WO2022218997A1

WO2022218997A1 - Novel universal vaccine presenting system

Info

Publication number: WO2022218997A1
Application number: PCT/EP2022/059786
Authority: WO
Inventors: Pascal Fender; Marie-Claire DAGHER; Solène BESSON
Original assignee: Centre National De La Recherche Scientifique (Cnrs); Commissariat A L'energie Atomique Et Aux Energies Alternatives; Universite Grenoble Alpes
Priority date: 2021-04-12
Filing date: 2022-04-12
Publication date: 2022-10-20

Abstract

The present invention provides novel engineered protein comprising: (i) a adenovirus penton base protomer comprising a peptide tag in the variable loop and/or in the RGD loop, and (ii) at least one a protein or at least one protein fragment fused to a binding partner of the peptide tag; where the peptide tag of (i) and the binding partner of the peptide tag of (ii) are covalently bound to each other via an isopeptide bond. The present invention further concerns the uses and therapeutic uses of the engineered protein.

Description

NOVEL UNIVERSAL VACCINE PRESENTING SYSTEM

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of virus like particles and vaccines.

The present invention notably provides novel engineered protein comprising:

(i) a adenovirus penton base protomer comprising a peptide tag in the variable loop and/or in the RGD loop, and

(ii) at least one protein or at least one protein fragment fused to a binding partner of the peptide tag ; where the peptide tag of (i) and the binding partner of the peptide tag of (ii) are covalently bound to each other via an isopeptide bond. The present invention further concerns the uses and therapeutic uses of the engineered protein.

BACKGROUND ART

Infectious diseases and cancers continue to plague and decimate populations world-wide. Among the means at our disposal to counter these threats, vaccination has proven to be exceptionally powerful (especially for infectious diseases). Small-pox has been eradicated, measles, polio and tetanus constrained from the world by vaccination. Nonetheless, severe threats continue to challenge human health, notably from emergent viruses that have adapted and result in new diseases, or pathogenic strains possessing attributes facilitating pathogenicity.

Recent such examples are the severe threat posed by coronaviruses, including SARS-CoV2. Global epidemics caused by these emergent viruses can incur severe costs for affected communities and economies.

A potent vaccination strategy to counter these emerging threats would be highly desirable. However, powerful and adaptative vaccines are utterly lacking to date.

Self-assembling protein-based nanoparticles are highly attractive tools for a broad range of biomedical applications, including vaccine development and cancer therapy. They form supramolecular architectures with unique properties (D. Diaz, A. Care, A. Sunna, Bioengineering strategies for protein-based nanoparticles Genes 9, E370 (2018)) including spontaneous self-organization from simple precursor protomers amenable to engineering. Moreover, the particle size is generally in the range of that of pathogens, notably viruses, against which the immune system has evolved to strongly react (M. F. Bachmann, G. T. Jennings, Vaccine delivery: A matter of size, geometry, kinetics and molecular patterns. Nat. Rev. Immunol. 10, 787-796 (2010); F. Zabel, T. M. KOndig, M. F. Bachmann, Virus-induced humoral immunity: On how B cell responses are initiated. Curr. Opin. Virol. 3, 357-362 (2013). Protein-based nanoparticles often adopt quasi-spherical shapes encapsulating a central cavity that can carry cargos, rendering them suitable to deliver drugs (U. Unzueta, M. V. Cespedes, E. Vazquez, N. F. Miralles, R. Mangues, A. Villaverde, Towards protein-based viral mimetics for cancer therapies. Trends Biotechnol. 33, 253-258 (2015)). Virus-like particles (VLPs) are made of many copies of identical building blocks resulting in highly repetitive surfaces, providing opportunities to display pathogen-derived or cancer- derived epitopes. These are often oligopeptide sequences, generally not immunogenic enough on their own to elicit a strong immune response resulting in protection (Yuan, M. et al. Structural basis of a shared antibody response to SARS-CoV-2. Science 369, 1119-1123 (2020); Zhou, P. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature Mar;579(7798):270-273. doi: 10.1038/s41586-020-2012-7 (2020)). However, if coupled to self-assembling protomers forming the VLP, then peptide epitopes can reach very high densities on the VLP, potentially able to trigger B cell receptor clustering and cross-presentation for facilitation of a strong immune response. Notably, VLPs can act as self-adjuvants, alleviating the need to supplement vaccine formulations with additional adjuvanting reagents that can have undesired side effects (N. Petrovsky, Comparative safety of vaccine adjuvants: A summary of current evidence and future needs. Drug Saf. 38, 1059-1074 (2015)).

The ADDomer (Adenovirus dodecahedron derived multimer) is a synthetic scaffold derived from a virus like particle (VLP) that occurs during the human adenovirus serotype 3 (HAd3) natural replication cycle, helping in viral internalization. ADDomer is a non-infectious 30 nm nano particle, formed of 60 copies of a single adenovirus protein, the penton base (also called penton base protomer), retaining the aptitude to autonomously self-assemble into a dodecahedron and the adenovirus-like ability to penetrate epithelial cells (Fender et al., 2012; Fender et al., 2005; WO2017167988A1). ADDomer is uniquely suited to display multiple peptide and protein epitopes by means of fully flexible, solvent exposed loops. Among these loops, the variable loop and the RGD loop allow the insertion of foreign proteins including antigens, and their presentation in 60 copies on the surface of the ADDomer. These loops offer convenient options to insert, using any method of synthetic biology, multiple copies of highly immunogenic peptide epitopes, derived for example from viral pathogens or tumour antigens. This antigen presentation system has additional adjuvant properties, which is beneficial in vaccination (Fender, P., et al. Impact of human adenovirus type 3 dodecahedron on host cells and its potential role in viral infection (2012) J Virol 86, 5380-5385; P. Fender, A. Boussaid, P. Mezin, J. Chroboczek, Synthesis, cellular localization, and quantification of penton-dodecahedron in serotype 3 adenovirus-infected cells. Virology 340, 167-173 (2005)). However, this system has several limitations: 1) the size and the structure complexity of the inserted foreign proteins, which can interfere with the stability of the ADDomer and may destroy the global architecture of the ADDomer particles; and 2) the impossibility to deliver post-translationally modified proteins such as glycoproteins, which are interesting targets for example for vaccines against emerging viruses. For instance, the SARS-CoV2 'Spike' protein responsible for COVID-19 is a glycoprotein. Therefore, there is a pressing need to provide novel systems capable of presenting large proteins of vaccine interest (such as tumour or pathogen antigens) with their post-translational modifications, and having the ability of inducing strong immunogenic responses.

SUMMARY OF THE INVENTION

The present invention fulfils this need. Indeed, the present Inventors have developed an original and efficiently adaptable system combining the ADDomer protein presenting capacity with a highly modulable binary tag-tag partner, altogether having valuable properties, especially for vaccines. The Inventors have surprisingly demonstrated that a peptide tag can be efficiently inserted in the external loops of the penton base protomer constituting the ADDomer, without impairing the ADDomer structure. The data show for the first time that the inserted peptide tag retains its capacity to covalently make bond to its binding partner that has been fused to a protein or a protein fragment of therapeutic interest (called a cargo), and resulting in a stable and functional ADDomer structure decorated with the therapeutic protein or fragment (displaying them in up to 60 copies of the cargo on the surface of the ADDomer). This novel presenting system has been in particular validated with a wide variety of cargos, including: (i) peptide epitope (as herein demonstrated with A2L tumour epitope),

(ii) large well-folded functional protein (as herein exemplified using a fluorescent protein, mCherry),

(iii) large antigens (as herein demonstrated with "melan A" tumour antigen);

(iv) large well-folded post-translationally modified proteins such as viral antigens (herein illustrated with SARS-CoV2 glycosylated fragments of the spike protein and fragments thereof, including the glycosylated Spike Receptor Binding Domain (RBD)).

Importantly, the data demonstrate that it is possible to bind multiple distinct cargos on the same ADDomer particle, allowing for instance the display of several antigen variants, highly useful for e.g. multivalent vaccines, cancer vaccines or medicaments.

In addition, the data obtained by the Inventors confirmed that the cargos displayed by the ADDomer are fully functional and immunogen. Notably, the large viral SARS-CoV2-RBD displayed on the ADDomer (ADD- RBD) was capable of specifically recognizing and strongly and quasi-irreversibly binding to its natural cell receptor (the ACE2 receptor) and of being recognized by serum from Covid-19 convalescent patients or anti-RBD monoclonal antibodies. In addition, vaccination of animals with ADD-RBD induced a potent and specific anti-SARS-CoV2-RBD response, and particularly with an ADD pre-immunity beneficial to the response against the displayed antigen. Moreover, the Inventors showed that the antigens displayed on the ADDomer can be efficiently taken up by different subsets of dendritic cells, thus triggering a cellular immune response beneficial in both infectiology and vaccination against cancer. Altogether, the data reveal the biological significance of this novel and adaptable cargo-presenting system and validate crucial applications especially in vaccination, but also in targeted drug-delivery within cells, active compound screening, and antibody detection. In particular SARS-Cov2-RBD displayed on the ADDomer and ACE2 receptor immobilized on a surface can be directly used as a system for active compounds screening (e.g. medicaments).

Thus, the present invention concerns an engineered protein comprising:

(i) an adenovirus penton base protomer comprising a peptide tag in the variable loop and/or in the RGD loop, and

(ii) at least one protein or at least one protein fragment fused to a binding partner of the peptide tag ; where the peptide tag of (i) and the binding partner of the peptide tag of (ii) are covalently bound to each other via an isopeptide bond.

Advantageously, the peptide tag is SpyTag and/or the binding partner is SpyCatcher or any variant thereof. In particularly preferred embodiments, the protein or the protein fragment of (ii) has at least one post- translational modification. The protein of (ii) is preferably an antigen, or the protein fragment of (ii) is preferably a fragment of an antigen.

The invention further relates to an adenovirus penton base protomer comprising a SpyTag peptide in the variable loop and/or in the RGD loop.

In the context of the present invention, and for the sake of completeness, it is also presently described, but not as part of the invention, a post-translationally-modified antigen or a post-translationally-modified domain of an antigen, fused to SpyCatcher; wherein the post-translationally modified antigen is preferably a post-translationally-modified antigen of an enveloped virus, more preferably a post-translationally- modified receptor binding protein of an enveloped virus; or wherein the post-translationally-modified domain of an antigen is preferably a post-translationally-modified domain of an antigen of an enveloped virus, more preferably a post-translationally-modified domain of a receptor binding protein of an enveloped virus; preferably wherein the post-translational modification is selected from glycosylation, phosphorylation, acylation, carboxylation, and any combination thereof, most preferably glycosylation.

The invention further concerns the engineered protein according to the invention or an immunogenic composition comprising said engineered protein, for use as a medicament.

The invention further concerns the engineered protein according to the invention or an immunogenic composition comprising said engineered protein, for use as a vaccine, preferably as a vaccine against cancer or infectious disease, in particular in a subject having already been exposed to an adenovirus.

The invention also relates to an in vitro use of the adenovirus penton base protomer of the invention for increasing immunogenicity of immunogenic protein(s), as far as said immunogenic protein(s) is(are) fused to the binding partner of the peptide tag(s) which is(are) in the adenovirus penton base protomer.

The invention also relates to an in vitro use of the engineered protein according to the invention for screening therapeutic molecules/compounds, preferably antiviral molecules/compounds.

The invention also relates to an In vitro use of the engineered protein according to the invention for detecting, in a biological sample from a subject, the presence of antibodies to a pathogen containing the protein or protein fragment as defined in (ii).

DETAILED DESCRIPTION OF THE INVENTION

The present Inventors have developed an original and efficiently adaptable system combining the ADDomer protein presenting capacity with a highly modulable binary tag-tag partner, altogether having valuable properties, especially for vaccines. The Inventors have surprisingly demonstrated that a peptide tag can be efficiently inserted in the external loops of the penton base protomer constituting the ADDomer, without impairing the ADDomer structure. The data show for the first time that the inserted peptide tag retains its capacity to covalently make bond to its binding partner that has been fused to a protein or a protein fragment of therapeutic interest (called a cargo), and resulting in a stable and functional ADDomer structure decorated with the therapeutic protein or fragment (displaying them in up to 60 copies of the cargo on the surface of the ADDomer). This novel presenting system has been in particular validated with a wide variety of cargos, including:

(i) peptide epitope (as herein demonstrated with A2L tumour epitope),

(iii) large antigens (as herein demonstrated with "melan A" tumour antigen);

(iv) large well-folded post-translationally modified proteins such as viral antigens (herein illustrated with SARS-CoV2 glycosylated fragments of the spike protein and fragments thereof, including the glycosylated Spike Receptor Binding Domain (RBD)). Importantly, the data demonstrate that it is possible to bind multiple distinct cargos on the same ADDomer particle, allowing for instance the display of several antigen variants, highly useful for e.g. multivalent vaccines, cancer vaccines or medicaments.

Definitions

Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in chemistry, biochemistry, cellular biology, molecular biology, and medical sciences.

The terms "virus" and "viral vector" are herein used interchangeably and are to be understood broadly as meaning a vehicle comprising at least one element of a wild-type virus genome that may be packaged into a viral particle or into the viral particle itself. These terms include viral vector (e.g. DNA viral vector) as well as viral particles generated thereof. Usually, a virus comprises a DNA or RNA viral genome packaged into a viral capsid and, in the case of an enveloped virus, lipids and other components (e.g. host cell membranes, etc). The terms "virus" and "viral vector" encompass wild-type and engineered viruses/modified viruses.

