is part of one or more expression cassettes. Sequences encoding heterologous proteins, and preferably expression cassettes containing such sequences encoding heterologous proteins, can be inserted, for example, into a deletion region of an adenovirus genome as defined herein.

[0047] In one preferred embodiment, the heterologous protein or fragment thereof is a coronavirus protein or fragment thereof, more preferably a SARS-CoV-2 protein or fragment thereof. The term “SARS-CoV-2” preferably means any coronavirus strain that has been classified as a strain of SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV). Furthermore or alternatively, it is a variant coronavirus having sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or preferably at least 99% (higher values ​​are preferred over earlier lower values) with the sequence of the original strain of the 2019 outbreak, “Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1” (based on Genbank Acc. NoMN908947, NCBI Reference Sequence NC_045512.2, version of March 30, 2020) or its sequence. In particular, the protein or fragment may be a coronavirus (preferably SARS-CoV-2) spike protein or fragment thereof, for example, the spike protein (or fragment thereof) comprises or consists of (i) the sequence or a variant thereof of SEQ ID NO: 30, and / or (ii) a polypeptide sequence encoded by the nucleotide sequence at positions 6-3824 of SEQ ID NO: 29. The variants of SEQ ID NO: 29 or 30 have sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (higher values ​​take precedence over earlier lower values) with respect to their respective SEQ ID NOs. The variants are preferably functional, i.e., capable of binding to the human ACE2 protein.

[0048] In one preferred embodiment, the SARS-CoV-2 protein or a variant thereof has one or more amino acid mutations (including substitutions and deletions): a) In sequence number 30, Asp 614 (or the Asp at the corresponding position in the mutant) is replaced with Gly (sequence number 24), b) In SEQ ID NO: 30, both amino acids Lys 986 and Val 987 (or Lys and Val at their corresponding positions in the mutant) are replaced with Pro (SEQ ID NO: 25), c) In SEQ ID NO: 30, amino acids 69, 70, and 144 are deleted, Asn 501 is replaced with Tyr, Ala 570 is replaced with Asp, Asp 614 is replaced with Gly, Pro 681 is replaced with His, Thr 716 is replaced with Ile, Ser 982 is replaced with Ala, and Asp 1118 is replaced with His. d) Sequence ID 30, with Leu 18 substituted with Phe, Asp 80 substituted with Ala, Asp 215 substituted with Gly, amino acids 242, 243, and 244 deleted, Lys 417 substituted with Asn, Glu 484 substituted with Lys, Asn 501 substituted with Tyr, Asp 614 substituted with Gly, and Ala 701 substituted with Val. e) In sequence number 30, Leu18 is replaced with Phe, Thr 20 is replaced with Asn, Pro26 is replaced with Ser, Asp138 is replaced with Tyr, Arg 190 is replaced with Ser, Lys 417 is replaced with Thr, Glu 484 is replaced with Lys, Asn501 is replaced with Tyr, Asp 614 is replaced with Gly, His 655 is replaced with Tyr, Thr 1027 is replaced with Ile, and Thr 1027 and Val 1176 are replaced with Phe. f) In sequence number 30, Ser 13 is replaced with Ile, Trp 152 is replaced with Cys, Leu 452 is replaced with Arg, and Asp 614 is replaced with Gly, g) The equivalent of SEQ ID NO: 30, with Gln 52 substituted with Arg, amino acids 69, 70 and 144 deleted, Glu 484 substituted with Lys, Gln 677 substituted with His, and Phe 888 substituted with Leu. h) In sequence number 30, Leu 5 is replaced with Phe, Thr 95 is replaced with Ile, Asp 253 is replaced with Gly, Asp 614 is replaced with Gly, Ala 701 is replaced with Val, and Glu 484 is replaced with Lys. i) In sequence number 30, Leu 5 is replaced with Phe, Thr 95 is replaced with Ile, Asp253 is replaced with Gly, Asp 614 is replaced with Gly, Ala 701 is replaced with Val, and Ser 477 is replaced with Asn. j) In sequence number 30, Thr 478 is replaced with Lys, Asp 614 is replaced with Gly, Pro 681 is replaced with His, and Thr 732 is replaced with Ala. k) In sequence number 30, Thr 95 is replaced with Ile, Gly 142 is replaced with Asp, Glu 154 is replaced with Lys, Leu 452 is replaced with Arg, Glu 484 is replaced with Gln, Asp 614 is replaced with Gly, Pro 681 is replaced with Arg, and Gln 1071 is replaced with His, and / or (preferably or) l) In sequence number 30, Thr 19 is replaced with Arg, Gly 142 is replaced with Asp, Glu 156 is replaced with Gly, amino acids 157 and 158 are deleted, Leu 452 is replaced with Arg, Thr 478 is replaced with Lys, Asp 614 is replaced with Gly, Pro 681 is replaced with Arg, and Asp 950 is replaced with Asn.

[0049] In other words, the SARS-CoV-2 protein may have the sequences described in items a) to l) above, or the sequences of its variants with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (maintaining substitutions / deletions of items a) to l). For example, the SARS-CoV-2 protein may have the sequence according to SEQ ID NO: 24 (with a substitution of Asp 614 to Gly), or a variant sequence of its own with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (maintaining substitutions), optionally accompanied by substitutions according to b), or the SARS-CoV-2 protein may have the sequence according to SEQ ID NO: 25 (with both Lys 986 and Val987 substituted to Pro), or a variant sequence of its own with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (maintaining substitutions).

[0050] In one embodiment, the polynucleotide encodes an adenovirus, preferably comprising an adenovirus genome containing the polynucleotide of the first embodiment. In one preferred embodiment, the adenovirus genome comprises the sequence shown in SEQ ID NOs: 1, 5, 8, 10, 14, 16, 18, 20, or 22, or the sequence of a variant having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (higher values ​​take precedence over lower values). In this regard, the term “encodes” does not require that the polynucleotide comprises only coding sequences, and may also include non-coding sequences of the adenovirus genome, particularly as preferably described herein. That is, the polynucleotide comprises both coding sequences and optionally non-coding sequences of the adenovirus.

[0051] In one preferred embodiment, the encoded adenovirus is a non-replicating adenovirus, preferably comprising the adenovirus genome specified above, but with deletions of one or more of the genomic regions / genes E1A, E1B, E2A, E2B, E3 and / or E4.

[0052] Most preferably, the encoded adenovirus encodes a recombinant adenovirus and preferably comprises an adenovirus genome shown in SEQ ID NOs: 1, 5, 8, 10, 14, 16, 18, 20, or 22 or a variant thereof as defined above, and preferably has one or more genes that encode one or more heterologous proteins or fragments thereof inserted (carrier adenovirus). Preferably, these one or more heterologous genes are inserted by substituting one or more genomic regions / genes E1A, E1B, E2A, E2B, E3 ORF1, E3 ORF2, E3 ORF3, E3 ORF4, E3ORF5, E3 ORF6, E3 ORF7, E3 ORF8, E3 ORF9, E4ORF6 / 7, E4 ORF6, E4 ORF5, E4 ORF4, E4 ORF3, E4ORF2, and / or E4 ORF1, more preferably E1, E3, and / or E4. Heterogenes are preferably inserted as part of an expression cassette. Optionally, the carrier adenovirus is also non-replicating as described herein, i.e., lacking one or more genomic regions / genes E1A, E1B, E2A, E2B, E3 and / or E4. For example, recombinant adenoviruses may be encoded by sequences according to SEQ ID NOs. 26, 27, or 28, or by sequences of mutants having at least 80% (preferably 80%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, where higher values ​​are preferred over earlier lower values) sequence identity with these sequences, and optionally, sequences of one or more genes encoding one or more heterogeneous proteins or fragments thereof are inserted.

[0053] In exemplary embodiments, the polynucleotides encode an adenovirus comprising polynucleotides of SEQ ID NO: 31 (optionally with substitutions resulting in a spike protein according to SEQ ID NO: 25, e.g., Pos 2487 C->T, Pos 2488 A->G, Pos 2489 C->G, Pos 2490 C->A, Pos 2491 T->G, and Pos 2492 T->G), SEQ ID NO: 32, or SEQ ID NO: 33, or polynucleotides of variants having sequence identity of at least 80%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% (where higher values ​​take precedence over earlier lower values). Preferably, deletions defined above for the adenovirus vector are considered, i.e., they do not contribute to a reduction in sequence identity.

[0054] In one embodiment, the polynucleotide encodes a recombinant adenovirus, wherein at least one adenovirus genomic region of the recombinant adenovirus is derived from an adenovirus (chimeric adenovirus) that does not contain one or more of the hexons, fibers, and / or penton proteins defined above. Preferably, the chimeric adenovirus is a chimeric form of one or more hexons, fibers, and / or penton proteins, or preferably only them. In other words, the polynucleotide encodes one or more of the hexons, fibers, and / or penton proteins defined above, but one or more, preferably all, of the other genomic regions are derived from a different adenovirus, particularly the adenoviruses shown in SEQ ID NOs: 1, 5, 8, 10, 14, 16, 18, 20, or 22. The different adenoviruses are preferably naturally occurring in different hosts, and more preferably human adenoviruses. The polynucleotide also preferably encodes one or more heterologous non-adenovirus proteins or fragments thereof, as defined above. Thus, one or more heterologous non-adenovirus genes are inserted into the adenovirus genome of the chimeric adenovirus. That is, the adenovirus genome of the chimeric adenovirus is derived from a non-monkey adenovirus, such as a human adenovirus, preferably a non-monkey (e.g., human) carrier adenovirus, except for the DNA encoding one or more of the hexons, fibers, and / or penton proteins defined above.

[0055] Generally, adenoviruses are preferably incapable of replication. To achieve this objective, adenoviruses are preferably deletions of one or more genomic regions E1A, E1B, E2A, E2B, E3 and / or E4, or preferably contain deletions and / or mutations that render the genomic region or the expression product encoded therein nonfunctional.

[0056] In one particular preferred embodiment, the polynucleotide may have a functionally impaired IVa2 gene, preferably a deletion or null mutation thereof, in all its variants described herein. This gene is involved in packing viral DNA, and its impairment results in the generation of virus-like particles. In this embodiment, the polynucleotide of the first embodiment preferably encodes one or more non-adenovirus B cell epitopes and / or T cell epitopes.

[0057] Second aspect In this invention, the present invention provides a hexone polypeptide encoded by a polynucleotide as defined in A), B), C), D), E), or F) of the first embodiment. Preferably, the hexone polypeptide is an isolated polypeptide.

[0058] Third aspect In this context, the present invention provides an adenovirus capsid comprising a hexon protein encoded by a polynucleotide of the first embodiment and preferably one or both of a fiber protein and a penton protein encoded by a polynucleotide of the first embodiment. Preferably, the adenovirus capsid is an isolated adenovirus capsid.

[0059] Adenovirus capsid polypeptides and capsids can be obtained by intracellular expression. The expressed polypeptides can optionally be purified using standard techniques. For example, cells may be lysed by mechanical or osmotic shock before being subjected to precipitation and chromatography steps, the nature and order of which depend on the specific recombinant material being recovered. Alternatively, the expressed polypeptides can be secreted and recovered from the culture medium in which the recombinant cells were cultured, as is known in the field of protein expression.

[0060] Fourth aspectIn this context, the present invention provides an adenovirus (also referred to herein as an adenovirus vector or vector of an adenovirus) comprising (i) an adenonucleotide encoded by a polynucleotide of the first embodiment, (ii) a polynucleotide of the first embodiment, and / or (iii) a polynucleotide of the first embodiment and / or (iii) a hexone polypeptide of the second embodiment or an adenovirus capsi of the third embodiment. Preferably, the adenovirus is an isolated adenovirus.

[0061] In other words, the adenovirus may be, for example, an adenovirus encoded by sequence numbers 1, 5, 8, 10, 14, 16, 18, 20, or 22, or a recombinant adenovirus such as a carrier adenovirus or chimeric adenovirus as defined above.

[0062] In exemplary embodiments, the present invention provides an adenovirus comprising a polynucleotide represented by any one of SEQ ID NOs: 26-28 and 31-33, or a variant thereof as defined above.

[0063] The adenovirus may or may not contain the polynucleotides of the first embodiment. If the polynucleotides are not contained in the adenovirus, they are preferably provided in trans (i.e., by a genetic element other than the adenovirus genome incorporated into the adenovirus). They are typically provided by a helper construct (e.g., a plasmid or virus) or by the genome or helper construct of a packaging host cell (complementing cell as described herein). The polynucleotides provided in trans are those not contained in the genome incorporated into the adenovirus and are more preferably homologous or other sequence variants of these polynucleotides. For example, if the polynucleotides provided in trans include hexone, penton and / or fiber genes, the genome incorporated into the adenovirus does not contain the polynucleotides encoding the hexone, penton and / or fiber proteins, respectively. Most preferably, the polynucleotides provided in trans encode at least one (preferably all) adenovirus capsid polypeptides as defined herein.

[0064] A series of adenovirus nucleic acid sequences can be used in the construction of adenovirus vectors for delivering genes to a host (e.g., human or other mammalian cells). For example, all or part of the adenovirus delayed early gene E3 may be removed from the adenovirus sequence that forms part of the recombinant virus. The function of monkey E3 is considered to be independent of the function and production of recombinant virus particles. In some embodiments, the adenovirus vector may also be constructed with a deletion of at least the ORF6 region of the E4 gene, more preferably the entire E4 region, due to redundancy in the function of this region. Yet another vector of the present invention may include a deletion in the delayed early gene E2A. Deletions can be made in any of the late genes L1 through L5 of the monkey adenovirus genome. Similarly, deletions in the metaphase genes IX and IVa2 may be useful for several purposes. Other deletions can be made in other structural or non-structural adenovirus genes. The above deletions can be used individually, i.e., the adenovirus sequences used in the present invention may contain deletions in only a single region. Alternatively, deletions of whole or partial genes that are effective in disrupting their biological activity may be used in any combination. For example, an adenovirus sequence may have deletions in the E1 and E4 regions, or the E1, E2a and E3 regions, or the E1 and E3 regions, or the E1, E2A and E4 regions, with or without the E3 deletion. Such deletions may be used in combination with other adenovirus gene mutations, such as temperature-sensitive mutations, to achieve the desired result.

[0065] Adenovirus vectors lacking any essential adenovirus sequence (e.g., a region selected from E1A, E1B, E2A, E2b, E4 ORF6, L1, or L4) can be cultured in a state where the adenovirus gene product necessary for viral infectivity and adenovirus particle replication is deficient. These helper functions may be provided by culturing the adenovirus vector in the presence of one or more helper constructs (e.g., plasmids or viruses) or packaging host cells (complementary cells as defined herein). See, for example, the techniques described in WO96 / 13597 for the preparation of “minimal” human adenovirus vectors.

[0066] A useful helper construct comprises a selected adenovirus gene sequence that complements the respective genes deleted and / or not expressed by the vector and the cells to which the vector is transfected. In one embodiment, the helper construct is replication-deficient and comprises essential and optional further adenovirus genes.

[0067] The helper construct may be formed into a polycation conjugate, as described in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); KJ Fisher and JM Wilson, Biochem. J., 299: 49 (April 1, 1994). The helper construct may optionally contain a reporter gene. Many such reporter genes are known in the art. The presence of a reporter gene on the helper construct, distinct from the transgene on the adenovirus vector, makes it possible to independently monitor both the adenovirus and the helper construct. This second reporter can be used to facilitate the separation of the resulting recombinant adenovirus from the helper construct during purification. A preferred helper construct is a helper virus.

