VIRUSES WITH MODIFIED CAPSID PROTEINS

Abstract

This invention relates to preparations comprising adenoviral vectors with modified capsid proteins. These modified capsid proteins enable customisable decoration of the adenoviral vector to be performed, enabling diverse applications from personalised cancer vaccines to targeted gene therapy vectors, and mixtures of the same. In particular, the adenoviral vectors with modified capsid proteins may be modified in the hexon and/or pIX capsid proteins. The invention makes use of peptide pairs to provide a “primed” adenovirus which is ready for decoration.

Claims

1. An adenoviral vector for preparation of a prophylactic or therapeutic composition, comprising at least one modification in a capsid protein, wherein said modification comprises the inclusion of a first peptide partner in a capsid protein, and said first peptide is capable of forming a covalent bond with a second peptide partner.

2. An adenoviral vector according to claim 1 where said second peptide partner is attached to an entity, optionally an antigen, a targeting moiety or a shielding entity.

3. An adenoviral vector according to claim 1 or claim 2 wherein at least one modification in a capsid protein is in a major capsid protein or in a minor capsid protein.

4. An adenoviral vector according to any one of claims 1 to 3 wherein at least one modification in a major capsid protein is in the hexon protein, optionally in a HVR loop.

5. An adenoviral vector according to any one of claims 1 to 3 wherein at least one modification in a minor capsid protein is in the pIX protein.

6. An adenoviral vector according to any one of claims 1 to 5 wherein at least one modification to a capsid protein is the insertion or fusion of said first peptide partner to the capsid protein.

7. An adenoviral vector according to any one of claims 1 to 6 wherein said covalent bond is an isopeptide.

8. An adenoviral vector according to claim 4 wherein the first peptide partner is DogTag and wherein the second peptide partner is DogCatcher or SnoopTagJr or SnoopTag.

9. An adenoviral vector according to claim 5 wherein a) a first peptide partner is SpyCatcher, optionally wherein the second peptide partner is SpyTag; b) a first peptide partner is SnoopCatcher, optionally wherein the second peptide partner is SnoopTagJr or SnoopTag; c) a first peptide partner is DogCatcher, optionally wherein the second peptide partner is DogTag; c) a first peptide partner is SnoopTagJr, optionally wherein the second peptide partner is SnoopCatcher; or d) a first peptide partner is SpyTag, optionally wherein the second peptide partner is SpyCatcher.

10. An adenoviral vector according to any one of claims 2 to 9 wherein the second peptide is attached to an antigen via genetic fusion or chemically attached to an antigen.

11. An adenoviral vector according to claim 10 wherein said antigen is a tumour-associated antigen, such as a neoepitope or neoantigen, a self-antigen, or an antigen from a pathogen, such as a virus, bacterium, parasite or fungus, optionally wherein the virus is SARS-CoV-2.

12. An adenoviral vector according to any preceding claim wherein the size of said second peptide partner attached to an entity is over 15 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa or 100 kDa in size.

13. An adenoviral vector according to claim 12 wherein said second peptide partner attached to an entity shields the adenovirus from antibody binding to the capsid.

14. An adenoviral vector according to any preceding claim for use in the manufacture of a vaccine comprising the addition of a second peptide partner attached to an antigen to said adenoviral vector.

15. A vaccine comprising the adenoviral vector of any preceding claim, wherein an antigen is attached to the adenoviral vector via the second peptide partner.

16. A vaccine as claimed in claim 15 wherein said antigen comprises at least one antigen specifically expressed on a tumour or cancer cell, or virus, optionally SARS-Cov-2.

17. A method of producing an adenoviral vector as claimed in any one of claims 1 to 14 comprising: introducing a nucleic acid which encodes a first peptide partner into the nucleic acid encoding a capsid protein; inserting the modified capsid gene into the genome of an appropriate adenovirus; and infecting a cell with said adenovirus and collecting the released progeny virus.

18. A vaccine comprising an adenoviral vector as claimed in claim 15 or 16 and an attached antigen for use in the treatment or prevention of a disease.

19. A kit comprising an adenoviral vector as claimed in any one of claims 1 to 14.

20. A vaccine comprising an adenoviral vector as claimed in claim 1, wherein a. said first peptide partner is SpyCatcher which is fused to the pIX capsid protein, and said second peptide partner is SpyTag, which is attached to an antigen, and wherein SpyCatcher and SpyTag are bound covalently through an isopeptide bond; b. said first peptide partner is SnoopCatcher which is fused to the pIX capsid protein, and said second peptide partner is SnoopTagJr, or SnoopTag which is attached to an antigen, and wherein SnoopCatcher and SnoopTagJr or SnoopTag are bound covalently through an isopeptide bond; or c. said first peptide partner is DogCatcher which is fused to the pIX capsid protein, and said second peptide partner is DogTag which is attached to an antigen, and wherein DogCatcher and DogTag are bound covalently through an isopeptide bond; or d. said first peptide partner is SnoopTagJr which is fused to the pIX capsid protein, and said second peptide partner is SnoopCatcher, which is attached to an antigen, and wherein SnoopTagJr and SnoopCatcher are bound covalently through an isopeptide bond; e. said first peptide partner is SpyTag which is fused to the pIX capsid protein, and said second peptide partner is SpyCatcher, which is attached to an antigen, and wherein SpyTag and SpyCatcher are bound covalently through an isopeptide bond.

21. A vaccine comprising an adenoviral vector as claimed in claim 1, wherein said first peptide partner is DogTag which is inserted into HVR loops of the hexon protein, and said second peptide partner is DogCatcher or SnoopTagJr, said second peptide partner being attached to an antigen, and wherein DogTag and DogCatcher/SnoopTagJr are bound covalently through an isopeptide bond.

22. An oncolytic virus preparation comprising an adenoviral vector as claimed in claim 1, wherein said second peptide partner is attached to a shielding entity or a targeting moiety.

23. An adenoviral vector as claimed in any one of claims 1 to 14 wherein said adenovirus is any serotype or species, optionally a human adenovirus.

24. An adenoviral vector as claimed in any one of claims 1 to 14 wherein said adenovirus is replication-competent, optionally modified to replicate only in selective cells.

25. An adenoviral vector as claimed in any one of claims 1 to 14 wherein said adenovirus is replication-incompetent.

26. An adenoviral vector as claimed in any one of claims 1 to 14 wherein said adenovirus is genetically modified to encode a transgene and optionally surface decorated via an isopeptide linkage.

27. An adenoviral vector capable of surface decoration via an isopeptide linkage via a modified capsid protein.

Description

Detailed Description of the Invention

FIGURES

[0095] FIG. 1 (A-D). Modular covalent decoration of the adenovirus capsid via insertion of SpyTag or DogTag into hexon HVR loops.

[0096] FIG. 1 shows four representations of embodiments of the present invention. FIG. 1A) Reaction of SpyCatcher with SpyTag via an isopeptide bond. FIG. 1B) Reaction of DogCatcher with DogTag via an isopeptide bond. FIG. 1C) Coupling reaction between SnoopTagJr and DogTag, catalysed by SnoopLigase. FIG. 1D) Modular hexon decoration, showing surface display of DogTag on the viral capsid and subsequent coupling of peptides or protein antigens via SnoopTagJr or DogCatcher. FIG. 1E) Multiple sequence alignment of the adenovirus hexon protein indicating location of hypervariable region (HVR) loops 1, 2 and 5 and the deletions generated at the insertion site prior to insertion of SpyTag or DogTag at each locus. Shaded residues indicate differing residue identity to Ad5. Hexon sequences from the following Human adenovirus serotypes are shown; Ad5 and Ad2 (species C), Ad12 and Ad18 (species A), Ad3 and Ad35 (species B), Ad4 (species E), Ad40 and Ad41 (species F). Note that the insertions sites indicated are identical between SpyTag and DogTag recombinants at each of the three HVRs tested. At each locus, SpyTag or DogTag sequences are flanked by GSGGSG sequences.

[0097] FIG. 2 (A-D). Reactivity of SpyTag following insertion into Ad5 HVR loops. FIG. 2A: SDS-PAGE and Coomassie staining analysis of Ad5 displaying SpyTag in the loop HVR1, HVR2 or HVR5 (1E+10 viral particles) incubated with SpyCatcher at 4° C. for 16 h. FIG. 2B: Vector infectivity assay of the samples shown in FIG. 2A. Data show infectious units (ifu) per ml mean+range for duplicate wells. FIG. 2(C-D): Ad5(GFP) vectors displaying SpyTag at HVR1 (Ad5(GFP)HVR1-SpyTag) (1E+10 viral particles) were incubated with biotinylated SpyCatcher at 15 μM or 40 μM under different conditions as shown. FIG. 2C: SDS-PAGE Coomassie staining analysis of Ad5 displaying SpyTag incubated with SpyCatcher at 20° C. or 37° C. for 3 hours or at 20° C. or 4° C. for 16 hours. FIG. 2D: Vector infectivity assay was performed on the same samples shown in FIG. 2C. Data show mean+range for duplicate wells.

[0098] FIG. 3 (A-D). Reactivity of DogTag following insertion into Ad5 HVR loops. FIG. 3A: Yield comparison of Ad5 vectors displaying DogTag on hexon surfaces (in HVR 1, 2 or 5) versus Ad5 vectors with an unmodified hexon (WT). P:I ratios (ratio of total viral particles calculated by UV spectrophotometry to infectious units calculated by GFP focus assay) for each vector batch are indicated above each bar. FIG. 3B: SDS-PAGE and Coomassie staining analysis of Ad5 displaying DogTag at HVR1, HVR2 or HVR5 (1E+10 viral particles) incubated with DogCatcher (5 μM) at 4° C. for 16 h. FIG. 3C: Vector infectivity assay was performed on the samples shown in FIG. 3B. Data show mean+SD of triplicate wells. FIG. 3D: SDS-PAGE and Coomassie staining of Ad5 displaying DogTag incubated with DogCatcher for 0.1, 1 or 16 hours at 4° C.

[0099] FIG. 4 (A-C). Reactivity of Ad5 HVR-DogTag with SnoopTagJr directed by SnoopLigase. FIG. 4A: Reactivity of SnoopTagJr fused to an affibody to HER2 (SnJr-AffiHER2) with DogTag inserted into different HVR loops, catalysed by SnoopLigase, assessed by SDS-PAGE and Coomassie staining. FIG. 4B-C: Temperature-dependence of reactivity of SnJr-AffiHER2 with Ad5(GFP)HVR5-DogTag. FIG. 4B: SDS-PAGE and Coomassie staining analysis of the reaction between SnJr-AffiHER2 and Ad5(GFP)HVR5-DogTag, catalysed by SnoopLigase at 4° C. or 20° C. FIG. 4C: A vector infectivity assay was performed on the same samples described in FIG. 4B. Data show mean+range of duplicate wells. FIG. 4D: SDS-PAGE and Coomassie staining analysis on the effect of glycerol (15% w/v) and salt addition on SnoopLigase-mediated reactivity.

[0100] FIG. 5 (A-B). Assessment of vector infectivity using an anti-hexon immunostaining assay for non-fluorescent recombinant vectors. FIG. 5A: Anti-hexon immunostaining of HEK293A cells infected with Ad5 vectors with a WT hexon protein or Ad5 vectors displaying DogTag at HVR5. Brightfield microscopy images using a 4× objective, with hexon-positive cells shown in black. Scale bar=1000 μm. FIG. 5B: Comparison of the infectious titre for Ad5(GFP) hexon WT and Ad5(GFP) HVR5 DogTag using both GFP fluorescence and hexon immunostaining assays. Both assays were performed using the same wells on the same plate, n=4. A two-tailed t-test was performed, but no statistically significant difference was observed between groups. Bars show mean±SD; dots represent spot counts from individual wells.

[0101] FIG. 6 (A-F). Reactivity of DogTag inserted into hexon loops with SnoopTagJr tagged peptides. FIG. 6A: Illustration of a competition assay using DogCatcher (DC) to assess the efficiency of SnoopTagJr-peptide (SnJr-peptide) coupling to hexon-DogTag (hexon-DT), catalysed by SnoopLigase (SnL). FIG. 6B: SDS-PAGE and Coomassie analysis of the coupling efficiency of SnoopTagJr-hTERT peptide to hexon-DT using the DogCatcher competition assay. FIG. 6C: SDS-PAGE and Coomassie analysis of the coupling efficiency of SnoopTagJr-SIINFEKL peptide (PEP1; SnJr-GGS-SIINFEKL. PEP2; SnJr-AAY-SIINFEKL) to hexon-DT using the DogCatcher competition assay. FIG. 6D: Illustration of assessment of SnJr-peptide coupling to hexon-DT using monovalent streptavidin (mSA) direct gel shift assay with Western blotting using anti-hexon antibody. If added after boiling, the biotin-streptavidin interaction is stable on SDS-PAGE. FIG. 6E: SDS-PAGE and Coomassie analysis of SnJr-biotin coupling to Ad5-DT using the direct gel shift assay with mSA. FIG. 6F: A vector infectivity assay was performed on the same samples shown in FIG. 6C. Bars show mean and range of duplicate wells.

[0102] FIG. 7 (A-C). Coupling of Ad5 HVR DogTag to DogCatcher fusion proteins. FIG. 7A: Illustration of three DogCatcher fusion constructs; NANP9, NANP18, and NANPD, derived from the Plasmodium falciparum circumsporozoite protein (PfCSP), isolate 3D7. NDVP and NANP tetrapeptide repeats are illustrated. FIG. 7B: SDS-PAGE and Coomassie analysis of the reactivity of DogCatcher-NANPn with DogTag inserted into hexon HVR5 loop. FIG. 7C: A vector infectivity assay was performed on the same samples shown in FIG. 7B. Bars show mean and range of duplicate wells. Sizes of surface decoration: DogCatcher-NANP9 (.sup.˜25-30 kDa), DogCatcher-NANP18 (.sup.˜35-40 kDa) and DogCatcher-NANP Domain (.sup.˜60 kDa)

[0103] FIG. 8 (A-C). Coupling via isopeptide bonds to the surface of Ad5 via hexon reduces the potency of virus-neutralising antibodies. FIG. 8A: Coupling via SnoopLigase to the surface of Ad5 via hexon reduces the potency of virus-neutralising antibodies. Adenovirus was added to cells in the presence of varying concentration of the monoclonal antibody targeting the adenovirus hexon (mAb 9C12). Productive adenovirus infection was detected from the fluorescence of adenovirus-encoded GFP expressed in the cells. Intensity of fluorescence of Ad5-DT alone, Ad5-DT+SnJr-AffiHER2 and Ad5-DT+SnL+SnJr-AffiHER2. FIG. 8B: Coupling of DogCatcher-NANPn to the surface of Ad5 via hexon reduces the potency of virus-neutralising antibodies, analysed as in FIG. 8A. Intensity of fluorescence from Ad5-DT alone, or DogCatcher-NANPn coupled to Ad5 DogTag. FIG. 8C: Adenovirus was added to cells in the presence of varying dilutions of adenovirus-neutralising serum. Productive adenovirus infection was detected from the fluorescence of adenovirus-encoded GFP expressed in the cells. Intensity of fluorescence of Ad5-DT alone, or DogCatcher-NANPn coupled to Ad5 DogTag. In A-C, bars indicate mean and range of duplicate values.

[0104] FIG. 9: Human coagulation Factor X-dependent vector transduction of SKOV3 cells. Ad5(GFP)-DogTag (Ad5) or Ad5(GFP)-DogTag:DogCatcher-NANP18 (Ad5-NANP18) vectors (2E+9 viral particles) were incubated in the presence or absence of human coagulation Factor X (8 μg/mL) on SKOV3 cells for 2 h at 37° C. in serum-free media. Then media was replaced with fresh complete media and plates were incubated for a further 48 h. Infectious titres were then calculated by enumeration of GFP-positive foci by fluorescence microscopy. Samples were plated in duplicate and bars show mean and range of data values.

[0105] FIG. 10 (A-C). Ad5(GFP) vectors displaying SIINFEKL peptides on the capsid surface generate CD8+T cell responses against both the capsid-displayed peptide and internally-encoded GFP antigens. FIG. 10A: Design and immunisation schedule for mouse immunogenicity experiment to assess CD8.sup.+ T cell responses to Ad5 surface-displayed peptide antigens. To confirm the importance of physical attachment of peptide epitopes to the capsid surface, SIINFEKL specific T cell responses after administration of Ad5(GFP) HVR5 DogTag with SIINFEKL attached (Ad5-DT:SIINFEKL) were compared to co-administration of Ad5(GFP) HVR5 DogTag (Ad5-DT) with free SIINFEKL peptide (Groups 1 and 2, vs Group 3), and to SIINFEKL peptide with poly I:C adjuvant (Group 4). FIG. 10B: Spleen ex vivo IFNγ-ELISPOT responses to SIINFEKL (spot forming cells per million splenocytes). FIG. 10C: Spleen ex vivo IFNγ-ELISPOT responses to the epitope DTLVNRIEL (EGFP.sub.118-126). In FIG. 10B and FIG. 10C, spots represent responses in individual animals, while bars represent median values with range.

[0106] FIG. 11 (A-D). Modular covalent decoration of the adenovirus capsid via fusion of SpyCatcher at the C-terminus of pIX. FIG. 11A: Modular adenovirus capsid decoration, showing surface display of SpyCatcher on the viral capsid via fusion to pIX, and subsequent coupling of peptide or protein antigens via SpyTag. FIG. 11B: SDS-PAGE analysis of Ad5(GFP) pIX-SpyCatcher coupled with SpyTag-Maltose binding protein (SpyTag-MBP, 5 μM) or SpyTag-Human cytomegalovirus (HCMV) pentamer (SpyTag-Pentamer, 2.5 μM) for 16 h at 4° C. gH is the pentamer subunit containing the SpyTag. Proteins detected by Western blotting using anti-SpyCatcher polyclonal mouse sera. FIG. 11C: SDS-PAGE analysis of Ad5(GFP) pIX-SpyCatcher coupled to SpyTag-peptides. Monovalent streptavidin (mSA) was added to induce a clear gel shift, and proteins detected by Western blotting using anti-SpyCatcher polyclonal mouse sera. FIG. 11D: Vector infectivity assay was performed on the same samples as shown in FIG. 11B and FIG. 11C. Bars show mean and SD of triplicate wells.

[0107] FIG. 12 (A-B). Viability, genetic stability, and yield of different recombinant adenovirus vectors with SpyCatcher, SnoopCatcher or DogCatcher fused to the C-terminus of pIX. FIG. 12A: Schematic representation of the pIX amino acid sequence for different Ad5(GFP) vectors with versions of SpyCatcher, SnoopCatcher or DogCatcher fused to the C-terminus of pIX, with corresponding information regarding vector viability in HEK293A cells and genetic stability (after >3 passages). SpyCatcher dN1 (delta N1) refers to the same N-terminally truncated version of SpyCatcher used in the other Figures from this document. SpyCatcher dN1dC2 (delta N1, delta C2) refers to a shorter version of SpyCatcher with an additional C terminal truncation. FIG. 12B: Yield comparison of viable Ad5 vectors (as indicated in FIG. 12A) displaying SpyCatcher, SnoopCatcher or DogCatcher at the C-terminus of pIX. Yields of vectors with pIX-Catcher fusions are compared to Ad5(GFP) HVR5 DogTag, using equivalent volumes of cultured cells and the same vector harvest/purification protocols. For SnoopCatcher and DogCatcher fusions, bars indicate mean and range of n=2 batches.