As used herein, "adenovirus" or "Ad" refers to a group of viruses belonging to the Adenoviridae family. Generally speaking, adenoviruses are non-enveloped and their genome consists of a single molecule of linear, double stranded DNA that codes for more than 30 proteins including the regulatory early proteins participating in the replication and transcription of the viral DNA which are distributed in 4 regions designated El to E4 (E denoting "early") dispersed in the adenoviral genome and the late (L) structural proteins (see e.g. Evans and Hearing, 2002, in "Adenoviral Vectors for Gene Therapy" pp 39-70, eds. Elsevier Science). El, E2 and E4 are essential to the viral replication whereas E3 is dispensable and appears to be responsible for inhibition of the host's immune response in the course of adenovirus infection. Adenoviruses can be found in human and various animals (e.g. canine, ovine, bovine, simian, etc.). Adenoviruses for use herein can be obtained from a variety of human or animal adenoviruses (e.g. canine, ovine, simian, etc.) and any serotype can be employed. It can also be a chimeric adenovirus (W02005/001103). A skilled person will recognize that elements derived from multiple serotypes can be combined in a single adenovirus. Desirably, the adenoviral vector originates from a human Ad, including those of rare serotypes, or from a primate (e.g. chimpanzee, gorilla). Representative examples of human adenoviruses include subgenus C (e.g. Ad2 Ad5 and Ad6), subgenus B (e.g. Ad3, Ad7, Adll, Adl4, Ad34, Ad35 and Ad50), subgenus D (e.g. Adl9, Ad24, Ad26, Ad48 and Ad49) and subgenus E (Ad4). Representative examples of chimp Ad include without limitation AdCh3 (Peruzzi et al., 2009, Vaccine 27: 1293-300) and AdCh63 (Dudareva et al, 2009, Vaccine 27: 3501-4) and any of those described in the art (see for example, W02010/086189; W02009/105084; W02009/073104; W02009/073103;

W02005/071093; and W003/046124). An exemplary genome sequence of human adenovirus type 5 (Ad5) is found in GenBank Accession M73260 and in Chroboczek et al. (1992, Virol. 186: 280-5).

The terms "enveloped virus" herein refers to a virus having a viral envelope, i.e. an outermost layer (generally a lipid bilayer) protecting the genetic material in their life-cycle when traveling between host cells. The viral envelopes are typically derived from portions of the host cell membranes (phospholipids and proteins), but include some viral glycoproteins. The glycoproteins may help viruses avoid the host immune system. Glycoproteins on the surface of the envelope serve to identify and bind to receptor sites on the host's membrane. The viral envelope then fuses with the host's membrane, allowing the capsid and viral genome to enter and infect the host. Generally, enveloped viruses also have a capsid (another protein layer), between the envelope and the genome. The lipid bilayer envelope is relatively sensitive to desiccation, heat, and amphiphiles such as soap and detergents, making enveloped viruses generally easier to sterilize than non-enveloped viruses, and having limited survival outside host environments. Enveloped viruses possess great adaptability and can change in a short time in order to evade the immune system. Enveloped viruses can cause persistent infections. Enveloped viruses include:

DNA viruses (such as Herpesviruses (belonging to the Herpesviridae family, which is a large family of DNA viruses comprising more than 130 known herpesviruses, some of them from mammals (among which nine herpesvirus types are known to primarily infect humans, including herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (or HHV-3), Epstein-Barr virus (EBV or HHV-4), and human cytomegalovirus (HCMV or HHV-5), birds, fish, reptiles, amphibians, and mollusks); Poxviruses (belonging to the Poxviridae family, comprising four genera that may infect humans: orthopoxvirus, parapoxvirus, yatapoxvirus, molluscipoxvirus); Hepadnaviruses (belonging to the family of Hepadanviridae, comprising Hepatitis B virus); Asfarviridae (a family of double-stranded DNA viruses comprising African swine fever virus (ASFV)); etc. );

RNA viruses (such as Flaviviruses (belonging to the Flaviviridae family, which is a family of enveloped positive-strand RNA viruses which mainly infect mammals and birds, comprising flavivirus); Alphaviruses (belonging to the Togaviridae family and comprising e.g. Venezuelan equine encephalitis virus (VEEV), chikungunya virus (CHIKV), Ross River virus (RRV), o'nyong- nyong virus (ONNV), Mayaro virus (MAYV), eastern and western equine encephalitis, Sindbis viruses, etc.); Arenaviruses (belonging to the Arenaviridae family and comprising Gairo virus, Gbagroube virus, Ippy virus, Kodoko virus, Lassa virus, Lujo virus, Luna virus, Lunk virus, Lymphocytic choriomeningitis virus, Merino Walk virus, Menekre virus, Mobala virus, Morogoro virus, Mopeia virus, Wenzhou virus, Tacaribe virus, Amapari virus, Chapare virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Patawa virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, etc.);; Coronaviruses (belonging to the Coronaviridae family); Hepatitis D; Orthomyxoviruses (belonging to the Orthomyxoviridae family, which is a family of negative-sense RNA viruses including seven genera: Alphainfluenzavirus, Betainfluenzavirus, Deltainfluenzavirus, Gammainfluenzavirus, Isavirus, Thogotovirus, and Quaranjavirus); Paramyxoviruses (belonging to the Paramyxoviridae family, which is a family of negative-strand RNA viruses); Rhabdovirus (belonging to the Rhabdoviridae family, which is a family of negative-strand RNA viruses); Bunyaviruses (belonging to the order of Bunyavirales, which is an order of segmented negative- strand RNA viruses); Filoviruses (belonging to the Filoviridae family); etc.) and

Retroviruses (belonging to the Retroviridae family, which is a family of viruses that transcribes back its RNA genome into DNA with a reverse transcriptase).

As used herein, "coronavirus" refers to a group of viruses belonging to the Coronaviridae family. Generally speaking, coronaviruses are enveloped viruses with a helically symmetrical capsid. They have a single- stranded, positive-sense RNA genome and are capable of infecting cells in birds and mammals. The morphology of the virions is typical, with a halo of protein protuberances ('Spike') which gave them their name of 'crown virus'. Among the four genera of coronaviruses (Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus), the Betacoronavirus genus (b-CoVs or Beta-CoVs) comprises viruses infecting animals and/or humans. Coronaviruses usually comprise four structural proteins, including spike (S or S protein), envelope (E), membrane (M), and nucleocapsid (N) proteins.

As used herein, the term "betacoronavirus" designates any virus belonging to the Betacoronavirus genus (b-CoVs or Beta-CoVs) within the Coronaviridae family, in particular any betacoronavirus belonging to one of the four lineages designated as A, B, C and D. It designates a betacoronavirus infecting animals (preferably a mammal) and/or humans. In particular, this designation includes the betacoronaviruses infecting human organisms selected from the group consisting of OC43, FIKU1, SARS-CoV-1, SARS-CoV-2 and MERS-CoV. The Betacoronavirus genus is subdivided into four lineages designated as A, B, C and D: Lineage A (also designated as subgenus Embecovirus) includes FICoV-OC43 and FICoV-FIKUl, virus able to infect various species; Lineage B (also designated as subgenus Sarbecovirus) includes SARS-CoV-1, SARS- CoV-2, and Bat SL-CoV-WIVl; Lineage C (also designated as subgenus Merbecovirus) includes Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4), Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), and MERS-CoV, able to infect notably camels and humans; Lineage D (also designated as subgenus Nobecovirus) includes Rousettus bat coronavirus HKU9 (BtCoV-HKU9).

In humans, coronavirus infections can cause respiratory pathologies associated with symptoms similar to the common cold, bronchiolitis and more serious diseases such as the Severe Acute Respiratory Syndrome caused by SARS-CoV-1, which generated an epidemic in 2003, and the Middle Eastern Respiratory Syndrome caused by MERS-CoV, which generated an epidemic in 2012. SARS-CoV-2 (and its variants) is the betacoronavirus causing the coronavirus epidemic of 2019-2021 (ongoing), generating the form of pneumonia known as coronavirus disease 2019 or COVID-19. According to the World Health Organization (WHO), by March 28, 2021, 126372442 cases of COVID-19 have been confirmed and 2 769 696 patients have died worldwide. This epidemic was declared a public health emergency of international concern by WHO on January 30, 2020.

"SARS-CoV2" or "SARS-CoV-2" or "SARS-CoV2 virus" herein refers to Coronavirus 2 which causes Severe Acute Respiratory Syndrome. SARS-CoV2 belongs to the species Coronavirus, in the genus Betacoronavirus and family Coronaviridae. "SARS-CoV2" herein means the SARS-CoV2 virus as originally identified as well as all SARS-CoV2 variants. As used herein, the SARS-CoV2 virus as originally identified refers to the SARS-CoV2 virus identified for the first time in Wuhan, China, and sequenced in early 2020 by a team from Fudan University in Shanghai (Zhou, P. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature Mar;579(7798):270-273. doi: 10.1038/s41586-020-2012- 7.(2020)). As used herein, the SARS-CoV2 virus variants refer to viruses related to this first identified viral strain, which appeared subsequently, and in particular the following SARS-CoV2 variants defined with reference to this first identified viral strain in Wuhan: i. the "Wuhan-like" strains; ii. the hCoV-19/France/ARA-104350/2020 strain (GISAID ID: EPI_ISL_683350) of lineage B.l (this strain has at least the D614G mutation in its spike protein; it is considered today as the wild type strain circulating in Europe, in comparison with the variants cited below); iii. the British variant strain hCoV-19/France/ARA-SC2118/2020 (GISAID: EPI_ISL_900512) of lineage B.l.1.7 (or alpha variant); iv. the South African strain (501Y.V2.FIV001) of lineage B.l.351 (or beta variant); v. the Brazilian variant strains of lineages B.l.1.28 and P.l (or gamma variant); vi. the Indian variant strains of lineages B.l.617.2 (or delta variant); vii.the variant strains of lineages B.l.1.529 (or omicron variant); viii. the SARS-CoV2 variant N501Y; ix. the SARS-CoV2 variant E484K; x. the SARS-CoV2 variant K417N; xi. the SARS-CoV2 variant K417T; xii. the SARS-CoV2 variant T547K; xiii. any other SARS-CoV2 variants yet to be identified; xiv. any combination thereof of i. to xiii

By "Spike" or "S protein" or "S" it is herein referred to a structural protein of a coronavirus. The spike protein is generally composed of two subunits, SI and S2, that are derived from a single protein by proteolytic cleavage. The SI subunit contains a receptor-binding domain (RBD) that recognizes and binds to the host receptor angiotensin-converting enzyme 2 (ACE2), while the S2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. The spike protein thus plays a key role in the receptor recognition, cell membrane fusion process, and entry in the host cell. With a size of 180-200 kDa, the S protein comprise an extracellular N-terminus, a transmembrane (TM) domain anchored in the viral membrane, and a short intracellular C-terminal segment. The S protein is preferably post-translationally modified, preferably by glycosylation (glycosylated S protein). S protein trimers visually form a characteristic bulbous, crown-like halo surrounding the viral particle. The Spike protein of SARS-COV2 has been well characterized. The total length of SARS-CoV2 S is 1273 amino acids and consists of a signal peptide (amino acids 1-13) located at the N-terminus, the SI subunit (amino acids 14-685), and the S2 subunit (amino acids 686-1273); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the SI subunit, there is an N-terminal domain (amino acids 14-305) and a receptor-binding domain (RBD, amino acids 319-541); the fusion peptide (FP) (amino acids 788-806), heptapeptide repeat sequence 1 (HR1) (amino acids 912-984), HR2 (amino acids 1163-1213), TM domain (amino acids 1213-1237), and cytoplasm domain (amino acids 1237-1273) comprise the S2 subunit. By "RBD domain of the Spike protein", it is herein referred to a receptor-binding domain (RBD) (i.e. a domain that recognizes and binds to the host cell receptor) of the S protein of any virus, preferably of any coronavirus, preferably of any betacoronavirus, more preferably of any SARS-CoV virus. The RBD domain is preferably post-translationally modified, preferably by glycosylation (glycosylated RBD domain).

The terms "variant", or "mutant", "derivative" can be used interchangeably to generally refer to a component or a specie (protein, protein fragment, polypeptide, polynucleotide, oligonucleotide, nucleoside, nucleotide, vector, virus, etc.) exhibiting one or more modification(s) with respect to a reference component (e.g. the wild-type component as found in nature as originally identified, i.e. the "original" corresponding component). A nucleotide or nucleoside variant can have a modified base and/or a modified sugar and/or a modified linkage. With respect to polypeptide and polynucleotide variants, any modification(s) can be envisaged, including substitution, insertion, deletion, and any combination thereof, of one or more nucleotide/amino acid residue(s). With respect to viruses, any modification(s) of the genome and/or proteome can be envisaged, including substitution, insertion, deletion, and any combination thereof, of one or more nucleotide/amino acid residue(s). The variant can be naturally occurring or artificial (e.g. mutated and/or engineered). As used herein, variants are preferably naturally occurring. Examples of such naturally occurring variants in the context of viruses includes naturally occurring variants of enveloped virus (Coronaviridae viruses, Flaviviridae viruses, Alphaviruses, Orthomyxoviridae (in particular Alphainfluenzaviruses), Filoviridae, Bunyaviridae, Arenaviridae, Retroviridae, etc.), in particular coronavirus variants (notably SARS-CoV virus variants, in particular SARS CoV2 variants, such as SARS-CoV2 variants listed above, including "Wuhan-like" strains, the hCoV- 19/France/ARA-104350/2020 strain (GISAID ID: EPI_ISL_683350) of lineage B.l, the British variant strain hCoV-19/France/ARA-SC2118/2020 (GISAID: EPI_ISL_900512) of lineage B.l.1.7, the South African strain (501Y.V2.FIV001) of lineage B.1.351, the Brazilian variant strains of lineages B.l.1.28 and P.1, SARS-CoV2 N501Y, SARS-CoV2 E484K, SARS-CoV2 K417N, SARS-CoV2 K417T, any combination of those variants, and any other SARS-CoV2 variants yet to be identified).