[0068] To produce recombinant adenovirus (Ad) lacking any of the genes described in connection with preferred embodiments herein, if the function of the deleted gene region is essential for viral replication and infectivity, the recombinant virus is preferably supplied by a helper construct or cell (i.e., a complement cell or packaging cell). Often, constructs / cells expressing human E1 can be used to transcomplement the vector used to produce recombinant adenovirus. This is because there is diversity between the polynucleotide sequences of the present invention and the human adenovirus E1 sequences found in currently available packaging constructs / cells, and the use of current human E1-containing constructs / cells is particularly advantageous because it prevents the production of adenoviruses capable of replication during the replication and production process. However, under certain conditions, it is desirable to utilize constructs / cells expressing the E1 gene product for the production of E1-deleted recombinant adenovirus.

[0069] If necessary, the sequences provided herein can be used to generate helper constructs / cells or cell lines expressing at least the adenovirus E1 gene from the adenovirus shown in SEQ ID NOs: 1, 5, 8, 10, 14, 16, 18, 20, or 22 under transcriptional control of a promoter for expression in a selected parental cell line (e.g., HeLa cells). For this purpose, inductive or constitutive promoters can be used. Examples of promoters are provided, for example, in the examples herein. Such E1-expressing cells are useful for generating recombinant adenovirus E1 deletion vectors. Furthermore, or alternatively, the present invention provides constructs / cells expressing one or more adenovirus gene products, e.g., E1A, E1B, E2A, and / or E4 ORF6, preferably Ad5 E4 ORF6, which can be constructed using essentially the same procedure as that used for generating recombinant adenovirus vectors. Such constructs / cells can be used to transcomplement adenovirus vectors lacking essential genes encoding their products, or to provide the helper functions necessary for packaging helper-dependent viruses (e.g., adeno-associated viruses).

[0070] Generally, when adenovirus vectors are delivered by transfection, the vector is approximately 1 x 10⁻⁶ 4 cells ~approx. 1 x 10 3 cells, and preferably about 10 5 The amount of DNA delivered to the cells is approximately 0.1 μg to approximately 100 μg, preferably approximately 10 to approximately 50 μg. However, the relative amount of vector DNA to the host cell may be adjusted considering factors such as the selected vector, the delivery method, and the selected host cell. The introduction of the vector into the host cell may be achieved by any means known in the art or disclosed herein, including transfection and infection using, for example, CaPO4 transfection or electroporation.

[0071] For the construction and assembly of a target recombinant adenovirus, in one example, an adenovirus vector can be transfected in vitro into a packaging cell line in the presence of a helper construct, allowing homologous recombination to occur between the helper and the adenovirus vector sequence, replicating the adenovirus transgene sequence in the vector and packaging it into a viral particle (virion) capsid, thereby obtaining recombinant viral vector particles well known in the art. The recombinant adenovirus of the present invention is useful, for example, for introducing a selected transgene into a selected host cell.

[0072] In one preferred embodiment, the adenovirus of the fourth embodiment has a seroprevalence of less than 5% of human subjects, preferably no seroprevalence in human subjects, most preferably a non-human great ape adenovirus, more preferably no seroprevalence in human subjects that have not previously been in contact with one or more adenoviruses represented by SEQ ID NOs: 1, 5, 8, 10, 14, 16, 18, 20 and / or 22. In this context, the human subjects preferably belong to an ethnic group selected from the group consisting of Europeans, Indigenous Africans, Asians, Indigenous Americans, and Indigenous Oceania. Methods for identifying the ethnic origin of human subjects are included in the Art (see, for example, WO 2003 / 102236).

[0073] In one further preferred embodiment of the recombinant adenovirus, the adenovirus can invade target cells in mammals, i.e., it is infectious. The infectious recombinant adenovirus of the present invention can be used as a vaccine and for gene therapy as described herein. Thus, in another embodiment, the recombinant adenovirus preferably includes a molecule for delivery to target cells. Preferably, the target cells are mammalian cells, e.g., non-human great ape cells, rodent cells, or human cells. For example, the molecule for delivery to target cells may be a polynucleotide (i.e., a heterogene) encoding a heterogeneous protein as defined herein, preferably within an expression cassette. Methods for introducing expression cassettes into the genome of adenoviruses are well known in the art. In one embodiment, for example, the recombinant adenovirus of the present invention, including an expression cassette encoding a heterogene, can be produced by replacing a genomic region of an adenovirus selected from E1A, E1B, E2A, E2B, E3, and / or E4 with the expression cassette. The adenovirus genomic regions E1A, E1B, E2A, E2B, E3, and E4 of the present invention can be readily identified by alignment with known and annotated adenovirus genomes such as human Ad5 (see BirgittTaeuber and Thomas Dobner, Oncogene (2001) 20, p. 7847-7854; and Andrew J. Davison, et al., Journal of General Virology (2003), 84, p. 2895-2908).

[0074] The molecule for delivery to target cells is preferably a heterologous polynucleotide, but may also be a polypeptide or small molecule compound, preferably having therapeutic or diagnostic activity. In one particular preferred embodiment, the molecule for delivery to target cells is a heterologous polynucleotide containing the 5' inverted terminal sequence (ITR) and 3'ITR of the adenovirus. It will be apparent to those skilled in the art that, if the recombinant adenovirus is produced, for example, in packaging cells, the molecular size of the molecule must be selected so that a capsid forms around the molecule and the molecule can be packaged. Therefore, preferably, the heterologous gene is a minigene, which may have, for example, up to 7000 base pairs or up to 8000 base pairs.

[0075] Fifth aspect In this context, the present invention provides (i) a virus-like particle (VLP) encoded by a polynucleotide of the first embodiment, and / or (ii) a virus-like particle (VLP) comprising a hexone polypeptide of the second embodiment or a capsid of the third embodiment. Preferably, the VLP is an isolated VLP.

[0076] In one embodiment, the polynucleotide encoding the VLP has either a deleted IVa2 gene or a null mutation in the IVa2 gene.

[0077] According to the definition of VLP below, the VLP of the fifth embodiment substantially does not contain adenovirus genomic DNA. VLPs containing adenovirus VLPs have been used for vaccination, gene therapy, or direct drug delivery, such as anticancer drugs (Chroboczek et al., ACTA ABP BIOCHIMICA POLONICA, Vol. 61, No. 3 / 2014). That is, the VLP of the fourth embodiment may contain one or more heterologous genes as defined above, or one or more B cell and / or T cell epitopes thereof. In another embodiment, it may contain one or more non-adenovirus genes for gene therapy, and / or one or more pharmaceuticals (e.g., anticancer drugs). In one embodiment, the VLP incorporates and preferably presents one or more heterologous proteins or fragments thereof (preferably B cell and / or T cell epitopes) as defined above.

[0078] Sixth aspectIn this invention, the present invention provides a vector comprising a polynucleotide of the first embodiment. Preferably, the vector is an isolated vector. In one preferred embodiment, the vector is a plasmid vector, for example, an expression vector. Plasmid vectors can be advantageously used to generate recombinant adenoviruses described herein. Since sequence information for novel hexone, penton, and fiber proteins of the present invention is provided, the recombinant adenovirus can be obtained, for example, by constructing a recombinant adenovirus encoded by the polynucleotide of the first embodiment and any other adenovirus genomic region. Methods for constructing recombinant adenoviruses are well known in the art. Techniques useful for the preparation of recombinant adenoviruses are outlined, for example, in Graham & Prevec, 1991 In Methods in Molecular Biology: Gene Transfer and Expression Protocols, (Ed. Murray, EJ.), p. 109; and Hitt et al., 1997, Advances in Pharmacology 40:137-206. Further methods are described in WO 2006 / 086284.

[0079] To express the polynucleotide of the first embodiment, the polynucleotide can preferably be subcloned into an expression vector containing a potent promoter that directs transcription, using an expression cassette. Suitable bacterial promoters are well known in the art, for example, Escherichia coli, Bacillus sp., and Salmonella, and kits of such expression systems are commercially available. Similarly, eukaryotic cell expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. For details on expression cassettes, see below.

[0080] The specific expression vector useful for transporting genetic information into cells is not particularly important. Any conventional vector used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include pBR322-based plasmids, plasmids such as pSKF and pET23D, and fusion expression systems such as GST and LacZ, but there are many more known to those skilled in the art that can be usefully used. Expression vectors containing regulatory elements derived from eukaryotic viruses are typically used in eukaryotic expression vectors, such as the SV40 vector, papillomavirus vector, and Epstein-Barr virus-derived vector. Examples of other eukaryotic vectors include pMSG and pAV009 / A + pMTO10 / A + This includes pMAMneo-5, baculovirus pDSVE, pcDNA3.1, pIRES, and any other vectors that enable protein expression under the direction of promoters such as the HCMV pre-early promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary cancer virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or any other promoters that have been shown to be effective for expression in eukaryotic cells. Some expression systems have markers that provide gene amplification, such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high-yield expression systems that do not involve gene amplification are also suitable. Elements that can be included in the expression vector include replicons that function in E. coli, genes encoding drug resistance that allow selection of bacteria that possess the recombinant plasmid, and intrinsic restriction sites in the non-essential region of the plasmid that allow insertion of eukaryotic sequences. The specific drug resistance gene selected is not critical, and any of the many drug resistance genes known in the art would be suitable. The prokaryotic sequence is optionally selected so as not to interfere with DNA replication in eukaryotic cells.

[0081] Seventh aspectIn this regard, the present invention provides a composition comprising (i) an adjuvant, (ii) a polynucleotide of the first embodiment, a hexone polypeptide of the second embodiment, an adenovirus capsid of the third embodiment, an adenovirus of the fourth embodiment, a virus-like particle of the fifth embodiment, or a vector of the sixth embodiment, and optionally (iii) a pharmaceutically acceptable excipient.

[0082] Preferably, the adjuvant is an agonist for a receptor selected from the group consisting of type I cytokine receptors, type II cytokine receptors, TNF receptors, vitamin D receptors that act as transcription factors, and Toll-like receptors 1 (TLR1), TLR-2, TLR3, TLR4, TLR5, TLR-6, ​​TLR7, and TLR9.

[0083] Compositions containing adjuvants can be used, for example, as vaccines for human subjects. For example, activation of certain receptors can stimulate an immune response. Such receptors are known to those skilled in the art and include, for example, cytokine receptors, particularly type I cytokine receptors, type II cytokine receptors, TNF receptors; and vitamin D receptors that act as transcription factors; and Toll-like receptors 1 (TLR1), TLR-2, TLR3, TLR4, TLR5, TLR-6, ​​TLR7, and TLR9. Agonists for such receptors have adjuvant activity, i.e., are immunostimulant. In one preferred embodiment, the adjuvant of the composition may be one or more Toll-like receptor agonists. In one more preferred embodiment, the adjuvant is a Toll-like receptor 4 agonist. In one particularly preferred embodiment, the adjuvant is a Toll-like receptor 9 agonist. See below for examples of adjuvants. Also, preferred pharmaceutically acceptable excipients are described below.

[0084] Eighth aspectIn this context, the present invention provides cells comprising a polynucleotide of the first embodiment, a hexone polypeptide of the second embodiment, an adenovirus capsid polypeptide of the third embodiment, an adenovirus of the fourth embodiment, a virus-like particle of the fifth embodiment, or a vector of the sixth embodiment. Preferably, the cells are isolated cells.

[0085] Preferably, the cell is a host cell expressing at least one adenovirus gene, or preferably all adenovirus genes, which are deleted or inactivated as described above, in order to render the adenovirus incapable of replication. The expression of this at least one gene allows the host cell to preferably replicate the otherwise incapable adenovirus. In one embodiment, a host cell expressing at least one adenovirus gene selected from the group consisting of E1A, E1B, E2A, E2B, E3, and E4. In particular, this at least one adenovirus gene is deleted or inactivated in the adenovirus genome. Such a complementary cell can be used for the proliferation and rescue of an incapable adenovirus, for example, because it is missing one of the gene products described above.

[0086] Cells can be selected from bacterial cells such as E. coli cells, yeast cells such as Saccharomyces cerevisiae or Pichia pastoris, plant cells, insect cells such as SF9 or Hi5 cells, or mammalian cells. Preferred examples of mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK 293) cells, HELA cells, human hepatoma cells (e.g., Huh7.5), Hep G2 human hepatoma cells, and Hep 3B human hepatoma cells.

[0087] If a cell contains a polynucleotide according to the first embodiment, this polynucleotide may be present in the cell either by (i) being freely dispersed as is, or by (ii) being incorporated into the cell genome or mitochondrial DNA.

[0088] In a more preferred embodiment, the cells are host cells expressing at least one adenovirus gene selected from the group consisting of E1A, E1B, E2A, E2B, E4, L1, L2, L3, L4, and L5, preferably HEK 293 cells or PER.C6TM cells.

[0089] Standard transfection methods can be used to generate bacterial, mammalian, yeast, or insect cell lines. Any well-known procedure for introducing foreign polynucleotide sequences into host cells can be used. For example, commercially available liposome-based transfection kits such as Lipofectamine® (Invitrogen), commercially available lipid-based transfection kits such as Fugene (Roche Diagnostics), polyethylene glycol-based transfection, calcium phosphate precipitation, biolistic gene guns, electroporation, or viral infection, and any other well-known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells can be used. The only requirement is that the specific genetic engineering procedure used can successfully introduce at least one gene into a host cell capable of expressing the receptor.

[0090] Further embodiments of the cells are described in relation to the third aspect of the present invention described above.

[0091] Ninth aspect In this regard, the present invention provides a polynucleotide of a first aspect, a hexone polypeptide of a second aspect, an adenovirus capsid polypeptide of a third aspect, an adenovirus of a fourth aspect, a virus-like particle of a fifth aspect, or a vector of a sixth aspect, a composition of a seventh aspect, and / or a cell of an eighth aspect for use in the treatment or prevention of disease.

[0092] In one embodiment, treatment or prevention is by vaccination. In another embodiment, treatment is by gene therapy. With regard to vaccination, the disease is preferably an infectious disease caused by a pathogen described herein, or preferably a non-infectious disease characterized by disease cells expressing antigens not expressed by healthy cells (e.g., tumor cells expressing tumor-associated antigens). With regard to gene therapy, the disease is a genetic disease caused by one or more somatic mutations leading to loss or gain of function of a gene or protein. In one preferred embodiment, the use is for the treatment or prevention of coronavirus disease. The term “coronavirus disease” is distinguished herein from coronavirus infection (entry of coronavirus into at least one cell of a subject and replication thereof in at least one cell) by the presence of at least one symptom of coronavirus disease. Unless the infection is accompanied by at least one symptom of coronavirus disease, it (or the subject) is asymptomatic (including pre-symptomatic). As used herein, the term coronavirus disease requires the presence of coronavirus infection and at least one symptom of coronavirus disease (also referred herein as symptomatic infection). Coronavirus symptoms include a dry cough, fever (37.8°C or higher), runny nose and / or nasal congestion, fatigue, difficulty breathing, pneumonia, organ dysfunction (e.g., heart, lungs, liver and / or kidneys), itchy throat, headache, joint pain, nausea, diarrhea, shivering, lymphopenia, loss of smell and / or taste. Preferably, coronavirus disease is characterized by the presence of two or more, three or more, or four or more symptoms, preferably one or more of the following: dry cough, fever (37.8°C or higher), difficulty breathing, and loss of smell and / or taste. Coronavirus disease is preferably a respiratory disease (e.g., SARS or MERS), more preferably SARS, and most preferably Covid-19.