[0108] FIG. 13 (A-C). Reactivity of SnoopCatcher and DogCatcher fused to the C-terminus of pIX. FIG. 13A: Reactivity of Ad5(GFP) pIX-SnoopCatcher vector with SnoopTagJr-fused to the full length spike protein from SARS CoV2 (Spike) or SnoopTagJr fused to the receptor binding domain (RBD) of SARS CoV2 spike. Ad5(GFP) pIX-SnoopCatcher (3E+9 viral particles) was incubated alone or co-incubated with Spike-SnoopTagJr (0.75 μM) or RBD-SnoopTagJr (5 μM) for 16 h at 4° C. Samples were run on SDS-PAGE and western blotting performed using polyclonal mouse sera with reactivity against SnoopCatcher. Species representing pIX-SnoopCatcher (pIX-SnC), pIX-SnoopCatcher:SnoopTagJr-RBD (pIX-SnC:SnJr-RBD) and pIX-SnoopCatcher:SnoopTagJr-Spike (pIX-SnC:SnJr-Spike) are indicated. FIG. 13B: Vector infectivity assay was performed using the same samples shown in FIG. 13A. Bars show mean and SD of triplicate samples. FIG. 13C: Reactivity of Ad5(GFP) pIX-DogCatcher vector with DogTag-fused to Small Ubiquitin Modifier (SUMO). Ad5(GFP) pIX-DogCatcher (1E+9 viral particles) was incubated alone or co-incubated with SUMO-DogTag (17 μM) for 16 h at 4° C. Samples were run on SDS-PAGE and western blotting performed using polyclonal mouse sera with reactivity against DogCatcher. Species representing pIX-DogCatcher (pIX-DC) and pIX-DogCatcher:DogTag-SUMO (pIX-DC:DT-SUMO) are indicated.

[0109] FIG. 14 (A-C). Reactivity of SnoopTagJr and SpyTag fused to the C-terminus of pIX. FIG. 14A: Yield comparison of Ad5 vectors displaying SnoopTagJr or SpyTag fused to the C-terminus of pIX. Both vectors have GGS (EAAAK)3 GS linker sequences between pIX and Tag. Yields of vectors in HEK293A cells with pIX-Catcher fusions are compared to Ad5(GFP) HVR5 DogTag, using equivalent volumes of cultured cells and the same vector harvest/purification protocols. FIG. 14B: Reactivity of Ad5(GFP) pIX-SnoopTagJr with SnoopCatcher fused to SARS CoV2 RBD and reactivity of Ad5(GFP) pIX-SpyTag with SpyCatcher fused to SARS CoV2 RBD. Vectors (1E+10 viral particles) were incubated alone, or co-incubated with RBD-SnoopCatcher, SnoopCatcher-RBD, or RBD-SpyCatcher (all 3.5 μM) for 16 h at 4° C. Samples were run on SDS-PAGE and western blotting performed using a polyclonal anti-RBD antibody. Species representing pIX-SnoopTagJr:SnoopCatcher-RBD (pIX-SnJr:SnC-RBD), pIX-SpyTag:SpyCatcher-RBD (pIX-ST:SC-RBD) and non-coupled SnoopCatcher-RBD/SpyCatcher-RBD (SnC/SC-RBD) are indicated and labelled (*). FIG. 14C: Vector infectivity assay was performed using the same samples shown in FIG. 14B. Bars show mean and range of duplicate samples.

ADENOVIRUS

[0110] Adenoviruses (Ad) are a non-enveloped double stranded DNA virus with a genome of approximately 36 kilobases (kb). There are over 60 human adenovirus serotypes grouped into species A-G. Each group comprises of a number of adenoviral serotypes, for example, the subgroup species C includes Ad5 and Ad2. Ad5 is the most extensively studied serotype, and the most widely used platform for the development of oncolytic viruses. In the development of oncolytic viruses, it is desirable to be able to target particular tissues, and therefore the tropism may be altered. A major issue with using some adenovirus serotypes, including Ad5, in clinical settings is the pre-existing immunity in humans. Adenoviruses are typically 70-90 nm in size with an icosahedral capsid shape. The outer capsid structure, also known as ‘capsid protein’ comprises three major types of protein (hexon, fiber and penton base). There are additional minor proteins in the outer capsid including VI, VIII, IX, IIIa and IVa2. Hexon is the major component of the adenoviral capsid accounting for more than 83% of the capsid protein. Hexon modification has been shown to allow for circumvention of pre-existing neutralising antibodies in some circumstances, including the swapping of HVR from different serotypes.

Adenovirus for Modification

[0111] Adenovirus can be replication-defective: certain genes are deleted from the genome in order to ensure that when the adenovirus is used as a therapeutic, it is no longer capable of replication. This may result from the deletion of a set of genes from the genome, and is within the skills of those working with adenoviruses. This may be an advantage for use in vaccines, where the aim of the adenoviral vector is to present the antigen to the immune system in a format that makes it highly immunogenic, while limiting cytotoxicity. However, for other applications such as oncolytic viruses, being replication-competent is key. Replication-competent adenoviruses may still contain some modifications to prevent replication in normal cells, for example by deletion of a key gene. Oncolytic Ad vectors lyse cancer cells at the end of their life cycle and it is important that the progeny do not infect normal tissue. Cancer cells are generally more permissive for adenoviral replication, whilst normal cells require the adenovirus to have a full complement of genes to assist replication.

[0112] The adenovirus may be from any serotype or strain of adenovirus. Therefore, suitable adenoviruses for modification may come from those that infect mammals other than humans, in order to minimise prior exposure effects. The capsid structure is strongly conserved, and therefore the adenoviral serotypes and species may be interchangeable.

[0113] The adenovirus may be any modified adenovirus. Thus, the modified adenovirus may additionally encode antigens for example. These encoded antigens would then be expressed after infection. This provides the possibility of a multi-faceted prophylactic or therapeutic, such that an antigen can be displayed on the surface of the virus and another antigen expressed upon vector transduction using host cell machinery. Thus, the adenovirus may be genetically modified, such that, for example it includes a transgene. This transgene is designed for delivery to the host cell and may be a gene encoding an antigen, for example.

Adenovirus-Mediated Infectivity

[0114] Adenovirus infectivity in cells that express the Coxsackievirus and adenovirus receptor (CAR) is mediated via the fiber protein. An example of a cell line that expresses the CAR receptor is HEK293 cells. Fiber binds to the CAR receptor on the surface of cells and this mediates the initial attachment of the virus. However, it was recently demonstrated that instead of a fiber-mediated entry of the adenovirus, Factor X (FX)—a coagulation factor present in human serum can bind to the hexon proteins of some adenovirus serotypes to facilitate the entry of the virus in some cell types. An example of a cell line that mediates infection via the hexon protein is SKOV3. It is believed that FX mediated infection via the adenovirus hexon can enhance liver tropism of adenovirus vectors in vivo. Modifications of the hexon protein such as insertion of DogTag and coupling to an antigen reduces hexon-mediated infectivity of the cells, as demonstrated in the Examples. This is a desirable effect as the natural tropism of adenovirus when injected intravenously can cause liver toxicity in patients at very high doses. Reduction of hexon-mediated infectivity to reduce liver toxicity would be advantageous to the present invention.

Hexon Capsid Protein

[0115] The hexon capsid protein is approximately 100 kDa in size, with 720 monomers per virion. Hexon monomers organise into trimers so that 12 lie on each of the 20 facets, resulting in 240 trimers per virion. Hexon sequences contain hypervariable regions (HVR) corresponding to loops on the external surface on the virus and therefore cover almost the entire surface of the virus. Each monomer has seven HVRs identified as HVR1-HVR7 which are serotype specific. As the loops are on the external surface of the virus, hexon loops are the main antigen recognition site, a target for host immune responses. Hexon protein varies in length, for example, Ad2 is the longest known hexon protein with a length of 968 amino acids (UniProt ID: P03277). Ad5, the most commonly used adenovirus for gene therapy has a length of 952 amino acids (UniProt ID: P04133). Modifying hexon HVRs which contain the serotype-specific epitope seems to be a promising approach to overcome the host neutralisation response. Any one of the HVRs could be modified. Exemplified herein, modifications were successfully made to HVR1, HVR2 and HVR5, surprisingly using DogTag. When the hexon protein was modified according to the invention and an antigen was attached via the peptide partner pair, neutralisation by anti-adenovirus neutralising antibodies was reduced.

pIX Capsid Protein

[0116] pIX protein is a minor capsid protein which is approximately 14.3 kDa in size. There are approximately 240 pIX monomers per virion. The pIX protein functions to stabilise the hexons on the viral surface. The C-terminus of the pIX protein is exposed on the surface of the virus and is therefore a desirable site for fusion of small and large peptides. Ad5 pIX has two domains connected by a flexible linker. The Ad5 pIX protein has a length of 196 amino acids (UniProt ID: Q2KS03).

Modifications to the Capsid Proteins

[0117] Modification to the capsid proteins can be genetic or non-genetic, including chemical. The capsid proteins can be genetically modified through the incorporation of antigens into the capsid. Alternatively, the viral particle surface may be directly modified. Modification of all three major capsid proteins has been demonstrated previously. However, the results from these modifications has been mixed, and there is a major obstacle in the size of the insert that the most promising approaches offer, particularly regarding modification of hexon.

[0118] “At least one modification” as used herein refers to the inclusion of a first peptide partner insertion into the viral capsid protein using any appropriate means. For example, the insertion of the first peptide partner into the adenoviral hexon loops or the fusion of a first peptide partner to the adenoviral pIX minor capsid protein. This modification may be made genetically through gene fusion, for example, or chemically.

Insertion

[0119] The first peptide partner may be directly inserted into the relevant capsid protein. As discussed herein, such an insertion is achieved by genetic manipulation. Alternatively, the first peptide partner can be inserted with a peptide sequence of any suitable length which separates the first peptide partner from the capsid protein. This peptide sequence may be described as a linker sequence, a spacer sequence, a structural sequence such as a helix, or even a hinge sequence. Linker or spacer sequences may simply separate the capsid protein and the first peptide partner and act as a “link” between the two entities. A structural sequence may provide a physical separation of the first peptide partner from the capsid protein. A hinge sequence may act as a linker between the capsid protein and the first peptide partner but permit a degree of motion to occur, such that the first peptide partner can move relative to the capsid protein. Various linkers, spacers and hinge sequences are exemplified herein, most notably those depicted in FIG. 12.

[0120] Where the insertion is into a loop of sequence, the first peptide partner may be separated from the capsid protein on one or both sides, i.e. it may be flanked by sequences which are linkers, spacers, structures or hinges. Each flanking side may be the same or different.

Peptide Partner Pairs

[0121] Proteins that are capable of spontaneous isopeptide bond formation (so-called “isopeptide proteins”) have been advantageously used to develop peptide partner pairs (i.e. two-part linkers) which covalently bind to each other and provide irreversible interactions (see e.g. WO2011/098772 and WO 2016/193746 both herein incorporated by reference, together with WO2018/189517 and WO2018/197854 both incorporated herein by reference). In this respect, proteins which are capable of spontaneous isopeptide bond formation may be expressed as separate fragments, to give a first peptide partner and a second peptide partner which is the peptide binding partner for the first peptide partner, where the two fragments are capable of covalently reconstituting by isopeptide bond formation. This covalent reconstitution links molecules or components fused to the second peptide partner and the requisite first peptide partner. The isopeptide bond formed by the peptide partner pair is stable under conditions where non-covalent interactions would rapidly dissociate, e.g. over long periods of time (e.g. weeks), at high temperature (to at least 95° C.), at high force, or with harsh chemical treatment (e.g. pH 2-11, organic solvent, detergents or denaturants).

[0122] Isopeptide bonds are amide bonds formed between carboxyl/carboxamide and amino groups, where at least one of the carboxyl or amino groups is outside of the protein main-chain (the backbone of the protein). Such bonds are chemically irreversible under typical biological conditions and they are resistant to most proteases. As isopeptide bonds are covalent in nature, they result in some of the strongest measured protein-protein interactions.

[0123] In brief, a two-part linker, i.e. a peptide partner pair (a so-called peptide tag/binding partner or catcher pair) may be derived from a protein capable of spontaneously forming an isopeptide bond (an isopeptide protein), wherein the domains of the protein are expressed separately to produce a peptide “tag” that comprises one of the residues involved in the isopeptide bond (e.g. an aspartate or asparagine, or a lysine) and a peptide or peptide binding partner (or “catcher”) that comprises the other residue involved in the isopeptide bond (e.g. a lysine, or an aspartate or asparagine) and at least one other residue required to form the isopeptide bond (e.g. a glutamate). Mixing the peptide tag and binding/catcher partner results in the spontaneous formation of an isopeptide bond between the tag and binding partner. Thus, by separately incorporating the peptide tag and binding partner into different molecules or components, e.g. proteins, it is possible to covalently link said molecules or components together via an isopeptide bond formed between the peptide tag and binding partner, i.e. to form a linker between the molecules or components incorporating the peptide tag and binding partner.

[0124] The spontaneous formation of the isopeptide bond may be in isolation, and not require the addition of any other entity. For some peptide tag and binding/catcher partner pairs, the presence of a third or helper entity, such as a ligase, may be required in order to generate the isopeptide bond.

[0125] A peptide tag/binding partner pair (two-part linker), termed SpyTag/SpyCatcher, has been derived from the CnaB2 domain of the Streptococcus pyogenes FbaB protein (Zakeri et al., 2012, Proc Natl Acad Sci USA 109, E690-697) and used in diverse applications including vaccine development (Brune et al., 2016, Scientific reports 6, 19234; Thrane et al., 2016, Journal of Nanobiotechnology 14, 30).

[0126] Variants, derivatives and modifications of the binding pairs may be made by any suitable means. Variants, derivatives and functionally operative modifications may involve amino acid additions, substitutions, alterations or deletions that retain the same function in relation to the ability to form an isopeptide bond with the relevant binding partner.

[0127] For some of the binding pairs, mediation by a third entity such as an enzyme is required. For example, SnoopLigase may be used to mediate the bond formation between SnoopTagJr/SnoopTag and DogTag. Thus, the pairing may require the assistance of an enzyme such as a ligase.

[0128] It will be understood that as used herein, either the first peptide partner or the second peptide partner may be the peptide “tag” and the other is the “binding partner/catcher”.

[0129] Suitably, the first and second peptide partners form the peptide partner pair termed SpyTag/SpyCatcher. Suitably, the SpyCatcher component is DeltaN1 (ΔN1) SpyCatcher (as described in Li, L., Fierer, J. O., Rapoport, T. A. & Howarth, M. Structural analysis and optimization of the covalent association between SpyCatcher and a peptide Tag. J. Mol. Biol. 426, 309-317 (2014)) which has a 23 amino acid truncation at the N-terminus compared to “SpyCatcher”.

[0130] In other embodiments, the first and second peptide partners form a peptide partner pair which is a mutated version of SpyTag/SpyCatcher displaying an increased rate of reaction for isopeptide bond formation such as, for example, those described in co-pending application, GB1706430.4. In some embodiments, these mutated forms may be useful for the attachment of large proteins (e.g. >50 kDa or >100 kDa, such as the >160 kDa HCMV pentameric protein exemplified herein) and/or where slow reactions or steric hindrance may be an issue.

[0131] In other embodiments, the isopeptide proteins forming the peptide partner pair may include SnoopTag/SnoopCatcher, described, for example in WO 2016/193746.

[0132] In some embodiments, one or both of the isopeptide proteins forming the peptide partner pair may have N- or C-terminal truncations, whilst still retaining the reactivity of the isopeptide bond.

[0133] Exemplary first and second peptide partner pairs (peptide tag/binding partner pairs; reactive pairs) are described in the following table:

TABLE-US-00001 Reactive pairs (a) SpyTag SpyCatcher SpyTag002 SpyTag002 RG T3H (b) SpyTag SpyCatcher002 SpyTag002 SpyTag002 RG T3H (c) SpyTag SpyCatcher002 D5A A92P Q100D SpyTag002 SpyTag002 RG T3H (d) SnoopTag SnoopCatcher SnoopTagJr (e) RrgATag RrgACatcher - denoted here also as RrgATag2 DogCatcher DogTag (f) Isopeptag Pilin-C (g) Isopeptag-N Pilin-N (h) PsCsTag PsCsCatcher (i) SnoopTagJr DogTag [mediated by SnoopLigase] SnoopTag
These entities are described, for example, in WO2011/098772, WO2016/193746, GB1706430.4 GB 1705750.6 or Li L., et al., J. Mol. Biol. 426, 309-317 (2014).

[0134] Variants, derivatives and modifications of the binding pairs may be made by any suitable means. Variants, derivatives and functionally operative modifications may involve amino acid additions, substitutions, alterations or deletions that retain the same function in relation to the ability to form an isopeptide bond with the relevant binding partner.

[0135] For some of the binding pairs, mediation by a third entity such as an enzyme is required. For example, SnoopLigase may be used to meditate the bond formation between SnoopTagJr and DogTag. Thus, the pairing may require the assistance of an enzyme such as a ligase.

Antigen

[0136] An antigen as used herein refers to any molecule that is capable of inducing immune responses. An antigen can be a self-antigen, cancer antigen, allergenic antigen, tumour antigen, viral antigen, bacterial antigen, parasitic antigen or fungal antigen. A tumour antigen includes tumour-specific antigen, tumour-associated antigen and neoantigens, newly formed antigens by cancerous cells. “Tumour-specific antigen” refers to antigens that are only found on tumour cells. “Tumour-associated antigen” refers to antigens presented by both tumour and normal cells. “Neoantigen” refers to newly formed antigens by tumour cells. “Antigen” as used herein includes peptides and epitopes, variants and derivatives thereof.

[0137] Tumour-associated antigens include, but are not limited to adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (CEA), CAGE 1 to 8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin DI, Cyclin-AI, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (ETA), ETV6-AML1 fusion protein, EZH2, ErbB receptors, E6, E7, FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100/Pmel 17, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7 antigen, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A 10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class 1, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-I/LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (PEM), PPPIR3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB 38/N Y-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, R F43, RU2AS, SAGE, secernin 1, SIRT2, SNRPDI, SOX10, Spl7, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-I/gp75, TRP-2, TRP2-INT2, tyrosinase (TYR), VEGF, WT1, XAGE-Ib/GAGED2a,

[0138] Those skilled in the art of identifying tumour-associated antigens will appreciate that new antigens, including neoantigens, are continually identified, and as such this list is not exhaustive.