When several mutations are contemplated, they can concern consecutive residues and/or non- consecutive residues. Preferred are variants (e.g. respectively protein variants, protein fragment variants, virus variants) that retain a degree of sequence identity of at least 80% with the reference component (e.g. respectively the corresponding "original" protein, the corresponding "original" protein fragment, the corresponding "original" virus). For illustrative purposes, "at least 80% identity" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiment, at least 80% identity also encompasses 100% identity.

The term "naturally occurring" or "native" or "wild type" is used to describe a biological molecule or organism that can be found in nature as distinct from being artificially produced by human. For example, a naturally occurring, native or wild-type virus, as well as a virus variant, refers to a virus (in particular an adenovirus) which can be isolated from a source in nature (infected subject or infected tissue/cells from an infected subject) or which has previously been isolated from a source in nature and can now be obtained from specific collections (e.g. ECCAC, ATCC, CNCM, etc) in which it has been deposited. A biological molecule or an organism which has been intentionally modified by human intervention in the laboratory is not naturally occurring. Representative examples of "non-naturally occurring viruses" include, among many others, mutated viruses and engineered viruses. Representative examples of "non- naturally occurring proteins" include, among others, engineered proteins.

As used herein, "engineered protein" means a protein which has been modified by one or more substitution(s) (including point substitution(s) as well as substitution of more than one amino acid residue), insertion(s), deletion(s), fusion(s) and any combination thereof. Substitution comprises replacement of at least one amino acid residue by at least one distinct amino acid residue, preferably by one or more foreign protein, by one or more foreign protein fragment, or by one or more foreign polypeptide (i.e. wherein the protein, protein fragment, or polypeptide originates from another species). Insertion comprises insertion (addition) of at least one amino acid residue, preferably insertion of one or more foreign protein, of one or more foreign protein fragment, or of one or more foreign polypeptide (i.e. wherein the protein, protein fragment, or polypeptide originates from another species). Fusion comprises covalently bonding at least one amino acid residue, preferably covalently bonding one or more foreign protein(s), one or more foreign protein fragment(s), or one or more foreign polypeptide(s) (i.e. wherein the protein, protein fragment, or polypeptide originates from another species), using a linker sequence or not. Preferably, the "foreign protein", "foreign protein fragment", or "foreign polypeptide" that is inserted and/or fused in the engineered protein is not expressed by a naturally-occurring adenovirus genome. More specifically, it can be of human origin or not (e.g. of animal (preferably mammalian), bacterial, yeast, or viral origin except from the same adenovirus serotype), preferably human. Such a foreign protein/protein fragment/polypeptide may be a native protein/protein fragment/polypeptide or any variant thereof obtained by mutation, deletion, substitution, insertion, fusion, and any combination thereof, of one or more amino acid (and/or one or more modified amino acid).

Substitution(s), insertion(s), deletion(s), and fusion(s) can be generated by a number of ways known to those skilled in the art, such as site-directed mutagenesis, PCR mutagenesis, DNA shuffling, chemical synthetic techniques (e.g. resulting in a synthetic nucleic acid molecule encoding the engineered protein), and/or using restriction sites, etc.

As used herein, "identity" or "sequence identity" means an exact sequence match between two polypeptides or amino acids, or between two nucleic acid molecules or oligonucleotides. The percent identities referred to in the context of the disclosure of the present invention are determined after optimal alignment of the sequences to be compared, which may therefore comprise one or more insertions, deletions, truncations and/or substitutions. This percent identity may be calculated by any sequence analysis method well-known to the person skilled in the art. The percent identity may be determined after global alignment of the sequences to be compared taken in their entirety over their entire length. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch (A general method applicable to the search for similarities in the amino acid sequence of two proteins, J. Mol. Biol., 1970, Mar;48(3):443-453). For nucleotide sequences, the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: "Gap open" equal to 10.0, "Gap extend" equal to 0.5, and the EDNAFULL matrix (NCBI EMBOSS Version NUC4.4). For amino acid sequences, the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: "Gap open" equal to 10.0, "Gap extend" equal to 0.5, and the BLOSUM62 matrix. Preferably, the percent identify as defined in the context of the present invention is determined via the global alignment of sequences compared over their entire length. For illustrative purposes, "at least 80% identity" herein means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

The terms "polynucleotide", "nucleic acid molecule", and "nucleic acid" are used interchangeably herein and are understood as a polymeric or oligomeric macromolecule made from nucleotide monomers (preferably from at least 5 nucleotide monomers, also called nucleotide residues). Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. Nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids (mixed polyribo-polydeoxyribonucleotides). These terms encompass single or double-stranded, linear or circular, natural or synthetic, unmodified or modified versions thereof (e.g. genetically modified polynucleotides; optimized polynucleotides), sense or antisense polynucleotides, chimeric mixture (e.g. RNA-DNA hybrids). Moreover, a polynucleotide may comprise non-naturally occurring nucleotides and may be interrupted by non-nucleotide components. Exemplary DNA nucleic acids include without limitations, complementary DNA (cDNA), genomic DNA, plasmid DNA, DNA vector, viral DNA (e.g. viral genomes, viral vectors), oligonucleotides, probes, primers, satellite DNA, microsatellite DNA, coding DNA, non-coding DNA, antisense DNA, and any mixture thereof. Exemplary RNA nucleic acids include, without limitations, messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), RNA vector, viral RNA, guide RNA (gRNA), antisense RNA, coding RNA, non-coding RNA, antisense RNA, satellite RNA, small cytoplasmic RNA, small nuclear RNA, etc. Polynucleotides described herein may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as those that are commercially available from Biosearch, Applied Biosystems, etc.) or obtained from a naturally occurring source (e.g. a genome, cDNA, etc.) or an artificial source (such as a commercially available library, a plasmid, etc.) using molecular biology techniques well known in the art (e.g. cloning, PCR, etc.). The nucleic acids, can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).

The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bond- linked polymer of amino acids, regardless of length or post-translational modification. These terms preferably refer to polymers of amino acid residues comprising at least six amino acids covalently linked by peptide bonds. The polymer can be linear, branched or cyclic. The polymer may comprise naturally occurring and/or amino acid analogues and it may be interrupted by non-amino acids. No limitation is placed on the maximum number of amino acids comprised in a polypeptide. As a general indication, the term refers to both short polymers (typically designated in the art as peptide, or protein fragment) and to longer polymers (typically designated in the art as polypeptide or protein). This term encompasses native polypeptides, modified polypeptides (also designated derivatives, analogues, variants, mutants), polypeptide fragments, polypeptide multimers (e.g. dimers), mutated polypeptides, engineered polypeptides, fusion polypeptides among others. A polypeptide is understood to be any translational product of a polynucleotide regardless of size, and whether glycosylated or not, and includes peptides and proteins. Polypeptides/Proteins usable herein (including protein derivatives, protein variants, protein fragments, protein domains, protein epitopes and protein domains) can be further modified by chemical or enzymatic modification. This means that such a chemically modified polypeptide or enzymatically modified polypeptide comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such chemical or enzymatic modifications include post-translational modifications. Chemical or enzymatic modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, increased water solubility, increased activity, enhanced properties, labelling, etc. As a general indication and without being bound therein, if the amino acid polymer contains more than 50 amino acid residues, it is preferably referred to as a polypeptide or a protein, whereas if the polymer consists of 50 or fewer amino acids, it is preferably referred to as a "peptide". The reading and writing senses of an amino acid sequence of a polypeptide, protein and peptide as used herein are the conventional reading and writing senses. The reading and writing convention for amino acid sequences of a polypeptide, protein and peptide places the amino terminus on the left, with the sequence then being written and read from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), from left to right.

The terms "peptide" or "protein fragment" or "part of a peptide or protein" or "protein domain" herein mean a portion of a peptide or protein, i.e., a portion of the sequence of consecutive amino acids making up said peptide or protein (referred to as the peptide or protein from which the fragment is derived). When the fragment is a peptide, protein, the fragment preferably comprises at least 6 consecutive amino acids of the peptide or protein from which it is derived; more preferably at least 8 consecutive amino acids, more preferably at least 10 consecutive amino acids, more preferably at least 12 consecutive amino acids, more preferably at least 15 consecutive amino acids, more preferably at least 20 consecutive amino acids, more preferably at least 30 consecutive amino acids of the peptide or protein from which it is derived. When the fragment is a peptide or protein fragment, the fragment preferably has a three- dimensional structure, under non-denaturing conditions (e.g., conditions that are usually non-denaturing for proteins, especially in the absence of denaturing and/or chaotropic agents). When the fragment is a peptide or protein fragment, the fragment is preferably a functional fragment. "Functional fragment" means any peptide or protein fragment, having at least one of the original functions of the peptide or protein from which said fragment is derived. Preferably, the functional fragment performs said function with an efficiency equal to at least 30% of that of said peptide or protein or molecule, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, preferably at least 100% of the efficacy of said peptide or protein. Examples of protein fragments (in particular of functional fragments) include e.g. protein domains, protein epitopes, etc. Fragments usable herein (including protein fragments, peptide fragment, protein domain, protein epitopes and protein domains) can be further modified by chemical or enzymatic modification (e.g. post-translational modification(s)). When the fragment is a peptide or protein fragment, a chemically/enzymatically modified fragment comprises other chemical groups than the 20 naturally occurring amino acids (e.g. can comprise post-translational modification(s)).

By "three-dimensional structure" or "tertiary structure" it is herein referred to the intrinsic folding of a molecule in space. A molecule with a three-dimensional structure is a molecule with a stable spatial configuration, (commonly called folding), which is its own and which is, in general, intimately linked to its function. When this structure is dissociated by the use of denaturing or chaotropic agents, the molecule is said to be denatured and loses its function. In particular, a three-dimensional structure is a structure with little flexibility. In contrast, a non three-dimensional structure can adopt a dynamic set of configurations that constantly change overtime. In the case of a protein, a peptide, or a fragment of these, the three-dimensional structure is the folding of the polypeptide chain in space. In this case, the three- dimensional structure is not a linear chain of amino acids that can adopt a dynamic set of configurations constantly changing over time (it is not a linear succession of amino acids without any spatial configuration). The three-dimensional structure of proteins, peptides, mixed molecules comprising a protein or a peptide, or fragments of these, is maintained by different interactions which can be: covalent interactions (disulfide bridges between cysteines); electrostatic interactions (ionic bonds, hydrogen bonds); van der Waals interactions; interactions with the solvent and the environment (ions, lipids...).

As used herein, "post-translational modification" refers to a chemical or enzymatic modification occurring naturally or not on a protein or a protein fragment, after or concomitantly to protein translation (e.g. biological or biochemical synthesis, e.g. using cellular machinery), or after or concomitantly to protein synthesis (e.g. artificial and/or chemical synthesis). This means that at least one of the naturally occurring amino acid of the protein or protein fragment is modified by the addition of at least one chemical group and/or the modification (including, but not limited to, the removal) of at least one chemical group of the naturally occurring amino acid. Examples of such chemical or enzymatic modifications include without limitation glycosylation, phosphorylation, acylation, carboxylation, acetylation, biotinylation, hydroxylation, lipoylation, amidation, ubiquitination, sumoylation, deamination, etc. By "post-translationally modified protein" it is herein referred to a protein having at least one (i.e. one or more) post-translational modification. By "post-translationally modified protein fragment" it is herein referred to a protein fragment having at least one (i.e. one or more) post- translational modification.

The term "penton base protein" or "penton base protomer" as used herein refers to an adenoviral protein that assembles into the so called "penton protein". Each penton protein comprises five penton base proteins. The penton base protein is one of the three proteins forming the adenoviruses coat. The other proteins are hexon and fibre. The penton base protein comprises flexible loops having the characteristic of being exposed to the surface. Among these loops, are the variable loop and the RGD loop. Since these loops are not conserved, they have the property to be dispensable and therefore may be targets for inserting "foreign sequence" or may even be replaced by "foreign sequence". Penton base proteins that are used in the present invention originate from adenovirus specific to any mammalian species. Preferably the adenovirus is a human or non-human great ape adenovirus (the latter preferably selected from Chimpanzee (Pan), Gorilla (Gorilla), orangutans (Pongo), Bonobo (Pan paniscus), and common Chimpanzee (Pan troglodytes)). It is understood by the skilled person that the penton base proteins of different adenoviruses may vary in their amino acid sequence. All such naturally occurring variants are encompassed by the term "penton base protein". Additionally, the term encompasses artificial variants that comprise insertion, deletions, substitutions, mutations, and any combination thereof, of the naturally occurring penton base protein sequence. Such mutations are in addition to the modifications of the variable loop and/or RGD loop described in more detail below. Any such artificial variants are comprised herein as long as the artificially modified penton base protein assembles into the penton protein and preferably as long as said penton protein assemble into VLPs. Preferably, the artificial variants of the penton base protein have at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity to a naturally occurring penton base protomer. Preferred penton base proteins are those having at least 85%, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 1 to 11. Most preferred penton base proteins are those indicated in SEQ ID NOs: 1 to 6 and 11, more preferably those indicated in SEQ ID NOs: 1 to 6. An engineered penton base protein, such as the one used in the engineered protein of the present invention, differ in sequence from naturally occurring penton base proteins by amino acid insertions, deletions and mutations as outlined in more detail below.