[0093] Adenoviruses are well known to be useful in gene therapy and vaccines. Preclinical and clinical studies have demonstrated the feasibility of vector design, robust antigen expression, and protective immunity using this system. Therefore, one preferred embodiment of its use is, for example, in vaccination of human subjects. Detailed instructions on how to use and prepare adenoviruses for vaccination are included in the art and are readily available in the literature known to those skilled in the art. For example, viral vectors based on non-human great ape adenoviruses are an alternative to the use of human-derived Ad vectors for the development of gene vaccines (Farina SF, J Virol. 2001 Dec;75(23):11603-13.; Fattori E, GeneTher. 2006 Jul;13(14):1088-96). Adenoviruses isolated from non-human great apes are closely related to adenoviruses isolated from humans, as evidenced by their efficient replication in human-derived cells. However, since human and non-human ape adenoviruses are related, there may be some degree of serological cross-reactivity between the two virus species, or none at all. This assumption was confirmed when chimpanzee adenoviruses were isolated and characterized. Thus, the non-human mega ape adenoviruses according to the present invention provide a basis for mitigating the adverse effects associated with existing human immunity to common serotypes of human adenoviruses, thereby providing a valuable medical tool that can be used, for example, in immunotherapy and / or gene therapy.

[0094] This is due to a novel sequence of adenovirus capsid proteins including hexon, penton, and fiber protein. That is, neutralizing antibodies specific to the capsid proteins according to the present invention are expected to be absent or present very little in human blood serum. Therefore, one advantage of the novel sequence is that it can be used, for example, for medical purposes, to enhance engineered prior art adenoviruses. As an example, for instance, this sequence can be used to exchange / replace one or more of the major structural capsid proteins of different adenoviruses (e.g., prior art adenoviruses) to obtain improved recombinant adenoviruses (chimeric adenoviruses) with reduced serum sickness rate in humans. The novel sequence, and thus the adenoviruses redesigned as described, when administered, do not encounter significant inhibitory immune responses in humans, thus enhancing their overall transduction efficiency and infectivity. Thus, such improved adenoviruses are expected to be more effective vaccines as their entry into host cells and antigen expression are not hindered by significant titers of neutralizing antibodies.

[0095] The vaccine preferably contains an adjuvant. Preferred immunological adjuvants are described herein and can be used in such vaccines.

[0096] When the use is vaccination, the recombinant adenovirus of the present invention is preferably from 1x10 8 to 1x10 11 viral particles (i.e., 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 2.5 x 10 10 or 5 x 10 10 particles) and can be administered at an immunologically and / or prophylactically effective dose.

[0097] Furthermore, in cases of vaccination requiring additional immunization, it is preferable to apply the “heterogeneous prime boost” methodology. In vaccination, one of the agents from the first to ninth embodiments (polynucleotide, hexone polypeptide, adenovirus capsid polypeptide, adenovirus, VLP, vector, composition, and cell, respectively) can be used for priming or boosting, particularly for heterogeneous prime boost vaccination. In a preferred embodiment of heterogeneous prime boost, two different vaccines, such as adenoviruses, can be used, and in humans, for example, it is particularly advantageous that one of the agents from the first to ninth embodiments is used as a booster vaccine due to antibody deficiency or neutralization.

[0098] Recombinant adenoviruses prepared using polynucleotides or recombinant adenovirus proteins or fragments thereof according to the present invention can be used to transfect host cells with polynucleotides (e.g., DNA). Thus, an infective (i.e., capable of entering host cells) but preferably replication-deficient adenovirus can be prepared to express any custom protein or polypeptide in host cells. Thus, in one preferred embodiment, the treatment referred to in the use of the present invention is gene therapy. Gene therapy may be in vivo, ex vivo, or in vitro gene therapy. Preferably, it is somatic cell gene therapy. When any one of the agents of the first to ninth embodiments is used in gene therapy and administered to a target of treatment, it is preferable that it is administered in a sufficiently large dose so that the treatment transfects, i.e., transfects, one or more cells of the patient. When the recombinant adenovirus, VLP and / or pharmaceutical composition according to the present invention is administered by any of the preferred administration methods disclosed herein, preferably 1 x 10 8 From 5x10 11 Virus particles (i.e., 1 x 10⁻⁶ 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10, 2.5 x 10 10 , 5 x 10 10 , 1 x 10 11 Or most preferably 5 x 10 11 This is an effective amount of particles. In a preferred embodiment, the preferred heterologous polynucleotide contained in the recombinant adenovirus of the present invention is capable of expressing a protein or polypeptide in a target host cell, wherein the protein or polypeptide includes a signal peptide that results in the secretion of the protein or polypeptide from the host cell. For example, a patient requiring a specific protein can be treated using the adenovirus of the present invention, which contains cDNA encoding the secreted form of that protein.

[0099] In further embodiments of the use of the present invention, any one of the first to ninth embodiments of the drug (hereinafter also referred to as the drug according to the present invention) is formulated to further include one or more pharmaceutically acceptable diluents, carriers; excipients including fillers, binders, lubricants, flow promoters, disintegrants, and adsorbents; and / or preservatives.

[0100] The pharmaceuticals according to the present invention can be administered via various well-known routes, such as intravenous, intramuscular, intranasal, intradermal, subcutaneous, and similar routes of administration, including oral, rectal, gastric, and parenteral administration. Parenteral, intramuscular, and intravenous administration are preferred. Preferably, the pharmaceuticals according to the present invention are formulated as syrups, infusions or injection solutions, tablets, capsules, caplets, lozenges, liposomes, suppositories, adhesive bandages, band-aids, delayed capsules, powders, or sustained-release formulations. The diluent is preferably water, a buffer solution, a buffered salt solution, or a salt solution, and the carrier is preferably selected from the group consisting of cocoa butter and vitebesol.

[0101] A particularly preferred pharmaceutical form for administering the pharmaceutical according to the present invention during use is a form suitable for injectable use, comprising a sterile aqueous solution or dispersion, and a sterile powder for immediate preparation of a sterile injectable solution or dispersion. Typically, such a solution or dispersion comprises a solvent or dispersion medium, such as a water-buffered aqueous solution, such as a biocompatible buffer, ethanol, glycerol, polyols such as propylene glycol and polyethylene glycol, a suitable mixture thereof, a surfactant, or a vegetable oil.

[0102] Infusions or injectable solutions can be achieved by any number of technically recognized techniques, including, but not limited to, the addition of preservatives such as antimicrobial or antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, or thymelsar. Furthermore, isotonic agents such as sugars or salts, particularly sodium chloride, can be incorporated into the infusion or injectable solution.

[0103] Preferred diluents for the present invention are water, a physiologically acceptable buffer solution, a physiologically acceptable buffer salt solution, or a salt solution. Preferred carriers are cocoa butter and vitebesol. Excipients that can be used with various pharmaceutically acceptable forms of the pharmaceutical according to the present invention can be selected from the following non-limiting list: a) Binders such as lactose, mannitol, crystalline sorbitol, dibasic phosphate, calcium phosphate, sugars, microcrystalline cellulose, carboxymethylcellulose, hydroxyethylcellulose, and polyvinylpyrrolidone; b) Lubricants such as magnesium stearate, talc, calcium stearate, zinc stearate, stearic acid, hydrogenated vegetable oil, leucine, glycerides, and sodium stearyl fumarate. c) Disintegrants such as starch, crocaramelose, sodium methylcellulose, agar, bentonite, alginic acid, carboxymethylcellulose, and polyvinylpyrrolidone.

[0104] Other suitable excipients are listed in the Handbook of Pharmaceutical Excipients published by the American Pharmaceutical Association.

[0105] A specific amount of the pharmaceutical according to the present invention is preferable for the treatment or prevention of a disease. However, it is understood that different doses of the pharmaceutical according to the present invention may be required to produce a therapeutic or preventive effect, depending on the severity of the disease, the type of disease, and the individual patient to be treated, for example, depending on the patient's general health condition. The determination of the appropriate dosage is within the discretion of the attending physician. When the pharmaceutical according to the present invention is used preventively, it may be prescribed as a vaccine. In this case, the pharmaceutical according to the present invention is preferably administered in the preferred doses and particularly preferred doses outlined above. Preferably, the administration of the vaccine is repeated at least two, three, four, five, six, seven, eight, nine, or at least ten times over a specified period until the vaccinated subject produces sufficient antibodies against the pharmaceutical according to the present invention and the risk of developing each disease is reduced. The period in this case usually varies depending on the antigenicity of the vaccine. Preferably, this period is within four weeks, three months, six months, or three years. In one embodiment, when the adenovirus according to the present invention is used for vaccination purposes, at least one of the hypervariable regions of the hexon protein may be replaced by an immunogenic epitope of each pathogen targeted for vaccination. The vaccine typically contains one or more adjuvants as outlined above. Detailed summaries of the use of adenoviruses for vaccination and related methods are provided below: Bangari DS and Mittal SK (2006) Vaccine, 24(7), p. 849-862; also Zhou D, et al., Expert Opin Biol Ther. 2006 Jan;6(1):63-72; and Folgori A, et al., Nat Med. 2006 Feb;12(2):190-7.; further, Draper SJ, et al., Nat Med. 2008 Aug;14(8):819-21. Epub 2008 Jul 27.

[0106] Tenth aspectIn this context, the present invention relates to an in vitro method for generating adenoviruses or adenovirus-like particles, the in vitro method comprising the following steps: (i) A step of expressing a polynucleotide of the first aspect in a cell so that adenoviruses or adenovirus-like particles aggregate within the cell, (ii) A step of isolating adenoviruses or adenovirus-like particles from cells or the culture medium surrounding the cells.

[0107] This method optionally includes a further step prior to step (i) of introducing a polynucleotide of the first embodiment or a vector of the sixth embodiment into cells (for example, as described above).

[0108] It is generally preferable that the polynucleotide encodes an adenovirus of the fourth aspect or a virus-like particle of the fifth aspect. The adenovirus is preferably non-replicating. The cell is preferably a cell of the eighth aspect. If the polynucleotide encodes a non-replicating adenovirus, the cell is preferably a helper cell or a helper construct (e.g., a helper plasmid or helper virus, e.g., transduced with a helper construct and preferably infected with a helper virus before or during step (i)), and the helper cell or helper construct each expresses a gene / genomic region that renders the adenovirus non-replicating.

[0109] Step (i) “so that the adenovirus or adenovirus-like particles assemble within the cell” means that all genes necessary to assemble the adenovirus or adenovirus-like particles are expressed within the cell, as described herein. This includes, in the case of assembling an adenovirus, all genes necessary to package the adenovirus (i.e., package the genome into a viral capsid).

[0110] In one further embodiment, the present invention relates to: (i) Isolated polynucleotides encoding adenovirus, (ii) Isolated adenovirus, (iii) Virus-like particles (VLPs) containing adenovirus capsids, (iv) Isolated vector containing (i), (v) Isolated cells containing any one of (i)~(iv), (vi) A composition comprising an adjuvant, one of (i) to (v), and optionally (iii) a pharmaceutically acceptable excipient. (vii) Any one of (i) to (vi) for use in the treatment or prevention of coronavirus disease, and (viii) An in vitro method for generating adenovirus or adenovirus-like particles, comprising the following steps: (a) Expressing polynucleotides encoding adenoviruses within cells so that adenoviruses or adenovirus-like particles can aggregate within the cells. (b) Isolating adenoviruses or adenovirus-like particles from cells or the culture medium surrounding the cells. In the above, adenovirus, the polynucleotide encoding it, or VLP includes the coronavirus spike gene or protein as defined above. Preferably, the coronavirus spike gene is contained within the adenovirus genome.

[0111] Adenovirus vectors can be derived from any adenovirus, including, but not limited to, those mentioned herein, such as Ad5, Ad11, Ad26, Ad35, Ad49, ChAd3, ChAd4, ChAd5, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63, and ChAd82 (for example, Ad5 and Ad11 may preferably be non-replicating, or Ad4 and Ad7 may be replicating).

[0112] To the extent that they are applicable to any adenovirus containing a coronavirus spike gene or protein, all embodiments and definitions set forth above and below in this specification also apply to this further aspect of the invention.

[0113] Definitions and further embodiments of the present invention The following are some definitions of terms that are frequently used herein. In each example of their use, these terms have their defined and preferred meanings in the remainder of this specification.

[0114] As used herein, the term “isolated” means a molecule that is substantially free from other naturally occurring molecules. In particular, isolated means that the molecule is not in an animal body or a sample of an animal body. Therefore, an isolated molecule is free from other molecules that it might encounter or come into contact with within an animal. Isolated does not mean isolated from other related components as described herein, for example, isolated from other components of a composition containing the molecule, or isolated from a vector or cell containing the molecule.

[0115] The term "polynucleotide" is intended to refer to nucleic acids, i.e., biomolecules composed of multiple nucleotides. This includes DNA, RNA, and synthetic analogs, such as PNA, with DNA being preferred.

[0116] The term "open reading frame" (ORF) refers to a sequence of nucleotides that can be translated into amino acids. Typically, an ORF contains a start codon, and the subsequent region is usually a multiple of three nucleotides in length, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) within a given reading frame. ORFs encode proteins in which the translated amino acids form peptide chains.

[0117] As used herein, the terms “protein,” “peptide,” “polypeptide,” “peptide (plural),” and “polypeptide (plural)” are used interchangeably throughout. These terms refer to both naturally occurring peptides (e.g., naturally occurring proteins) and synthetic peptides, which may contain naturally occurring or unnaturally occurring amino acids. Peptides can also be chemically modified by modifying the side chains or free amino or carboxyl termini of naturally occurring or unnaturally occurring amino acids. This chemical modification includes the addition of further chemical moieties and modification of functional groups of amino acid side chains, such as glycosylation. Peptides are polymers having at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 amino acids, most preferably at least 8 or at least 30 amino acids. Since the polypeptides and proteins disclosed herein are derived from adenoviruses, it is preferable that the molecular weight of the isolated polypeptides or proteins used herein does not exceed 200 kDa.

[0118] Adenoviruses (Ad) are non-enveloped icosahedral viruses that have been identified in several avian and mammalian hosts. Human adenoviruses (hAds) belong to the genus Mastadenovirus, which includes many Ads of known human and animal origins (e.g., cattle, pigs, dogs, mice, horses, monkeys, and sheep). Human adenoviruses are generally classified into six subgroups (A-F) based on numerous biological, chemical, immunological, and structural criteria, including hemagglutination ability in rats and rhesus monkeys, DNA homology, restriction enzyme cleavage patterns, G+C content, and carcinogenicity (Straus, 1984; in The Adenoviruses, ed. H. Ginsberg, pps. 451-498, New York: PlenusPress, and Horwitz, 1990; in Virology, eds. BN Fields and DM Knipe, pps. 1679-1721).