[0139] Viral antigens include, but are not limited to antigens of the following viruses or class of viruses; Human Papilloma Viruses (HPV), Human Immunodeficiency virus (HIV), Herpes Simplex Virus (HSV2/HSV1), Influenza virus (types A, B and C), Polio virus, Respiratory Syncitial Virus (RSV), Rhinoviruses, Rotaviruses, Hepatitis A virus, Norwalk Virus Group, Enteroviruses, Astroviruses, Measles virus, Parainfluenza virus, Mumps virus, Varicella-Zoster virus, Human Cytomegalovirus (HCMV), Epstein-Barr virus, Adenoviruses, Rubella virus, Human T-cell Lymphoma type I virus (HTLV-I), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Poxviruses, Marburg virus and Ebola virus, SARS-CoV-2

Bacterial antigens include, but are not limited to antigens of the following bacteria: Mycobacterium tuberculosis, Chlamydia, Neisseria gonorrhoeae, Shigella, Salmonella, Vibrio cholerae, Treponema pallidum, Pseudomonas, Bordetella pertussis, Brucella, Francisella tularensis, Helicobacter pylori, Leptospira interrogans, Legionella pneumophila, Yersinia pestis, Streptococcus (types A and B), Pneumococcus, Meningococcus, Haemophilus influenzae (type b), Toxoplasma gondii, Campylobacter, Moraxella catarrhalis, Klebsiella granulomatis and Actinomyces.

[0140] Fungal antigens include, but are not limited to antigens of the following fungal pathogens: Candida and Aspergillus, Cryptococcus, Histoplasma and Pneumocystis.

[0141] Parasitic antigens include, but are not limited to antigens of the following parasitic pathogens: Taenia, Flukes, Roundworms, Plasmodium, Amoeba, Giardia, Cryptosporidium, Schistosoma, Trichomonas, Trypanosoma and Trichinella.

[0142] In order to enhance expression of the antigen-second peptide partner prior to coupling, a leader sequence may be used. Those skilled in the art are aware of appropriate leader sequences to enhance expression. Such are exemplified herein.

Pharmaceutical Composition and Use

[0143] The compositions of the invention may be incorporated into a vaccine or therapeutic composition. Suitably, a vaccine or immunogenic composition will comprise particles of the invention in an immunogenic dose.

[0144] A pharmaceutical composition may comprise a particle or composition in accordance with the invention provided with a pharmaceutically acceptable carrier. Suitable carriers are well known to those skilled in the art. In one embodiment a pharmaceutical composition comprises a buffer, excipient or carrier. Suitably a pharmaceutical composition may comprise suitable excipients and formulations to maintain stability of the composition. Suitably the formulation may comprise an adjuvant. In one embodiment, the formulation may comprise AddaVax™ or a similar squalene-based oil-in-water nano-emulsion with a formulation similar to MF59®. Other suitable adjuvants include liposome-based adjuvants such as Matrix M and AS01. Other suitable adjuvants include aluminium-based formulations such as Alhydrogel®. In one embodiment the formulation may comprise EDTA, for example at a concentration of 5 mM. Suitable excipients or formulations may depend on the properties of the particle or immunogenic composition; for example, the choice of expression system may affect the stability, glycosylation or folding of the proteins of the composition, which may in turn affect the optimal formulation of the composition. Methods of determination of a suitable excipient, formulation or adjuvant will be known to those skilled in the art.

Vaccine

[0145] A vaccine is a preparation that comprises a fragment or entire entity against which it is possible to raise an immune response. It is an entity such as a protein, peptide, lipoprotein, glycoprotein or fragments thereof that are capable of inducing an immune response. For example, the vaccine may comprise micro-organisms or a part thereof capable of inducing an immune response against said micro-organism. A vaccine comprising an immunogenic adenoviral vector in accordance with the invention can be used against any pathogen for which the antigen displayed is crucial for the induction of an immune response. Further, the vaccine may comprise an immunogenic adenoviral vector in accordance with the invention displaying tumour related antigens. These tumour-related antigens may be modified self-proteins and the like. The vaccine may therefore raise an immune response to the tumour cells.

[0146] Such vaccine compositions (or other immunogenic) are formulated in a suitable delivery vehicle. Generally, doses for the immunogenic compositions are within the ranges defined for therapeutic compositions. Optionally, a vaccine composition of the invention may be formulated to contain other components, including, for example, adjuvants, stabilizers, pH adjusters, preservatives and the like. Such components are well known to those skilled in the art of vaccines. Examples of suitable adjuvants include, without limitation, liposomes, alum, monophosphoryl lipid A, and any biologically active factor, such as a cytokine, an interleukin, a chemokine and optimally combinations thereof.

[0147] The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, rectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, locally and/or using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly or via a catheter and/or lavage.

[0148] Vaccines for the treatment or prevention of a disease as used herein includes but is not limited to urogenital cancers (such as prostate cancer, renal cell cancers, bladder cancers), gynaecological cancers (such as ovarian cancers, cervical cancers, endometrial cancers), lung cancer, gastrointestinal cancers (such as non-metastatic or metastatic colorectal cancers, pancreatic cancer, gastric cancer, oesophageal cancers, hepatocellular cancers, cholangiocellular cancers), head and neck cancer (e.g. head and neck squamous cell cancer), malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer (e.g. hormone refractory metastatic breast cancer), malignant melanoma, Merkel Cell Carcinoma or bone and soft tissue sarcomas, and haematologic neoplasias, such as multiple myeloma, acute myelogenous leukaemia, chronic myelogenous leukaemia, myelodysplastic syndrome and acute lymphoblastic leukaemia. In a preferred embodiment, the disease is non-small cell lung cancer (NSCLC), breast cancer (e.g. hormone refractory metastatic breast cancer), head and neck cancer (e.g. head and neck squamous cell cancer), hormone sensitive or hormone refractory prostate cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, soft tissue sarcoma, or small cell lung cancer.

[0149] The vaccine may be used to treat or prevent infection with any one of the disease-causing pathogens hereinbefore described.

Ad-DogTag

[0150] The “Ad-DogTag” viral vector comprises the insertion of DogTag into surface loops of the hexon capsid protein enabling display of up to .sup.˜720 ligands/virion. Coupling of an antigen to hexon-DogTag has been achieved by the present inventors using SnoopTagJr-tagged antigens (using SnoopLigase as a catalyst) or directly via DogCatcher linked antigens. Previous technologies have only been capable of inserting small immunogenic T cell or B cell epitopes with a length of <100 amino acids into adenovirus hexon loops. The present invention demonstrates the coupling of peptides of 10-60 kDa to hexon, which has not previously been possible to achieve. This represents a big step forwards in the development of vaccines based upon adenovirus in particular.

Ad-SpyCatcher

[0151] The “Ad-SpyCatcher” viral vector comprises the fusion of SpyCatcher onto the C-terminus of adenovirus minor capsid protein pIX. The recent invention was successful in modifying the pIX minor capsid protein without loss of viral infectivity.

Ad-SnoopCatcher

[0152] The “Ad-SnoopCatcher” viral vector comprises the fusion of SnoopCatcher onto the C-terminus of adenovirus minor capsid protein pIX. The work here shows success in modifying the pIX minor capsid protein without loss of viral infectivity.

Ad-DogCatcher

[0153] The “Ad-DogCatcher” viral vector comprises the fusion of DogCatcher onto the C-terminus of adenovirus minor capsid protein pIX. The work here shows success in modifying the pIX minor capsid protein without loss of viral infectivity.

Ad-SnoopTagJr

[0154] The “Ad-SnoopTagJr” viral vector comprises the fusion of SnoopTagJr onto the C-terminus of adenovirus minor capsid protein pIX. The work here shows success in modifying the pIX minor capsid protein without loss of viral infectivity.

Ad-SpyTag

[0155] The “Ad-SpyTag” viral vector comprises the fusion of SpyTag onto the C-terminus of adenovirus minor capsid protein pIX. The work here shows success in modifying the pIX minor capsid protein without loss of viral infectivity.

[0156] All references mentioned herein are incorporated by reference where permitted.

EXAMPLES

[0157] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

Examples—Materials and Methods

[0158] Generating a Bacterial Artificial Chromosome (BAC)-Derived Replication-Defective Molecular Clone of Ad5 Expressing GFP

[0159] Plasmid pAd-PL-DEST, an E1/E3-deleted (and therefore replication-defective) molecular clone of Ad5, was obtained from Invitrogen. An expression construct, consisting of an immediate early cytomegalovirus promoter (CMVp) driving expression of enhanced green fluorescent protein (EGFP), was cloned into shuttle vector pENTR4 (Invitrogen). The CMVp EGFP expression construct was then inserted into the Ad5 E1 locus using Invitrogen Gateway site-specific recombination (LR clonase) technology. BAC sequences from pBELOBAC11 (NEB) were amplified using forward (5′-TTAATTAAcgtcgaccaattctcatg) and reverse (5′-TTAATTAAgtcgacagcgacacacttg) primers to introduce PacI sites at either end of the BAC cassette. The entire Ad5(GFP) genome sequence was subsequently cloned into the BAC with PacI, to generate pBAC-Ad5(GFP).

[0160] Genetic Modification of PBAC-Ad5(GFP) to Insert Protein Superglue Technology into Viral Capsid Proteins Using BAC GalK Recombineering

[0161] SW102, an E. coli strain required for GalK recombineering, was obtained from the National Cancer Institute, National Institutes of Health, USA. Modified from DH10B, SW102 cells contain A-Red-encoded recombination genes (exo, bet, gam) under the control of a temperature-sensitive repressor with a deleted galactokinase (GalK) gene (which is necessary for bacterial growth using galactose as the sole carbon source). The GalK recombineering system enables the GalK gene to be used for both positive and negative selection, and GalK recombineering was performed exactly as described in Warming et al, 2005 [Warming S, Costantino N, Court D L, Jenkins N A, Copeland N G. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 2005; 33(4):e36]. Insertion sites were created in hexon HVR loops (as described in FIG. 1E) or at the C-terminus of pIX by insertion of the Ga/K gene at these specific loci by recombineering followed by positive selection for the presence of Ga/K. Insertion of SpyTag/DogTag/SnoopTagJr/SpyCatcher/SnoopCatcher/DogCatcher sequences was performed by recombineering to replace Ga/K, and subsequent selection against the presence of the Ga/Kgene using 2-deoxygalactose.

[0162] Rescue of Recombinant Adenoviruses Incorporating Protein Superglue Technology

[0163] BAC DNA from recombinant adenovirus molecular clones was linearised with PacI to release left and right viral inverted terminal repeats (ITRs). Linearised DNA was transfected into E1-complementing Human Embryonic Kidney (HEK) 293A cells (Invitrogen) in 25 cm.sup.2 flasks (T25) using Lipofectamine 2000 reagent (Invitrogen). After cytopathic effect (CPE) was observed, the cells were harvested, subjected to three cycles of freeze-thaw, and the virus amplified further in HEK293A cells. Upon infection of 10×150 cm.sup.2 flasks (T150), virus was harvested from infected cells after 48 hours and purified by CsCl gradient ultracentrifugation according to standard protocols. Purified virus was dialysed against sucrose storage buffer (10 mM Tris-HCl, 7.5% w/v sucrose, pH 7.8) and stored at −80° C.

[0164] Estimation of Viral Particle Count for Purified Viral Vector Preparations The number of adenovirus particles in a purified preparation can be estimated by measuring viral DNA content by spectrophotometric absorption at 260 nm as described by Maizel et al, 1968. [J. Maizel, D. White, M. Scharff, The polypeptides of adenovirus: I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology, Volume 36, Issue 1, September 1968, Pages 115-125]. Briefly, samples were diluted 1:10 in virus storage buffer containing 1% w/v sodium dodecyl sulphate (SDS) to release viral DNA from capsids and absorbance at 260 nm was measured using a spectrophotometer. An absorbance of 1.00 (AU, 1 cm path length) at 260 nm corresponds to 1.1×10.sup.12 viral particles/mL.

[0165] Infectious Titration of Recombinant Adenoviruses in HEK293A Cells

[0166] Infectious titre of vector preparations was assessed by single cell infectivity assay on HEK293A cells. For vectors expressing EGFP, infected HEK293 cells were visualised and enumerated directly by fluorescent microscopy. An alternative assay for vectors without a fluorescent marker, by immunostaining for expression of the hexon capsid protein, was also tested (FIG. 5). Tenfold serial dilutions of virus in complete media (Dulbecco's Modified Eagles Medium, plus 1×GlutaMAX and 10% v/v foetal bovine serum) were performed in sterile 96-well deep well plates. Two or three serial dilutions were performed per virus, and 50 μL of each dilution (10.sup.3 to 10.sup.10 dilution) was added per well of a 96-well plate containing adherent HEK293 cells at 80-90% confluency. For hexon immunostaining, 96-well plates pre coated with poly-L-Lysine were used to improve cell adhesion to the plate surface. Plates were incubated for 48 hours at 37° C., 5% CO.sub.2. For titration of EGFP-expressing vectors, 48 hours post infection, single GFP-positive cells were enumerated by fluorescence microscopy, and an infectious titre was calculated in infectious units (ifu) per mL. For hexon immunostaining, media was aspirated from the cell monolayer and cells were fixed with ice-cold methanol. Plates were then washed three times in Dulbecco's phosphate buffered saline (PBS 1X, Gibco) before blocking for an hour with 3% w/v low-fat milk (Marvel). Detecting mouse monoclonal anti-hexon antibody (B025/AD51, Thermo-Fisher) was added at 1:1000 dilution in 1% w/v milk in PBS and incubated for an hour at 25° C. After incubation of the primary antibody, cells were washed a further three times with 1% w/v milk in PBS prior to addition of a secondary goat anti-mouse alkaline phosphatase (ALP) conjugated antibody (STAR117A, BioRad) at 1:1000 dilution in 1% w/v milk in PBS. After a further hour incubation, plates were washed five times in PBS prior to development. To develop, 100 μL of freshly prepared SIGMAfast BCIP/NBT solution (Sigma) was added to each well and plates incubated at 25° C. until the appearance of dark stained foci, representing single infected cells. P:I ratios for CsCl-purified vector preparations were calculated by dividing the estimated no. of viral particles per mL with the no. of infectious units (ifu) per mL.

[0167] Assessment of Coagulation Factor X-Mediated Vector Transduction of SKOV3 Cells

[0168] SKOV3 cells (human ovary adenocarcinoma) were obtained from Public Health England and cultured in McCoy's 5a media with 2 mM Glutamine and 15% v/v foetal bovine serum (complete McCoy's media). For the assay, GFP-expressing vectors were serially diluted (1:10 to 1:107) in serum free media. Human coagulation Factor X (FX) was added to diluted vectors at a final concentration of 8 μg/mL (control samples without addition of FX were included). Vector-FX mixtures were added to monolayers of SKOV3 cells (80-90% confluent) in 96-well plates, and incubated with cells for 2 h at 37° C. and 5% CO.sub.2. After 2 h, vector-FX mixtures were replaced with complete McCoy's media, and plates incubated at 37° C., 5% CO.sub.2 for a further 48 h. Infectivity was assessed after 48 h by enumeration of GFP-positive foci as described above.

[0169] Production of Protein and Peptide Ligands

[0170] SpyTag- and SnoopTagJr-fused peptide ligands were produced using solid-phase synthesis techniques by Insight Biotechnology at >95% purity. Peptides were quality control tested by HPLC and mass spectrometry.

[0171] DNA constructs for expression of polyhistidine-tagged recombinant DogCatcher-NANP fusion proteins were cloned into expression plasmid pET45(+) (EMD Millipore) for protein production in BL21(DE3) E. coli. (NEB). DNA sequences for DogCatcher and Plasmodium falciparum circumsporozoite protein (PfCSP) from the 3D7 strain of malaria were synthesised separately (GeneArt, Thermo Fisher), DNA fragments required for individual constructs amplified by PCR, and assembled in pET45(+) by restriction cloning. Recombinant proteins were purified using affinity Ni-NTA resin (Qiagen) according to a previously published protocol [SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation. Buldun C M, Jean J X, Bedford M R, Howarth M. J Am Chem Soc. 2018 Feb. 28; 140(8):3008-3018. doi: 10.1021/jacs.7b13237], dialysed into PBS, and stored at −80° C.

[0172] SpyCatcher (GenBank: AFD50637.1) and SpyTag-MBP (Addgene Plasmid #35050) were expressed in E. coli and purified by Ni-NTA exactly as described (Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Zakeri B, Fierer J O, Celik E, Chittock E C, Schwarz-Linek U, Moy V T, Howarth M. Proc Natl Acad Sci USA. 2012 Mar. 20; 109(12):E690-7. doi: 10.1073/pnas.1115485109).

[0173] Monovalent streptavidin (mSA) was expressed in E. coli, refolded from exclusion bodies and purified by ion-exchange chromatography exactly as described (Plug-and-play pairing via defined divalent streptavidins. Fairhead M, Krndija D, Lowe E D, Howarth M. J Mol Biol. 2014 Jan. 9; 426(1):199-214. doi: 10.1016/j.jmb.2013.09.016).

[0174] SnoopLigase (GenBank: AVD97783.1), SnoopTagJr-AffiHER2, and SUMO-DogTag (GenBank:MG867376) were expressed in E. coli and purified by Ni-NTA exactly as described (SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation. Buldun C M, Jean J X, Bedford M R, Howarth M. J Am Chem Soc. 2018 Feb. 28; 140(8):3008-3018. doi: 10.1021/jacs.7b13237).

[0175] DogCatcher (previously termed RrgACatcher in the patent “Methods and products for fusion protein synthesis” Howarth M, Veggiani G, Gayet R. 2015, United Kingdom Patent application WO2016193746A1) was expressed in E. coli and purified by Ni-NTA following standard protocols (Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Zakeri B, Fierer J O, Celik E, Chittock E C, Schwarz-Linek U, Moy V T, Howarth M. Proc Natl Acad Sci U S A. 2012 Mar. 20; 109(12):E690-7. doi: 10.1073/pnas.1115485109).

[0176] HCMV pentamer (with SpyTag-gH) is described in patent application PCT/GB2019/051245. Protein was expressed in suspension 293F cells by transient transfection using five separate plasmids (sequences provided). Protein was harvested, concentrated by tangential flow filtration, affinity purified by C-tag affinity resin (Thermo Fisher), and further purified by size exclusion chromatography on a Superdex 200 column (GE) using an AKTA chromatography system (GE).