As used herein, the term "RGD-loop" refers to a polypeptide sequence of between 10 to 200 amino acids that is located in the adenovirus penton base protomer and contains the RGD motif. As used herein, the "RGD-motif" is a three amino acid long polypeptide composed of arginine, glycine and aspartic acid. This motif was originally identified in fibronectin as mediating binding to integrins. The RGD-motif is also present in many other receptors and mediates both cell-substrate and cell-cell interactions.

Preferred RGD loop sequences are those having at least 85%, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 17 to 27. Most preferred RGD loop sequences are those indicated in SEQ ID NOs: 17 to 22 and 27, more preferably in SEQ ID NOs: 17 to 22. A particularly preferred RGD loop sequence in the one located between amino acid residues 310 and 349 in the penton base protomer of SEQ ID NO 1.

The RGD-loop can be divided in three consecutive subloops: the "first RGD-subloop", the "enlarged RGD motif", and the "second RGD-subloop". The RGD-loop may thus be represented by the formula "First RDG- subloop - Enlarged RGD motif - Second RDG-subloop". The "first RGD-subloop" herein refers to a polypeptide sequence of between 10 to 40 amino acids that is located directly N-terminally to the "enlarged RGD motif" comprised in the penton base protomer i.e. directly adjacent to the "enlarged RGD motif" with not any one amino acids in between. Its C-terminal end within the penton base protomer is thus determined by the beginning of the enlarged RGD motif. Preferred first RGD-subloop sequences are those having at least 85%, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 63 to 73. Most preferred first RGD-subloop sequences are those indicated in SEQ ID NOs: 63 to 68 and 73, more preferably in SEQ ID Nos: 63 to 68. The term "second RGD-subloop" as used herein refers to a polypeptide sequence of between 10 to 35 amino acids that is located directly C-terminally to the "enlarged RGD motif" comprised in the penton base protomer ie directly adjacent to the "enlarged RGD motif" with not any one amino acids in between. Its C-terminal end within the protomer can thus be defined by the sequence that is located C-terminally of its C-terminal end, which is conserved among different adenoviruses. Preferred second RGD-subloop sequences are those having at least 85%, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 85 to 95. Most preferred second RGD-subloop sequences are those indicated in SEQ ID NOs: 85 to 90 and 95, more preferably in SEQ ID Nos: 85 to 90. The term "enlarged RGD motif" as used herein refers to a polypeptide sequence of 14 amino acids encompassing the RGD motif. The enlarged RGD motif is defined by a sequence encompassing the RGD motif and comprising the 5 amino acids located N-terminally of the R of the RGD motif (i.e. the 5 N-terminal amino acids directly adjacent to the RGD motif) and the 6 amino acids located C-terminally of the D of the RGD motif (i.e. the 6 C-terminal amino acids directly adjacent to the RGD motif). Preferred enlarged RGD motif sequences are those having at least 85%, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 74 to 84. Most preferred second RGD-subloop sequences are those indicated in SEQ ID NOs: 74 to 79 and 84, more preferably in SEQ ID Nos: 74 to 79. The enlarged RGD-motif in the penton base protomers of the engineered polypeptides of the present invention may be intact, absent or mutated in a way that the penton base protomer does no longer bind to integrins. In specific embodiments, the RGD-motif and/or the enlarged RGD-motif of the penton base protomers of the engineered polypeptides of the present invention no longer binds to integrins, in particular when a foreign sequence is inserted in the first RGD- subloop, or in the second RGD-subloop, or in the enlarged RGD motif, or in the RGD motif, or in place of the RGD-motif, or any combination thereof).

The term "variable loop" or "V loop" as used herein corresponds to a polypeptide sequence of between 10 to 80 amino acids, located between the beta sheet b3 and the beta sheet b4 of the adenovirus penton base protein. Both the length and the amino acids composition of this loop are very variable amongst serotypes. Preferred V loop sequences are those having at least 85%, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more at least preferably 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 28 to 38. Most preferred V loop sequences are those indicated in SEQ ID NOs: 28 to 33 and 38, more preferably in SEQ ID Nos: 28 to 33.

The expression "between XX and YY amino acids" (XX and YY being integers) as used in the context of the present invention is meaning "from XX to YY amino acids", thus including the lower XX and the upper YY bounds of the interval.

The phrase "engineered polypeptide/protein capable of assembling into VLPs" or "assembles into a VLP" as used interchangeably in the context of the present invention refers to the ability of five penton base protomers to self-assemble into a penton protein and subsequently of twelve penton proteins to self-assemble into a spherically shaped particle, i.e. a virus-like particle (VLP). The ability to assemble and to maintain the penton protein or preferably the VLP structure can be ascertained by methods known in the art and described herein, in particular by electron microscopy (EM). Preferred conditions at which the capability to assemble into VLPs is assessed is 20°C and physiologic buffer conditions. In a further preferred embodiment the term encompasses engineered polypeptides that not only assemble into VLPs but maintain the quasi-spherical shape at temperatures above 20°C, preferably at temperatures above 30°C, preferably at temperatures above 40°C, more preferably above 45°C and even more preferably above 50°C. The integrity of the spherical shape can be assessed by EM, preferably under physiological buffer conditions. As used herein, "peptide tag" refers to a peptide sequence of between 6 to 400 amino acids (preferably between 8 to 300 amino acids, more preferably between 10 to 200 amino acids, more preferably between 12 to 82 amino acids). Examples of peptide tags include affinity tags, solubilization tags, chromatography tags, epitope tags, fluorescence tags, etc. Affinity tags are generally appended to proteins so that they can be purified from their crude biological source using an affinity technique. These include chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), poly(His) tag, etc. Solubilization tags are especially used for proteins expressed in chaperone-deficient species such as E. coli, to assist in the proper folding in proteins and keep them from precipitating. These include thioredoxin (TRX) and poly(NANP). Some affinity tags have a dual role as a solubilization agent, such as MBP, and GST. Chromatography tags are used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique. Often, these consist of polyanionic amino acids, such as FLAG-tag. Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes. Epitope tags include ALFA-tag, V5-tag, Myc-tag, FIA-tag, Spot-tag, T7-tag, NE-tag, etc. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification and any other appropriate technique. Fluorescence tags are notably used to give visual readout on a protein. GFP and its variants are the most commonly used fluorescence tags. More advanced applications of GFP include using it as a folding reporter (fluorescent if folded, colourless if not). Protein tags may allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FIAsFI-EDT2 for fluorescence imaging). Often tags are combined, in order to connect proteins to multiple other components. Peptide tag also herein include covalent peptide tags. Examples of covalent peptide tags include, but are not limited to:

Isopeptag (a peptide tag which binds covalently to pilin-C protein), preferably having the amino acid sequence shown in SEQ ID NO: 39 (TDKDMTITFTNKKDAE);

SpyTag (a peptide tag which binds covalently to SpyCatcher protein; WO2011098772) and variants thereof, preferably having an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 40 (AHIVMVDAYKPTK), the amino acid sequence shown in SEQ ID NO: 41 (VPTIVMVDAYKRYK; SpyTag002), the amino acid sequence shown in SEQ ID NO: 42 (RGVPHIVMVDAYKRYK; SpyTag003);

SnoopTag (a peptide tag which binds covalently to SnoopCatcher protein), preferably having the amino acid sequence shown in SEQ ID NO: 43 (KLGDIEFIKVNK);

SnoopTagJr (a peptide tag which binds to either SnoopCatcher protein or DogTag (mediated by SnoopLigase)), preferably having the amino acid sequence shown in SEQ ID NO: 44 (KLGSIEFIKVNK);

DogTag (a peptide tag which covalently binds to SnoopTagJr, mediated by SnoopLigase), preferably having the amino acid sequence shown in SEQ ID NO: 45 (DIPATYEFTDGKFIYITNEPIPPK). DogTag may be used both as peptide tag and as a binding partner (see below);

SdyTag (a peptide tag which binds covalently to SdyCatcher protein), preferably having the amino acid sequence shown in SEQ ID NO: 46 (DPIVMIDNDKPIT).

The peptide tag as used herein is preferably a covalent peptide tag, preferably selected in the group consisting of SpyTag, Isopeptag, SnoopTag, SnoopTagJr, DogTag, and SdyTag; more preferably the peptide tag is SpyTag. As used herein, "binding partner of a peptide tag" or "binding partner" refers to a peptide/polypeptide sequence of between 10 to 300 amino acids (preferably between 12 to 288 amino acid, more preferably between 20 to 200 amino acids). The peptide tag is preferably genetically grafted (i.e. fused) onto an engineered protein/protein fragment. The binding partner of a peptide tag spontaneously reacts with said peptide tag, to form an intermolecular isopeptide bond between the pair peptide tag-binding partner. Examples of binding partners of peptide tags include, but are not limited to: pilin-C protein and variants thereof (a binding partner which binds covalently to Isopeptag), preferably having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 47, more preferably having the amino acid sequence shown in SEQ ID NO: 47;

SpyCatcher (a binding partner which binds covalently to SpyTag) and variants thereof (such as SpyCatcher 002 and SpyCatcher 003; Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, Hytonen VP, Howarth M. Proc Natl Acad Sci U S A. 2019 Dec 10;116(52):26523-33. doi: 10.1073/pnas.1909653116.), preferably having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO: 48, SEQ ID NO:49, and SEQ ID NO:50, more preferably having the amino acid sequence shown in any of SEQ ID NO: 48, SEQ ID NO:49, and SEQ ID NO:50;

SnoopCatcher and variants thereof (a peptide tag which binds covalently to SnoopTag or SnoopTagJr), preferably having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 51, more preferably having the amino acid sequence shown in SEQ ID NO: 51;

DogTag and variants thereof (a binding partner which covalently binds to SnoopTagJr, mediated by SnoopLigase), preferably having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 45, more preferably having the amino acid sequence shown in SEQ ID NO: 45. DogTag may be used both as binding partner and as a peptide tag (above);

SdyCatcher and variants thereof (a binding partner which binds covalently to SdyTag), preferably having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 52, more preferably having the amino acid sequence shown in SEQ ID NO: 52.

The binding partner of a peptide tag as used herein is preferably selected in the group consisting of pilin- C protein, SpyCatcher, SnoopCatcher, DogTag, SdyCatcher, and any variant thereof; more preferably the binding partner is SpyCatcher or any variant thereof (Hatlem et al., Int. J. Mol. Sci. 2019, 20, 2129; doi:10.3390/ijms20092129; Keeble et al., Chem. Sci., 2020, 11, 7281-7291 DOI: 10.1039/d0sc01878c). The variants of the binding partner are well described in the literature and are available to the skilled person and do not need to be described in detail herein.

The term "fusion" or "fusion protein" as used herein refers to the combination of two or more polypeptides/peptides in a single polypeptide chain (i.e. at least one polypeptide/peptide is "fused to" an identical or distinct polypeptide/peptide). The fusion can be direct (i.e. without any additional amino acid residues in between) or through a linker (e.g. 3 to 30 amino acids long peptide composed of amino acid residues such as glycine, serine, threonine, asparagine, alanine and/or proline). It is within the reach of the skilled person to define accordingly the need and location of the translation-mediating regulatory elements (e.g. the initiator Met and codon STOP). For example, multi-epitopes from the same or different antigen(s) may be envisaged as well.

By "cargo" it is herein referred to a protein or a protein fragment comprised in the engineered protein of the invention. The cargo is preferably fused to the binding partner of the peptide tag as defined herein. The cargo is preferably selected from the group consisting of antigens, enzymes, hormones, ligands (including signals, such as transport or targeting or addressing signals), receptors, toxins, antibodies, any fragment thereof (preferably a functional fragment), and any combination thereof. The size of the cargo ranges preferably from 6 amino acids to 8000 amino acids, more preferably from 8 amino acids to 7000 amino acids, more preferably from 10 amino acids to 6000 amino acids, more preferably from 12 amino acids to 5000 amino acids, more preferably from 15 amino acids to 4000 amino acids, more preferably from 20 amino acids to 3000 amino acids, more preferably from 25 amino acids to 2000 amino acids, more preferably from 40 amino acids to lOOOamino acids, more preferably from 50 amino acids to 500 amino acids, more preferably from 100 amino acids to 400 amino acids. Otherwise stated, the size of the cargo ranges preferably from 0,4kDa to 800 kDa, more preferably from 0,6 kDa to 700 kDa, more preferably from 0,7 kDa to 600 kDa, more preferably from 0,8 kDa to 500 kDa, more preferably froml,2 kDa to 400 kDa, more preferably from 2 kDa to 300 kDa, more preferably from 2,5 kDa to 200 kDa, more preferably from 4 kDa to 100 kDa, more preferably from 5 kDa to 50 kDa, more preferably from 10 kDa to 40 kDa.