[0119] Adenovirus viral particles (virions) have icosahedral symmetry and a diameter of 60–90 nm depending on the serotype. The icosahedral capsid contains three main proteins: hexon (II), penton base (III), and knob-like fiber (IV) proteins (WC Russel, J. Gen.Virol., 81: 2573-2604 (2000)). More specifically, the adenovirus capsid contains 252 capsomeres, of which 240 are hexons and 12 are pentons. The hexons and pentons are derived from three different viral polypeptides. The hexons contain three identical polypeptides, namely polypeptide II. The pentons contain a penton base that provides a binding site to the capsid and a trimer fiber protein that non-covalently binds to the penton base and protrudes from it. Other proteins, namely proteins IX, VI, and IIIa, are also normally present in the adenovirus capsid. These proteins are thought to stabilize the viral capsid.

[0120] One aspect of the existing immunity observed in humans is humoral immunity, which can lead to the production and persistence of antibodies specific to adenovirus proteins. The humoral response induced by adenoviruses is directed towards the capsid. Adenoviruses isolated from non-human apes are closely related to adenoviruses isolated from humans, as demonstrated by their efficient replication within human-derived cells.

[0121] The capsid can be modified as described herein by incorporating a non-adenoviral polypeptide, such as a T cell and / or B cell epitope.

[0122] The term "hexon protein" refers to the hexon (II) protein contained in adenoviruses. The hexon protein or its variants according to the present invention have the same function as the hexon protein or its fragments in infectious adenovirus virions. Therefore, adenoviruses containing the hexon or its variants, preferably as a capsid protein, can invade host cells. A suitable method for generating variants of the hexon protein is described in U.S. Patent 5,922,315. In this method, at least one loop region of the adenovirus hexon is modified with at least one loop region of another adenovirus serotype. It is easy to determine whether recombinant adenoviruses can invade host cells. For example, after contacting host cells with adenovirus, the recombinant host cells can be washed and lysed, and it can be determined whether adenovirus RNA and / or DNA are found in the host cells, for example, using a suitable hybridization probe specific to adenovirus RNA and / or DNA. Alternatively, or additionally, host cells after contact with recombinant adenovirus can be washed, lysed, and probed with adenovirus-specific antibodies, for example, using Western blotting. Yet another method allows observation, for example in vivo, of whether host cells express gene products, for example, whether they express fluorescent proteins upon infection with recombinant adenovirus containing an expression cassette suitable for expressing gene products within host cells.

[0123] "Adenovirus penton protein" refers to the penton-based (III) protein contained in adenoviruses. Adenovirus penton proteins are characterized by their localization to the icosahedral symmetry corners of the capsid. The penton protein or its variants according to the present invention have the same function as the penton protein in infectious adenovirus virions. Therefore, adenoviruses containing the penton or its variants preferably as the capsid protein can enter host cells, which can be tested as described above. Furthermore, functional penton has affinity for adenovirus fiber proteins. The average person skilled in the art is well familiar with methods for testing protein-protein affinity. To determine whether a first protein can bind to a second protein, a person skilled in the art can use, for example, a genetic yeast two-hybrid assay, or a biochemical assay such as pull-down, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) based assay, or plasmon resonance assay. When using pull-down assays or plasmon resonance assays, it is useful to fused at least one protein to an affinity tag such as an HIS tag or GST tag, as is well known in the field of biochemistry.

[0124] The term "fiber protein" refers to the fibrous (IV) protein containing rhizomes found in adenoviruses. The fiber protein or its variants according to the present invention have the same function as the fiber protein or its fragments in infectious adenovirus virions. Therefore, adenoviruses containing the fiber or fiber variants, preferably as capsid proteins, can enter host cells, which can be tested as described above. Furthermore, functional fiber proteins have affinity for the penton protein of adenoviruses. In addition, the glycosylated form of functional adenovirus fiber protein can trimerize. Thus, it is also preferable that its variants can be glycosylated and / or trimerize. Affinity, including trimerization, can be tested as described above, and glycosylation assays are also well known in the art.

[0125] In the context of polynucleotide, polypeptide, or protein sequences, the term "identity" or "identical" refers to the number of residues in two sequences that are identical when aligned to the maximum extent possible. Specifically, the percentage sequence identity of two sequences, whether nucleic acid or amino acid sequences, is calculated by dividing the exact number of matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Alignment tools that can be used to align two sequences are well known to those skilled in the art and are available, for example, on the World Wide Web, such as Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ) or MUSCLE (http: / / www.ebi.ac.uk / Tools / msa / muscle / ) for polypeptide alignment, MAFFT (http: / / www.ebi.ac.uk / Tools / msa / mafft / ) for polynucleotide alignment, or WATER (http: / / www.ebi.ac.uk / Tools / psa / emboss_water / ) for polynucleotide and polypeptide alignment. Alignment between two sequences can be performed using default parameter settings, for example, preferably for MAFFT: Matrix: Blosum62, Gap Open: 1.53, Gap Extend: 0.123, and for the WATER polypeptide: MATRIX: DNAFULL, Gap Open: 10.0, Gap Extend: 0.5. Those skilled in the art will understand that it may be necessary to introduce gaps in either sequence to produce a satisfactory alignment. The "best sequence alignment" is defined as the alignment that produces the maximum number of aligned identical residues while having the minimum number of gaps. Preferably, this is a global alignment and includes all residues of all sequences within the alignment.

[0126] The term "mutant" in relation to polypeptides generally refers to a modified version of a polypeptide, e.g., a mutation, in which one or more amino acids in the polypeptide may be deleted, inserted, altered, and / or substituted. Generally, a mutant is functional, meaning that an adenovirus containing a functional mutant can infect host cells. More specific functions are defined herein and take precedence over the general definition. A "mutation" or "amino acid mutation" may be a substitution, deletion, and / or insertion of an amino acid (the "and" may apply if multiple mutations exist). Preferably, it is a substitution (i.e., a conserved or non-conserved amino acid substitution), and more preferably a conserved amino acid substitution. In some embodiments, substitutions also include the exchange of a naturally occurring amino acid with an amino acid that does not naturally exist. A conservative substitution includes the substitution of an amino acid with another amino acid having similar chemical properties to the amino acid being substituted. Preferably, a conservative substitution is a substitution selected from the group consisting of: (i) Substitution of a basic amino acid with another different basic amino acid; (ii) Substitution of one different acidic amino acid with another acidic amino acid; (iii) Substitution of one aromatic amino acid with another different aromatic amino acid; (iv) Substitution of another different nonpolar aliphatic amino acid with a nonpolar aliphatic amino acid; and (v) Substitution of one polar uncharged amino acid with another polar uncharged amino acid.

[0127] Basic amino acids are preferably selected from the group consisting of arginine, histidine, and lysine. Acidic amino acids are preferably aspartic acid or glutamic acid. Aromatic amino acids are preferably selected from the group consisting of phenylalanine, tyrosine, and tryptophan. Nonpolar aliphatic amino acids are preferably selected from the group consisting of glycine, alanine, valine, leucine, methionine, and isoleucine. Polar uncharged amino acids are preferably selected from the group consisting of serine, threonine, cysteine, proline, asparagine, and glutamine. In contrast to conservative amino acid substitutions, nonconservative amino acid substitutions are the exchange of one amino acid with any amino acid that does not fall under the conservative substitutions (i) to (v) outlined above.

[0128] The means for determining sequence identity are described above.

[0129] The amino acids in proteins can also be modified, for example, chemically. For instance, the side chains or free amino or carboxyl termini of amino acids in a protein or polypeptide can be modified, for example, by glycosylation, amidation, phosphorylation, or ubiquitination. Chemical modifications can occur in vivo, for example, in a host cell, as is well known in the art. For example, a protein can be glycosylated by an appropriate chemical modification motif, such as a glycosylation sequence motif present in the amino acid sequence of a protein. Unless the modification results in a change in the identity of the modified amino acid (e.g., substitution or deletion), the modified polypeptide is within the range of polypeptides referred to with respect to a particular sequence number, i.e., not a variant as defined herein.

[0130] The term “mutant” in relation to polynucleotides generally refers to a modified version of a polynucleotide, e.g., a mutation, in which one or more nucleotides of the polynucleotide may be deleted, inserted, altered, and / or substituted. Generally, a mutant is functional, meaning that an adenovirus containing a functional mutant can infect a host cell. More specific functions are defined herein and take precedence over the general definition. A “mutation” can be a substitution, deletion, and / or insertion of a nucleotide (the “and” may apply if multiple mutations are present). Preferably, it is a substitution, more preferably an amino acid substitution, most preferably a conserved amino acid substitution.

[0131] An antigenic protein or fragment thereof (the fragment itself is antigenic) can trigger an immune response in mammals. Preferably, it is a tumor antigen or an antigen derived from a pathogen. The term “pathogen” refers to any organism that can cause disease in a subject. Pathogens include, but are not limited to, bacteria, protozoa, fungi, nematodes, viroids, viruses, and parasites, each pathogen, alone or in combination with another pathogen, can cause disease in vertebrates, including, but not limited to, mammals, including, but not limited to, humans. As used herein, the term “pathogen” also includes organisms that may not normally be pathogenic in an immunodeficient host but are pathogenic in an immunodeficient host.

[0132] Generally, adenovirus genomes are well-characterized. The overall structure of adenovirus genomes exhibits general conservation in that certain open reading frames are located in similar positions, such as the locations of the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes for each virus. Each end of the adenovirus genome contains a sequence known as the inverted end sequence (ITR), which is necessary for viral replication. Viruses also contain proteases encoded by the virus, which are required to process some of the structural proteins necessary for the production of infectious viral particles (virions). The structure of the adenovirus genome is described based on the order in which viral genes are expressed after transduction into a host cell. More specifically, viral genes are called early (E) or late (L) genes, depending on whether transcription occurs before or after the initiation of DNA replication. In the initial stages of transduction, the adenovirus E1A, E1B, E2A, E2B, E3, and E4 genes are expressed, preparing the host cells for viral replication. In the later stages of infection, the expression of late-stage genes L1-L5, which encode structural components of the viral particle, is activated.

[0133] As used herein, the term “vector” includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phages, adenovirus (Ad) vectors (e.g., as exemplified in further embodiments of the present invention described above), adeno-associated virus (AAV) vectors (e.g., AAV type 5), alphavirus vectors (e.g., Venezuelan encephalitis virus (VEE), Sindbisvirus (SIN), Semryki Forest virus (SFV), and VEE-SIN chimeras), herpesvirus vectors, measles virus vectors, poxvirus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC (vaccinia copenhagen strain), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), and vesicular stomatitis virus vectors, virus-like particles, or bacterial spores. Vectors include expression vectors, cloning vectors, and vectors useful for generating recombinant adenoviruses in host cells.

[0134] As described above, “heterogeneous protein or fragment thereof” may be a non-adenovirus protein or fragment thereof, particularly an antigenic protein or fragment thereof. For this purpose, the polynucleotide encoding the heterogeneous protein may be a molecule delivered to the target cell, for example, an antigenic protein or fragment thereof, preferably an antigenic protein or fragment thereof of a pathogen such as a pathogenic virus, bacterium, fungus, protozoan or parasite, or a polynucleotide encoding a tumor antigen. “Antigen” refers to any protein or peptide that can induce an immune response in mammals. The antigen preferably contains at least 8 amino acids, most preferably 8 to 12 amino acids.

[0135] The term “expression cassette” refers to a nucleic acid molecule containing at least one nucleic acid sequence to be expressed, along with its transcriptional and translational regulatory sequences. Modifying an expression cassette directs the expression of a different sequence or combination of sequences in the vector into which it is incorporated. Expression cassettes can be easily inserted, removed, or replaced with another cassette, as restriction sites are preferably designed to be located at the 5' and 3' ends. Preferably, an expression cassette includes cis-regulatory elements for the efficient expression of a given gene, such as a promoter, an start site, and / or a polyadenylation site. More specifically with respect to the present invention, an expression cassette includes all the additional elements necessary for the expression of a polynucleotide of the first embodiment in a host cell. Thus, a typical expression cassette includes a promoter operably linked to the polynucleotide of the first embodiment, as well as signals necessary for efficient polyadenylation of the transcript, a ribosome binding site, and translation termination. Additional elements of an expression cassette may include, for example, an enhancer. An expression cassette should also include a transcription termination region downstream of the structural gene to provide efficient termination. The stop region may be obtained from the same gene as the promoter sequence, or from a different gene.

[0136] As used herein, the term “minigene” refers to a heterogeneic construct in which one or more functionally non-essential segments of a gene are deleted relative to a naturally occurring gene. A “minigene cassette” is an expression cassette containing a minigene for expression.

[0137] The term “replication-competent” recombinant adenovirus (AdV) refers to an adenovirus that can replicate in a host cell in the absence of any recombinant helper proteins present in the host cell. Preferably, a “replication-competent” adenovirus contains the following complete or functional essential initial genes: E1A, E1B, E2A, E2B, E3, and E4. Wild-type adenoviruses isolated from a particular animal are replication-competent in that animal.

[0138] The term “replication-defective” or “replication-incompetent” refers to an adenovirus that has been made non-replicating by being manipulated to include at least a functional deletion, i.e., a deletion that impairs the function of a gene without completely removing the gene, such as the introduction of an artificial stop codon, a deletion or mutation of an active site or interaction domain, a mutation or deletion of a gene's regulatory sequence, or the complete removal of a gene encoding a gene product essential for viral replication, such as one or more adenovirus genes selected from E1, E2, E3, and E4. The recombinant adenovirus viruses of the present invention are preferably replication-defective.

[0139] The term “recombinant adenovirus” specifically refers to an adenovirus modified to contain heterologous polynucleotide and / or polypeptide sequences. “Heterologous” can mean a strain of adenovirus, particularly one derived from a different host (e.g., a human host, i.e., human adenoviruses such as Ad3 or Ad5), or one derived from a non-adenoviral organism, such as a pathogen-derived antigen or human tumor antigen as described herein. Thus, the term includes chimeric adenoviruses and carrier adenoviruses, respectively. Recombinant adenoviruses can contain heterologous polynucleotide and / or polypeptide sequences derived from both other adenoviruses and non-adenoviral organisms; i.e., they can be both chimeric adenoviruses and carrier adenoviruses.

[0140] As used herein, the term “virus-like particle” or “VLP” in this context refers to a non-replicating, empty viral shell derived from an adenovirus. VLPs generally consist of one or more viral proteins, including but not limited to proteins called capsid, coat, shell, surface, and / or envelope proteins. They contain functional viral proteins responsible for viral cell permeability, ensuring efficient cell entry. VLPs are formed spontaneously during the recombinant expression of proteins in appropriate expression systems. Methods for producing specific VLPs are known in the art. In particular, adenovirus VLPs can be produced by impairing their function, such as by deleting or introducing a null mutation in the adenovirus’s Iva2 gene, which is involved in viral DNA packing (Ostapchuk et al. J Virol. 2011 Jun; 85(11): 5524-5531). The presence of VLPs can be detected using conventional techniques known in the art, such as electron microscopy and X-ray crystallography. See, for example, Baker et al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J.Virol. (1994) 68:4503-4505. For example, cryo-electron microscopy can be performed on a vitrified aqueous sample of the VLP preparation in question, and images are recorded under appropriate exposure conditions.

[0141] "Substantially free of adenovirus genomic DNA" in a VLP means either that such genomic DNA is not present in the VLP, or that the VLP does not have enough DNA to enable viral replication in cells infected with the VLP, and therefore does not express DNA to complement the DNA in the VLP to allow viral replication.