[0177] DNA constructs for expression of SARS CoV2 Spike and RBD fusion proteins were cloned into mammalian protein expression plasmid pcDNA3.4. DNA sequences for SARS CoV2 Spike and RBD were synthesised separately (GeneArt, Thermo Fisher), and assembled in frame with SnoopTagJr, SpyCatcher, or SnoopCatcher by PCR amplification and restriction cloning. To facilitate secretion of RBD fusion constructs, a leader sequence was introduced at the N-terminus (for RBD-SnoopTagJr, RBD-SpyCatcher and RBD-SnoopCatcher the leader sequence from SARS CoV2 spike MFVFLVLLPLVSSQC was used, for SnoopCatcher-RBD the IgK leader sequence METDTLLLWVLLLWVPGSTGD was used). Spike-SnoopTagJr and RBD-SnoopTagJr proteins were expressed in suspension Expi293F cells, and SnoopCatcher-RBD, RBD-SnoopCatcher and RBD-SpyCatcher proteins were expressed in suspension ExpiCHO-S cells. Protein was harvested from culture supernatant, affinity purified using C-tag affinity resin (Thermo Fisher) using an AKTA chromatography system (GE), and dialysed into tris-buffered saline (TBS) pH 7.4.

[0178] Coupling Reactions

[0179] For in vitro assays, coupling reactions between SpyCatcher and SpyTag, DogCatcher and DogTag, SnoopCatcher and SnoopTagJr, and SnoopTagJr and DogTag (catalysed by SnoopLigase) partners were performed by co-incubation of spontaneously reacting components in a total volume of 20-25 μL, with individual components at concentrations described in the figure legends. Reactions between Ad5-HVR-DogTag and DogCatcher (including DogCatcher fusions), between Ad5-HVR-SpyTag or Ad5-pIX-SpyTag and SpyCatcher (including SpyCatcher fusions), between Ad5-pIX-SpyCatcher and SpyTag (including SpyTag fusions), between Ad5-pIX-SnoopCatcher and SnoopTagJr (including SnoopTagJr fusions), between Ad5-pIX-DogCatcher and DogTag (including DogTag fusions) and between Ad5-pIX-SnoopTagJr and SnoopCatcher (including SnoopCatcher fusions) were incubated for 16 h at 4° C. Reactions between Ad5-HVR-DogTag and SnoopTagJr (including SnoopTagJr fusions) catalysed by SnoopLigase were incubated for 48 h at 4° C. SnoopLigase catalysed reactions were performed in buffers containing a final concentration of 15% v/v glycerol and minimal salt to increase the efficiency of coupling.

[0180] Peptide-decorated vector batches for immunisation studies were prepared by co-incubating 5E+11 viral particles of Ad5(GFP)-HVR5-DogTag with 35 μM SnoopTagJr-GGSSIINFEKL, 30 μM SnoopLigase, and 15% v/v glycerol in a total volume of 400 μL for 48 hours at 4° C. To remove excess peptide and SnoopLigase (FIG. 10A, Group 1) coupled vectors were dialysed into sucrose storage buffer using SpectraPor dialysis cassettes (100 kDa MWCO). No excess peptide was detectable post-dialysis on a coomassie-stained SDS-PAGE gel. Peptide-decorated vectors were stored at −80° C., endotoxin tested, and infectious titration of stored batches was repeated prior to immunisation.

[0181] Assessment of Coupling Efficiency by SDS-PAGE

[0182] Coupling reactions were performed as described above and stopped by addition of SDS loading buffer (BioRad, 31.5 mM Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 0.005% Bromophenol Blue, 300 mM DTT). Samples were boiled at 95° C. for 5 min and loaded on SDS-PAGE (NuPAGE 4-12% Bis-Tris, Invitrogen) gels. For direct gel shift assays to assess coupling of ligands to Ad5-HVR-DogTag or Ad5-HVR-SpyTag, proteins were resolved by SDS-PAGE (200V, 40-55 min) and visualized by Coomassie staining [16 h staining with Quick Coomassie (Generon), destained with water]. Coupling efficiency was assessed by comparing band intensities of uncoupled hexon-DogTag/hexon-SpyTag with hexon-DogTag:DogCatcher/hexon-SpyTag:SpyCatcher using Image J (For hexon-SpyTag:SpyCatcher coupling efficiency, % hexon coupled=band intensity of hexon-SpyTag:SpyCatcher divided by the sum of band intensities of hexon-SpyTag:SpyCatcher and uncoupled hexon-SpyTag, multiplied by 100).

[0183] For assessment of coupling of Ad5-HVR-DogTag to SnoopTagJr fused peptides, a competition assay using DogCatcher (DC) protein was performed due to the fact that coupling of low molecular weight peptides could not be accurately assessed using the direct gel shift assay. After coupling, an excess of DogCatcher protein (30 μM) was added to the reaction and samples incubated at 4° C. for a further 24 hours. Since DC binds .sup.˜100% of free (uncoupled) hexon-DogTag molecules on the surface of Ad5 (see FIG. 3B) the proportion of hexon-DogTag coupled to SnoopTagJr-peptide can be estimated to be the proportion of hexon that does not undergo a gel shift of .sup.˜20 kDa (molecular weight of DogCatcher) on Coomassie-stained SDS-PAGE after incubation with DogCatcher.

[0184] An alternative assay to the DogCatcher competition assay, enabling assessment of SnoopTagJr-peptide coupling to hexon-DogTag by direct gel shift, exploited the high affinity biotin:streptavidin interaction (stable in room temperature SDS loading buffer). Biotinylated SnoopTagJr-peptide was incubated with SnoopLigase and Ad5-HVR-DogTag as described previously. After 48 hours, the coupling reaction was stopped by boiling in SDS loading buffer to denature all proteins and virion structures. Samples were briefly cooled on ice, before incubation with an excess of monovalent streptavidin (mSA, 2-fold excess over biotin peptide) for 30 min at 25° C. Samples were run on SDS-PAGE, transferred to nitrocellulose, and Western blotting performed using an anti-hexon primary mouse monoclonal antibody (clone 65H6, ThermoFisher). Migration of the hexon protein was visualised using a goat anti-mouse-alkaline phosphatase secondary (1:1000 dilution) followed by BCIP/NBT substrate for development (Sigma). A gel-shift (.sup.˜50 kDa) was observed for hexon-DogTag protein coupled to SnoopTagJr-biotin by virtue of the interaction between mSA and biotin.

[0185] For assessment of coupling of protein ligands to Ad5-pIX-SpyCatcher, Ad5-pIX-SnoopCatcher, Ad5-pIX-DogCatcher, Ad5-pIX-SpyTag or Ad5-pIX-SnoopTagJr, samples were run on SDS-PAGE and then transferred to nitrocellulose for Western blotting. Protein species covalently coupled to pIX-fusions were detected using primary antibodies or mouse antisera and alkaline-phosphatase conjugated secondary antibodies as described in figure legends. Western blots were developed as described above.

[0186] Antibody Neutralisation Assay

[0187] For assessment of vector neutralisation by potent neutralising mouse monoclonal antibody (mAb) 9C12 (Developmental Studies Hybridoma Bank, University of Iowa), Ad5(GFP) vectors were incubated with serially diluted mAb 9C12 antibody at a 1:1 ratio in complete media for 1 hour at 37° C. The vector-antibody mix was then added to an 80% confluent monolayer of HEK293A cells in a 96-well plate format (cells were infected at a multiplicity of infection of 200 ifu/cell). Cells were incubated with the vector-antibody mix for 2 hours at 37° C. 5% CO.sub.2, before the mix was replaced with fresh media and the plates returned to 37° C. 5% CO.sub.2 for a further 24 h. After 24 h, GFP expression within HEK293A cells was used as a readout of vector infectivity; bulk fluorescence was measured on a fluorimeter (Tecan) using an excitation wavelength of 395 nm and emission wavelength of 509 nm.

[0188] For assessment of vector neutralisation by Ad5-positive serum, serum samples were obtained by immunising mice with 1E+8 ifu of an Ad5 vector expressing ovalbumin (vector had an unmodified hexon). Serum was harvested two-weeks post immunization, stored at −80° C., and then serially diluted for the neutralisation assay (two-fold dilutions were prepared from 1:8 to 1:1024 in complete media, to give a final range of 1:16 to 1:2048 on cell monolayers). Diluted serum was incubated with Ad5(GFP) vectors, the mix incubated on HEK293 cells and bulk GFP fluorescence read 24 h later exactly as described above.

[0189] Mouse Immunisations

[0190] All mouse procedures were performed in accordance with the terms of the UK Animals (Scientific Procedures) Act Project Licence (PA7D20B85) and approved by the Oxford University Ethical Review Body. Female C57BL/6 mice (6 weeks of age, Charles River), housed in specific-pathogen free environments, were immunised intramuscularly by injection of 50 μL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 μL total). Adenoviral vectors were administered at a dose of 5E+9 viral particles, peptides administered at a dose of 5 μg, and poly I:C (InvivoGen) administered at a dose of 10 μg. Endotoxin dose was <1 EU per mouse. Experiments were performed at Biomedical Services, University of Oxford, and completed two-weeks post-immunisation.

[0191] Ex-Vivo IFN-Gamma ELISPOT

[0192] Spleen ex vivo interferon-gamma (IFN-γ) ELISpot was performed according to standard protocols as described previously [Larsen K C, Spencer A J, Goodman A L, Gilchrist A, Furze J, Rollier C S, Kiss-Toth E, Gilbert S C, Bregu M, Soilleux E J, Hill A V, Wyllie D H, Expression of tak1 and tram induces synergistic pro-inflammatory signalling and adjuvants DNA vaccines. Vaccine. 2009 Sep. 18; 27(41):5589-98]. To measure antigen specific responses, cells were re-stimulated for 18-20 hours with peptides at a final concentration of 5 μg/mL. To measure SIINFEKL-specific responses, SIINFEKL peptide (Cambridge Bioscience) was used. For GFP-specific responses, EGFP peptide DTLVNRIEL (EGFP.sub.118-126) (synthesised by Insight Biotechnology) was used. Spot forming cells (SFC) were measured using an automated ELISpot reader system (AID).