The term "antigen" as used herein refers to any structure recognized by and/or selectively bound by molecules of the immune response, e.g. antibodies, immune cells receptors (e.g. T cell receptors (TCRs), B cell receptors (BCRs), etc.), and the like. "Antigen" herein means a natural or synthetic molecule which, when recognized by antibodies or cells of the immune system of an organism, is capable of triggering an immune response in it. Antigens are recognized by highly variable antigen receptors (such as B-cell receptor or T-cell receptor) of the adaptive immune system and may elicit a humoral or cellular immune response. Antigens that elicit such a response are also referred to as immunogens. An antigen may be foreign or toxic to the body or may be a cellular molecule (e.g. protein) that is associated with a particular disease. Antigens are usually peptides, proteins, sugars (such as polysaccharides or polyosides) and their lipid derivatives (lipids). Antigens can also be nucleic acids, or haptens (i.e. fragments of antigens). Suitable antigens include, but not limited to, biological components (e.g. peptides, polypeptides, post translational modified polypeptides and polynucleotides); complex components (e.g. cells, cell mixtures, live or inactivated organisms such as bacteria, viruses, fungi, prions, etc...), and combinations thereof. In the case of protein antigens, the part of the antigen recognized by an antibody or a lymphocyte receptor is called an "epitope" or "antigenic determinant". The same antigen can have several epitopes (identical or different) and thus induce a varied immune response. Antigen recognition by lymphocytes depends on the nature of the epitope. B lymphocytes bind directly to conformational epitopes through their membrane immunoglobulins. T cells recognize sequential epitopes presented by antigen presenting cells. The antigen may be exogenous, i.e. foreign to the individual (in this case, it may be allogeneic: from an individual of the same species; or xenogeneic: from other species), or it may be endogenous, i.e. an antigen unique to the host (self-antigens). Preferred antigens for use herein are cancer/tumour antigens and antigens of pathogens (the latter preferably selected from antigens of eukaryotic or prokaryotic pathogens). The antigen is preferably a microorganism, plant, alga, microalgae, bacterium, virus, parasite, yeast, fungus, insect, animal, cancer, or tumour antigen; The antigen is preferably a protein, lipid, or sugar antigen of bacteria, virus, parasite, yeast, fungus, cancer, or tumour. The various categories of antigens are well known to the person skilled in the art, who can refer in particular to reference works in the field (such as G. J. V. Nossal, G L Ada, Antigens, Lymphoid Cells and the Immune Response, Academic Press, 1971; Marc H. V. Van Regenmortel, Structure of Antigens, Volume 3 CRC Press, Dec. 20, 1995; Edouard Drouhet, Garry T. Cole, Louis De Repentigny, Jean Latge, Fungal Antigens: Isolation, Purification, and Detection, Springer Science & Business Media, Nov. 11, 2013; Graziano D.F., Finn O.J. (2005) Tumor Antigens and Tumor Antigen Discovery. In: Khleif S.N. (eds) Tumor Immunology and Cancer Vaccines. Cancer Treatment and Research, vol 123. Springer, Boston, MA; Wang M, Claesson MH, Methods Mol Biol. 2014;1184:309-17. Classification of human leukocyte antigen (HLA) supertypes; as well as specialized databases as described in Galperin, Fernandez-Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes, NAR, Volume 45, Issue Dl, January 2017, Pages Dl- Dll; in particular the PMAPP database, a database of human autoantigens (available at aagatlas.ncpsb.org, among others). It is contemplated that the term antigen encompasses native antigens and derivatives thereof (e.g. mutated and/or engineered antigens), provided that such derivative is capable of being the target of an immune response. As used herein, an "antigen fragment" is any portion of an antigen, preferably provided that such fragment/portion is capable of being the target of an immune response (e.g. epitopes, immunogenic domains, etc.). In the case of a protein antigen, the antigen fragment preferably comprises at least 6 consecutive amino acid residues of the antigen (preferably at least 8 consecutive amino acid residues of the antigen, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the antigen).

As used herein, the term "epitope", also known as antigenic determinant, is the part of a molecule that is recognized by the immune system, notably by antibodies (e.g. an antibody or antigen-binding fragment thereof) and/or immune cells (such as lymphocytes, e.g. B cells, T cells, etc.). Epitopes usually consist of chemically active surface groupings of molecules such as amino acids and/or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. In case of protein epitopes, there are sequential epitopes, corresponding to a sequence of amino acids, and conformational epitopes, linked to the structure of the protein and therefore sensitive to denaturation. Conformational and non-conformational epitopes can be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. It is contemplated that the term epitope encompasses native epitopes and derivatives thereof (e.g. mutated and/or engineered epitopes), provided that such derivative is capable of being the target of an immune response The term "ligand" generally refers to a substance that binds to a receptor of a cell and induces a biological signal. The term ligand encompasses "addressing or targeting or transport signal", "signalling molecule", "signal", "cellular signal". Examples of ligands include, but are not limited to, peptide and protein addressing sequences (Waehler et al., doi:10.1038/nrg2141), oligosaccharides, molecules allowing transport and/or cellular internalization, neurotransmitters, as well as receptor ligands (receptors being as defined below) and cellular recognition molecules such as Toll Like Receptor Ligands or C-type lectin receptor ligands (Hennessy et al., doi:10.1038/nrd3203; Lepenies et al., http://dx.doi.Org/10.1016/j.addr.2013.05.007). An addressing sequence is a short amino acid sequence, usually located at the N-terminal end of the protein, used to indicate their destination. An addressing or targeting or transport signal can be an addressing or targeting or transport signal to/from the nucleus; an addressing or targeting or transport signal to/from the cytoplasm; an addressing or targeting or transport signal to/from the cytosol; an addressing or targeting or transport signal to/from the cell membrane; an addressing or targeting or transport signal to/from the mitochondria; an addressing or targeting or transport signal to/from the peroxisomes; a signal for addressing or targeting or transport to/from lysosomes; a signal for addressing or targeting or transport to/from the endoplasmic reticulum; a signal for addressing or targeting or transporting secretory pathways. The ligand may be a ligand of a receptor (for example SARS CoV-2 RBD is a ligand of ACE2, Yan, R. et al. Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2. Science (2020) doi:10.1126/science.abb2762), preferably of a membrane or transmembrane receptor, preferably of a membrane or transmembrane receptor of a membrane selected from a cell membrane, an extracellular membrane, a cytoplasmic membrane or a nuclear membrane. The ligand/signal may comprise at least: a peptide, protein, glycoprotein, sugar, oside, lipid, nucleic acid, or any combination thereof. Preferably, the ligand/signal comprises at least one peptide, protein, glycoprotein, or nucleic acid. The ligand is preferably a prokaryotic, eukaryotic, or viral ligand/signal, preferably a ligand/signal from an animal, plant, alga, microalgae, microorganism, bacterium, parasite, yeast, fungus, insect, virus, or cancer; more preferably a mammalian ligand, such as a human ligand. The various categories of ligands are well known to the skilled person, who will be able to refer in particular to reference works in the field (such as Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, 1 Nov. 2016; Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007/978-1-4419-0919-0, Springer- Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, Aug 22, 2014). It is contemplated that the term ligand encompasses native ligands and derivatives thereof (e.g. mutated and/or engineered ligands), provided that such derivative is capable of inducing a biological signal. A "ligand fragment" is any portion of a ligand, preferably provided that such fragment/portion is capable of binding to a receptor of a cell and inducing a biological signal. In the case of a protein ligand, the ligand fragment preferably comprises at least 6 consecutive amino acid residues of the ligand (preferably at least 8 consecutive amino acid residues of the ligand, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the ligand).

By "toxin" it is herein meant a substance that is toxic to one or more living organisms. A toxin is typically synthesized by a living organism (e.g. bacteria, poisonous fungus, insect or venomous snake or mollusc, etc.), to which it confers its pathogenic power. Toxins produced by bacteria are called bacteriotoxins, those produced by fungi are called mycotoxins, those produced by plants are called phytotoxins, those produced by algae are called phycotoxins, those produced by animals are called animal toxins. The toxin can be a chemical molecule, a peptide, a protein, a glycoprotein, a sugar, an oside, a lipid, a nucleic acid, or any combination of these Several families of bacteria secrete biotoxins (exotoxins) in the tissues they colonize. Other bacteria (Gram-negative) retain most of the toxic compounds within themselves, which are released only during cell lysis, by chemical, physical or mechanical means (endotoxins). Toxic plants produce toxins via their secondary metabolites: these are molecules which, unlike primary toxins (proteins, lipids, carbohydrates, amino acids ...) are produced outside the metabolic pathways necessary to ensure survival (thus primary metabolites). Plant toxins can be classified into three groups: phenols, nitrogenous and terpenes. The toxin can be a neurotoxin (a toxin acting on the nervous system), a myotoxin (acting on the contraction of muscles, including cardiotoxins on the heart and others such as strychnine on respiratory muscles), a hemotoxin (acting on the blood), a cytotoxin (acting on the cells), a dermatotoxin (acting on the skin and mucous membranes), a hepatotoxin (acting on the liver), a nephrotoxin (acting on the kidney), an enterotoxin (acting on the digestive tract) etc.. The toxin may be an anatoxin; that is, a toxin that has been treated so as to retain its antigenic power and lose its toxicity. The toxin is preferably a toxin of a microorganism, plant, alga, microalgae, bacterium, virus, parasite, yeast, fungus, insect, animal, or tumour; preferably a toxin of a eukaryotic or prokaryotic pathogen, or of a cancer. The various categories of toxins are well known to the skilled person, who will be able to refer in particular to reference works in the field (such as Michael W. Parker, Protein Toxin Structure, Springer Science & Business Media, June 29, 2013; Michael R. Dobbs, Clinical Neurotoxicology E-Book: Syndromes, Substances, Environments, Elsevier Health Sciences, July 22, 2009; Walker AA, Robinson SD, Yeates DK, Jin J, Baumann K, Dobson J, Fry BG, King GF. Entomo-venomics: The evolution, biology and biochemistry of insect venoms. Toxicon. 2018 Nov;154:15-27; Vilarino N, Louzao MC, Abal P, Cagide E, Carrera C, Vieytes MR, Botana LM. Human Poisoning from Marine Toxins: Unknowns for Optimal Consumer Protection. Toxins (Basel). 2018 Aug 9;10(8); and specialized databases as described in Galperin, Fernandez-Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes, NAR, Volume 45, Issue Dl, January 2017, Pages Dl-Dll; in particular the Comparative Toxicogenomics Database, as described in Davis, Grondin Murphy, Johnson, Lay, Lennon-Hopkins, Saraceni-Richards, Sciaky, King, Rosenstein, Wiegers, Mattingly, The Comparative Toxicogenomics Database: 2013 update, NAR, Volume 41, Issue Dl, 1 January 2013, Pages D1104-D1114 (available in part at ctdbase. org)). It is contemplated that the term toxin encompasses native toxins and derivatives thereof (e.g. mutated and/or engineered toxins), provided that such derivative is capable of capable of being toxic to an organism and/or a cell. As used herein, a "toxin fragment" is any portion of a toxin, preferably provided that such fragment/portion is capable of being toxic to an organism and/or a cell. In the case of a protein toxin, the toxin fragment preferably comprises at least 6 consecutive amino acid residues of the toxin (preferably at least 8 consecutive amino acid residues of the toxin, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the toxin).

As used herein, a "receptor" is a molecule in the cell membrane or cytoplasm or cell nucleus that binds specifically to a specific factor (e.g. a ligand (such as a neurotransmitter), a pathogenic agents (e.g. a viruses such as SARS-CoVl and SARS-CoV2 which interact with ACE2 (Li et al, 10.1038/nature02145, Zhang et al., 10.1007/s00134-020-05985-9) an hormone, or other substances), inducing a cellular response to that ligand. Ligand-induced changes in receptor behaviour lead to physiological changes that constitute the "biological effects" of the ligand. Receptors can include at least: a peptide, a protein, a glycoprotein, a sugar, an oside, a lipid, a nucleic acid, or any combination of these. Receptors are typically proteins or mixed proteins (proteins modified and/or associated with another molecule). The receptor can be a receptor of the external part of the plasma membrane, a transmembrane receptor embedded in the lipid bilayer of cell membranes (usually a transmembrane protein, acting for example as a receptor for hormones and neurotransmitters - these receptors are either coupled to a G protein or carry an enzymatic or ion channel activity that allows the activation of metabolic signal transduction pathways in response to ligand binding), or an intracellular receptor (these receptors can sometimes enter the nucleus of the cell to modulate the expression of specific genes, in response to activation by the ligand). The receptor is preferably a microorganism, plant, alga, microalgae, bacterium, virus, parasite, yeast, fungus, insect, animal, or tumor receptor or cell-specific receptor; preferably a eukaryotic or prokaryotic pathogen or cancer receptor; preferably a bacterial, viral, parasite, yeast, fungus, or tumour protein or glycoprotein receptor. The various categories of receptors are well known to the skilled person, who will be able to refer in particular to reference works in the field (such as Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, 1 Nov. 2016; Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10. 1007/978-1-4419-0919-0, Springer- Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, Aug. 22, 2014; and specialized databases as described in Galperin, Fernandez-Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: A look back and upcoming changes, NAR, Volume 45, Issue Dl, January 2017, Pages Dl-Dll; in particular the GPCRdb database, , as described in Isberg V. , Mordalski S., Munk C., Rataj K., Harpsoe K., Hauser A.S., Vroling B., Bojarski A.J., Vriend G., Gloriam D.E.. GPCRdb: an information system for G protein-coupled receptors. Nucleic Acids Res. 2016; 44:D356-D364 (available notably at gpcrdb.org)). It is contemplated that the term receptor encompasses native receptors and derivatives thereof (e.g. mutated and/or engineered antigens), provided that such derivative is capable of binding specifically to a specific factor (e.g. a ligand, an hormone, or other substance). As used herein, an "receptor fragment" is any portion of an antigen, preferably provided that such fragment/portion is capable of binding specifically to a specific factor (e.g. a ligand, an hormone, or other substance). In the case of a protein receptor, the receptor fragment preferably comprises at least 6 consecutive amino acid residues of the receptor (preferably at least 8 consecutive amino acid residues of the receptor, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the receptor).