[0142] In addition to the above, an "epitope," also known as an antigenic determinant, is a segment of a macromolecule recognized by the immune system, particularly antibodies, B cells, or T cells. In the context of this invention, the term "epitope" preferably refers to a segment of a protein or polyprotein recognized by the immune system. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural properties and specific charge properties. Stereoconformal epitopes and non-stereoconformal epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not.

[0143] A "non-adenovirus T cell epitope" is an epitope that can be presented on the surface of an antigen-presenting cell, where the epitope is bound to an MHC molecule. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, while most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides with a length of 8-11 amino acids, while MHC class II molecules present longer peptides with a length of 13-17 amino acids.

[0144] A "non-adenovirus B cell epitope" is an epitope that is recognized by B cells as a three-dimensional structure on the surface of a native antigen.

[0145] B cell and T cell epitopes can be predicted using in silico tools such as the online B cell or T cell prediction tools available on the IEDB Analysis Resource.

[0146] The term "presenting one or more non-adenoviral B cell epitopes" means that one or more epitopes are incorporated into the capsid so that they can be recognized by B cells. The term "incorporating one or more non-adenoviral B cell / T cell epitopes" means that the epitopes are either included in the VLP without being incorporated into the capsid, or they are incorporated into the capsid. When incorporated into the capsid, they may or may not be externally presented for recognition by immune cells.

[0147] An "immune adjuvant," or simply an "adjuvant," is a substance that accelerates, prolongs, and / or enhances the quality and / or intensity of the immune response to an antigen / immunogen compared to the administration of the antigen alone, and therefore reduces the amount of antigen / immunogen required for a given vaccine, and / or the frequency of injections required to produce an adequate immune response to the antigen / immunogen of interest. Examples of adjuvants that may be used in connection with the compositions according to the present invention include: gel-like precipitates of aluminum hydroxide (alum); AlPO4; alhydrogels; bacterial products derived from the outer membrane of Gram-negative bacteria, particularly monophosphoryl lipid A (MPLA), lipopolysaccharides (LPS), muramyl dipeptides and their derivatives; Freund's incomplete adjuvants; liposomes, particularly neutral liposomes, compositions and optionally liposomes containing cytokines; nonionic block copolymers; ISCOMATRIX adjuvants (Drane et al., 2007); unmethylated DNA containing CpG dinucleotides (CpG motifs), particularly CpG ODNs with a phosphorothioate (PTO) backbone (CpG PTO ODN) or a phosphodiester (PO) backbone (CpG PO ODN); synthetic lipopeptide derivatives, particularly Pam3Cys; lipoarabinomannan; peptidoglycan; zymosan; heat shock proteins (HSPs), particularly HSP 70; dsRNA and its synthetic derivatives, particularly poly-I:poly-C; polycationic peptides, particularly poly-L-arginine; taxol; fibronectin; flagellin; imidazoquinoline; cytokines with adjuvant activity, particularly GM-CSF, interleukin-(IL-)2, IL-6, IL-7, IL-18, type I and type II interferons, particularly interferon-γ, TNF-α; 25-dihydroxyvitamin D3 (calcitriol); and synthetic oligopeptides, particularly MHCII-presenting peptides. Nonionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP) (e.g., POE-POP-POE block copolymers) can be used as adjuvants (Newman et al., 1998). This type of adjuvant is particularly useful in compositions containing nucleic acids as active ingredients.

[0148] In relation to the present invention, the term "vaccination" is active immunity, which is the induction of a specific immune response by administering an antigen (a substance recognized as foreign by the immune system of a vaccinated individual and being immunogenic) in a suitable immunogenic preparation (e.g., subcutaneously, intradermally, intramuscularly, orally, or nasally) to the immune system. Thus, the antigen is used as a trigger for the immune system to construct a specific immune response to the antigen. Vaccination within the scope of the present invention can, in principle, be carried out in both therapeutic and prophylactic senses. This includes vaccination against pathogens described herein for the treatment or prevention of infectious diseases, or vaccination for the treatment or prevention of non-infectious diseases such as cancer. In the case of non-infectious diseases, the antigen is preferably a cell membrane antigen, and in particular an antigen expressed only by diseased cells and not by non-disease cells. One example is a tumor-associated antigen. In this context, the term "tumor-associated antigen" means a structure that is primarily presented by tumor cells, thereby enabling differentiation from non-malignant tissue. Preferably, such tumor-associated antigens are located on or within the cell membrane of tumor cells. Tumor-associated antigens are described, for example, in DeVita et al. (Eds., "Biological Therapy of Cancer", 2nd Edition, Chapter 3: Biology of Tumor Antigens, Lippincott Company, ISBN 0-397-51416-6 (1995)).

[0149] As used herein, “priming” refers to the administration of a vaccine to induce / generate an immune response in a mammal, and “boosting” refers to the administration of a vaccine to enhance an immune response in a mammal. The term “heterologous prime-boost” means that the vaccine used to induce / generate an immune response (priming) in a mammal is different from the vaccine used to enhance an immune response (boosting) in a mammal. Heterologous prime-boost is useful when the subject, e.g., a patient, has produced antibodies against a first vector and a boost is needed. In this context, the first (prime) vaccine and the second (boost) vaccine, e.g., adenovirus, are sufficiently different if the antibody response induced during priming with the first vaccine does not prevent 70% or more, preferably 80% or more, of the second vaccine particles administered for boosting from entering the cell nuclei of the primed and boosted animal.

[0150] The term “gene therapy” can be broadly defined as the concept of directly introducing foreign genetic material into cells, tissues, or organs to correct defective genes for the purpose of improving a patient’s clinical condition. As used herein, the term “gene therapy” preferably refers to “somatic therapy” and not to “germline therapy” which induces genetic changes that are passed from generation to generation, and somatic therapy is limited to the individual who receives the treatment. Gene therapy, preferably somatic therapy, can be further distinguished by rapid and easy direct gene transfer into an organism (“in vivo”) or by sophisticated, but more specific and controllable, gene transfer into cells or tissues that are transplanted outside the body (“ex vivo” or “in vitro”) and subsequently re-transplanted after treatment.

[0151] The term "neutralizing antibody" refers to an antibody that binds to an adenovirus epitope, preventing the adenovirus from causing a proliferative infection in a host cell, or prevents the transduction of a target cell by a non-replicating vector that expresses a transgene (e.g., adenovirus DNA that can invade cells, especially host cells).

[0152] The terms “SARS-CoV-2,” “SARS-COV2,” “SARS-CoV-2,” “Severe acute respiratory syndrome coronavirus 2,” and “2019-nCoV” are used interchangeably throughout this specification and refer to the virus that causes coronavirus disease of 2019 (COVID-2019 or COVID-19).

[0153] Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the present invention has been described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described embodiments for carrying out the invention, which will be apparent to those skilled in the art, are intended to be covered by the invention.

[0154] The present invention will be described with reference to the following examples, but these examples are merely illustrative and should be interpreted as not limiting the scope of the present invention. [Examples]

[0155] Example 1: GRAd32, GRAd23, and GRAd21 The construction of the pGRAd vectors was carried out following the procedure detailed below. The pGRAd32, pGRAd23, and GRAd21 vectors were derived from wild-type adenovirus strains isolated from fecal samples obtained from healthy gorillas using standard procedures. The wild-type virus was isolated by seeding fecal extracts onto a monolayer of A549 cells. The cell monolayer was observed daily for the appearance of cytopathic effects. Samples recorded as positive by microscopic observation were collected, and the cells were lysed by freeze-thaw cycles (-80°C / 37°C). The clarified cell lysates were then used for viral amplification by infecting a monolayer of fresh cells. After two passages of viral amplification, the adenovirus was purified using standard procedures.

[0156] The viral genome (GRAd32, SEQ ID NO: 1; GRAd23, SEQ ID NO: 22; GRAd21, SEQ ID NO: 10) was extracted from purified viruses by SDS / proteinase K digestion followed by phenol-chloroform extraction. The purified adenovirus DNA was cloned into a shuttle plasmid vector and further modified by introducing the following deletions into the viral genome:

[0157] GRAd32: 1) Deletion of the E1 region of the viral genome (from bp 445 to bp 3403) 2) Deletion of the E3 region of the viral genome (from bp 28479 to bp 32001) 3) Deletion of the E4 region of the viral genome (from bp 34144 to bp 36821).

[0158] GRAd23: 1) Deletion of the E1 region of the viral genome (from bp 451 to bp 3403) 2) Deletion of the E3 region of the viral genome (from bp 28494 to bp 32016) 3) Deletion of the E4 region of the viral genome (from bp 34159 to bp 36836).

[0159] GRAd21: 1) Deletion of the E1 region of the viral genome (from bp 456 to bp 3403) 2) Deletion of the E3 region of the viral genome (from bp 28343 to bp 31875) 3) Deletion of the E4 region of the viral genome (from bp 34005 to bp 36681).

[0160] GRAd Shuttle Vector The gorilla group C adenovirus shuttle vector was constructed following these steps: The first step was the construction of a plasmid pGRAd ITRs-only shuttle: the leftmost GRAd was amplified by PCR using the plasmid "pUC57-GRAd ends" (SEQ ID NO: 34) as a template with the following primers: Fw: 5' - cca ggc cgt gcc ggc acg ttc - 3' (SEQ ID NO: 70) Rev: 5' - att acc ctg tta tcc cta cgt c - 3' (SEQ ID NO: 71)

[0161] The rightmost GRAd molecule was amplified by PCR using the plasmid "pUC57-GRAd ends" (SEQ ID NO: 34) as a template with the following primers: Fw: 5' - gta ggg ata aca ggg taa tgc a - 3' (Sequence ID 72) Rev: 5' - aaa cat gag aat tgg tcg acg g - 3' (Sequence ID 73)

[0162] The leftmost and rightmost ends of GRAd were cloned into pBeloBAC11 (SEQ ID NO: 35), which had been pre-digested with HpaI / SfiI, according to the Gibson assembly method, to obtain the "pGRAd ITRs-only shuttle" (SEQ ID NO: 36).

[0163] The second step was the construction of the plasmid "pDE1_GRAd_shuttle": The hCMVtetO-GAG-bGHpolyA cassette was amplified by PCR using the plasmid "phCMVtetO-GAG-bGHpolyA" (SEQ ID NO: 37) as a template, the Gag antigen encoded by nucleotides 1220-2719 of SEQ ID NO: 37, and the following primers: Fw: 5' - gtt ttt att gtc gcc gtc atc tga cgg gcc gcc att gca tac gtt gta tccata tc -3' (SEQ ID NO: 74) Rev: 5' - aag cgc gat cgc ggc cgc ggc cat aga gcc cac cgc atc c - 3' (SEQ ID NO:75)

[0164] GRAd fragments containing the pIX coding region were amplified by PCR using the plasmid "pGRAd pIX" (SEQ ID NO: 38) as a template and the following primers: Fw: 5' - ccg cgg ccg cga tcg cgc tta ggc ctg acc atc tgg - 3' (Sequence ID 76) Rev: 5' - ctg tta tcc cta ggc gcg cct tag ggg gag gca agg ctg - 3' (SEQ ID NO: 77)

[0165] The Amp-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ggc gcg cct agg gat aac agg gta ata ccc cta ttt gtt tat ttt tct aa -3' (SEQ ID NO: 78) Rev: 5' - tgc tgg tgc tgt gag agt gcg act cgg gtc tag gcg cgc cat tac cct gtt atccct att att tgt taa ctg tta att gt - 3' (SEQ ID NO: 79)

[0166] The hCMVtetO::GAG-bGHpolyA cassette, the fragment containing pIX, and the AmpR-LacZ-SacB selected cassette were cloned into a "pITRs-only GRAd shuttle" pre-digested by I-SceI using the Gibson assembly method to generate a "pDE1 GRAd shuttle" (SEQ ID NO: 40).

[0167] The shuttle plasmid was designed to contain restriction enzyme sites (PmeI) present only at the ends of both ITRs, enabling the release of viral DNA from the plasmid DNA. See schematic diagram Figure 2.

[0168] Example 2: GRAd23 vector construction GRAd23 DE1 Vector GRAd23 wt genomic DNA (SEQ ID NO: 22) was isolated by proteinase K digestion followed by phenol / chloroform extraction and inserted into the pDE1 GRAd shuttle by homologous recombination in E. coli strain BJ5138 to obtain the pGRAd23 vector. Homologous recombination between the pIX gene, the right ITR DNA sequence located at the end of the shuttle (digested with I-SceI), and the viral genomic DNA enabled insertion into the shuttle vector. Simultaneously, the E1 region substituted in the expression cassette was deleted, ultimately generating the "pGRAd23 DE1 GAG" BAC vector (SEQ ID NO: 41). A schematic diagram of the pGRAd23 DE1 GAG BAC is shown in Figure 3.

[0169] GRAd23 DE1 Leftward Vector The construction strategy was based on two different processes: Step 1: Replacement of the E1 region using the AmpR-LacZ-SacB selected cassette. The AmpR-LacZ-SacB selective cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - gtt ccg ggt caa agt ctc cgt ttt tat tgt cgccgt cat ctg acg ggc cga ccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 80) Rev: 5' - tgg tgc agg cca gca cca gat ggt cag gcc taagcg cga tcg cgg ccc ggt tat ttg tta act gtt aat tgt cc -3' (SEQ ID NO: 81) The DNA fragment obtained by PCR was cloned into "pGRAd23 DE1 GAG" BAC (SEQ ID NO: 41) using recombineering, and "pGRAd23 DE1 A / L / S" BAC (SEQ ID NO: 42) was obtained. Step 2: Deletion of the AmpR-LacZ-SacB selection cassette and insertion of hCMVtetO::GAG-bGHpA at left-facing E1. The hCMVtetO-GAG-bGHpolyA cassette was amplified by PCR using the plasmid "phCMVtetO-GAG-bGHpolyA" (SEQ ID NO: 37) as a template and the following primers: Fw: 5' - gtt ccg ggt caa agt ctc cgt ttt tat tgt cgccgt cat ctg acg ggc cgc cat aga gcc cac cgc atc - 3' (SEQ ID NO: 82) Rev: 5' - tgg tgc agg cca gca cca gat ggt cag gcc taagcg cga tcg cgg ccc ggc cat tgc ata cgt tgt atc cat -3' (SEQ ID NO: 83) The DNA fragment obtained by PCR was cloned into "pGRAd23 DE1 A / L / S" BAC (SEQ ID NO: 42) by recombinant engineering, and "pGRAd23 DE1L GAG" BAC (SEQ ID NO: 43) was obtained.

[0170] GRAd23 DE1DE3 Vector The construction strategy was based on two different processes; Step 1 - Replacement of the E3 region using the AmpR-LacZ-SacB selected cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmp-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ctg tca ttt gtg tgc tga gta taa taa agg ctgaga tca gaa tct act cga ccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 84) Rev: 5' - agt gat ttt tta ttg att aca gtt atg atc aattga aag gga taa ggt ctt att tgt taa ctg tta att gtc c -3' (SEQ ID NO: 85) DNA fragments obtained by PCR were inserted into "pGRAd23 DE1" BAC (SEQ ID NO: 41) using recombinant engineering to obtain "pGRAd23 DE1 GAG DE3 A / L / S" BAC (SEQ ID NO: 44).