TABLE-US-00002 Modified adenovirus sequences: Ad5-HVR-SpyTag sequences Ad5-HVR1-SpyTag Hexon sequence Amino acid (SEQ ID NO: 1): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLA VGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAGSGGSGAHIVMVDAYKPTKGSGGSGTHVFGQAPYSG INITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQQNGKLESQVE MQFFSTTEATAGNGDNLTPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAG QASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQEN GWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLVDCYINLGARW SLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFD SICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPY YTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPE SYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQKKFLCDRTLWR IPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHRPHRGVIETVYLRTPFSAGNATT DNA (incl. STOP) (SEQ ID NO: 2): ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTG CAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACA GACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCT GTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTAC TCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGGCAGCGGAGGATCCGGCGCC CATATCGTGATGGTGGACGCCTACAAGCCTACCAAAGGCTCTGGCGGAAGCGGCACTCACGTATTTGGGCAGGCGCCTTATTCTGGT ATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAATATGCCGATAAAACATTTCAACCTGAACCTCAA ATAGGAGAATCTCAGTGGTACGAAACTGAAATTAATCATGCAGCTGGGAGAGTCCTTAAAAAGACTACCCCAATGAAACCATGTTAC GGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAAGTGGAA ATGCAATTTTTCTCAACTACTGAGGCGACCGCAGGCAATGGTGATAACTTGACTCCTAAAGTGGTATTGTACAGTGAAGATGTAGAT ATAGAAACCCCAGACACTCATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAATGGGCCAACAATCTATGCCC AACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAACAGCACGGGTAATATGGGTGTTCTGGCGGGC CAAGCATCGCAGTTGAATGCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTCCATTGGTGAT AGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGAT GAACTTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTAAAACCTAAAACAGGTCAGGAAAAT GGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAATAATTTTGCCATGGAAATCAATCTAAATGCC AACCTGTGGAGAAATTTCCTGTACTCCAACATAGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCT GATAACCCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGTTAGTGGACTGCTACATTAACCTTGGAGCACGCTGG TCCCTTGACTATATGGACAACGTCAACCCATTTAACCACCACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGT CGCTATGTGCCCTTCCACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCATACACCTACGAG TGGAACTTCAGGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGCATTAAGTTTGAT AGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCTTGAGGCCATGCTTAGAAACGACACCAACGAC CAGTCCTTTAACGACTATCTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATATCCATCCCCTCC CGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGGGCTACGACCCTTAT TACACCTACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAGGTGGCCATTACCTTTGACTCT TCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTT GCCCAGTGTAACATGACCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTACAACATTGGCTACCAGGGCTTCTATATCCCAGAG AGCTACAAGGACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACTAAATACAAGGACTAC CAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTTGGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCT GCTAACTTCCCCTATCCGCTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGCACCCTTTGGCGC ATCCCATTCTCCAGTAACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCAACTCCGCCCACGCGCTA GACATGACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCGG CCGCACCGCGGCGTCATCGAAACCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAA Ad5-HVR2-SpyTag Hexon sequence Amino acid (SEQ ID NO: 3): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLA VGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEEDDDNEDEVDEQAEQQKTHVFGQAPY SGINITKEGIQIGVGSGGSGAHIVMVDAYKPTKGSGGSGPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKP TNENGGQGILVKQQNGKLESQVEMQFFSTTEATAGNGDNLTPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRPNYI AFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYC FPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTY DYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKD VNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAF RGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMT KDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYP LIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHRPHRGVI ETVYLRTPFSAGNATT DNA (incl. STOP) (SEQ ID NO: 4): ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTG CAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACA GACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCT GTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTAC TCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGCTACTGCTCTTGAAATAAAC CTAGAAGAAGAGGACGATGACAACGAAGACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTAT TCTGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGGCAGCGGAGGATCCGGCGCCCATATCGTGATGGTGGACGCCTAC AAGCCTACCAAAGGCTCTGGCGGAAGCGGCCCTAAATATGCCGATAAAACATTTCAACCTGAACCTCAAATAGGAGAATCTCAGTGG TACGAAACTGAAATTAATCATGCAGCTGGGAGAGTCCTTAAAAAGACTACCCCAATGAAACCATGTTACGGTTCATATGCAAAACCC ACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAAGTGGAAATGCAATTTTTCTCAACT ACTGAGGCGACCGCAGGCAATGGTGATAACTTGACTCCTAAAGTGGTATTGTACAGTGAAGATGTAGATATAGAAACCCCAGACACT CATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAATGGGCCAACAATCTATGCCCAACAGGCCTAATTACATT GCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAACAGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAAT GCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGTACTTTTCT ATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAAATTACTGC TTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTAAAACCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGATGCT ACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAATAATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAGAAATTTC CTGTACTCCAACATAGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAACCCAAACACCTAC GACTACATGAACAAGCGAGTGGTGGCTCCCGGGTTAGTGGACTGCTACATTAACCTTGGAGCACGCTGGTCCCTTGACTATATGGAC AACGTCAACCCATTTAACCACCACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTCCAC ATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCAGGAAGGAT GTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGCATTAAGTTTGATAGCATTTGCCTTTACGCC ACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCTTGAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTAT CTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCGGCTTTC CGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGGGCTACGACCCTTATTACACCTACTCTGGCTCT ATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAGGTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGC AATGACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATGACC AAAGACTGGTTCCTGGTACAAATGCTAGCTAACTACAACATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAGGACCGCATG TACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACTAAATACAAGGACTACCAACAGGTGGGCATCCTA CACCAACACAACAACTCTGGATTTGTTGGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTATCCG CTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGCACCCTTTGGCGCATCCCATTCTCCAGTAAC TTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCAACTCCGCCCACGCGCTAGACATGACTTTTGAGGTG GATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCGGCCGCACCGCGGCGTCATC GAAACCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAA Ad5-HVR5-SpyTag Hexon sequence Amino acid (SEQ ID NO: 5): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLA VGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEEDDDNEDEVDEQAEQQKTHVFGQAPY SGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQQNGKLESQ VEMQFFSGSGGSGAHIVMVDAYKPTKGSGGSGPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRPNYIAFRDNFIGL MYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINT ETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVA PGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSL GNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLK TKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQML ANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDS ITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHRPHRGVIETVYLRTPF SAGNATT DNA (incl. STOP) (SEQ ID NO: 6): ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTG CAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACA GACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCT GTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTAC TCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGCTACTGCTCTTGAAATAAAC CTAGAAGAAGAGGACGATGACAACGAAGACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTAT TCTGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAATATGCCGATAAAACATTTCAACCTGAA CCTCAAATAGGAGAATCTCAGTGGTACGAAACTGAAATTAATCATGCAGCTGGGAGAGTCCTTAAAAAGACTACCCCAATGAAACCA TGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAA GTGGAAATGCAATTTTTCTCAGGCAGCGGAGGATCCGGCGCCCATATCGTGATGGTGGACGCCTACAAGCCTACCAAAGGCTCTGGC GGAAGCGGCCCTAAAGTGGTATTGTACAGTGAAGATGTAGATATAGAAACCCCAGACACTCATATTTCTTACATGCCCACTATTAAG GAAGGTAACTCACGAGAACTAATGGGCCAACAATCTATGCCCAACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTA ATGTATTACAACAGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAATGCTGTTGTAGATTTGCAAGACAGAAAC ACAGAGCTTTCATACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGCTAT GATCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACA GAGACTCTTACCAAGGTAAAACCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAATGAAATA AGAGTTGGAAATAATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAGAAATTTCCTGTACTCCAACATAGCGCTGTATTTG CCCGACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAACCCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCT CCCGGGTTAGTGGACTGCTACATTAACCTTGGAGCACGCTGGTCCCTTGACTATATGGACAACGTCAACCCATTTAACCACCACCGC AATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTCCACATCCAGGTGCCTCAGAAGTTCTTTGCC ATTAAAAACCTCCTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCAGGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTA GGAAATGACCTAAGGGTTGACGGAGCCAGCATTAAGTTTGATAGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACC GCCTCCACGCTTGAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTATCTCTCCGCCGCCAACATGCTCTACCCT ATACCCGCCAACGCTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTCACGCGCCTTAAG ACTAAGGAAACCCCATCACTGGGCTCGGGCTACGACCCTTATTACACCTACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTAC CTCAACCACACCTTTAAGAAGGTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCAACGAG TTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATGACCAAAGACTGGTTCCTGGTACAAATGCTA GCTAACTACAACATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAGGACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCC ATGAGCCGTCAGGTGGTGGATGATACTAAATACAAGGACTACCAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTT GGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTATCCGCTTATAGGCAAGACCGCAGTTGACAGC ATTACCCAGAAAAAGTTTCTTTGCGATCGCACCCTTTGGCGCATCCCATTCTCCAGTAACTTTATGTCCATGGGCGCACTCACAGAC CTGGGCCAAAACCTTCTCTACGCCAACTCCGCCCACGCGCTAGACATGACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTT TATGTTTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCGGCCGCACCGCGGCGTCATCGAAACCGTGTACCTGCGCACGCCCTTC TCGGCCGGCAACGCCACAACATAA Ad5-HVR-DogTag sequences Ad5-HVR1-DogTag Hexon sequence Amino acid (SEQ ID NO: 7): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLA VGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAGSGGSGDIPATYEFTDGKHYITNEPIPPKGSGGSGT HVFGQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQ QNGKLESQVEMQFFSTTEATAGNGDNLTPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRPNYIAFRDNFIGLMYYN STGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLT KVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLV DCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDL RVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKET PSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYN IGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQK KFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHRPHRGVIETVYLRTPFSAGN ATT DNA (incl. STOP) (SEQ ID NO: 8): ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTG CAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACA GACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCT GTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTAC TCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGGCAGCGGAGGATCCGGCgat attccggctacatatgaatttaccgatggtaaacattatatcaccaatgaaccgataccgccgaaaGGCTCTGGCGGAAGCGGCACT CACGTATTTGGGCAGGCGCCTTATTCTGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAATAT GCCGATAAAACATTTCAACCTGAACCTCAAATAGGAGAATCTCAGTGGTACGAAACTGAAATTAATCATGCAGCTGGGAGAGTCCTT AAAAAGACTACCCCAATGAAACCATGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAA CAAAATGGAAAGCTAGAAAGTCAAGTGGAAATGCAATTTTTCTCAACTACTGAGGCGACCGCAGGCAATGGTGATAACTTGACTCCT AAAGTGGTATTGTACAGTGAAGATGTAGATATAGAAACCCCAGACACTCATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCA CGAGAACTAATGGGCCAACAATCTATGCCCAACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAAC AGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAATGCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCA TACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTT AGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACC AAGGTAAAACCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAAT AATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAGAAATTTCCTGTACTCCAACATAGCGCTGTATTTGCCCGACAAGCTA AAGTACAGTCCTTCCAACGTAAAAATTTCTGATAACCCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGTTAGTG GACTGCTACATTAACCTTGGAGCACGCTGGTCCCTTGACTATATGGACAACGTCAACCCATTTAACCACCACCGCAATGCTGGCCTG CGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTCCACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTC CTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCAGGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTA AGGGTTGACGGAGCCAGCATTAAGTTTGATAGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCTT GAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTATCTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAAC GCTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACC CCATCACTGGGCTCGGGCTACGACCCTTATTACACCTACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACC TTTAAGAAGGTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAG CGCTCAGTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATGACCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTACAAC ATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAGGACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAG GTGGTGGATGATACTAAATACAAGGACTACCAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTTGGCTACCTTGCC CCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTATCCGCTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAA AAGTTTCTTTGCGATCGCACCCTTTGGCGCATCCCATTCTCCAGTAACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAAC CTTCTCTACGCCAACTCCGCCCACGCGCTAGACATGACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTT GAAGTCTTTGACGTGGTCCGTGTGCACCGGCCGCACCGCGGCGTCATCGAAACCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAAC GCCACAACATAA Ad5-HVR2-DogTag Hexon sequence Amino acid (SEQ ID NO: 9): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLA VGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEEDDDNEDEVDEQAEQQKTHVFGQAPY SGINITKEGIQIGVGSGGSGDIPATYEFTDGKHYITNEPIPPKGSGGSGPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPM KPCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSTTEATAGNGDNLTPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQ QSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENH GTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSN VKISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGS YTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPI SIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGE GYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREG QAYPANFPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVV RVHRPHRGVIETVYLRTPFSAGNATT DNA (incl. STOP) (SEQ ID NO: 10): ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTG CAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACA GACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCT GTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTAC TCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGCTACTGCTCTTGAAATAAAC CTAGAAGAAGAGGACGATGACAACGAAGACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTAT TCTGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGGCAGCGGAGGATCCGGCgatattccggctacatatgaatttacc gatggtaaacattatatcaccaatgaaccgataccgccgaaaGGCTCTGGCGGAAGCGGCCCTAAATATGCCGATAAAACATTTCAA CCTGAACCTCAAATAGGAGAATCTCAGTGGTACGAAACTGAAATTAATCATGCAGCTGGGAGAGTCCTTAAAAAGACTACCCCAATG AAACCATGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAA AGTCAAGTGGAAATGCAATTTTTCTCAACTACTGAGGCGACCGCAGGCAATGGTGATAACTTGACTCCTAAAGTGGTATTGTACAGT GAAGATGTAGATATAGAAACCCCAGACACTCATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAATGGGCCAA CAATCTATGCCCAACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAACAGCACGGGTAATATGGGT GTTCTGGCGGGCCAAGCATCGCAGTTGAATGCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGAT TCCATTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAATCAT GGAACTGAAGATGAACTTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTAAAACCTAAAACA GGTCAGGAAAATGGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAATAATTTTGCCATGGAAATC AATCTAAATGCCAACCTGTGGAGAAATTTCCTGTACTCCAACATAGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCAAC GTAAAAATTTCTGATAACCCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGTTAGTGGACTGCTACATTAACCTT GGAGCACGCTGGTCCCTTGACTATATGGACAACGTCAACCCATTTAACCACCACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTG CTGGGCAATGGTCGCTATGTGCCCTTCCACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCA TACACCTACGAGTGGAACTTCAGGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGC ATTAAGTTTGATAGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCTTGAGGCCATGCTTAGAAAC GACACCAACGACCAGTCCTTTAACGACTATCTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATA TCCATCCCCTCCCGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGGGC TACGACCCTTATTACACCTACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAGGTGGCCATT ACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAG GGTTACAACGTTGCCCAGTGTAACATGACCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTACAACATTGGCTACCAGGGCTTC TATATCCCAGAGAGCTACAAGGACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACTAAA TACAAGGACTACCAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTTGGCTACCTTGCCCCCACCATGCGCGAAGGA CAGGCCTACCCTGCTAACTTCCCCTATCCGCTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGC ACCCTTTGGCGCATCCCATTCTCCAGTAACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCAACTCC GCCCACGCGCTAGACATGACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGTCTTTGACGTGGTC CGTGTGCACCGGCCGCACCGCGGCGTCATCGAAACCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAA Ad5-HVR5-DogTag Hexon sequence Amino acid (SEQ ID NO: 11): MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLA VGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEEDDDNEDEVDEQAEQQKTHVFGQAPY SGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQQNGKLESQ VEMQFFSGSGGSGDIPATYEFTDGKHYITNEPIPPKGSGGSGPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRPNY IAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNY CFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEINLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNT YDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRK DVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAA FRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNM TKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPY PLIGKTAVDSITQKKFLCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHRPHRGV IETVYLRTPFSAGNATT DNA (incl. STOP) (SEQ ID NO: 12): ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTG CAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACA GACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCT GTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTAC TCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGCTACTGCTCTTGAAATAAAC CTAGAAGAAGAGGACGATGACAACGAAGACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTAT TCTGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAATATGCCGATAAAACATTTCAACCTGAA CCTCAAATAGGAGAATCTCAGTGGTACGAAACTGAAATTAATCATGCAGCTGGGAGAGTCCTTAAAAAGACTACCCCAATGAAACCA TGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAA GTGGAAATGCAATTTTTCTCAGGCAGCGGAGGATCCGGCgatattccggctacatatgaatttaccgatggtaaacattatatcacc aatgaaccgataccgccgaaaGGCTCTGGCGGAAGCGGCCCTAAAGTGGTATTGTACAGTGAAGATGTAGATATAGAAACCCCAGAC ACTCATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAATGGGCCAACAATCTATGCCCAACAGGCCTAATTAC ATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAACAGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTG AATGCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGTACTTT TCTATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAAATTAC TGCTTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTAAAACCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGAT GCTACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAATAATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAGAAAT TTCCTGTACTCCAACATAGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAACCCAAACACC TACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGTTAGTGGACTGCTACATTAACCTTGGAGCACGCTGGTCCCTTGACTATATG GACAACGTCAACCCATTTAACCACCACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTC CACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCAGGAAG GATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGCATTAAGTTTGATAGCATTTGCCTTTAC GCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCTTGAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGAC TATCTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCGGCT TTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGGGCTACGACCCTTATTACACCTACTCTGGC TCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAGGTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCT GGCAATGACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATG ACCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTACAACATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAGGACCGC ATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACTAAATACAAGGACTACCAACAGGTGGGCATC CTACACCAACACAACAACTCTGGATTTGTTGGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTAT CCGCTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGCACCCTTTGGCGCATCCCATTCTCCAGT AACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCAACTCCGCCCACGCGCTAGACATGACTTTTGAG GTGGATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCGGCCGCACCGCGGCGTC ATCGAAACCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAA Ad5-pIX-SpyCatcher sequences pIX-(EAAAK3)-SpyCatcher sequence: Amino acid (SEQ ID NO: 13): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVGGSEAAAKEAAAKEAAAKGSDSATHIKFSKRDED GKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI DNA (incl. STOP) (SEQ ID NO: 14): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGGAggctccGAAGCC GCCGCTAAAGAAGCTGCTGCCAAAGAGGCCGCTGCAAAGggatccGACAGCGCCACACACATCAAGTTCAGCAAGAGAGATGAGGAC GGCAAAGAGCTGGCTGGCGCAACAATGGAACTGAGAGATAGCAGCGGCAAGACCATCAGCACCTGGATCTCCGACGGCCAAGTGAAG GACTTCTATCTGTACCCCGGCAAGTACACCTTCGTGGAAACCGCCGCTCCTGACGGATATGAAGTGGCCACCGCCATCACCTTCACC GTGAATGAGCAGGGACAAGTGACCGTGAACGGCAAGGCCACAAAAGGCGACGCCCACATTTAA Ligands for capsid decoration described in this patent: Recombinant Proteins: DogCatcher Amino acid (SEQ ID NO: 15): MGSSHHHHHHSSGLVPRGSHMKLGDIEFIKVNKNDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKY RLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQ DNA (incl. STOP) (SEQ ID NO: 16): ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAAACTGGGCGATATTGAATTTATT AAAGTGAACAAAAACGATAAAAAGCCGCTGCGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGATATCTATGGC GCGATTGATCAGAATGGGACCTATCAAAATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTAAGAATCTGAGCGATGGCAAATAT CGCCTGTTTGAAAATAGCGAACCCGCTGGCTATAAACCGGTGCAGAATAAGCCGATTGTGGCGTTTCAGATTGTGAATGGCGAAGTG CGTGATGTGACCAGCATTGTGCCGCAGTAA DogCatcher-NANP9 Amino acid (SEQ ID NO: 17): MAHHHHHHVGTGKLGDIEFIKVNKNDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPA GYKPVQNKPIVAFQIVNGEVRDVTSIVPQGSGGSGGSNANPNANPNANPNANPNANPNANPNANPNANPNANP DNA (incl. STOP) (SEQ ID NO: 18): ATGGCACATCACCACCACCATCACGTGGGTACCGGTAAACTGGGCGATATTGAATTTATTAAAGTGAACAAAAACGATAAAAAGCCG CTGCGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGATATCTATGGCGCGATTGATCAGAATGGGACCTATCAA AATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTAAGAATCTGAGCGATGGCAAATATCGCCTGTTTGAAAATAGCGAACCCGCT GGCTATAAACCGGTGCAGAATAAGCCGATTGTGGCGTTTCAGATTGTGAATGGCGAAGTGCGTGATGTGACCAGCATTGTGCCGCAG GGCTCTGGCGGAAGCGGCggatccAATGCGAACCCTAATGCGAATCCCAATGCAAATCCCAATGCGAACCCTAACGCAAATCCGAAC GCAAACCCTAACGCGAACCCTAATGCTAATCCTAACGCCAATCCTtaa DogCatcher-NANP18 Amino acid (SEQ ID NO: 19): MAHHHHHHVGTGKLGDIEFIKVNKNDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPA GYKPVQNKPIVAFQIVNGEVRDVTSIVPQGSGGSGGSNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNA NPNANPNANPNANPNANPNANP DNA (incl. STOP) (SEQ ID NO: 20): ATGGCACATCACCACCACCATCACGTGGGTACCGGTAAACTGGGCGATATTGAATTTATTAAAGTGAACAAAAACGATAAAAAGCCG CTGCGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGATATCTATGGCGCGATTGATCAGAATGGGACCTATCAA AATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTAAGAATCTGAGCGATGGCAAATATCGCCTGTTTGAAAATAGCGAACCCGCT GGCTATAAACCGGTGCAGAATAAGCCGATTGTGGCGTTTCAGATTGTGAATGGCGAAGTGCGTGATGTGACCAGCATTGTGCCGCAG GGCTCTGGCGGAAGCGGCggatccAATGCTAACCCTAACGCTAACCCCAACGCCAATCCGAATGCGAATCCTAACGCCAATCCAAAT GCCAATCCGAACGCGAACCCAAACGCTAATCCAAACGCGAATCCAAATGCGAACCCTAATGCGAATCCCAATGCAAATCCCAATGCG AACCCTAACGCAAATCCGAACGCAAACCCTAACGCGAACCCTAATGCTAATCCTAACGCCAATCCTtaa DogCatcher-NANP Domain Amino acid (SEQ ID NO: 21): MAHHHHHHVGTGKLGDIEFIKVNKNDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPA GYKPVQNKPIVAFQIVNGEVRDVTSIVPQGSGGSGGSNANPNVDPNANPNVDPNANPNVDPNANPNANPNANPNANPNANPNANPNA NPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPN ANPNANPNANPNANPNANPNANPNANPNANP DNA (incl. STOP) (SEQ ID NO: 22): ATGGCACATCACCACCACCATCACGTGGGTACCGGTAAACTGGGCGATATTGAATTTATTAAAGTGAACAAAAACGATAAAAAGCCG CTGCGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGATATCTATGGCGCGATTGATCAGAATGGGACCTATCAA AATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTAAGAATCTGAGCGATGGCAAATATCGCCTGTTTGAAAATAGCGAACCCGCT GGCTATAAACCGGTGCAGAATAAGCCGATTGTGGCGTTTCAGATTGTGAATGGCGAAGTGCGTGATGTGACCAGCATTGTGCCGCAG GGCTCTGGCGGAAGCGGCGGATCCAATGCAAATCCGAATGTTGATCCGAACGCGAACCCGAACGTGGACCCTAACGCCAATCCTAAT GTGGACCCAAATGCGAATCCAAATGCTAACCCAAACGCAAACCCGAATGCGAACCCCAATGCCAATCCGAACGCTAATCCCAATGCT AATCCTAATGCAAATCCAAACGCGAATCCGAACGCCAATCCTAACGCAAACCCGAACGCAAATCCAAATGCAAACCCAAATGCTAAT CCTAATGCGAACCCGAATGCTAACCCGAATGCAAACCCTAACGTTGACCCTAATGCTAACCCTAACGCTAACCCCAACGCCAATCCG AATGCGAATCCTAACGCCAATCCAAATGCCAATCCGAACGCGAACCCAAACGCTAATCCAAACGCGAATCCAAATGCGAACCCTAAT GCGAATCCCAATGCAAATCCCAATGCGAACCCTAACGCAAATCCGAACGCAAACCCTAACGCGAACCCTAATGCTAATCCTAACGCC AATCCTTAA SpyTag-MBP Amino acid (SEQ ID NO: 23): MGSSHHHHHHSSGLVPRGSHMGAHIVMVDAYKPTKGSGESGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIP ALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKG ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSY EEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSS DNA (incl. STOP) (SEQ ID NO: 24): ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGGAGCCCACATCGTGATGGTGGAC GCCTACAAGCCGACGAAGggtagtggtgaaagtggtAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTAT AACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAA TTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTG TTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATT GCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCG GCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCT GCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGT CTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGC GAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACC TTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAG TTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTAC GAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATG TCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAG ACTAATTCGAGCTCGTAA SnoopLigase Amino acid (SEQ ID NO: 25): MGSWSHHHHHHSSGGSGVNKNDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPPGYKP VQNKPIVAFQIVNGEVRDVTSIVPPGVPATYEFT DNA (incl. STOP) (SEQ ID NO: 26): atgggcagctggagccatcatcatcatcatcacagctctggtggtagtggtgtgaataagaacgataaaaagccgctgcgtggtgcc gtgtttagcctgcagaaacagcatcccgactatcccgatatctatggcgcgattgatcagaatgggacctatcaaaatgtgcgtacc ggcgaagatggtaaactgacctttaagaatctgagcgatggcaaatatcgcctgtttgaaaatagcgaacccccgggctataaaccg gtgcagaataagccgattgtggcgtttcagattgtgaatggcgaagtgcgtgatgtgaccagcattgtgccgccgggtgtgccggct acatatgaatttacctaa SnoopTagJr-AffiHER2 Amino acid (SEQ ID NO: 27): MGSSHHHHHHSSGGKLGSIEFIKVNKGSGESGSGASMTGGQQMGRDPGVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPS QSANLLAEAKKLNDAQAPKGLE DNA (incl. STOP) (SEQ ID NO: 28): atgggcagcagccatcatcatcatcatcacagcagcggcgggaaactgggctctattgaatttattaaagtgaacaaaggcagtggt gagtcgggatccggagctagcatgactggtggacagcaaatgggtcgggatccgggcgtggacaacaaattcaacaaagaaatgagg aacgcttactgggagatagctcttttacccaacttaaacaatcaacagaaaagggctttcataaggtcgttatacgatgacccaagc caaagcgctaaccttttagcagaagctaaaaagctaaatgatgctcaggcgccgaaaggcctcgagtaa SpyCatcher Amino acid (SEQ ID NO: 29): MSYYHHHHHHDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDG QVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI DNA (incl. STOP) (SEQ ID NO: 30): ATGTCGTACTACCATCACCATCACCATCACGATTACGACATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGTTGATACC TTATCAGGTTTATCAAGTGAGCAAGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGT GATGAGGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGA CAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATT ACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCTCATATTTAA HCMV Pentamer HCMV gH-SpyTag Amino acid (SEQ ID NO: 31): MRPGLPSYLIILAVCLFSHLLSSRYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQCTYNSSLRNSTVVRENAISFNFFQSYNQY YVFHMPRCLFAGPLAEQFLNQVDLTETLERYQQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTP HGWTESHTTSGLHRPHFNQTCILFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVVTVSIDDDTPMLLIFGHLPRVLFKAP YQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALAL FAAARQEEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQITDITSLVRLVYILSKQNQQHLI PQWALRQIADFALKLHKTHLASFLSAFARQELYLMGSLVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPC SSSGRRDHSLERLTRLFPDATVPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYIVTNQYLIKGISYPVSTTVV GQSLIITQTDSQTKCELTRNMHTTHSITVALNISLENCAFCQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLM LLKNGTVLEVTDVVVDATDSRLLGSGGSGAHIVMVDAYKPTKHHHHHH DNA (incl. STOP) (SEQ ID NO: 32): ATGCGGCCAGGCCTCCCCTCCTACCTCATCATCCTCGCCGTCTGTCTCTTCAGCCACCTACTTTCGTCACGATATGGCGCAGAAGCC GTATCCGAACCGCTGGACAAAGCGTTTCACCTACTGCTCAACACCTACGGGAGACCCATCCGCTTCCTGCGTGAAAATACCACCCAG TGTACCTACAACAGCAGCCTCCGTAACAGCACGGTCGTCAGGGAAAACGCCATCAGTTTCAACTTTTTCCAAAGCTATAATCAATAC TATGTATTCCATATGCCTCGATGTCTTTTTGCGGGTCCTCTGGCGGAGCAGTTTCTGAACCAGGTAGATCTGACCGAAACCCTGGAA AGATACCAACAGAGACTTAACACTTACGCGCTGGTATCCAAAGACCTGGCCAGCTACCGATCTTTTTCGCAGCAGCTAAAGGCACAA GACAGCCTAGGTGAACAGCCCACCACTGTGCCACCGCCCATTGACCTGTCAATACCTCACGTTTGGATGCCACCGCAAACCACTCCA CACGGCTGGACAGAATCACATACCACCTCAGGACTACACCGACCACACTTTAACCAGACCTGTATCCTCTTTGATGGACACGATCTA CTATTCAGCACCGTCACACCTTGTTTGCACCAAGGCTTTTACCTCATCGACGAACTACGTTACGTTAAAATAACACTGACCGAGGAC TTCTTCGTAGTTACGGTGTCCATAGACGACGACACACCCATGCTGCTTATCTTCGGCCATCTTCCACGCGTACTTTTCAAAGCGCCC TATCAACGCGACAACTTTATACTACGACAAACTGAAAAACACGAGCTCCTGGTGCTAGTTAAGAAAGATCAACTGAACCGTCACTCT TATCTCAAAGACCCGGACTTTCTTGACGCCGCACTTGACTTCAACTACCTAGACCTCAGCGCACTACTACGTAACAGCTTTCACCGT TACGCCGTGGATGTACTCAAGAGCGGTCGATGTCAGATGCTGGACCGCCGCACGGTAGAAATGGCCTTCGCCTACGCATTAGCACTG TTCGCAGCAGCCCGACAAGAAGAGGCCGGCGCCCAAGTCTCCGTCCCACGGGCCCTAGACCGCCAGGCCGCACTCTTACAAATACAA GAATTTATGATCACATGCCTCTCACAAACACCACCACGCACCACGTTGCTGCTGTATCCCACGGCCGTGGACCTGGCCAAACGAGCC CTTTGGACACCGAATCAGATCACCGACATCACCAGCCTCGTACGCCTGGTCTACATACTCTCTAAACAGAATCAGCAACATCTCATC CCCCAATGGGCACTACGACAGATCGCCGACTTTGCCCTAAAACTACACAAAACGCACCTGGCCTCTTTTCTTTCAGCCTTCGCACGC CAAGAACTCTACCTCATGGGCAGCCTCGTCCACTCCATGCTGGTACATACGACGGAGAGACGCGAAATCTTCATCGTAGAAACGGGC CTCTGTTCATTGGCCGAGCTATCACACTTTACGCAGTTGTTAGCTCATCCACACCACGAATACCTCAGCGACCTGTACACACCCTGT TCCAGTAGCGGGCGACGCGATCACTCGCTCGAACGCCTCACGCGTCTCTTCCCCGATGCCACCGTCCCCGCTACCGTTCCCGCCGCC CTCTCCATCCTATCTACCATGCAACCAAGCACGCTGGAAACCTTCCCCGACCTGTTTTGCTTGCCGCTCGGCGAATCCTTCTCCGCG CTGACCGTCTCCGAACACGTCAGTTATATCGTAACAAACCAGTACCTGATCAAAGGTATCTCCTACCCTGTCTCCACCACCGTCGTA GGCCAGAGCCTCATCATCACCCAGACGGACAGTCAAACTAAATGCGAACTGACGCGCAACATGCATACCACACACAGCATCACAGTG GCGCTCAACATTTCGCTAGAAAACTGCGCCTTTTGCCAAAGCGCCCTGCTAGAATACGACGACACGCAAGGCGTCATCAACATCATG TACATGCACGACTCGGACGACGTCCTTTTCGCCCTGGATCCCTACAACGAAGTGGTGGTCTCATCTCCGCGAACTCACTACCTCATG CTTTTGAAAAACGGTACGGTACTAGAAGTAACTGACGTCGTCGTGGACGCCACCGACAGTCGTCTCCTCGGAAGCGGAGGCTCTGGT GCCCATATCGTGATGGTGgacgCCTACAAGCCTACCAAACATCATCACCATCACCACTAA HCMV gL Amino acid (SEQ ID NO: 33): MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEVFEGDKYESWLRPLVNVTGRDGPLSQLIR YRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIF TEHVLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTR VNLPAHSRYGPQAVDAR DNA (incl. STOP) (SEQ ID NO: 34): ATGTGCCGCCGCCCGGATTGCGGCTTCTCTTTCTCACCTGGACCGGTGATACTGCTGTGGTGTTGCCTTCTGCTGCCCATTGTTTCC TCAGCCGCCGTCAGCGTCGCTCCTACCGCCGCCGAGAAAGTCCCCGCGGAGTGCCCCGAACTAACGCGCCGATGCTTGTTGGGTGAG GTGTTTGAGGGTGACAAGTATGAAAGTTGGCTGCGCCCGTTGGTGAATGTTACCGGGCGCGATGGCCCGCTATCGCAACTTATCCGT TACCGTCCCGTTACGCCGGAGGCCGCCAACTCCGTGCTGTTGGACGAGGCTTTCCTGGACACTCTGGCCCTGCTGTACAACAATCCG GATCAATTGCGGGCCCTGCTGACGCTGTTGAGCTCGGACACAGCGCCGCGCTGGATGACGGTGATGCGCGGCTACAGCGAGTGCGGC GATGGCTCGCCGGCCGTGTACACGTGCGTGGACGACCTGTGCCGCGGCTACGACCTCACGCGACTGTCATACGGGCGCAGCATCTTC ACGGAACACGTGTTAGGCTTCGAGCTGGTGCCACCGTCTCTCTTTAACGTGGTGGTGGCCATACGCAACGAAGCCACGCGTACCAAC CGCGCCGTGCGTCTGCCCGTGAGCACCGCTGCCGCGCCCGAGGGCATCACGCTCTTTTACGGCCTGTACAACGCAGTGAAGGAATTC TGCCTGCGTCACCAGCTGGACCCGCCGCTGCTACGCCACCTAGATAAATACTACGCCGGACTGCCGCCCGAGCTGAAGCAGACGCGC GTCAACCTGCCGGCTCACTCGCGCTATGGCCCTCAAGCAGTGGATGCTCGCTAA HCMV UL131A Amino acid (SEQ ID NO: 35): MRLCRVWLSVCLCAVVLGQCQRETAEKNDYYRVPHYWDACSRALPDQTRYKYVEQLVDLTLNYHYDASHGLDNFDVLKRINVTEVSL LISDFRRQNRRGGTNKRTTFNAAGSLAPHARSLEFSVRLFAN DNA (incl. STOP)-contains intron (bold) (SEQ ID NO: 36): ATGCGGCTGTGTCGGGTGTGGCTGTCTGTTTGTCTGTGCGCCGTGGTGCTGGGTCAGTGCCAGCGGGAAACCGCGGAAAAAAACGAT TATTACCGAGTACCGCATTACTGGGACGCGTGCTCTCGCGCGCTGCCCGACCAAACCCGTTACAAGTATGTGGAACAGCTCGTGGAC CTCACGTTGAACTACCACTACGATGCGAGCCACGGCTTGGACAACTTTGACGTGCTCAAGAGGTGAGGGTACGCGCTAAAGATGCAT GACAACGGGAAGGTAAGGGCGAACGGGTAACGGGTAAGTAACCGCATGGGGTATGAAATGACGTTCGGAACCTGTGCTTGCAGAATC AACGTGACCGAGGTGTCGTTGCTCATCAGCGACTTTAGACGTCAGAACCGTCGCGGCGGCACCAACAAAAGGACCACGTTCAACGCC GCCGGTTCGCTGGCGCCACACGCCCGGAGCCTCGAGTTCAGCGTGCGGCTCTTTGCCAACTAG HCMV UL128 Amino acid (SEQ ID NO: 37): MSPKDLTPFLTALWLLLGHSRVPRVRAEECCEFINVNHPPERCYDFKMCNRFTVALRCPDGEVCYSPEKTAEIRGIVTTMTHSLTRQ VVHNKLTSCNYNPLYLEADGRIRCGKVNDKAQYLLGAAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAKMGYMLQ DNA (incl. STOP)-contains introns (bold) (SEQ ID NO: 38): ATGAGTCCCAAAGATCTGACGCCGTTCTTGACGGCGTTGTGGCTGCTATTGGGTCACAGCCGCGTGCCGCGGGTGCGCGCAGAAGAA TGTTGCGAATTCATAAACGTCAACCACCCGCCGGAACGCTGTTACGATTTCAAAATGTGCAATCGCTTCACCGTCGCGTACGTATTT TCATGATTGTCTGCGTTCTGTGGTGCGTCTGGATCTGTCTCTCGACGTTTCTGATAGCCATGTTCCATCGACGATCCTCGGGAATGC CAGAGTAGATTTTCATGAATCCACAGGCTGCGGTGTCCGGACGGCGAAGTCTGCTACAGTCCCGAGAAAACGGCTGAGATTCGCGGG ATCGTCACCACCATGACCCATTCATTGACACGCCAGGTCGTACACAACAAACTGACGAGCTGCAACTACAATCCGTAAGTCTCTTCC TGAGGGCCTTACAGCCTATGGGAGAGTAAGACAGAGAGGGACAAAACATCATTAAAAAAAAAAGTCTAATTTCACGTTTTGTACCCC CCTTCCCCTCCGTGTTGTAGGTTATACCTCGAAGCTGACGGGCGAATACGCTGCGGCAAAGTAAACGACAAGGCGCAGTACCTGCTG GGCGCCGCTGGCAGCGTTCCCTATcGATGGATCAATCTGGAATACGACAAGATAACCCGGATCGTGGGCCTGGATCAGTACCTGGAG AGCGTTAAGAAACACAAACGGCTGGATGTGTGCCGCGCTAAAATGGGCTATATGCTGCAGTGA HCMV UL130 Amino acid (SEQ ID NO: 39): MLRLLLRHHFHCLLLCAVWATPCLASPWSTLTANQNPSPPWSKLTYSKPHDAATFYCPFLYPSPPRSPLQFSGFQRVSTGPECRNET LYLLYNREGQTLVERSSTWVKKVIWYLSGRNQTILQRMPRTASKPSDGNVQISVEDAKIFGAHMVPKQTKLLRFVVNDGTRYQMCVM KLESWAHVFRDYSVSFQVRLTFTEANNQTYTFCTHPNLIVGGGGSGGGGSGGGGSEPEA DNA (incl. STOP) (SEQ ID NO: 40): ATGCTGCGGCTTCTGCTTCGTCACCACTTTCACTGCCTGCTTCTGTGCGCGGTTTGGGCAACGCCCTGTCTGGCGTCTCCGTGGTCG ACGCTAACAGCAAACCAGAATCCGTCCCCGCCATGGTCTAAACTGACGTATTCCAAACCGCATGACGCGGCGACGTTTTACTGTCCT TTTCTCTATCCCTCGCCCCCACGATCCCCCTTGCAATTCTCGGGGTTCCAGCGGGTATCAACGGGTCCCGAGTGTCGCAACGAGACC CTGTATCTGCTGTACAACCGGGAAGGCCAGACCTTGGTGGAGAGAAGCTCCACCTGGGTGAAAAAGGTGATCTGGTACCTGAGCGGT CGGAACCAAACCATCCTCCAACGGATGCCCCGAACGGCTTCGAAACCGAGCGACGGAAACGTGCAGATCAGCGTGGAAGACGCCAAG ATTTTTGGAGCGCACATGGTGCCCAAGCAGACCAAGCTGCTACGCTTCGTCGTCAACGATGGCACACGTTATCAGATGTGTGTGATG AAGCTGGAGAGCTGGGCTCACGTCTTCCGGGACTACAGCGTGTCTTTTCAGGTGCGATTGACGTTCACCGAGGCCAATAACCAGACT TACACCTTCTGCACCCATCCCAATCTCATCGTTGGAGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCTGAGCCCGAG GCCTAA Peptides (solid phase synthesized-amino acid sequences only): SnoopTagJr-hTERT (SEQ ID NO: 41): GKLGSIEFIKVNKGEARPALLTSRLRFIPK SnoopTagJr-GGS-SHNFEKL (PEP1) (SEQ ID NO: 42): GKLGSIEFIKVNKGGGSSIINFEKL SnoopTagJr-AAY-SHNFEKL (PEP2) (SEQ ID NO: 43): GKLGSIEFIKVNKGAAYSIINFEKL Biotin-SnoopTagJr (SEQ ID NO: 44): Biotin-GKLGSIEFIKVNK (N-terminal biotin) Biotin-SpyTag003 (SEQ ID NO: 45): Biotin-GSRGVPHIVMVDAYKRYK (N-terminal biotin) Modified adenovirus sequences: Ad5-pIX-SpyCatcher sequences Ad5-pIX-SpyCatcher (no linker) pIX sequence Amino acid (SEQ ID NO: 46): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI DNA (incl. STOP) (SEQ ID NO: 47): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGACAGCGCCACACAC ATCAAGTTCAGCAAGAGAGATGAGGACGGCAAAGAGCTGGCTGGCGCAACAATGGAACTGAGAGATAGCAGCGGCAAGACCATCAGC ACCTGGATCTCCGACGGCCAAGTGAAGGACTTCTATCTGTACCCCGGCAAGTACACCTTCGTGGAAACCGCCGCTCCTGACGGATAT GAAGTGGCCACCGCCATCACCTTCACCGTGAATGAGCAGGGACAAGTGACCGTGAACGGCAAGGCCACAAAAGGCGACGCCCACATT TAA Ad5-pIX-SpyCatcher (EAAAK3-GS linker) pIX sequence Amino acid (SEQ ID NO: 48): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVEAAAKEAAAKEAAAKGSDSATHIKFSKRDEDGKE LAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI DNA (incl. STOP) (SEQ ID NO: 49): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGAAGCCGCCGCTAAA GAAGCTGCTGCCAAAGAGGCCGCTGCAAAGggatCCGACAGCGCCACACACATCAAGTTCAGCAAGAGAGATGAGGACGGCAAAGAG CTGGCTGGCGCAACAATGGAACTGAGAGATAGCAGCGGCAAGACCATCAGCACCTGGATCTCCGACGGCCAAGTGAAGGACTTCTAT CTGTACCCCGGCAAGTACACCTTCGTGGAAACCGCCGCTCCTGACGGATATGAAGTGGCCACCGCCATCACCTTCACCGTGAATGAG CAGGGACAAGTGACCGTGAACGGCAAGGCCACAAAAGGCGACGCCCACATTTAA Ad5-pIX-SpyCatcher (GGS-EAAAK3 linker) pIX sequence Amino acid (SEQ ID NO: 50): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVGGSEAAAKEAAAKEAAAKDSATHIKFSKRDEDGK ELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI DNA (incl. STOP) (SEQ ID NO: 51): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGGAggCtCCGAAGCC GCCGCTAAAGAAGCTGCTGCCAAAGAGGCCGCTGCAAAGGACAGCGCCACACACATCAAGTTCAGCAAGAGAGATGAGGACGGCAAA GAGCTGGCTGGCGCAACAATGGAACTGAGAGATAGCAGCGGCAAGACCATCAGCACCTGGATCTCCGACGGCCAAGTGAAGGACTTC TATCTGTACCCCGGCAAGTACACCTTCGTGGAAACCGCCGCTCCTGACGGATATGAAGTGGCCACCGCCATCACCTTCACCGTGAAT GAGCAGGGACAAGTGACCGTGAACGGCAAGGCCACAAAAGGCGACGCCCACATTTAA Ad5-pIX-SpyCatcher (EAAAK3 linker, no GGS or GS hinges) pIX sequence Amino acid (SEQ ID NO: 52): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVEAAAKEAAAKEAAAKDSATHIKFSKRDEDGKELA GATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI DNA (incl. STOP) (SEQ ID NO: 53): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGAAGCCGCCGCTAAA GAAGCTGCTGCCAAAGAGGCCGCTGCAAAGGACAGCGCCACACACATCAAGTTCAGCAAGAGAGATGAGGACGGCAAAGAGCTGGCT GGCGCAACAATGGAACTGAGAGATAGCAGCGGCAAGACCATCAGCACCTGGATCTCCGACGGCCAAGTGAAGGACTTCTATCTGTAC CCCGGCAAGTACACCTTCGTGGAAACCGCCGCTCCTGACGGATATGAAGTGGCCACCGCCATCACCTTCACCGTGAATGAGCAGGGA CAAGTGACCGTGAACGGCAAGGCCACAAAAGGCGACGCCCACATTTAA Ad5-pIX-SpyCatcher (GGS-EAAAK5-GS linker) pIX sequence Amino acid (SEQ ID NO: 54): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVGGSEAAAKEAAAKEAAAKEAAAKEAAAKGSDSAT HIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAH I DNA (incl. STOP) (SEQ ID NO: 55): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGGAGGCTCCGAAGCC GCCGCTAAAGAAGCTGCTGCCAAAGAGGCCGCTGCAAAGGAGGCCGCAGCTAAGGAAGCCGCCGCTAAGGGATCCGACAGCGCCACA CACATCAAGTTCAGCAAGAGAGATGAGGACGGCAAAGAGCTGGCTGGCGCAACAATGGAACTGAGAGATAGCAGCGGCAAGACCATC AGCACCTGGATCTCCGACGGCCAAGTGAAGGACTTCTATCTGTACCCCGGCAAGTACACCTTCGTGGAAACCGCCGCTCCTGACGGA TATGAAGTGGCCACCGCCATCACCTTCACCGTGAATGAGCAGGGACAAGTGACCGTGAACGGCAAGGCCACAAAAGGCGACGCCCAC ATTTAA Ad5-pIX-SpyCatcher(deltaN1deltaC2) pIX sequence Amino acid (SEQ ID NO: 56): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVGGSEAAAKEAAAKEAAAKGSDSATHIKFSKRDED GKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG DNA (incl. STOP) (SEQ ID NO: 57): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGGAggctccGAAGCC GCCGCTAAAGAAGCTGCTGCCAAAGAGGCCGCTGCAAAGggatccGACAGCGCCACCCACATCAAGTTCAGCAAGAGGGACGAGGAC GGCAAGGAGCTGGCCGGCGCAACAATGGAGCTGAGGGACAGCAGCGGCAAGACCATCAGCACCTGGATCAGCGACGGCCAGGTGAAG GACTTCTACCTGTACCCCGGCAAGTACACCTTCGTGGAGACCGCCGCCCCCGACGGCTACGAGGTGGCCACCGCCATCACCTTCACC GTGAACGAGCAGGGCCAGGTGACCGTGAACGGCTAA Ad5-pIX-SnoopCatcher sequences Ad5-pIX-SnoopCatcher pIX sequence Amino acid (SEQ ID NO: 58): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVGGSEAAAKEAAAKEAAAKGSHMKPLRGAVFSLQK QHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTN GKHYITNEPIPPK DNA (incl. STOP) (SEQ ID NO: 59): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGGAggctccGAAGCC GCCGCTAAAGAAGCTGCTGCCAAAGAGGCCGCTGCAAAGggatccCACATGAAGCCCCTGAGGGGCGCCGTGTTCAGCCTGCAGAAG CAGCACCCCGACTACCCCGACATCTACGGCGCCATCGACCAGAACGGCACCTACCAGAACGTGAGGACCGGCGAGGACGGCAAGCTG ACCTTCAAGAACCTGAGCGACGGCAAGTACAGGCTGTTCGAGAACAGCGAGCCCGCCGGCTACAAGCCCGTGCAGAACAAGCCCATC GTGGCCTTCCAGATCGTGAACGGCGAGGTGAGGGACGTGACCAGCATCGTGCCCCAGGACATCCCCGCCACCTACGAGTTCACCAAC GGCAAGCACTACATCACCAACGAGCCCATCCCCCCCAAGTAA Ad5-pIX-DogCatcher sequences Ad5-pIX-DogCatcher pIX sequence Amino acid (SEQ ID NO: 60): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVGGSEAAAKEAAAKEAAAKGSKLGDIEFIKVNKND KKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSI VPQ DNA (incl. STOP) (SEQ ID NO: 61): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGGAggctccGAAGCC GCCGCTAAAGAAGCTGCTGCCAAAGAGGCCGCTGCAAAGggatccAAACTGGGCGATATTGAATTTATTAAAGTGAACAAAAACGAT AAAAAGCCGCTGCGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGATATCTATGGCGCGATTGATCAGAATGGG ACCTATCAAAATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTAAGAATCTGAGCGATGGCAAATATCGCCTGTTTGAAAATAGC GAACCCGCTGGCTATAAACCGGTGCAGAATAAGCCGATTGTGGCGTTTCAGATTGTGAATGGCGAAGTGCGTGATGTGACCAGCATT GTGCCGCAGTAA Ad5-pIX-SpyTag sequences Ad5-pIX-SpyTag pIX sequence Amino acid (SEQ ID NO: 62): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVGGSEAAAKEAAAKEAAAKGSAHIVMVDAYKPTK DNA (incl. STOP) (SEQ ID NO: 63): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGGAGGCTCCGAAGCC GCCGCTAAAGAAGCTGCTGCCAAAGAGGCCGCTGCAAAGGGATCCGCCCACATCGTTATGGTGGATGCCTACAAGCCCACCAAATAA Ad5-pIX-SnoopTagJr sequences Ad5-pIX-SnoopTagJr pIX sequence Amino acid (SEQ ID NO: 64): MSTNSFDGSIVSSYLTTRMPPWAGVRQNVMGSSIDGRPVLPANSTTLTYETVSGTPLETAASAAASAAAATARGIVTDFAFLSPLAS SAASRSSARDDKLTALLAQLDSLTRELNVVSQQLLDLRQQVSALKASSPPNAVGGSEAAAKEAAAKEAAAKGSKLGSIEFIKVNK DNA (incl. STOP) (SEQ ID NO: 65): ATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTG ATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAG ACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGC AGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTC GTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTGGAGGCTCCGAAGCC GCCGCTAAAGAAGCTGCTGCCAAAGAGGCCGCTGCAAAGGGATCCaaactgggctctattgaatttattaaagtgaacaaaTA Ligands for capsid decoration described in this application: Recombinant Proteins: SARS CoV2 Spike-SnoopTagJr Amino acid (SEQ ID NO: 66): MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQP FLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAG AAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFA SVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA TVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVA VLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPGSASSVASQSIIAYT MSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMI AQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQ ALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCG KGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIG IVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGIVQQQNN LLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSGGSGKLGSIEFIKV NKEPEA DNA (incl. STOP) (SEQ ID NO: 67): ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGACCACCAGAACACAGCTGCCTCCAGCCTACACC AACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCAGGACCTGTTCCTGCCTTTC TTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGTCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAAC GACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGC CTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTCTACTAC CACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCT TTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATC TACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGC ATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGT GCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGAT TGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGG GTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGATTCGCC TCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCTTC AAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAGATGAA GTGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCC TGGAACAGCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTC GAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAG TCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCC ACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTG CTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAG ACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCA GTGCTGTACCAGGACGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACC GGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGCACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGC GCTGGCATCTGTGCCAGCTACCAGACACAGACAAACAGCCCTGGCAGCGCCTCTTCTGTGGCCAGCCAGAGCATCATTGCCTACACA ATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAG ATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTG CAGTACGGCAGCTTCTGCACCCAGCTGAATAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCC CAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGGACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCC AGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTG GGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATC GCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCTGGCGCCGCTCTGCAGATCCCCTTTGCT ATGCAGATGGCCTACAGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAAC AGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCAGAATGCCCAG GCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAACTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCT CCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCC GCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGC AAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAG AATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGG TTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGC ATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACA AGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTG GCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAAGGCTCTGGAATTGTGCAGCAGCAAAACAAT CTGCTGCGGGCCATCGAGGCTCAGCAGCATCTGCTGCAGCTGACCGTGTGGGGAATCAAGCAGCTCCAGGCCAGAATCCTGGCTGGT GGATCTGGCGGCCATACCACCTGGATGGAATGGGACAGAGAGATCAACAACTACACCAGCCTGATCCACAGCCTGATTGAGGAATCC CAGAATCAGCAAGAGAAGAACGAGCAAGAACTGCTGGAAGGAAGCGGAGGCTCTGGTaaactgggctctattgaatttattaaagtg aacaaaGAGCCCGAAGCCTAA SARS CoV2 Spike RBD-SnoopTagJr Amino acid (SEQ ID NO: 68): MFVFLVLLPLVSSQCRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL CFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS TPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGSGGSGKLGSIEFIKVNKEPEA DNA (incl. STOP) (SEQ ID NO: 69): ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATC ACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGC GTGGCCGACTACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTG TGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCC GACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACTCCAAAGTCGGCGGCAAC TACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAGC ACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAG CCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATGGAAGCGGA GGCTCTGGTaaactgggctctattgaatttattaaagtgaacaaaGAGCCCGAAGCCTAA SARS CoV2 Spike RBD-SnoopCatcher Amino acid (SEQ ID NO: 70): MFVFLVLLPLVSSQCRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL CFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS TPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGSGGSGGSGGSGHMKPLRGAVFSLQKQHPDYP DIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNGKHYIT NEPIPPKEPEA DNA (incl. STOP) (SEQ ID NO: 71): ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATC ACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGC GTGGCCGACTACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTG TGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCC GACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACTCCAAAGTCGGCGGCAAC TACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAGC ACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAG CCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATGGAAGCGGA GGCTCTGGTGGATCCGGTGGATCTGGACACATGAAGCCTCTGAGAGGCGCCGTGTTCAGCCTGCAGAAACAGCACCCTGACTACCCC GATATCTACGGCGCCATCGACCAGAACGGCACCTACCAGAATGTTCGGACAGGCGAGGATGGCAAGCTGACCTTCAAGAACCTGAGC GACGGCAAGTACCGGCTGTTCGAGAATTCTGAGCCTGCCGGCTACAAGCCCGTGCAGAACAAACCTATCGTGGCCTTCCAGATCGTG AACGGCGAAGTGCGGGATGTGACCAGCATCGTGCCTCAGGATATCCCCGCCACCTACGAGTTCACCAACGGCAAGCACTACATCACC AACGAGCCCATTCCTCCAAAAGAGCCCGAAGCCTAA SnoopCatcher-SARS CoV2 Spike RBD Amino acid (SEQ ID NO: 72): METDTLLLWVLLLWVPGSTGDGHMKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYK PVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNGKHYITNEPIPPKGSGGSGGSRVQPTESIVRFPNITNLCPFGEVFNATRFA SVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA TVCGPKKSTNEPEA DNA (incl. STOP) (SEQ ID NO: 73): atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacGGTCACATGAAGCCCCTGAGGGGC GCCGTGTTCAGCCTGCAGAAGCAGCACCCCGACTACCCCGACATCTACGGCGCCATCGACCAGAACGGCACCTACCAGAACGTGAGG ACCGGCGAGGACGGCAAGCTGACCTTCAAGAACCTGAGCGACGGCAAGTACAGGCTGTTCGAGAACAGCGAGCCCGCCGGCTACAAG CCCGTGCAGAACAAGCCCATCGTGGCCTTCCAGATCGTGAACGGCGAGGTGAGGGACGTGACCAGCATCGTGCCCCAGGACATCCCC GCCACCTACGAGTTCACCAACGGCAAGCACTACATCACCAACGAGCCCATCCCCCCCAAGGGCTCTGGCGGAAGCGGCggatccCGG GTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGATTCGCC TCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCTTC AAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAGATGAA GTGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCC TGGAACAGCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTC GAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAG TCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCC ACAGTGTGCGGCCCTAAGAAAAGCACCAATGAGCCCGAGGCCTAA SARS CoV2 Spike RBD-SpyCatcher Amino acid (SEQ ID NO: 74): MFVFLVLLPLVSSQCRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL CFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS TPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGSGGSGGSGGSGDSATHIKFSKRDEDGKELAG ATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIEPEA DNA (incl. STOP) (SEQ ID NO: 75): ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATC ACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGC GTGGCCGACTACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTG TGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCC GACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACTCCAAAGTCGGCGGCAAC TACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAGC ACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAG CCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATGGAAGCGGA GGCTCTGGTGGATCCGGTGGATCTGGCGATTCTGCCACACACATCAAGTTCAGCAAGCGCGACGAGGACGGCAAAGAACTGGCTGGC GCTACCATGGAACTGAGAGACAGCAGCGGCAAGACCATCAGCACCTGGATCTCCGACGGCCAAGTGAAGGACTTCTATCTGTACCCC GGCAAGTACACCTTCGTGGAAACCGCCGCTCCTGACGGATATGAAGTGGCCACCGCCATCACCTTCACCGTGAATGAGCAGGGACAA GTGACCGTGAACGGCAAGGCCACAAAAGGCGACGCCCACATTGAGCCCGAAGCCTAA