"Enzyme" herein refers to a protein with catalytic properties (enzymatic properties). Virtually all biomolecules capable of catalysing chemical reactions in cells are enzymes; however, some catalytic biomolecules are made of RNA and are therefore distinct from enzymes: these are ribozymes. An enzyme works by lowering the activation energy of a chemical reaction, which increases the speed of the reaction. The enzyme is not modified during the reaction. The initial molecules are the substrates of the enzyme, and the molecules formed from these substrates are the products of the reaction. Enzymes are characterized by their very high specificity. Moreover, an enzyme has the characteristic of being reusable. Enzymes are generally globular proteins that act alone or in complexes of several enzymes or subunits. Like all proteins, enzymes consist of one or more polypeptide chains folded to form a three-dimensional structure corresponding to their native state.

Enzymes are much larger molecules than their substrates. Their size can vary from about 50 residues to more than 2000 residues. Only a very small part of the enzyme - between two and four residues most often, sometimes more - is directly involved in catalysis, the so-called catalytic site (or catalytic domain). The catalytic site may be located in the vicinity of one or more binding sites, at which the substrate(s) is (are) bound and oriented to catalyse the chemical reaction. The catalytic site and the binding sites form the active site of the enzyme.

Enzymes perform a large number of functions in living organisms. For example, they can be involved in signal transduction and regulation of cellular processes, in the generation of movement, in active transmembrane transport, in digestion, in metabolism, in the immune system, in nucleic acid digestion or cleavage mechanisms or in nucleic acid production (referred to here as "nucleic acid-acting enzymes"), in prodrug conversion mechanisms (prodrug-to-drug conversion). The enzyme is preferably a prokaryotic, eukaryotic or viral enzyme, preferably an enzyme from an animal, plant, alga, microalgae, insect, microorganism, bacterium, parasite, yeast, fungus or virus, more preferably a mammalian enzyme, such as a human enzyme. The various categories of enzymes are well known to the person skilled in the art, who can refer in particular to reference works in the field (such as Schomburg D., Schomburg I., Springer Handbook of Enzymes. 2 edn. Heidelberg: Springer; 2001-2009; Liebecq C., IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN) and Nomenclature Committee of IUBMB (NC-IUBMB) Biochem. Mol. Biol. Int. 1997;43:1151-1156; IUBMB (1992), Enzyme Nomenclature 1992, Academic Press, San Diego; and specialized databases as described in Schomburg D, Schomburg I. Methods Mol Biol. 2010;609:113-28. Enzyme databases; in particular, the BRENDA database (available, inter alia, at brenda- enzymes.org), as described, for example, by Chang A, Schomburg I, Placzek S, Jeske L, Ulbrich M, Xiao M, Sensen CW, Schomburg D, Nucleic Acids Res. 2015 Jan;43. Epub 2014 Nov 5. BRENDA in 2015: exciting developments in its 25th year of existence). It is contemplated that the term enzyme encompasses native enzymes and derivatives thereof (e.g. mutated and/or engineered enzymes), provided that such derivative is capable of having an enzymatic activity. As used herein, an "enzyme fragment" is any portion of an enzyme, preferably provided that such fragment/portion is capable of having an enzymatic activity. In the case of a protein enzyme, the enzyme fragment preferably comprises at least 6 consecutive amino acid residues of the enzyme (and is preferably an enzyme catalytic site) (preferably at least 8 consecutive amino acid residues of the enzyme, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the enzyme).

As used herein, an "hormone" is a biologically active chemical substance, usually synthesized by a glandular cell (usually following a stimulation and/or an inhibition) and secreted into the internal environment where it circulates (by blood, lymph or sap). It transmits a message in chemical form (generally by acting on specific receptors of a target cell) and thus plays a role of messenger in the body. It is able to act at very low doses. The hormone is advantageously a plant or animal hormone. Plant hormones are also called phytohormones or growth factors. They often have the function of ensuring the growth of the plant or its morphogenesis. Animal hormones are in most cases produced by the endocrine system (an endocrine gland or endocrine tissue).

Advantageously, the hormone is a vertebrate hormone, preferably selected from the following chemical classes:

Amino-derived hormones, which consist of a single amino acid (tyrosine or tryptophan) but in a derived form.

Peptide hormones, which are chains of amino acids, hence proteins, called peptides for the shorter ones.

Steroid hormones, which are steroids derived from cholesterol.

Hormones based on lipids and phospholipids.

The hormone is preferably selected from peptide or protein hormones, amine-derived hormones, steroid hormones and lipid hormones. The hormone is preferably an animal or plant hormone, preferably a mammalian hormone, preferably a human hormone. The different categories of hormones are well known to the skilled person, who can refer to reference books in the field (such as Davies P.J. (2010) The Plant Hormones: Their Nature, Occurrence, and Functions. In: Davies P.J. (eds) Plant Hormones. Springer, Dordrecht; AW Norman, G Litwack, Hormones, Academic Press, 1997; A Kastin, Handbook of biologically active peptides, Academic Press, 2013). It is contemplated that the term hormone encompasses native hormones and derivatives thereof (e.g. mutated and/or engineered hormones), provided that such derivative is capable of stimulating and/or inhibiting a biological process. As used herein, an "hormone fragment" is any portion of an hormone, preferably provided that such fragment/portion is capable of stimulating and/or inhibiting a biological process. In the case of a protein hormone, the hormone fragment preferably comprises at least 6 consecutive amino acid residues of the hormone (preferably at least 8 consecutive amino acid residues of the hormone, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the hormone).

The term "antibody" as used herein refers to a protein or a glycoprotein belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are used interchangeably. An antibody is produced by plasma cells and is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, its antigen. The antibody can be polyclonal or monoclonal. Monoclonal antibodies are antibodies that recognize only one type of epitope on a given antigen. They are by definition all identical and produced by a single plasma cell clone. Polyclonal antibodies are a mixture of antibodies recognizing different epitopes on a given antigen, each idiotype being secreted by a different plasma cell clone. During the immune response, an organism synthesizes antibodies directed against several epitopes of an antigen: the response is called polyclonal. As used herein, the term antibody encompasses minibodies, diabodies, nanobodies, and the like. Preferably, the antibody is an antibody from an animal, preferably a mammalian antibody, more preferably a human or humanized antibody. Advantageously, the antibody is a monoclonal antibody. The various categories of antibodies are well known to the person skilled in the art, who will be able to refer in particular to reference works in the field (such as Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, 1 Nov. 2016; Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007/978-1-4419-0919-0, Springer- Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, Aug 22, 2014; Bayer V., An Overview of Monoclonal Antibodies. Semin Oncol Nurs. 2019 Sep 2:150927; Wang W, Wang EQ, Balthasar JP. Monoclonal antibody pharmacokinetics and pharmacodynamics. Clin Pharmacol Ther. 2008 Nov;84(5):548-58). It is contemplated that the term antibody encompasses native antibodies and derivatives thereof (e.g. mutated and/or engineered antibodies as well as antibody mimetics), provided that such derivative is capable of specifically binding to an antigen. The term "antibody fragment" as used herein, refers to one or more portion or fragment of an antibody, retaining the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antibody fragment" include, but are not limited to, a fragment antigen binding (Fab) fragment, a Fab' fragment, a F(ab')₂ fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a scAb (single chain antibody fragment), a nanobody, a minibody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, an alternative scaffold protein, and any combination thereof (e.g. a fusion protein thereof).

The term "diabody" as used herein refers to a fusion protein or a bivalent antibody which can bind different antigens. A diabody is composed of two single protein chains which comprise fragments of an antibody, namely variable fragments. Diabodies comprise a heavy chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL, or VL-VH). By using a short peptide connecting the two variable domains, the domains are forced to pair with the complementary domain of another chain and thus, create two antigen-binding sites. Diabodies can target the same (monospecific) or different antigens (bispecific).

The term "single domain antibody" as used herein refers to antibody fragments consisting of a single, monomeric variable domain of an antibody. Simply, they only comprise the monomeric heavy chain variable regions of heavy chain antibodies produced by camelids or cartilaginous fish. Due to their different origins, they are also referred to VHH or VNAR (variable new antigen receptor)-fragments. Alternatively, single-domain antibodies can be obtained by monomerization of variable domains of conventional mouse or human antibodies by the use of genetic engineering. They show a molecular mass of approximately 12-15 kDa and thus, are the smallest antibody fragments capable of antigen recognition. Further examples include nanobodies or nanoantibodies.

The term "antibody mimetic" as used herein refers to compounds which can specifically bind antigens, similar to an antibody, but are not structurally related to antibodies. Usually, antibody mimetics are artificial peptides or proteins with a molar mass of about 3 to 20 kDa which comprise one, two or more exposed domains specifically binding to an antigen. Examples include inter alia the LACI-D1 (lipoprotein- associated coagulation inhibitor); affilins, e.g. human-y B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domain of Fyn; Kunits domain of protease inhibitors; monobodies, e.g. the 10^th type III domain of fibronectin; adnectins: knottins (cysteine knot miniproteins); atrimers; evibodies, e.g. CTLA4- based binders, affibodies, e.g. three-helix bundle from Z-domain of protein A from Staphylococcus aureus; Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-ll; affilins; armadillo repeat proteins; DARPins (Designed Ankyrin Repeat Proteins); etc. Nucleic acids and small molecules are sometimes considered antibody mimetics as well (aptamers), but not artificial antibodies, antibody fragments and fusion proteins composed from these. Common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs.

"DARPin" or "Designed Ankyrin Repeat Protein" herein refers to genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat proteins, one of the most common classes of binding proteins in nature, which are responsible for diverse functions such as cell signalling, regulation and structural integrity of the cell. DARPins comprise, or consist essentially of, or consist of, at least three repeat motifs or modules, of which the most N- and the most C-terminal modules are referred to as "caps", since they shield the hydrophobic core of the protein. The number of internal modules is indicated as number (e.g. NIC, N2C, N3C, ...) while the caps are indicated with "N" or "C", respectively.

As used herein, "specific binding" means that a binding moiety (e.g. an antibody) binds stronger to a target (first target), such as an epitope, for which it is specific compared to the binding to another target (second target, non-specific). The binding is "stronger" if the binding moiety binds to the first target with a affinity higher than to the second target (i.e. if the binding moiety binds to the first target with a dissociation constant (K_d) which is lower than the dissociation constant for the second target). The ligand binding to its respective target generally results in a biological effect. Preferably, the specific binding occurs with a high affinity, preferably with K_d of less than 10⁷, 10⁸, 10⁹, 10¹⁰ M or less. Such affinity is preferably measured at 37°C. The person skilled in the art knows the assays that are suitable to determine/measure the affinity. Suitable assays include, but are not limited to, surface plasmon resonance measurements (e.g. Biacore), quartz crystal microbalance measurements (e.g. Attana), and competition assays.

As used herein, a "subject" or an "individual" is an animal, preferably a mammal, including, but not limited to, human, ovine, bovine, canine, feline, monkeys (including e.g. dog, cat, cattle, goat, pig, swine, sheep, etc.). More preferably, the subject is a human subject. A human subject can be known as a patient. As used herein, "subject of interest" or "subject in need thereof" refers to a subject who has been diagnosed with a disease, or a subject who is susceptible or suspected to suffer from a disease.

By "disease" or "disorder" or "pathology" it is herein meant an alteration in the functions or health of a living organism. It refers to both the disease, which refers to all alterations in health, and a disease, which refers to a particular entity characterized by its own causes, symptoms, course and therapeutic possibilities.

"Infection" or "infectious disease" herein refers to a disease caused by the transmission of a micro organism or infectious agent: virus, bacterium, parasite, fungus, protozoa, etc.

As used herein, "cancer" or "cancerous disease" means a disease caused by the transformation of cells that become abnormal and proliferate excessively (we can speak of anarchic proliferation). These disordered cells can eventually form a mass called a tumour (usually a malignant tumour). Cancer cells usually tend to invade nearby tissues and break away from the tumour. They can then migrate through blood vessels and lymphatic vessels to form another tumour: this is called metastasis. Cancers include a wide range of pathologies with very different forms and consequences, but they all share a very typical set of characteristics, regardless of the cancer concerned. The following histological elements can be found in most cancers:

- an independence of the cancer cells from the signals that normally stimulate cell multiplication;

- insensitivity of cancer cells to antiproliferative signals and mechanisms;

- a proliferative capacity that is no longer limited (infinite growth, often resulting in neoplasms);

- the disappearance of the apoptosis phenomenon in these same cancer cells, in other words a form of aggressive "immortality" at the expense of the patient;

- the regression or dedifferentiation of cells towards a form more and more reminiscent of embryonic stem cells (the cancer cell can thus pass from a specialized/differentiated cell state to an unspecialized, immature, multipotent or pluripotent cell state);

- an abnormal ability to induce angiogenesis;

- often the acquisition of invasiveness in advanced stages;

- lesions in the surrounding tissue (necrosis), whether or not there is tissue invasion;

- with very rare exceptions, cells from the individual affected by this cancer (these are cells of the Self).

The term "tumour" refers to an increase in volume of a tissue, without specifying the cause. It is a neoformation of body tissue (neoplasia) that occurs as a result of a disturbance in cell growth, either benign or malignant (when it is a malignant tumour, it is called cancer). A neoplasia can involve any type of tissue. Depending on the location of the tumour and the function of the affected tissue, it can lead to organ dysfunction and affect the entire body, even causing death. Tumours can occur in all multicellular organisms, including plants. A distinction is made between benign and malignant tumours:

Benign tumours are tumours that are generally not serious, i.e., cannot give rise to daughter tumours (metastases), as is the case with warts or moles. However, a benign tumour can lead to serious complications (compression, inflammation, etc.) through its mechanical action.