[0171] Second step - E3 region deletion: The AmpR-LacZ-SacB selection cassette was deleted using the single-stranded oligonucleotide 5'-ctg tca ttt gtg tgc tga gta taa taa agg ctg aga tca gaa tct actcgg acc tta tcc ctt tca att gat cat aac tgt aat caa taa aaa atc ​​act-3' (SEQ ID NO: 86). Using a single-stranded DNA fragment oligo, the selection cassette was recombinantly replaced with "pGRAd23DE1 GAG DE3A / L / S"BAC (SEQ ID NO: 44) to create "pGRAd23DE1 GAG DE3"BAC (SEQ ID NO: 45). This method resulted in a deletion of the E3 region from bp 28494 to bp 32016 of the GRAd 23 wild-type genome. A schematic diagram is shown in Figure 4.

[0172] E1E4 deletion GRAd23 vector The strategy for constructing the GRAd23 vector backbone, including the deletion of the native E4 region, and replacing it in the Ad5 E4 orf6 code region was based on two different steps: Step 1 - Replacement of the E4 region using the AmpR-LacZ-SacB selected cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ccc ttc cac ata gct taa att atc acc agt gcaaat gga aaa aaa atc ​​aaa ccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 87) Rev: 5' - cgg cac ttg gcc ttt ttc aca ctc tga tta gtgctg gtg ctg tga gag tgt tat ttg tta act gtt aat tgt cc -3' (SEQ ID NO: 88) Next, the DNA fragment obtained by PCR was inserted into the native GRAd23 E4 region of the "pGRAd23 DE1 GAG" (SEQ ID NO: 41) BAC by recombinant engineering, thereby obtaining the "pGRAd23DE1 GAG DE4 A / L / S" BAC (SEQ ID NO: 46).

[0173] Step 2 - Deletion of the AmpR-LacZ-SacB selected cassette for E4 region deletion: The AmpR-LacZ-SacB selection cassette was deleted and replaced with human adenovirus 5 E4orf6, which was amplified by PCR using the following primers with the genome of purified wild-type human adenovirus 5 (SEQ ID NO: 47) as a template. Fw: 5' - ccc ttc cac ata gct taa att atc acc agt gcaaat gga aaa aaa atc ​​aac tac atg ggg gta gag tca ta - 3' (SEQ ID NO: 89) Rev: 5' - cgg cac ttg gcc ttt ttc aca ctc tga tta gtgctg gtg ctg tga gag tga tga ctac gtc cgg cgt tcc -3' (SEQ ID NO: 90) Next, a DNA fragment containing the human Ad5 E4 orf6 coding region obtained by PCR was inserted into the "pGRAd23 DE1 GAG DE4 A / L / S" BAC (SEQ ID NO: 46), and the AmpR-LacZ-SacB selective cassette was replaced by recombinant engineering. The final product, "pGRAd23 DE1 DE4 hAd5E4orf6" BAC (SEQ ID NO: 48), was obtained.

[0174] E1E3E4 deletion GRAd23 vector The strategy for constructing the GRAd23 vector backbone, which included both the E3 region deletion and the native E4 region deletion, and for the replacement in the Ad5 E4 orf6 code region, was based on two different processes: Step 1 - Replacement of the E3 region using the Amp-LacZ-SacB selected cassette: The Amp-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ctg tca ttt gtg tgc tga gta taa taa agg ctgaga tca gaa tct act cga ccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 91) Rev: 5' - agt gat ttt tta ttg att aca gtt atg atc aattga aag gga taa ggt ctt att tgt taa ctg tta att gtc c -3' (SEQ ID NO: 92) DNA fragments obtained by PCR were inserted into the "pGRAd23 DE1 DE4 hAd5E4 orf6" (SEQ ID NO: 48) BAC via recombinant engineering to obtain the "pGRAd23 DE1 GAG DE3 A / L / S DE4 hAd5 E4orf6" BAC (SEQ ID NO: 49).

[0175] Second step - E3 region deletion: The AmpR-LacZ-SacB selection cassette was deleted using the single-stranded oligonucleotide 5' - ctg tca ttt gtg tgc tga gta taa taa agg ctg aga tca gaa tct actcgg acc tta tcc ctt tca att gat cat aac tgt aat caa taa aaa atc ​​act - 3' (SEQ ID NO: 86). Using a single-stranded DNA fragment oligo, the selection cassette was recombinantly replaced with "pGRAd23 DE1GAG DE3 A / L / S DE4 hAd5 E4orf6" BAC (SEQ ID NO: 49) to create "pGRAd23 DE1 GAG DE3 DE4 hAd5 E4orf6" BAC (SEQ ID NO: 50). A schematic diagram is shown in Figure 5.

[0176] Example 3: Construction of a GRAd23 vector expressing the SARS-CoV-2 spike gene The construction of the pGRAd23 SARS-CoV-2 spike vector proceeded through the following steps outlined below.

[0177] Fabrication of phCMV-IntronA::I-SceI-WPRE-bGHpA First, the pCMV-IntronA::I-SceI-WPRE-bGHpA shuttle plasmid was constructed by modifying the "pUC19-hCMVtetO::SEAP-bGHpA" plasmid (SEQ ID NO: 51). The intron A-I-SceI cassette was amplified by PCR using the "pVIJnsA" plasmid (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - acc ggg acc gat cca gcc - 3' (SEQ ID NO: 93) Rev1: 5' - taa tcc aga ggt tga tta tta ccc tgt tat ccctag aat tct ttg cca aaa tga tgc tgc aga aaa gac cca tgg aa - 3' (SEQ ID NO: 94) Rev2: 5' - caa att ttg taa tcc aga ggt tga ttc ccg ggtaat cca gag gtt gat tat tac c - 3' (SEQ ID NO: 95) This PCR was performed using one forward primer and two reverse primers to leave space for inserting the I-SceI tag into the reverse primer.

[0178] The WPRE cassette was amplified by PCR using plasmid pCAG21 (SEQ ID NO: 53) as a template and the following primers: Fw: 5' - caa cct ctg gat tac aaa att tg - 3' (Sequence ID 96) Rev: 5' - acg cgg gga cca cgg gtt aac ccg ggg cgg gga ggc ggc cca aa - 3' (SEQ ID NO: 97) The intron A-I-SceI PCR product and the WPRE cassette PCR product were ligated into the plasmid "pUC19-hCMVtetO::SEAP-bGHpA" (SEQ ID NO: 51) previously digested with HindIII-SmaI according to the Gibson method to generate "phCMVtetO-IntronA::I-SceI-WPRE-bGHpA" (SEQ ID NO: 54).

[0179] Fabrication of phCMV-IntronA::SARS CoV-2 S-WPRE-bGHpA The full-length coding sequence of the surface glycoprotein S (Genbank Accession No. QHD43416 identical to YP_009724390) of SARS CoV-2 virus (Genbank Accession NC_045512.2 identical to MN908947) was codon-optimized by including a minimal Kozak sequence upstream of the first ATG, and a human influenza hemagglutinin (HA) TAG coding sequence was fused to the 3' end of the S gene (SEQ ID NO: 29) (Kozak: nucleotides 1-5, spike protein nucleotides 6-3824, HA TAG nucleotides 3825-3857, stop codon nucleotides 3858-3860), and chemically synthesized by Doulix (Via Torino, 107, 30172 Venezia VE). The modified S gene was cloned into the I-SceI site of "pCMV-IntronA::I-SceI-WPRE-bGHpA" (SEQ ID NO: 54) by Doulix using the Gibson assembly method to generate the plasmid "phCMVtetO-IntronA::SARS CoV-2S-WPRE-bGHpA" (SEQ ID NO: 55).

[0180] DE1L DE3 GRAd23 SARS CoV-2 S construction Insertion of the SARS-CoV-2 S gene expression cassette in the leftward direction into the DE1L DE3 deletion GRAd23 vector was obtained through the following steps:

[0181] First step - Replacement of the E3 region with the AmpR-LacZ-SacB selection cassette of the DE1L backbone: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ctg tca ttt gtg tgc tga gta taa taa agg ctg aga tca gaa tct act cgaccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 98) Rev: 5' - agt gat ttt tta ttg att aca gtt atg atc aat tga aag gga taa ggt cttatt tgt taa ctg tta att gtc c -3' (SEQ ID NO: 99) The DNA fragment obtained by PCR was inserted into the "pGRAd23 DE1L GAG" BAC (SEQ ID NO: 43) by recombinant engineering to obtain the "pGRAd23 DE1L GAG DE3 A / L / S" BAC (SEQ ID NO: 56).

[0182] Second step - E3 region deletion: The AmpR-LacZ-SacB selection cassette was deleted using the single-stranded oligonucleotide 5' - ctg tca ttt gtgtgc tga gta taa taa agg ctg aga tca gaa tct act cgg acc tta tcc ctt tca att gatcat aac tgt aat caa taa aaa atc ​​act - 3' (SEQ ID NO: 86). Using this single-stranded DNA fragment oligo, the selection cassette was replaced with "pGRAd23 DE1L GAG DE3A / L / S" BAC (SEQ ID NO: 56), and "pGRAd23 DE1L GAG DE3" BAC (SEQ ID NO: 57) was synthesized by recombinant engineering.

[0183] Third step - Replacement of left-facing GAG regions using the Amp-LacZ-SacB selection cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - gat ggc tgg caa cta gaa ggc aca gca gat cgc ggc cgc tgt cga ctg aattct gat ggg ctt tat ttt att att tgt taa ctg tta att gtc - 3' (SEQ ID NO: 100) Rev: 5' - cga tcc agc ctc cgc ggc cgg gaa cgg tgc att gga acg cgg att ccc cgtgcc aag agt gag atc tac cac ccc tat ttg ttt att ttt ct - 3' (SEQ ID NO: 101) The DNA fragment obtained by PCR was cloned into "pGRAd23 DE1L GAG DE3" BAC (SEQ ID NO: 57) using recombinant engineering, and "pGRAd23 DE1L A / L / S DE3" BAC (SEQ ID NO: 27) was obtained.

[0184] Step 4 - Deletion of the AmpR-LacZ-SacB selection cassette to replace E1's hCMVtetO-IntronA::SARS-CoV-2S-WPRE-bGHpA in a leftward orientation: The full-length cassette hCMVtetO-IntronA::kozak - SARS CoV-2 S - HA -WPRE - bGHpA cassette was obtained from the plasmid "phCMVtetO-IntronA::SARS CoV-2S-WPRE-bGHpA" (SEQ ID NO: 55) by SpeI / PacI digestion, cloned into "pGRAd23 DE1L A / L / S DE3" BAC (SEQ ID NO: 27), and then produced "pGRAd23 DE1L hCMVtetO-IntronA::SARS CoV-2 S-WPRE-bGHpA DE3" BAC (SEQ ID NO: 32).

[0185] Example 4: GRAd32 vector construction Construction of the GRAd32 DE1 vector GRAd32 wt genomic DNA (SEQ ID NO: 1) was isolated by proteinase K digestion followed by phenol / chloroform extraction and inserted into the pDE1 GRAd shuttle (SEQ ID NO: 40) by homologous recombination in E. coli strain BJ5138 to obtain the pGRAd32 vector. Homologous recombination between the pIX gene, the correct ITR DNA sequence present at the end of the shuttle (digested with I-SceI), and the viral genomic DNA allowed for simultaneous deletion of the E1 region substituted by the GAG ​​expression cassette, enabling insertion into the shuttle vector. This ultimately resulted in the creation of the "pGRAd32 DE1 GAG wrongITR-L" BAC vector (SEQ ID NO: 58), which retains the GRAd32 pIX and right ITR, and the left ITR of the shuttle BAC.

[0186] Correction of GRAd32 DE1 vector ITR-L The construction strategy was based on two different processes, which are described below: Step 1: Replacement of the ITR-L region using the AmpR-LacZ-SacB selected cassette. The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - tgt cct gct tat cca caa cat ttt gcg cac ggt tat gtg gac aaa ata cctggt tac ccc tat ttg ttt att ttt ct - 3' (SEQ ID NO: 102) Rev: 5' - gac atg agc caa tat aaa tgta cat att atg ata tgg ata caa cgt atg caatgg tta ttt gtt aac tgt taa ttg tc -3' (SEQ ID NO: 103) The DNA fragment obtained by PCR was cloned into "pGRAd32 DE1 GAG wrongITR-L" BAC (SEQ ID NO: 58) using recombinant engineering, and "pGRAd23 DE1 GAG wrongITR-L ALS in ITR-L" BAC (SEQ ID NO: 59) was obtained.

[0187] Step 2: Insertion of ITR-L to correct missing or damaged AmpR-LacZ-SacB selected cassette: ITR-L was amplified by PCR using GRAd32 genomic DNA (SEQ ID NO: 1) as a template and the following primers: Fw: 5' - tgt cct gct tat cca caa cat ttt gcg cac ggt tat gtg gac aaa ata cctggt tgc cgt tta aac cat cat caa taa tat acc tta ttt tg - 3' (SEQ ID NO: 104) Rev: 5' - gac atg agc caa tat aaa tgt aca tat tat gat atg gat aca acg tat gcaatg gcg gcc atg acg gtg aca ata aaa acg ga -3' (SEQ ID NO: 105). The DNA fragment obtained by PCR was then cloned into "pGRAd23 DE1 GAG wrongITR-L ALS in ITR-L" BAC (SEQ ID NO: 59) by recombinant engineering, and "pGRAd23 DE1 GAG" ITR-modified BAC (SEQ ID NO: 60) was obtained.

[0188] Construction of the GRAd32 DE3DE4 vector The construction strategy was based on four different steps, which are described below: Step 1 - Replacement of E3 region by AmpR-LacZ-SacB selected cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmp-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ctg tca ttt gtg tgc tga gta taa taa agg ctg aga tca gaa tct act cgaccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 106) Rev: 5' - agt gat ttt tta ttg att aca gtt atg atc aat tga aag gga taa ggt cttatt tgt taa ctg tta att gtc c -3' (SEQ ID NO: 107). DNA fragments obtained by PCR were inserted into "pGRAd32 DE1 GAG" BAC (SEQ ID NO: 60) using recombinant engineering to obtain "pGRAd32 DE1 GAG DE3 ALS" BAC (SEQ ID NO: 61).

[0189] Second step - E3 region deletion: The AmpR-LacZ-SacB selection cassette was deleted using the single-stranded oligonucleotide 5'- ctg tca ttt gtgtgc tga gta taa taa agg ctg aga tca gaa tct act cgg acc tta tcc ctt tca att gatcat aac tgt aat caa taa aaa atc act - 3' (SEQ ID NO: 86). Using a single-stranded DNA fragment oligo, the selection cassette was recombinantly engineered to replace the "pGRAd32 DE1GAG DE3 ALS" BAC (SEQ ID NO: 61) to generate the "pGRAd32DE1 GAG DE3" BAC (SEQ ID NO: 62). By this method, the E3 region from bp 28479 to bp 32001 of the GRAd32 wild-type genome was deleted.

[0190] Step 3 - Replacement of the E4 region with the AmpR-LacZ-SacB selection cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ccc ttc cac ata gct taa att atc acc agt gca aat gga aaa aaa atc aaaccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 108) Rev: 5' - cgg cac ttg gcc ttt ttc aca ctc tga tta gtg ctg gtg ctg tga gag tgttat ttg tta act gtt aat tgt cc -3' (SEQ ID NO: 109) The DNA fragment obtained by PCR was inserted by recombinantly engineering to replace the native GRAd32 E4 region of the "pGRAd32 DE1 GAG DE3" BAC (SEQ ID NO: 62) to obtain the "pGRAd32DE1 GAG DE3 DE4 ALS" BAC (SEQ ID NO: 63).