Example 1—Modular Covalent Decoration of the Adenovirus Capsid Via Insertion of SpyTag or DogTag into Hexon HVR Loops

[0193] In order to modify the hexon proteins of the adenovirus capsid, sequence alignment was undertaken to identify the location of hypervariable region (HVR) loops 1, 2 and 5 in FIG. 1E [Sequence comparison of the following adenoviruses; Ad5 and Ad2 (species C), Ad12 and Ad18 (species A), Ad3 and Ad35 (species B), Ad4 (species E), Ad40 and Ad41 (species F)]. Deletions were generated at the insertion sites of the HVR loops indicated in FIG. 1E. SpyTag and DogTag were inserted at each locus. The insertion sites indicated are identical between SpyTag and DogTag recombinants at each of the three HVRs tested. At each locus, SpyTag or DogTag sequences are flanked by GSGGSG sequences on each side.

Example 2—Coupling of SpyCatcher with SpyTag Inserted into HVR Loops

[0194] Ad5 vectors displaying SpyTag at HVR1, HVR2 or HVR5 were incubated with biotinylated SpyCatcher at 15 μM or 40 μM in order to assess the coupling reaction between SpyTag and SpyCatcher. Samples were run on SDS-PAGE and proteins visualised by Coomassie staining as shown in FIG. 2A.Coupling efficiency was assessed by comparing band intensities of uncoupled hexon-SpyTag with hexon-SpyTag:SpyCatcher using ImageJ software (% hexon coupled=band intensity of hexon-SpyTag:SpyCatcher divided by the sum of band intensities of hexon-SpyTag:SpyCatcher and uncoupled hexon-SpyTag, multiplied by 100).

[0195] Immediately after the coupling reaction described in FIG. 2A, a vector infectivity assay was undertaken with the same samples (FIG. 2B). Samples were serially diluted, applied to monolayers of HEK293A cells in a 96-well plate, and incubated for 48 h at 37° C. with 5% CO.sub.2. Infectious titres were calculated by enumeration of GFP fluorescent foci by fluorescence microscopy. Infectious titres were calculated as no. of infectious units (ifu) per ml.

[0196] Ad5(GFP) vectors displaying SpyTag at HVR1 (Ad5(GFP) HVR1 SpyTag) (1E+10 viral particles) were incubated with biotinylated SpyCatcher at 15 μM or 40 μM under different conditions (FIG. 2C). Samples were run on SDS-PAGE and proteins visualised by Coomassie staining. A vector infectivity assay was performed on the same samples as shown in FIG. 2C (FIG. 2D).

[0197] The results demonstrate that SpyTag is poorly reactive after insertion into Ad5 HVR loops, and that vector infectivity is substantially reduced (over 100-fold) upon coupling of SpyCatcher to >50% of hexon protein on the viral capsid.

Example 3—Coupling of DogCatcher with DogTag Inserted into HVR Loops

[0198] HEK293A cells were infected either with wild type Ad5(GFP) (native hexon protein) or DogTag inserted into HVR1 (HVR1DT), HVR2 (HVR2DT) or HVR5 (HVR5DT). Vector yield from 1500 cm.sup.2 of adherent HEK293A cells infected with Ad5 displaying DogTag on hexon surfaces is equivalent to vector yield with unmodified hexon Ad5 (FIG. 3A).

[0199] Ad5(GFP) vectors (1E+10 viral particles) displaying DogTag at either HVR1, HVR2 or HVR5 were incubated with 5 μM DogCatcher. Reactions were performed for 16 h at 4° C. as shown in FIG. 3B. Coupling efficiency was assessed by comparing band intensities of uncoupled hexon-DogTag with hexon-DogTag:DogCatcher using ImageJ as described in Example 2. FIG. 3C shows an infectivity assay performed on the same samples as in FIG. 3B.

[0200] Ad5(GFP) vectors displaying DogTag at either HVR1, HVR2 or HVR5 (1E+10 viral particles) were incubated with DogCatcher at 20 μM or 80 μM for 0.1, 1 or 16 hours at 4° C. as shown in FIG. 3D. Coupling efficiency was assessed as described above.

[0201] The data show that DogTag is highly reactive after insertion into Ad5 HVR loops and that vector infectivity is not inhibited by coupling to DogCatcher, despite coverage of >90% capsid hexon.

Example 4—Reactivity of SnoopTagJr with DogTag Inserted into HVR Loops

[0202] Ad5(GFP) vectors (1E+10 viral particles) displaying DogTag at either HVR1, HVR2 or HVR5 were incubated with SnoopTagJr-AffiHER2 (80 μM) and SnoopLigase (70 μM). Samples were run on SDS-PAGE and proteins visualised by Coomassie staining. Coupling efficiency was quantified by comparing band intensities of uncoupled hexon-DogTag with hexon-DogTag:SnJr-AffiHER2 using ImageJ (FIG. 4A).

[0203] To assess the temperature-dependence of hexon-DogTag coupling to SnoopTagJr-AffiHER2, Ad5(GFP) vectors displaying DogTag at HVR5 (1E+10 viral particles) were incubated with SnoopTagJr-AffiHER2 (80 μM) and SnoopLigase (70 μM) for 16 h at either 20° C. or 4° C. (FIG. 4B). A vector infectivity assay was performed as described in Example 3 on the samples used for FIG. 4B (FIG. 4C).

[0204] Ad5(GFP) vectors displaying DogTag at HVR5 (1E+10 viral particles) were incubated with SnoopTagJr-AffiHER2 (80 μM) and SnoopLigase (70 μM) for 16 h at 4° C. (FIG. 4D). Different concentrations of NaCl were added at either 150 mM or 75 mM, and glycerol was added as indicated at 15% (v/v). Samples were analysed by SDS-PAGE and Coomassie staining.

Example 5—Assessment of Vector Infectivity for Non-Fluorescent Recombinant Vectors

[0205] A hexon immunostaining assay to assess vector infectivity was performed with Ad5(GFP) (with a WT hexon) and Ad5(GFP) HVR5 DogTag. Both vectors were serially diluted, and 50 μl of each dilution was transferred to a monolayer of HEK293A cells in a poly-L-lysine coated 96-well plate. After a 48 h incubation at 37° C. with 5% CO.sub.2, GFP-positive cells were counted by fluorescence microscopy. For immunostaining, cells on the same plate were fixed with ice-cold methanol and an anti-hexon mouse monoclonal antibody (clone 65H6, ThermoFisher) was used to detect hexon protein within infected cells. Hexon-positive cells were subsequently stained using an alkaline phosphatase-conjugated secondary antibody, and BCIP/NBT substrate solution. The results demonstrated that HEK293A cells infected with Ad5 vectors with a WT hexon protein and Ad5 vectors displaying DogTag at HVR5 showed equivalent intensity of hexon immunostaining.

[0206] The infectious titres for Ad5(GFP) (hexon WT) and Ad5(GFP) HVR5 DogTag were compared using both GFP fluorescence and hexon immunostaining assays. Both assays were performed using the same wells on the same plate, n=4. Bars show mean+SD and dots represent spot counts from individual wells. The results were not significantly different (ns) as calculated by a two-tailed t-test.

Example 6—Reactivity of DogTag Inserted into Hexon Loops with SnoopTagJr Tagged Peptides

[0207] Peptides were synthesized consisting of the SnoopTagJr sequence fused to T cell epitopes. These SnoopTagJr tagged peptides were coupled to Ad5(GFP) HVR5 DogTag using SnoopLigase. A gel shift assay with Coomassie staining was not suitable to assess the coupling efficiency due to the low molecular weight of these short peptides (.sup.˜3 kDa). The efficiency of peptide coupling was instead assessed using a competition assay with DogCatcher (described in FIG. 6A). SnoopTagJr-peptide (SnJr-peptide) and hexon-DogTag (hexon-DT) are coupled using SnoopLigase (SnL). After coupling, an excess of DogCatcher (DC) protein is added to the reaction. DC binds >90% of free (uncoupled) hexon-DT molecules on the surface of Ad5 (see FIG. 3B), therefore the proportion of hexon-DT coupled to SnJr-peptide can be estimated to be the proportion of hexon that does not undergo a gel shift of .sup.˜20 kDa on SDS-PAGE after incubation with DC.

[0208] Coupling efficiency of SnoopTagJr-hTERT peptide (SnoopTagJr fused to a T cell epitope from human telomerase reverse transcriptase, EARPALLTSRLRFIPK) to hexon-DT was assessed using the DogCatcher competition assay described above. Ad5(GFP) HVR5 DogTag (Ad5-DT, 1E+10 viral particles) was incubated with different combinations of SnL (70 μM) and SnJr-hTERT (80 μM), and in some samples DogCatcher (20 μM, excess) was added after the peptide-peptide coupling reaction to assess coupling efficiency (FIG. 6B). All reactions were performed at 4° C. Samples were run on SDS-PAGE with subsequent Coomassie staining (FIG. 6B). The proportion of coupled hexon-DT:SnJr-hTERT compared to hexon-DT:DC (representing free hexon-DT after the initial SnL-catalysed reaction) was assessed by comparing band intensities as indicated. The optimum time interval for the hexon-DT:SnJr-hTERT reaction was determined to be 48 h.