Malignant tumours are often referred to as cancer. In addition to attacking surrounding tissue, they produce daughter tumours (metastases) that spread through the blood or lymph.

The term "tumour" is preferably used here to mean a malignant tumour.

The terms "prevention" or "prevention of a disease" or "prevention of the onset of a disease" means reducing the risk of the onset, development or amplification of a disease, the causes of a disease, the symptoms of a disease, the effects (or consequences, preferably the adverse, deleterious effects/consequences) of a disease, or any combination thereof; and/or delaying the onset, development or amplification of a disease, the causes of a disease, the symptoms of a disease, the effects (or consequences, preferably the deleterious effects/consequences) of a disease, or any combination thereof.

"Treatment" or "treatment of a disease" herein means the reduction, inhibition, and/or disappearance of a disease, the causes of a disease, the symptoms of a disease, the effects (or consequences, preferably the harmful, deleterious effects/consequences) of a disease, or any combination thereof.

"Medicament" or "drug" means any substance or composition represented as having curative or preventive properties with respect to human or animal disease. A medicament therefore includes any substance or composition that may be used in or administered to humans or animals for the purpose of making a medical diagnosis or restoring, correcting or modifying their physiological functions by exerting a pharmacological, immunological or metabolic action.

As used herein, "vaccine" or "vaccine composition" means a pathogenic or tumour-producing substance which, when inoculated into an individual in a preferably harmless form, confers immunity against a disease (or protection against a disease). In general, a vaccine stimulates the body's immune response. A vaccine can be preventive, allowing the prevention of a disease. A vaccine can also be therapeutic, helping the patient fight an ongoing disease. Accordingly, the term "vaccine" refers to any component or group of components which is expected to cause a biological response when delivered appropriately to a subject through the presence or expression of one or more biological substance(s) (e.g. a polypeptide such as an antigen, an enzyme, a cytokine, a SiRNA, etc.).

The terms "therapeutic" or "therapeutic uses" in the context of the present invention cover "prevention", "treatment" and "vaccine" uses.

A "therapeutically effective amount" corresponds to the amount of each active entity that is sufficient for producing a beneficial health result. An "immunologically effective amount" corresponds to the amount of each active entity that is sufficient for producing a detectable immune response. As used herein, a "pharmaceutically acceptable vehicle" is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like, compatible with administration in a subject and in particular in a human. For general guidance, appropriate carriers for use herein are well known the art (see for example the most current edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins).

As used herein, the term "screening" refers to a process for testing and selecting compounds/active agents for a specific effect/activity on a molecule, a virus, a parasite, a bacterium, a cell, a tissue, an organ, a disease (preferably an infectious disease and/or a cancer), an organism (human beings, human embryos and human embryonic stem cells excluded). For example, the compounds/active agents may be tested for an antiviral activity/effect (such as an anti-coronavirus activity/effect), for an antibacterial activity/effect, for an anti-tumoral and/or anti-cancer activity/effect, and any combination thereof.

Dendritic cells (DCs) are antigen presenting cells from the mammalian immune system. They are present in the skin and tissues in contact with foreign environment, where they recognize and process antigens while migrating to the lymph nodes where they present them to lymphoid B and T cells, thereby initiating a specific immune response. There are different subsets of DCs in humans, the major ones are conventional DCs, cDC2 and cDCl, and plasmacytoid DCs, pDCs. Many cancer immunotherapeutic strategies target DCs directly or indirectly for the induction of antigen-specific immune responses.

In the following detailed description, the embodiments may be taken alone or in any suitable combination by the skilled person, and the above definition apply to all embodiments described below.

Engineered protein

The present Inventors have developed an original and efficiently adaptable system combining the ADDomer protein presenting capacity with a highly modulable binary tag-tag partner, altogether having valuable vaccine properties. The Inventors have surprisingly demonstrated that a peptide tag can be efficiently inserted in the external loops of the penton base protomer constituting the ADDomer, without impairing the ADDomer structure. The data show for the first time that the inserted peptide tag retains its capacity to covalently bond to its binding partner that has been fused to a protein or a protein fragment of therapeutic interest (called a cargo), resulting in a stable and functional ADDomer structure fully decorated with the therapeutic protein or fragment (present them in up to 60 copies of the cargo are displayed on the surface of the ADDomer). This novel presenting system has been in particular validated with a wide variety of cargos, including:

(i) peptide epitope, such as tumour or viral epitopes, (as herein demonstrated with A2L tumour epitope),

(iii) large antigens (as herein demonstrated with "Melan A" tumour antigen);

(iv) large well-folded post-translationally modified proteins such as viral antigens (as herein illustrated with SARS-CoV2 glycosylated spike protein and fragments thereof, including the glycosylated Spike Receptor Binding Domain (RBD). Importantly, the data demonstrate that it is possible to bind multiple distinct cargos on the same ADDomer particle, allowing for instance the display of several antigen variants, highly useful for e.g. multivalent vaccines, cancer vaccines or medicaments.

In addition, the data obtained by the Inventors confirmed that the cargos displayed by the ADDomer are fully functional and immunogen. Notably, the large viral SARS-CoV2-RBD displayed on the ADDomer (ADD- RBD) was capable of specifically recognizing and strongly and quasi-irreversibly binding to its natural cell receptor (the ACE2 receptor) and of being recognized by serum from Covid-19 convalescent patients or anti-RBD monoclonal antibodies. In addition, vaccination of animals with ADD-RBD induced a potent and specific anti-SARS-CoV2-RBD response, and particularly with an ADD pre-immunity beneficial to the response against the displayed antigen. Moreover, the Inventors showed that the antigens displayed on the ADDomer can be efficiently taken up by different subsets of dendritic cells, thus triggering a cellular immune response beneficial in both infectiology and vaccination against cancer. Altogether, the data reveal the biological significance of this novel and adaptable cargo-presenting system and validate crucial applications in vaccination, targeted drug-delivery within cells, active compound screening, and antibody detection. In particular SARS-Cov2-RBD displayed on the ADDomer and ACE2 receptor immobilized on a surface can be directly used as a system for active compounds screening (e.g. medicaments).

Accordingly, the present invention relates to an engineered protein comprising, or consisting essentially of, or consisting of:

The protein or the protein fragment of (ii) (i.e. which is fused to a binding partner of the peptide tag) is also herein called a cargo.

The present invention also concerns an engineered protein comprising, or consisting essentially of, or consisting of an adenovirus penton base protomer comprising a peptide tag in the variable loop and/or in the RGD loop; wherein the peptide tag is covalently bound to a binding partner via an isopeptide bond; and wherein the binding partner is fused to at least one protein (herein called a cargo) or at least one protein fragment (herein called a cargo).

In one embodiment, the engineered protein comprises, or consists essentially of, or consists of:

(i) at least one adenovirus penton base protomer comprising a peptide tag in the variable loop and/or in the RGD loop, and

(ii) at least one protein or at least one protein fragment fused to a binding partner of the peptide tag; where the peptide tag of (i) and the binding partner of the peptide tag of (ii) are covalently bound to each other via an isopeptide bond. In a specific embodiment, at least one protein and at least one protein fragment are both fused to the binding partner of the peptide tag, in (ii).

Advantageously, two or more distinct proteins and/or two or more distinct protein fragments (i.e. two or more distinct cargos) are used in (ii) (or a plurality of distinct proteins and/or protein fragments are used in (ii) (i.e. a plurality of distinct cargos)). Thus, in one embodiment, the engineered protein comprises, or consists essentially of, or consists of:

(ii) two or more (preferably distinct) proteins and/or two or more (preferably distinct) protein fragments, fused to a binding partner of the peptide tag ; where the peptide tag of (i) and the binding partner of the peptide tag of (ii) are covalently bound to each other via an isopeptide bond.

Thus, in another embodiment, the engineered protein comprises, or consists essentially of, or consists of:

(i) two or more (preferably a plurality of) adenovirus penton base protomers comprising a peptide tag in the variable loop and/or in the RGD loop, and

(ii) two or more (preferably a plurality of) (preferably distinct) proteins and/or two or more (preferably a plurality of) (preferably distinct) protein fragments, fused to a binding partner of the peptide tag ; where the peptide tag of (i) and the binding partner of the peptide tag of (ii) are covalently bound to each other via an isopeptide bond ; and wherein each of the binding partner of (ii), covalently bound to the peptide tag of adenovirus penton base protomer of (i), is fused to only one of the protein of (ii) and/or to only one of the protein fragment of (ii) (or alternatively wherein each of the binding partner of (ii), covalently bound to the peptide tag of adenovirus penton base protomer of (i), is fused to two or more (preferably distinct) proteins of (ii) and/or to two or more (preferably distinct) protein fragments of (ii)). In an advantageous embodiment, the engineered protein comprises, or consists essentially of, or consists of, two or more adenovirus penton base protomers comprising a peptide tag in the variable loop and/or in the RGD loop; wherein the peptide tag is covalently bound to a binding partner via an isopeptide bond; and wherein the binding partner is fused to two or more (preferably distinct) proteins and/or to two or more (preferably distinct) protein fragments. In an advantageous embodiment, the engineered protein comprises, or consists essentially of, or consists of, two or more adenovirus penton base protomers comprising a peptide tag in the variable loop and/or in the "RGD loop"; wherein the peptide tag is covalently bound to a binding partner via an isopeptide bond; and wherein each of the binding partner covalently bound to the peptide tag is fused to only one protein and/or to only one protein fragment, preferably wherein the engineered protein comprises at least two distinct proteins each fused to a distinct binding partner and/or at least two distinct protein fragments each fused to a distinct binding partner.

Advantageously, the engineered protein comprises, or consists essentially of, or consists of, a plurality of adenovirus penton base protomers comprising a peptide tag in the variable loop and/or in the RGD loop; wherein the peptide tag is covalently bound to a binding partner via an isopeptide bond; and wherein the binding partner is fused to a plurality of proteins and/or to a plurality of protein fragments. The engineered protein is advantageously a viral mime. Indeed, in the one hand, the engineered protein mimics the virus for binding to the attachment receptor and in the other hand it is recognised by the host immune system as a pathogen.

The engineered protein is also advantageously a tumour antigen presenting system. Indeed, it enables entry into the dendritic cells and in turn allows lymphocyte cross-presentation.

Advantageously, the penton base protomer (used for and/or present in the engineered protein of the invention), can spontaneously pentamerize to form a pentamer. The pentamer (i.e. 12 pentamers) can advantageously auto-assemble in a dodecahedron. In a preferred embodiment, the engineered protein of the invention is capable of assembling into a Virus-Like-Particle (VLP).

In one embodiment, the peptide tag is inserted in the RGD loop, or in the variable loop, or in place of the RGD loop (i.e. in place of the entire RGD loop encompassing the first RGD-subloop, the enlarged RGD motif, and the second RGD-subloop), or in place of one or more consecutive amino acids of the RGD loop, or in place of the variable loop (i.e. in place of the entire variable loop), or in place of one or more consecutive amino acids of the variable loop, or any combination thereof.

In one embodiment, the peptide tag is inserted in the first RGD-subloop, or in the enlarged RGD motif, or in the second RGD-subloop, or in the RGD motif, or in place of the first RGD-subloop, or in place of the enlarged RGD motif, or in place of the second RGD-subloop, or in place of the RGD motif, or in place of one or more consecutive amino acids of the first RGD-subloop, or in place of one or more consecutive amino acids of the enlarged RGD motif, or in place of one or more consecutive amino acids of the second RGD-subloop, or in place of one or more consecutive amino acids of the RGD motif, or any combination thereof.

Preferred penton base proteins are those having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs:l to 11. Most preferred penton base proteins are those indicated in SEQ ID NOs: 1 to 6, or 11, more preferably those indicated in SEQ ID NOs: 1 to 6.

In preferred embodiments, the peptide tag is inserted in one selected from the group consisting of:

1. the RGD loop, between two consecutive amino acid residues located from position 310 to position 349 of SEQ ID NO: 1; or between two consecutive amino acid residues located from a position equivalent to position 310 to a position equivalent to position 349 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l;

2. the RGD loop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 310 to position 349 of SEQ ID NO: 1; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 310 to a position equivalent to position 349 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l; 3. the first RGD-subloop, between two consecutive amino acid residues located from position 310 to position 323 of SEQ ID NO: 1; or between two consecutive amino acid residues located from a position equivalent to position 310 to a position equivalent to position 323 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l;

4. the first RGD-subloop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 310 to position 323 of SEQ ID NO: 1; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 310 to a position equivalent to position 323 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l;

5. the second RGD-subloop, between two consecutive amino acid residues located from position 338 to position 349 of SEQ ID NO: 1; or between two consecutive amino acid residues located from a position equivalent to position 338 to a position equivalent to position 349 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l;

6. the second RGD-subloop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 338 to position 349 of SEQ ID NO: 1; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 338 to a position equivalent to position 349 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l;

7. the enlarged RGD motif, between two consecutive amino acid residues located from position 324 to position 337 of SEQ ID NO: 1; or between two consecutive amino acid residues located from a position equivalent to position 324 to a position equivalent to position 337 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l;

8. the enlarged RGD motif, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 324 to position 337 of SEQ ID NO: 1; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 324 to a position equivalent to position 337 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l;

9. in the variable loop (V-loop), between two consecutive amino acid residues located from position 150 to position 169 of SEQ ID NO: 1; or between two consecutive amino acid residues located from a position equivalent to position 150 to a position equivalent to position 169 of SEQ ID NO: 1, after optimal global alignment with SEQ ID NO:l;

10. in the variable loop (V-loop), instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 150 to position 169 of SEQ ID NO: 1; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 150 to a position equivalent to position 169 of SEQ ID NO:

I, after optimal global alignment with SEQ ID NO:l; and

11. any combination thereof.