[0191] Step 4 - Deletion of AmpR-LacZ-SacB Selected Cassette for E4 Region Deletion: The AmpR-LacZ-SacB selection cassette was deleted and replaced with human adenovirus 5 E4orf6, which was amplified by PCR using the following primers with the purified wild-type human adenovirus 5 (SEQ ID NO: 47) genome as a template: Fw: 5' - ccc ttc cac ata gct taa att atc acc agt gca aat gga aaa aaa atc ​​aac tacatg ggg gta gag tca ta - 3' (SEQ ID NO: 110) Rev: 5' - cgg cac ttg gcc ttt ttc aca ctc tga tta gtg ctg gtg ctgt gag agt gatgac tac gtc cgg cgt tcc -3' (SEQ ID NO: 111). Next, a DNA fragment containing the human Ad5 E4 orf6 coding region obtained by PCR was inserted into the "pGRAd32 DE1 GAG DE3 DE4 ALS" BAC (SEQ ID NO: 63), and the AmpR-LacZ-SacB selection cassette was replaced by recombinant engineering. The final product was the "pGRAd32 DE1 GAG DE3 DE4 hAd5E4orf6" BAC (SEQ ID NO: 64). This method resulted in a deletion of the E4 region from bp 34144 to bp 36821 of the GRAd32 wild-type genome.

[0192] Construction of the GRAd32 DE1DE3DE4 vector Replacement of the E1 region using the AmpR-LacZ-SacB selected cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - tta cgt gaa ttt ccg cgt tcc ggg tca aag tct ccg ttt tta ttg tca ccgtca tac ccc tat ttg ttt att ttt ct - 3' (SEQ ID NO: 112) Rev: 5' - gct aga ccc aaa ctc ggc cct ggt gca ggc cag cac cag atg gtc agg cct aagctt att tgt taa ctg tta att gtc -3' (SEQ ID NO: 113) The DNA fragment obtained by PCR was inserted by recombinant engineering by substituting the CMV::GAG-bGHpA cassette of the "pGRAd32 DE1 GAG DE3 DE4 hAd5E4orf6" BAC (SEQ ID NO: 64) to obtain the "pGRAd32 DE1 ALS DE3 DE4 hAd5E4orf6" BAC (SEQ ID NO: 26).

[0193] Example 5: Generation of pGRAd32 DE1 SARS-CoV2 DE3DE4 vector The full-length hCMVtetO-IntronA::kozak-SARSCoV-2S-HA-WPRE-bGHpA cassette was amplified by PCR using “phCMVtetO-IntronA::SARSCoV-2S-WPRE-bGHpA” (SEQ ID NO: 54) as a template with the following primers: Fw: 5' - tta cgt gaa ttt ccg cgt tcc ggg tca aag tct ccg ttt tta ttg tcg ccgtca tct gac ggg ccg cca tag agc cca ccg cat ccc cag cat gcc tgc tat t - 3' (SEQ ID NO: 114) Rev: 5' - gct aga ccc aaa ctc ggc cct ggt gca ggc cag cac cag atg gtc agg cctaag cgc gat cgc ggc ccg gcc att gca tac gtt gta tc - 3' (SEQ ID NO: 115). This PCR was performed to obtain "pGRAd32 DE1 ALS DE3 DE4 hAd5E4orf6" (SEQ ID NO: 26), which had been pre-digested with HpaI, and then cloned into E. coli strain BJ5138 by homologous recombination to obtain "pGRAd32 DE1SARS-COV2 DE3 DE4" (SEQ ID NO: 31).

[0194] Example 6: Immunogenicity of GRAd23 DE1 Gag GRAd23 DE1 cells expressing HIV-1 Gag antigen under the control of a tet operator (tetO) were rescued by transfecting GRAd23 DE1 Gag DNA (SEQ ID NO: 41) into a HEK 293-derived packaging cell line expressing a Tet repressor, and amplified by serial passage following a standard procedure. The purified virus was injected into mice in parallel with a human Ad5 vector expressing HIV-1 Gag antigen.

[0195] To evaluate the T cell response to the Gag antigen, groups of six mice were injected with 1x10^6 and 1x10^7 vp / mouse. The T cell response was assessed in spleen cells 3 weeks post-immunization by ex vivo interferon-γ enzyme-coupled immunospot (Elispot) assay using HIV Gag peptide T cell epitopes mapped in BALB / c mice.

[0196] The results are shown in Figure 6, expressed as IFN-γ spot-forming cells (SFCs) per million splenocytes. Each point represents the response of one mouse, and the lines correspond to the mean for each dose group. The dose injected, in terms of the number of viral particles, is shown on the x-axis. The results show that the GRAd23 vector exhibits higher immunological potency compared to the benchmark human Ad5 vector.

[0197] To evaluate the B cell response to the HIV-1 Gag antigen, groups of five mice were vaccinated by intramuscular injection of 5 x 10^8 viral particles of Ad5 or GRAd23 expressing the HIV-Gag antigen per mouse. B cell responses were measured 3 and 6 weeks after immunization by ELISA to assess the antibody response to HIV-1 Gag. The results are shown in Figure 7, demonstrating higher antibody titers in mice with the GRAd23 vector compared to the benchmark human Ad5 vector. Each point represents the response of one mouse, and the lines correspond to the mean for each dose group.

[0198] Example 7: Serum prevalence of GRAd23 and GRAd32 in humans This assay evaluated the effect of neutralizing antibody titers derived from human serum (40 samples) on the ability of human Ad5, gorilla GRAd23 (Figure 8), or gorilla GRAd32 (Figure 9) to transduce HEK 293 cells carrying the secreted alkaline phosphatase (SEAP) gene. SEAP expression in the supernatant of infected cells was revealed by a colorimetric assay. Neutralizing titer was defined as the dilution of human serum that yielded a 50% reduction in SEAP activity observed in a virus-positive control. The results showed low serum prevalence in both GRAd23 (Figure 8) and GRAd32 (Figure 9). The percentage of clinically relevant neutralizing titers (titers >200 that negatively impact human vaccination efficiency) was 67.5% for Ad5, compared to only 10% for GRAd23 and 0% for GRAd32.

[0199] Example 8: GRAd21 vector construction The GRAd21 wt genomic DNA (SEQ ID NO: 10) was isolated by proteinase K digestion followed by phenol / chloroform extraction and inserted into the pDE1 GRAd shuttle (SEQ ID NO: 40) by homologous recombination in E. coli strain BJ5138 to obtain the pGRAd21 vector. Homologous recombination between the pIX gene, the right ITR DNA sequence at the end of the shuttle (digested with I-SceI), and the viral genomic DNA enabled insertion into the shuttle vector. Simultaneously, the E1 region substituted in the expression cassette was deleted, ultimately generating the "pGRAd21 DE1 GAG" BAC vector (SEQ ID NO: 65).

[0200] Construction of GRAd21 DE1 GAG DE3DE4 The construction strategy was based on four different steps, which are described below: Step 1 - Replacement of the E3 region using the AmpR-LacZ-SacB selected cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmp-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ctg tca ttt gtg tgc tga gta taa taa agg ctg aga tca gaa tct act cgaccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 98) Rev: 5' - agt gat ttt tta ttg att aca gtt atg atc aat tga aag gga taa ggt cttatt tgt taa ctg tta att gtc c -3' (SEQ ID NO: 99). DNA fragments obtained by PCR were inserted into "pGRAd21 DE1 GAG" BAC (SEQ ID NO: 65) using recombinant engineering to obtain "pGRAd21 DE1 GAG DE3 ALS" BAC (SEQ ID NO: 66).

[0201] Second step - E3 region deletion: The AmpR-LacZ-SacB selection cassette was deleted using the single-stranded oligonucleotide 5'-ctg tca ttt gtg tgc tga gta taa taa agg ctg aga tca gaa tct actcgg acc tta tcc ctt tca att gat cat aac tgt aat caa taa aaa atc ​​act-3' (SEQ ID NO: 86). Using a single-stranded DNA fragment oligo, the selection cassette was recombinantly engineered to replace "pGRAd21 DE1 GAG DE3 ALS" BAC (SEQ ID NO: 66) to create "pGRAd21 DE1 GAG DE3" BAC (SEQ ID NO: 67). This method resulted in a deletion of the E3 region from bp 28343 to bp 31875 of the GRAd21 wild-type genome.

[0202] Third step - Replacement of the E4 region using the AmpR-LacZ-SacB selected cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - ccc ttc cac ata gct taa att atc acc agt gcaaat gga aaa aaa atc ​​aaa ccc cta ttt gtt tat ttt tct aa - 3' (SEQ ID NO: 87) Rev: 5' - cgg cac ttg gcc ttt ttc aca ctc tga tta gtgctg gtg ctg tga gag tgt tat ttg tta act gtt aat tgt cc -3' (SEQ ID NO: 88) Next, the DNA fragment obtained by PCR was recombinantly replaced and inserted into the native GRAd21 E4 region of "pGRAd21 DE1 GAG DE3" BAC (SEQ ID NO: 67) to obtain "pGRAd21 DE1GAG DE3 DE4 ALS" BAC (SEQ ID NO: 68).

[0203] Step 4 - E4 Region Deletion: Deletion of AmpR-LacZ-SacB Selective Cassette: The AmpR-LacZ-SacB selection cassette was deleted and replaced with human adenovirus 5 E4orf6, which was amplified by PCR using the following primers with the purified wild-type human adenovirus 5 (SEQ ID NO: 47) genome as a template: Fw: 5' - ccc ttc cac ata gct taa att atc acc agt gcaaat gga aaa aaa atc ​​aac taca tgg ggg tag agt cat a - 3' (SEQ ID NO: 89) Rev: 5' - cgg cac ttg gcc ttt ttc aca ctc tga tta gtgctg gtg ctgt gag agt gat gac tac gtc cgg cgt tcc -3' (SEQ ID NO: 90).

[0204] Next, a DNA fragment containing the human Ad5 E4 orf6 coding region obtained by PCR was inserted into the "pGRAd21 DE1 GAG DE3 DE4 ALS" BAC (SEQ ID NO: 68), and the AmpR-LacZ-SacB selection cassette was replaced by recombinant engineering. Finally, the "pGRAd21 DE1 GAG DE3 DE4 hAd5E4orf6" BAC (SEQ ID NO: 69) was obtained. This method resulted in a deletion of the E4 region from bp 34005 to bp 36681 of the GRAd21 wild-type genome.

[0205] Construction of an empty vector GRAd21DE1DE3DE4 Replacement of the E1 region using the AmpR-LacZ-SacB selected cassette: The AmpR-LacZ-SacB selection cassette was amplified by PCR using the plasmid "pAmpR-LacZ-SacB" (SEQ ID NO: 39) as a template and the following primers: Fw: 5' - tta cgt gaa ttt ccg cgt tcc ggg tca aag tct ccg ttt tta ttg tca ccgtca tac ccc tat ttg ttt att ttt ct - 3' (SEQ ID NO: 112) Rev: 5' - gct aga ccc aaa ctc ggc cct ggt gca ggc cag cac cag atg gtc agg cctaag ctt att tgt taa ctg tta att gtc -3' (SEQ ID NO: 113). By recombinantly replacing the CMV::GAG-bGHpA cassette of "pGRAd21 DE1 GAG DE3 DE4 hAd5E4orf6" BAC (SEQ ID NO: 68), a DNA fragment obtained by PCR was inserted, yielding "pGRAd21 DE1 ALS DE3 DE4 hAd5E4orf6" (SEQ ID NO: 28).

[0206] Vector creation for pGRAd21 DE1 SARS-CoV2 DE3 DE4 hAd5E4orf6 The full-length hCMVtetO-IntronA::kozak-SARSCoV-2S-HA-WPRE-bGHpA cassette was amplified by PCR using "phCMVtetO-IntronA::SARSCoV-2S-WPRE-bGHpA" (SEQ ID NO: 55) as a template and the following primers. Fw: 5' - acc caa act cgg ccc tgg tgc agg cca gca cca gat ggt cag gcc taa gcgaca ttg att att gac tag tta tta - 3' (SEQ ID NO: 116) Rev: 5' - tcc gcg ttc cgg gtc aaa gtc tcc gtt ttt att gtc gcc gtc atc tga cgtccc cag cat gcc tgc tat t - 3' (SEQ ID NO: 117). This PCR was cloned into "pGRAd21 DE1 ALS DE3 DE4 hAd5E4orf6" (SEQ ID NO: 28) using recombinant engineering with the E. coli strain SW102, and "pGRAd21 DE1 SARS-COV2 DE3 DE4" (SEQ ID NO: 33) was obtained.

[0207] Example 9: Construction of GRAd33, GRAd34, GRAd35, GRAd36, GRAd37, and GRAd38 vectors The GRAd33, GRAd34, GRAd35, GRAd36, and GRAd38 vector constructs were constructed by inserting the following segments of the GRAd33, GRAd34, GRAd35, GRAd36, and GRAd38 hexons into the GRAd23-derived target vector construct via standard homologous recombination: GRAd33 recombinant segment: Nucleotides 19381-21586 of SEQ ID NO: 16 GRAd34 recombinant segment: Nucleotides 19381-20491 of SEQ ID NO: 20 GRAd35 recombinant segment: Nucleotides 19381-20491 of SEQ ID NO: 18 GRAd36 recombinant segment: Nucleotides 19381-21591 of SEQ ID NO: 5 GRAd38 recombinant segment: Nucleotides 19381-20491 of SEQ ID NO: 8

[0208] The GRAd37 vector construct was constructed by inserting the following segments of the GRAd37 fiber into a target vector construct derived from GRAd21 using standard homologous recombination: GRAd37 recombinant segment: Nucleotides 33189-33779 of SEQ ID NO: 14

[0209] Example 10: Immunogenicity of GRAd21 DE1 Gag Immunogenicity of GRAd21 gorilla vector compared to human Ad5. Balb / c mice were 10% immunogenic to either hAd5 or GRAd21 vectors encoding the HIV-1 gag protein.6 and 10 7 Cells were immunized with viral particles (VP). Twenty-one days after priming, spleens were harvested and stimulated with gag peptide, after which T cell responses were measured by IFNg-ELISpot. Horizontal bars represent mean values. The immunogenicity of GRAd21 was comparable to that observed in human Ad5 (Figure 10).

[0210] Example 11: Expression and immunogenicity of GRAd32 DE1 spike Expression and immunogenicity of GRAd32 DE1, which encodes the SARS-CoV-2 spike antigen (GRAd32-S). Figure 11: Whole-cell FACS analysis of HeLa cells infected with GRAd32-S at MOI=250. Cells were isolated 48 hours after infection and stained with SinoBiologicals anti-S2 polyclonal antibody (40590-T62). Figure 12:10 7 , 10 6 , or 10 5 IFN-γ spleen ELISpot response after immunization with VP. T cell response to the peptide pool across full-length S proteins was assayed 2 weeks after immunization of Balb / c mice immunized intramuscularly. Figure 13: Serum antibody response to spike antigen after immunization with GRAd32-S in Balb / c mice was measured by ELISA in spike-coated 96-well plates. Data were collected 5 weeks after immunization. 9 VP and 10 8 This is expressed as the IgG endpoint titer of individual serum samples from animals immunized with VP's GRAd32-S.