[0209] FIG. 6C shows an assessment of coupling efficiency of SnoopTagJr-SIINFEKL peptides (SIINFEKL is a mouse CD8.sup.+ T cell epitope derived from ovalbumin) to hexon-DT using the DogCatcher competition assay described above. PEP1 corresponds to the peptide sequence SnJr-GGSSIINFEKL and PEP2 corresponds to the peptide sequence SnJr-AAYSIINFEKL. Ad5-DT (1E+10 viral particles) was incubated with SnJr-SIINFEKL peptides (PEP1 or PEP2, 80 μM) and SnL (70 μM) for 48 h at 4° C. DC (20 μM) was subsequently added. Samples were then incubated for a further 24 h at 4° C. Samples were run on SDS-PAGE and peptide coupling efficiency was assessed as described above.

[0210] Alternatively, a direct gel shift assay using monovalent streptavidin (mSA) can be used to assess coupling efficiency of short peptides as described in FIG. 6D. Biotinylated SnJr-peptide was incubated with SnL and Ad5-DT as described previously. After 48 h, the coupling reaction was stopped by boiling in sample buffer including sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) to denature all proteins and virion structures. Samples were briefly cooled on ice before incubation with an excess of monovalent streptavidin (mSA) for 30 min at 20° C. Samples were run on SDS-PAGE, transferred to nitrocellulose, and Western blotting performed using an anti-hexon primary mouse monoclonal antibody (clone 65H6, ThermoFisher). A gel-shift (.sup.˜50 kDa) is observed for hexon-DT protein coupled to SnJr-biotin by virtue of the strong interaction between mSA and biotin (hexon-DT:SnJr-biotin/mSA). FIG. 6E shows the SDS-PAGE of the mSA direct gel shift assay as described above with additional controls. Bands corresponding to hexon-DT:SnJr-biotin/mSA and hexon-DT species are indicated. FIG. 6F shows a vector infectivity assay (by GFP focus enumeration, as described in example 5) was performed on the same samples shown in FIG. 6C. The results demonstrate that Ad5-DT infectivity is not affected by surface display of SnJr-peptides.

Example 7—Coupling of Ad5 HVR DogTag to DogCatcher Fusion Proteins

[0211] The circumsporozoite protein (CSP) of Plasmodium falciparum (Pf) has been extensively studied as a malaria vaccine candidate antigen. The protein contains a highly immunogenic repetitive region, primarily consisting of repeats of the sequence NANP. Repeat sequences of varying lengths from this region were fused to the C-terminus of DogCatcher and the resulting fusion proteins coupled to Ad5(GFP) HVR5 DogTag. Three DogCatcher fusion constructs; NANP9, NANP18, and NANPD (consisting of the entire NANP repetitive domain of PfCSP from the 3D7 strain) are shown in FIG. 7A.

[0212] Reactivity of DogCatcher-NANPn with DogTag inserted into hexon HVR5 loop was assessed. Ad5(GFP) vectors (1E+9 viral particles) displaying DogTag at HVR5 were incubated with different DogCatcher-NANPn fusions (as indicated) at 15 μM. Reactions were performed for 16 h at 4° C. Samples were run on SDS-PAGE and proteins visualised by Coomassie staining (FIG. 7B). Coupling efficiency was assessed as described above. A vector infectivity assay (by GFP focus enumeration, as described above) was performed on the same samples shown in FIG. 7B (FIG. 7C).

Example 8—Coupling of DogCatcher-NANPn to the Surface of Ad5 Via Hexon Reduces the Potency of Virus Neutralising Antibodies

[0213] Ad5(GFP) HVR5 DogTag vectors (Ad5-DT, 1E+10 viral particles) were incubated with SnoopLigase (SnL) and SnJr-AffiHER2 (or SnJr-AffiHER2 alone) for 48 h at 4° C. After 48 h, approximately 60-65% of the hexon was coupled to SnJr-AffiHER2 (a comparable coupling efficiency to the experiment shown in FIG. 4B). Coupled vector was incubated with serially diluted virus-neutralising mouse monoclonal anti-hexon antibody mAb 9C12 (Developmental Studies Hybridoma Bank, University of Iowa) for 1 h at 37° C., before the vector-antibody mix was added to an 80% confluent monolayer of HEK293A cells in a 96-well plate format (cells were infected at a multiplicity of infection of 200 ifu/cell). Cells were incubated with the vector-antibody mix for 2 h at 37° C. with 5% CO.sub.2, before the mix was replaced with fresh media and the plates returned to 37° C. with 5% CO.sub.2 for a further 24 h. After 24 h, GFP expression within HEK293A cells was employed as a readout of infectivity—bulk fluorescence was measured on a fluorimeter using an excitation of 395 nm and emission of 509 nm. The data, shown in FIG. 8A, demonstrated an increase in the IC.sub.50 of a potent neutralising anti-hexon monoclonal antibody upon coupling of SnoopTagJr-AffiHER2 to Ad5 DogTag. IC.sub.50 for mAb 9C12 neutralisation increased from 2.5 ng/ml (Ad5-DT alone, and Ad5-DT+SnJr-AffiHER2 only) to 7 ng/ml with coupled vector (Ad5-DT+SnL+SnJr-AffiHER2).

[0214] In FIG. 8B, a similar experiment to that described above was performed using the DogCatcher-NANPn fusions described in FIG. 7A. Ad5(GFP) HVR5 DogTag vectors (Ad5-DT, 1E+9 viral particles) were incubated with DogCatcher-NANPn constructs for 16 h at 4° C., exactly as described in FIG. 7. Coupling efficiency of each of the constructs to Ad5-DT was similar to that shown in FIG. 7B. As described above, vector was incubated with mAb 9C12 for 1 h, the mix added to cells at a multiplicity of infection of 200 ifu/cell for 2 h, and vector replaced with fresh media prior to reading GFP fluorescence after a further 24 h. A substantial increase in IC.sub.50 for mAb 9C12 was observed with the DogCatcher-NANPn coupled vectors compared to Ad5-DT alone, implying shielding of the hexon from the neutralising monoclonal antibody (FIG. 8B). Ad5-DT:DC-NANP18 exhibited the greatest increase in IC.sub.50, >10-fold compared to Ad5-DT alone.

[0215] In FIG. 8C, a neutralisation assay with Ad5-positive mouse serum (instead of mAb 9C12) was performed on coupled Ad5-DT:DC-NANPn vectors. To generate Ad5-positive mouse serum, mice were immunised intramuscularly with 1E+8 ifu Ad5(Ovalbumin) (WT hexon) vector (Ad5 E1 and E3 deleted vector with an unmodified hexon expressing ovalbumin instead of GFP under the control of a constitutive CMV promoter encoded at E1 locus) and serum was harvested two-weeks post-immunisation. The results of the neutralization assay demonstrated that IC.sub.50 of the neutralizing anti-serum is increased with Ad5-DT:NANP9 and Ad5-DT:NANP18 compared to Ad5-DT alone (FIG. 8C).

Example 9—Human Coagulation Factor X-Dependent Vector Transduction of SKOV3 Cells

[0216] It has been demonstrated that, particularly after intravenous administration, some serotypes of adenovirus including Ad5 can transduce hepatocytes via an interaction between human coagulation Factor X (FX) and the hexon protein of the virus [Simon N. Waddington, John H. McVey, David Bhella, Menzo J. E. Havenga, Stuart A. Nicklin, Andrew H. Baker, Adenovirus Serotype 5 Hexon Mediates Liver Gene Transfer. Cell, 2008, Volume 132, Issue 3, P397-409]. In vitro, FX mediated infection can be assessed by measuring vector transduction of SKOV3 cells, a human ovarian cancer cell line, in the presence or absence of FX.

[0217] Ad5(GFP)-DogTag (Ad5) or Ad5(GFP)-DogTag:DogCatcher-NANP18 (Ad5-NANP18) vectors (2E+9 viral particles) were incubated in the presence or absence of human coagulation Factor X (8 μg/mL) on SKOV3 cells for 2 h at 37° C. in serum-free media. Then media was replaced with fresh complete media and plates were incubated for a further 48 h. Infectious titres were then calculated by enumeration of GFP-positive foci by fluorescence microscopy.

[0218] The data indicate that coupling of DogCatcher-NANP18 to the Ad5 hexon reduces FX dependent infection of SKOV3 cells, presumably by shielding the viral capsid from interaction with FX.

Example 10—Mouse Immunogenicity Experiment to Assess CD8.SUP.+ T Cell Responses to Ad5 Surface-Displayed Peptide Antigens

[0219] C57BL/6 mice (4/group) were immunised intramuscularly with either surface-display vector Ad5(GFP) HVR5-DT:SnJr-GGSSIINFEKL (5E+9 viral particles, dialysed to remove excess peptide and Snoopligase) (Group 1), the same SIINFEKL surface display vector as in Group 1 but without dialysis to remove excess peptide and Snoopligase (5E+9 viral particles, excess SIINFEKL peptide 0.7 μg) (Group 2), Ad5(GFP) vector (5E+9 viral particles) co-administered with SnJr-GGSSIINFEKL peptide (0.7 μg, not coupled to the vector) (Group 3), or SnJr-GGSSIINFEKL peptide (5 μg, co-administered with 10 μg poly I:C) (group 4) (FIG. 10A). The effective dose of SIINFEKL peptide displayed on the coupled vector from Group 1 was calculated to be approx. 10 ng, taking into account the molecular weight of SnJr-GGSSIINFEKL, a coupling efficiency of 80% hexon coverage (see FIG. 6C, PEP1), 720 copies of hexon per virion, and 5E+9 virions per vaccine dose. The dose of peptide in Group 4 is 500-fold higher than in Group 1. After 14 days post-immunisation, mice were sacrificed and CD8+T cell responses in the spleen were measured by overnight ex vivo IFNγ-ELISPOT.

[0220] Spleen ex vivo IFNγ-ELISPOT responses to SIINFEKL, a murine CD8+T cell epitope are shown in FIG. 10B. SIINFEKL responses in Group 1 and Group 2 were higher than those in Group 3, demonstrating that attachment of SnJr-GGSSIINFEKL to the vector capsid is required to achieve potent SIINFEKL T cell responses. Spleen ex vivo IFNγ-ELISPOT responses to DTLVNRIEL (EGFP.sub.118-126), a murine CD8.sup.+ T cell epitope present in the encoded GFP transgene expressed by the vector upon cell transduction, are shown in FIG. 10C. There were no significant differences in EGFP.sub.118-126 responses between Groups 1-3, demonstrating that surface decoration of the vector capsid did not impair T cell responses to the encoded GFP transgene antigen.

Example 11—Modular Covalent Decoration of the Adenovirus Capsid Via Fusion of SpyCatcher at the C-Terminus of pIX

[0221] An (EAAAK).sub.3 linker was inserted between the C-terminus of viral pIX ad the N-terminus of SpyCatcherΔN1 to facilitate successful rescue of the virus and efficient capsid display as shown in FIG. 11A.

[0222] Ad5(GFP) pIX-SpyCatcher vectors (3E+9 viral particles) were incubated with SpyTag-Maltose binding protein (SpyTag-MBP, 5 μM) or SpyTag-Human cytomegalovirus pentamer (SpyTag-Pentamer, 2.5 μM) for 16 h at 4° C. (FIG. 11B). Samples containing SpyTag-MBP or SpyTag-Pentamer alone were included as controls. After conjugation, samples were run on SDS-PAGE and transferred to nitrocellulose for Western blotting. pIX-SpyCatcher, pIX-SpyCatcher:SpyTag-MBP, and pIX-SpyCatcher:SpyTag-gH (from the HCMV Pentamer) species were detected using antisera from mice immunised with SpyCatcher protein (FIG. 11B).

[0223] Ad5(GFP) pIX-SpyCatcher vectors (3E+9 viral particles) were incubated with biotinylated SpyTag peptide (5 μM) for 16 h at 4° C. (FIG. 11C). To assess peptide coupling efficiency to pIX-SpyCatcher by gel-shift assay, vector material was denatured and boiled in sample buffer including sodium dodecyl sulfate (SDS) and DTT, before cooling and incubating with monovalent streptavidin (mSA) as described in FIG. 6D. pIX-SpyCatcher and pIX-SpyCatcher:SpyTag-biotin/mSA species were detected by SDS-PAGE and Western blotting with anti-SpyCatcher antisera as described in B.

[0224] A vector infectivity assay (by GFP focus enumeration, described previously) was performed on the same samples shown in FIGS. 11B and 11C (FIG. 11D). The results demonstrated that Ad5(GFP) pIX-SpyCatcher infectivity is not affected by surface display of SpyTag-protein fusions or peptides.

Example 12—Viability, Genetic Stability, and Yield of Different Recombinant Adenovirus Vectors with SpyCatcher, SnoopCatcher or DogCatcher Fused to the C-Terminus of pIX

[0225] Further research demonstrated that the vector construct described in Example 11 (SpyCatcher fused to the C-terminus of pIX via an EAAAK3 linker) while reactive, was not genetically stable. A range of alternative constructs with alternative linker sequences (GSGGSGGSG (GSG3), GGSEAAAKEAAAKEAAAKGS (EAAAK3), or GGSPANLKALEAQKQKEQRQAAEELANAKKLKEQLEKGS (a helical sequence from ribosomal protein L9, Veggiani et al, Proc Natl Acad Sci USA. 2016 Feb. 2; 113(5):1202-7. doi: 10.1073/pnas.1519214113) (HELIX)), GS hinge sequences either side of the linker, or alternative Catchers (SpyCatcher dN1, SpyCatcher dN1dC2, SnoopCatcher, DogCatcher) were constructed (FIG. 12A). Each vector DNA construct was linearised by PacI restriction digest to release the viral inverted terminal repeats (ITRs) prior to transfection into HEK293A cells. Viability of each construct was assessed by inspecting cell monolayers for development of cytopathic effect (CPE) which would indicate successful rescue of the recombinant virus. Viable viral vector preparations were passaged to increase virus titer, with three passages typically required to obtain sufficient material for purification by CsCl gradient ultracentrifugation (from initial transfection). A forth passage was required to obtain a second batch of each viable vector, and genetic stability was assessed after the third and fourth passage by sequencing of the pIX locus. To assess vector yield, viable vectors were amplified from a total of 10×T150 flasks (total surface area 1500 cm.sup.2 HEK293A cells), purified by CsCl ultracentrifugation, and infectious titer calculated by GFP infectivity assay as described previously (FIG. 12B).

Example 13—Reactivity of SnoopCatcher and DogCatcher Fused to the C-Terminus of pIX

[0226] To assess reactivity of Ad5(GFP) pIX-SnoopCatcher with SnoopTagJr-fused ligands, Ad5(GFP) pIX-SnoopCatcher vectors were incubated with tagged full-length SARS CoV2 Spike (Spike) or SARS CoV2 spike receptor binding domain (RBD) proteins. Both recombinant proteins were produced in mammalian suspension 293F cells, with SnoopTagJr fused at the C-terminus. After a 16 h coupling reaction at 4° C., samples were run on SDS-PAGE and a western blot performed using mouse sera with reactivity against SnoopCatcher (sera from mice immunised with IMX313-DogTag:SnoopTag-CIDR:SnoopLigase, Andersson, A. C., Buldun, C. M., Pattinson, D. J. et al. SnoopLigase peptide-peptide conjugation enables modular vaccine assembly. Sci Rep 9, 4625 (2019)) (FIG. 13A). Controls included vector alone (Vector), Spike alone (Spike) and RBD alone (RBD). Species including pIX-SnoopCatcher (pIX-SnC), and covalently coupled pIX-SnoopCatcher:SnoopTagJr-Spike (pIX-SnC:SnJr-Spike) and pIX-SnoopCatcher:SnoopTagJr-RBD (pIX-SnC:SnJr-RBD) are indicated. Immediately after the coupling reactions described in FIG. 13A, a vector infectivity assay (GFP foci) was undertaken with the same samples (FIG. 13B). The data indicate that SnoopCatcher is reactive when fused to the C-terminus of pIX, and that coupling of ligands to pIX does not impair vector infectivity in vitro (despite the size of the SARS CoV2 Spike trimer exceeding 500 kDa).

[0227] To assess reactivity of Ad5(GFP) pIX-DogCatcher with DogTag-fused ligands, Ad5(GFP) pIX-DogCatcher vectors were incubated with E coli expressed small ubiquitin modifier (SUMO) protein with DogTag fused at the C-terminus. After a 16 h coupling reaction at 4° C., samples were run on SDS-PAGE and a western blot performed using mouse sera with reactivity against DogCatcher (FIG. 13C). The same mouse sera used for the western blot described in FIG. 13A was also used in FIG. 13C, since DogCatcher shares a significant degree of sequence identity with SnoopCatcher. Controls included vector alone (Vector) and SUMO alone (SUMO). Species pIX-DogCatcher (pIX-DC) and pIX-DogCatcher:DogTag-SUMO (pIX-DC:DT-SUMO) are indicated.

Example 14—Reactivity of SnoopTagJr and SpyTag Fused to the C-Terminus of pIX

[0228] Ad5(GFP) vectors were constructed with SnoopTagJr or SpyTag fused to the C-terminus of pIX, with an EAAAK3 linker between pIX and tag flanked by GGS and GS hinges, similar to the SnoopCatcher and DogCatcher fusion constructs shown in FIG. 12A. Both Ad5 pIX-SnoopTagJr and Ad5 pIX-SpyTag vectors were transfected into HEK293A cells, passaged, and amplified into 10×T150 cm.sup.2 flasks. Infectious yield of both vectors was high, and comparable to Ad5(GFP) HVR5-DogTag (FIG. 14A).

[0229] To assess reactivity of SnoopTagJr and SpyTag displayed as a pIX fusion, Ad5 pIX-SnoopTagJr (Ad5 pIX-SnJr) or Ad5 pIX-SpyTag (Ad5 pIX-ST) vectors were co-incubated with recombinant mammalian (Chinese Hamster Ovary, CHO) cell expressed SARS CoV2 RBD proteins fused to either SnoopCatcher (RBD-SnC or SnC-RBD, C- or N-terminal fusions to RBD respectively) or SpyCatcher (RBD-SC, C-terminal fusion). After a 16 h coupling reaction at 4° C., samples were run on SDS-PAGE and a western blot performed using an anti-SARS CoV2 RBD polyclonal antibody (Sino Biological) (FIG. 14B). Protein species representing coupling between Tag and Catcher modules are indicated (*). Immediately after the coupling reactions described in FIG. 14B, a vector infectivity assay (GFP foci) was undertaken with the same samples (FIG. 14C). Coupling of ligands did not impair infectivity of either Ad5 pIX-SnoopTagJr or Ad5 pIX-SpyTag.