[0211] Example 12: In vitro expression of SARSCoV2 spike using different GRAd vectors. Antigen expression of GRAd23b-S2P, GRAd32b-S2P, GRAd34b-S2P, and GRAd39b-S2P, vectors encoding the prototype SARS-CoV-2 spike protein stabilized in the pre-fusion conformation (S2P). For all of these vectors, "b" indicates that both the E1 and E3 regions are deleted in their respective viral genomes. GRAD32b-S2P was constructed by standard homologous recombination, replacing the GAG ​​in "pGRAd32DE1 GAG DE3" (SEQ ID NO: 62) with a modified version of the SARS CoV2 spike protein (SEQ ID NO: 29) stabilized in the pre-fusion conformation by substituting the codons Lys986 and Val987 with Pro. Next, GRAd39b-S2P was constructed by standard homologous recombination, replacing the hexon-coding region of GRAD32b S2P with the GRAd34 hexon (nucleotides 19381-20491 of SEQ ID NO: 20). GRAD23b S2P was similarly constructed by standard homologous recombination, replacing the GAG ​​in "pGRAd23 DE1 GAG DE3 BAC" (SEQ ID NO: 45) with the S2P version of the spike protein. Although there were no statistically significant differences in the productivity levels of these vectors (virus particles generated per cell at a specific point in time after the onset of synchronous infection, data not shown), spike antigen expression showed an unexpected increase with one of the GRAd vectors. HeLa cells were infected with 50 MOI of each vector, and cell lysates were collected 48 hours after infection. Western blot analysis revealed that levels of antigen produced by cells were higher in samples infected with GRAd34b-S2P (Figure 14).

[0212] Example 13: In vivo expression of SARSCoV 2 spike using different GRAd vectors. GRAd32b-S2P, GRAd34b-S2P, and GRAd39b-S2P were further tested in mouse immunogenicity experiments. Wild-type BALB / c mice were infected with 10^8 or 10^7 viral particles of GRAd32b-S2P, GRAd34b-S2P, or GRAd39b-S2P, and serum was collected 2 or 5 weeks after vaccination. Figure 15 shows the endpoint titers of antibodies produced against the spike-2P antigen, as measured by ELISA of recombinant spike receptor-binding domain (RBD) protein. In this case as well, GRAD34b-S2P showed a clear improvement of approximately 2-3 times, even at low doses, compared to GRAD32b-S2P.

[0213] Example 14: Clinical trial of a GRAd vector expressing SARS-CoV-2 spike GRAD32b-S2P (hereinafter referred to as GRAd-COV2, which expresses a SARS-COV2 spike protein stabilized by two Pro mutations that produce the spike protein shown in SEQ ID NO: 25, but with the sequence shown in SEQ ID NO: 31, but with the substitutions Pos 2487C->T, Pos 2488 A->G, Pos2489 C->G, Pos 2490 C->A, Pos2491 T->G, and Pos 2492 T->G) was then subjected to a dose-escalation open-label clinical trial designed to confirm its safety and immunogenicity. This study included two age cohorts of adults: young adults (18-55) and older adults (65-85). Each cohort consisted of three groups of 15 volunteers each, evaluating single doses of GRAd-COV2 at three different dose levels: low dose (LD) 5 x 10^10; medium dose (ID) 1 x 10^11; and high dose (HD) 2 x 10^11 viral particles (vp). Safety and immunogenicity endpoints were collected in the first four weeks after vaccination for volunteers enrolled in both age cohorts. GRAd-COV2 was manufactured under good manufacturing practice (GMP) conditions and suspended in formulation buffer at a concentration of 2 x 10^11 vp / mL. Volunteers received a single intramuscular injection into the deltoid muscle. For HD, 1 ml of GRAd-COV2 was injected undiluted. For ID and LD, the vaccine was diluted with sterile saline to a final injection volume of 1 ml. For immunogenicity analysis, three independent anonymized sample sets (serum and PBMCs) from hospitalized or recovering COVID-19 patients, collected 20–60 days after symptom onset, were used. A positive control included human plasma from donors recovered from COVID-19, treated with an anti-SARS-CoV-2 Ab (NIBSC code 20 / 130) research reagent.

[0214] Antibody responses to GRAd-COV2 vaccination were monitored by clinically validated chemiluminescence immunoassay (CLIA), revealing similar dynamics of anti-S IgG induction in all study groups (Figure 16A). Importantly, high-dose vaccines provided similar IgG levels in both age cohorts 4 weeks post-vaccination (median IgG in the high-dose group was 61.8 in young adults and 56.3 in older adults). ELISA assays showed that 89 out of 90 volunteers (98.8%) expressed detectable levels of anti-S IgG (both antibodies against the entire spike protein and antibodies specific to RBD) (Figures 16B-C).

[0215] Neutralizing antibodies against SARS-CoV-2 were evaluated using two different in vitro assays with live SARS-CoV-2 virus. A microneutralization assay (MNA90) performed 4 weeks post-vaccination detected neutralizing antibodies in the serum of 25 / 44 (56.8%) of young adult volunteers and 33 / 45 (73.3%) of elderly volunteers (Figure 16D). A plaque reduction neutralization assay (PRNT50) revealed that SARS-CoV-2 neutralizing antibodies were detectable in 42 / 44 (92.5%) of young adult volunteers and 45 / 45 (100%) of elderly volunteers (Figure 16E). In all groups, the titers of binding and neutralizing antibodies induced by GRAd-COV2 vaccination were within the range measured in subjects who had recovered from mild COVID-19 (Figures 16A-D).

[0216] Next, the T cell response to newly isolated PBMCs from volunteers in both cohorts was evaluated using a quantitative IFNγ ELISpot assay. Administration of GRAd-COV2 at all three doses induced a potent S-specific IFNγ-producing T cell response in both cohorts (Figure 17A), with 80% of evaluable subjects across both age cohorts showing a response exceeding 1000 SFCs / 1 million PBMCs. There were no significant differences between the young adult and elderly study groups receiving the same vaccine dose (p values ​​were 0.116, 0.984, and 0.152 for LD, ID, and HD, respectively). All regions of the S protein showed similar immunogenicity in both age cohorts (Figure 17B). S-specific T cell responses were generally higher in GRAd-COV2 vaccinated subjects than in SARS-CoV-2 convalescent controls sampled 1–2 months after symptom onset. Intracellular cytokine staining (ICS) and FACS analysis revealed that vaccine-induced responses were involved in both S protein-specific CD4 and CD8 T lymphocytes in young adult and elderly volunteers (Figures 17C–D and E–F), with S-specific CD4 slightly higher than CD8 T cell responses. Importantly, among GRAd-COV2 vaccine-induced S-specific CD4, IFNγ production was more prominent than IL4 and IL17 in both age cohorts, indicating that the vaccine primarily induced T helper 1 (Th1) responses (Table below Figures 17C and 17E).

[0217] Taken together, this data indicates that GRAd-COV2 is an efficient vaccine vector that induces both antibody and T-cell responses across all age groups.

[0218] Drawing terminology Hexon Penton Fiber IFNγSFC / 10 6splenocytes IFNγSFC / 10 6 splenocytes vector dose anti-p24 GAG serum titers weeks post-immunization nAb titer nAb titer Human serum SFC / 10 6 splenocytes SFC / 10 6 splenocytes Negative staining control Uninfected cells GRAd32-2P infected cells GRAd32-2P infected cells #positive mice #positive mice Spike IgG titer IgG endpoint titer Endpoint titer (spike RBD) % of CD4 secreting cytokines % of CD8 secreting cytokines

Claims

1. A) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 2, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 2, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219–225 of SEQ ID NO: 2, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257–268 of SEQ ID NO: 2, or a variant thereof containing up to two mutations. (v) HVR5 containing the amino acid sequence from positions 276 to 290 of SEQ ID NO: 2, or a variant thereof containing up to two mutations. (vi) HVR6 containing the amino acid sequence at positions 314–322 Y of SEQ ID NO: 2, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 431–456 of SEQ ID NO: 2, or a variant thereof containing up to two mutations, B) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 9, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219–225 of SEQ ID NO: 9, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257–268 of SEQ ID NO: 9, or a variant thereof containing up to two mutations. (v) HVR5 containing the amino acid sequence from positions 276 to 290 of SEQ ID NO: 9, or a variant thereof containing up to two mutations. (vi) HVR6 containing the amino acid sequence at positions 314–322 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 431–456 of SEQ ID NO: 9, or a variant thereof containing up to two mutations, C) (i) HVR1 containing the amino acid sequence from positions 136 to 163 of SEQ ID NO: 11, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence at positions 182–196 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 214–220 of SEQ ID NO: 11, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 252–262 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence at positions 270–278 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, (vi) HVR6 containing the amino acid sequence at positions 302–310 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 419–442 of SEQ ID NO: 11, or a variant thereof containing up to two mutations, D) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 17, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219–225 of SEQ ID NO: 17, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257–267 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence at positions 275–289 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, (vi) HVR7 containing the amino acid sequence at positions 313–321 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 430–455 of SEQ ID NO: 17, or a variant thereof containing up to two mutations, E) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 19, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 19, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219–225 of SEQ ID NO: 19, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257–268 of SEQ ID NO: 19, or a variant thereof containing up to two mutations. (v) HVR5 containing the amino acid sequence from positions 276 to 290 of SEQ ID NO: 19, or a variant thereof containing up to two mutations. (vi) HVR6 containing the amino acid sequence at positions 314–322 of SEQ ID NO: 19, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 431-456 of SEQ ID NO: 19, or a variant thereof containing up to two mutations, F) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219–225 of SEQ ID NO: 21, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257–267 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence at positions 275–289 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, (vi) HVR6 containing the amino acid sequence at positions 313–321 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence at positions 430-455 of SEQ ID NO: 21, or a variant thereof containing up to two mutations, G) (i) HVR1 containing the amino acid sequence from positions 136 to 168 of SEQ ID NO: 23, or a variant thereof containing up to two mutations. (ii) HVR2 containing the amino acid sequence from positions 187 to 201 of SEQ ID NO: 23, or a variant thereof containing up to two mutations, (iii) HVR3 containing the amino acid sequence at positions 219–225 of SEQ ID NO: 23, or a variant thereof containing up to two mutations. (iv) HVR4 containing the amino acid sequence at positions 257–268 of SEQ ID NO: 23, or a variant thereof containing up to two mutations, (v) HVR5 containing the amino acid sequence from positions 276 to 290 of SEQ ID NO: 23, or a variant thereof containing up to two mutations. (vi) HVR6 containing the amino acid sequence at positions 314–322 of SEQ ID NO: 23, or a variant thereof containing up to two mutations, and (vii) HVR7 containing the amino acid sequence from positions 431 to 456 of SEQ ID NO: 23, or a variant thereof containing up to two mutations. An isolated polynucleotide encoding an adenovirus hexone protein, wherein the polynucleotide encoding the adenovirus hexone protein described in G) further encodes the adenovirus fiber protein of SEQ ID NO: 6, or a variant thereof containing up to two mutations.

2. A) The hexone protein described above includes a variant having the amino acid sequence of SEQ ID NO: 2 or at least 80% sequence identity thereof. The hexone protein described in B) includes a variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

9. The hexone protein described in C) includes the amino acid sequence of SEQ ID NO: 11 or a variant having at least 80% sequence identity, The hexone protein described in D) includes the amino acid sequence of SEQ ID NO: 17 or a variant having at least 80% sequence identity thereto, and / or The hexone protein described in E) includes a variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

19. The hexone protein described in F) includes a variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 21, and / or The hexone protein described in G) includes a variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

23. The isolated polynucleotide according to claim 1.

3. Regarding A), further encoding an adenovirus fiber protein, including the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6, or a variant having at least 80% sequence identity thereof, With respect to B), D), E), and / or F), further encoding an adenovirus fiber protein, including the amino acid sequence shown in SEQ ID NO: 6, or a variant having at least 80% sequence identity thereof, and / or Regarding C), further encoding an adenovirus fiber protein, including the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 15, or a variant having at least 80% sequence identity thereof, The isolated polynucleotide according to claim 1 or 2.

4. Regarding A), further encoding an adenovirus penton protein, including the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 7, or a variant having at least 80% sequence identity thereof, With respect to B), D), E), F), and / or G), further encoding an adenovirus penton protein, including the amino acid sequence shown in SEQ ID NO: 7, or a variant having at least 80% sequence identity thereof, and / or Regarding C), the adenovirus penton protein further encodes the amino acid sequence shown in SEQ ID NO: 13, or a variant having at least 80% sequence identity thereof. An isolated polynucleotide according to any one of claims 1 to 3.

5. The isolated polynucleotide according to any one of claims 1 to 4, wherein the adenovirus comprises a non-adenovirus gene, protein or fragment thereof, and optionally, the non-adenovirus gene or protein is a coronavirus gene or protein, preferably a SARS-CoV-2 gene or protein.

6. The isolated polynucleotide according to claim 5, wherein the non-adenovirus gene or protein is a coronavirus gene or protein, and the coronavirus gene or protein is a spike gene or protein, preferably comprising the sequence shown in SEQ ID NO: 30 or a variant having at least 80% sequence identity thereof.

7. An isolated hexone polypeptide encoded by a polynucleotide as defined in A), B), C), D), E), or F) of claim 1, or An isolated hexone polypeptide encoded by a polynucleotide as defined in A), B), C), D), E), or F) of claim 2.

8. An isolated adenovirus capsid comprising a hexone encoded by an isolated polynucleotide according to any one of claims 1 to 4, and preferably also comprising a fiber and / or penton protein.

9. (i) an adenovirus encoded by a polynucleotide according to any one of claims 1 to 6, (ii) an adenovirus comprising a polynucleotide as described in any one of claims 1 to 6, and / or (iii) An adenovirus comprising the hexone polypeptide described in claim 7 or the adenovirus capsid described in claim 8.

10. A virus-like particle encoded by a polynucleotide according to any one of claims 1 to 6.

11. A vector comprising a polynucleotide according to any one of claims 1 to 6.

12. A composition comprising (i) an adjuvant, (ii) a polynucleotide according to any one of claims 1 to 6, a hexone polypeptide according to claim 7, an adenovirus capsid according to claim 8, an adenovirus according to claim 9, a virus-like particle according to claim 10, or a vector according to claim 11, and optionally (iii) a pharmaceutically acceptable excipient.

13. Isolated cells comprising a polynucleotide according to any one of claims 1 to 6, a hexone polypeptide according to claim 7, an adenovirus capsid according to claim 8, an adenovirus according to claim 9, a virus-like particle according to claim 10, or a vector according to claim 11.

14. A polynucleotide according to any one of claims 1 to 6, a hexone polypeptide according to claim 7, an adenovirus capsid according to claim 8, an adenovirus according to claim 9, a virus-like particle according to claim 10, a vector according to claim 11, a composition according to claim 12, and / or a cell according to claim 13, for use in the treatment or prevention of a disease, preferably coronavirus disease, more preferably Covid-19.

15. An in vitro method for producing adenovirus or adenovirus-like particles, (i) A step of expressing a polynucleotide according to any one of claims 1 to 6 in a cell so that adenoviruses or adenovirus-like particles assemble inside the cell, (ii) An in vitro method comprising the step of isolating adenoviruses or adenovirus-like particles from cells or the culture medium surrounding the cells.

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