Thus, such insertion(s) may delete all or part of the respectively indicated amino acid residues located in the RGD loop, the first RGD-subloop, the second RGD-subloop, the RGD motif, the enlarged RGD motif, the V-loop, or any combination thereof.

In other preferred embodiments, the peptide tag is inserted in one selected from the group consisting of:

1. the RGD loop, between two consecutive amino acid residues located from position 317 to position 364 of SEQ ID NO: 11; or between two consecutive amino acid residues located from a position equivalent to position 317 to a position equivalent to position 364 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll;

2. the RGD loop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 317 to position 364 of SEQ ID NO: 11; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 317 to a position equivalent to position 364 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll;

3. the first RGD-subloop, between two consecutive amino acid residues located from position 317 to position 334 of SEQ ID NO: 11; or between two consecutive amino acid residues located from a position equivalent to position 317 to a position equivalent to position 334 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll;

4. the first RGD-subloop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 317 to position 334 of SEQ ID NO: 11; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 317 to a position equivalent to position 334 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll;

5. the second RGD-subloop, between two consecutive amino acid residues located from position 349 to position 364 of SEQ ID NO: 11; or between two consecutive amino acid residues located from a position equivalent to position 349 to a position equivalent to position 364 of SEQ ID NO:

II, after optimal global alignment with SEQ ID NO:ll;

6. the second RGD-subloop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 349 to position 364 of SEQ ID NO: 11; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 349 to a position equivalent to position 364 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll; 7. the enlarged RGD-motif, between two consecutive amino acid residues located from position 335 to position 348 of SEQ ID NO: 11; or between two consecutive amino acid residues located from a position equivalent to position 335 to a position equivalent to position 348 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll;

8. the enlarged RGD-motif, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 335 to position 348 of SEQ ID NO: 11; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 335 to a position equivalent to position 348 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll;

9. in the variable loop (V-loop), between two consecutive amino acid residues located from position 150 to position 178 of SEQ ID NO: 11; or between two consecutive amino acid residues located from a position equivalent to position 150 to a position equivalent to position 178 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll;

10. in the variable loop (V-loop), instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from position 150 to position 178 of SEQ ID NO: 11; or instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) located from a position equivalent to position 150 to a position equivalent to position 178 of SEQ ID NO: 11, after optimal global alignment with SEQ ID NO:ll; and

11. any combination thereof.

1. the RGD loop, between two consecutive amino acid residues of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 17 to 27;

2. the RGD loop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 17 to 27; the first RGD-subloop, between two consecutive amino acid residues of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 63 to 73; the first RGD-subloop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 63 to 73; the second RGD-subloop, between two consecutive amino acid residues of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 85 to 95; the second RGD-subloop, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 85 to 95; the enlarged RGD motif, between two consecutive amino acid residues of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 74 to 84; the enlarged RGD motif, instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 74 to 84; 9. in the variable loop (V-loop), between two consecutive amino acid residues of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 28 to 38;

10. in the variable loop (V-loop), instead of at least one amino acid residues (e.g. instead of one amino acid residue, or instead of at least two consecutive amino acid residues) of an amino acid sequence having at least 85% sequence identity, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99%, more preferably 100% sequence identity, with any of the sequences indicated in SEQ ID NOs: 28 to 38; and

11. any combination thereof.

In a preferred embodiment, the peptide tag (further comprising linker sequences or not) is inserted instead of the amino acid residues located from position 159 to position 163 of SEQ ID NO :11; or instead of the amino acid residues located from a position equivalent to position 159 to a position equivalent to position 163 of SEQ ID NO :11, after optimal global alignment with SEQ ID NO:ll. Alternatively or in combination, in a preferred embodiment, the peptide tag (further comprising linker sequences or not) is inserted instead of the amino acid residues located from position 335 to position 348 of SEQ ID NO :11; or instead of the amino acid residues located from a position equivalent to position 335 to a position equivalent to position 348 of SEQ ID NO :11, after optimal global alignment with SEQ ID NO:ll.

Particularly preferred sequences of the adenovirus penton base protomer comprising a peptide tag in the variable loop and/or in the RGD loop, are selected from the amino acid sequences having at least 85%, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 13 and 15, more preferably selected from the amino acid sequences SEQ ID NOs: 13 and 15.

The peptide tag is preferably a covalent peptide tag, preferably selected in the group consisting of Isopeptag, SpyTag, SnoopTag, SnoopTagJr, DogTag, and SdyTag (all as defined above in the definition section); more preferably the peptide tag is SpyTag.

In an advantageous embodiment, the peptide tag to be inserted in adenovirus penton base protomer further comprises at least one linker sequence in N-Ter, or at least one linker sequence in C-Ter, or at least one linker sequence in N-Ter and at least one linker sequence in C-Ter, in particular consisting of between 3 to 15 amino acids. The linker sequence may be selected from the group consisting of sequences of between 3 to 15 amino acids chosen among G, S, A, P, in any combination, preferably 3 to 15 amino acids chosen among G and S in any combination, and more preferably the GSG peptide.

The binding partner of the peptide tag is preferably selected in the group consisting of pilin-C protein, SpyCatcher, SnoopCatcher, DogTag, SdyCatcher and any variant thereof (all as defined above in the definition section); more preferably the binding partner is SpyCatcher or any variant thereof (more preferably SpyCatcher).

In one embodiment, the protein and/or the protein fragment of (ii) (i.e. the cargo) is fused to N-terminal position (i.e. to the N-terminus) or the C-terminal position (i.e. to the C-terminus) of the binding partner of the peptide tag (preferably to the N-terminal position (i.e. to the N-terminus) of the binding partner of the peptide tag). Indeed, the Inventors have shown that fusing a cargo to the N-ter of the binding partner is more efficient than fusing a cargo in C-ter of the binding partner.

In a preferred embodiment, the protein or the protein fragment of (ii) (i.e. the cargo) has at least one post-translational modification, preferably selected from glycosylation, phosphorylation, acylation, carboxylation, acetylation, biotinylation, hydroxylation, lipoylation, amidation, ubiquitination, sumoylation, deamination, and any combination thereof; more preferably selected from glycosylation, phosphorylation, acylation, carboxylation, and any combination thereof; most preferably glycosylation.

In a preferred embodiment, the protein or the protein fragment of (ii) (i.e. the cargo) is selected from the group consisting of antigens, enzymes, hormones, ligands (including signals, such as transport or targeting or addressing signals), receptors, toxins, antibodies, any fragment thereof (preferably a functional fragment) (all as defined in the definition section above), and any combination thereof. The protein of (ii) (i.e. the cargo) is more preferably an antigen and/or the protein fragment is more preferably an antigen fragment.

In a particularly preferred embodiment of the engineered protein of the invention, the protein of (ii) (i.e. the cargo) is a glycosylated antigen, preferably a glycosylated antigen of an enveloped virus, more preferably a glycosylated receptor binding protein of an enveloped virus; and/or the protein fragment of (ii) (i.e. the cargo) is a glycosylated domain of an antigen, preferably a glycosylated domain of an antigen of an enveloped virus, more preferably a glycosylated domain of a receptor binding protein of an enveloped virus.

The enveloped virus is preferably selected from the group consisting of Coronaviridae viruses, Flaviviridae viruses, Alphaviruses, Orthomyxoviridae (in particular Alphainfluenza viruses), Filoviridae, Bunyaviridae, Arenaviridae, Retroviridae, more preferably from the group consisting of Coronaviridae viruses, more preferably from the group consisting of SARS-CoV viruses, more preferably from the group consisting of SARS-CoV2 and variants thereof.

The protein of (ii) is more preferably the Spike protein of SARS-CoV2 or any variant thereof, preferably the glycosylated Spike protein of SARS-CoV2 or any variant thereof. The protein fragment of (ii) is more preferably a fragment of the Spike protein of SARS-CoV2 or any variant thereof, preferably a glycosylated domain of Spike protein of SARS-CoV2, and more preferably the glycosylated RBD domain of the Spike protein of SARS-CoV2 or any variant thereof. Advantageously, the adenovirus penton base protomer of the engineered protein of the invention further comprises a cleaving site of an enzyme (preferably a TEV site) in the variable loop and/or in the RGD loop, flanking the peptide Tag. Indeed, such a cleaving site advantageously allows to increase accessibility of the peptide Tag and/or to increase the efficacy in the formation of the covalent bond with the binding partner fused to the cargo. In one embodiment, the cleaving site of an enzyme is inserted upstream the enlarged RGD motif of the RGD loop, or downstream the enlarged RGD motif of the RGD loop, or in the enlarged RGD motif, or instead of the enlarged RGD motif, or any combination thereof. In one embodiment, the cleaving site of an enzyme is inserted in the first RGD-subloop, or in the enlarged RGD motif, or in the second RGD-subloop, or any combination thereof.

The present invention further concerns a pentamer comprising the engineered protein of the invention (i.e. a pentamer formed by pentamerisation of the engineered protein of the invention), as well as a dodecahedron comprising the engineered protein of the invention (i.e. a dodecahedron formed by auto assembling of 12 pentamers formed by the engineered protein of the invention), as well as the Virus-Like- Particle (VLP) comprising the engineered protein of the invention (i.e. a VLP formed by auto-assembling the engineered protein of the invention).

The present invention further concerns a composition comprising, or consisting essentially of, the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof. The composition can be an immunogenic (immunostimulating) composition or an immunosuppressive composition. The composition can be a vaccine composition. The composition is preferably an immunogenic composition and/or a vaccine. Advantageously, the composition further comprises a pharmaceutically acceptable vehicle. In some embodiments, the composition may further comprise additional therapeutic/vaccine compounds.

Preferably, the composition is a pharmaceutical composition which comprises a therapeutically effective amount of the active agent(s) (engineered protein of the invention), and one or more pharmaceutically acceptable vehicle(s).

Desirably, the composition of the invention is formulated appropriately to ensure its stability under the conditions of manufacture and long-term storage (i.e. for at least 6 months, with a preference for at least two years) at freezing (e.g. -70°C, -20°C), refrigerated (e.g. 4°C) or ambient (e.g. 20-25°C) temperature and it must also be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The composition can be formulated to provide quick, sustained, or delayed release of the active agent(s) after administration. Suitable examples of sustained-release compositions include semipermeable matrices of solid hydrophobic polymers containing the drug, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

In one embodiment, the composition is formulated for oral administration, e.g. under forms including solid, semi-solid and liquid systems such as tablets, soft or hard capsules containing multi- or nanoparticulates, liquids, powders, chews, gels, fast dispersing dosage forms, films, ovules, sprays and buccal/mucoadhesive patches; or for topical or mucosal administration.

In another and preferred embodiment, the composition is formulated for parenteral administration. Sterile injectable solutions can be prepared by incorporating the active agent (i.e. the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof; and optionally at least one additional therapeutic/vaccine compounds) in the required amount in an appropriate solvent, followed by filtered sterilization. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Each unit contains a predetermined quantity of the active agent calculated to produce the desired therapeutic effect in association with the selected pharmaceutically acceptable vehicle(s).

The amount of the engineered protein in the composition of the present invention, the administration route and the periods of time necessary to achieve the desired result (immunosuppressive or immunogenic/immunostimulating effect, therapeutic effect) can be routinely defined by medical staff considering the various factors mentioned above.

For illustrative purposes, a therapeutically effective amount of the engineered protein, or the pentamer, or the dodecahedron, or the VLP, or any combination thereof, to be included in the composition described herein (individual doses), in particular in a vaccine composition, would be in the range from about 1 microgram to about 1000 micrograms, more preferably from 5 to 100 micrograms.

The skilled person in the art is capable of defining appropriate therapeutically effective amount of the engineered protein, or the pentamer, or the dodecahedron, or the VLP, or any combination thereof to be included in the composition described herein.

Engineered adenovirus penton base protomer and post-translationally-modified protein

The Inventors have surprisingly demonstrated that a peptide tag can be efficiently inserted in the external loops of the penton base protomer constituting the ADDomer, without impairing the ADDomer structure, thus providing an original and efficiently adaptable system of peptide tag presentation. Such a system is convenient for various applications, including applications involving modulable binary tag-tag partner.

The Inventors have also shown that more than one peptide tag can be efficiently inserted in the external loops of the penton base protomer, still without impairing the ADDomer structure, thus providing an adenovirus penton base protomer which is able to be used in combination with at least one protein or at least one protein fragment fused to the respective binding partners of the peptide tags. This allows controlled display of two sets of proteins or protein fragments, which is advantageous for example in vaccination, to broaden the protection spectrum of the vaccine against a family of viruses and/or a series of serotypes or tumor antigens. With certain viruses, it might also be interesting to cover all serotypes at once, to prevent the adverse Antibody Dependent Enhancement (ADE) phenomenon.

Accordingly, the present invention further concerns an adenovirus penton base protomer comprising a peptide tag inserted in the variable loop and/or in the "RGD loop". The peptide tag is preferably a covalent peptide tag, preferably selected in the group consisting of Isopeptag, SpyTag, SnoopTag, SnoopTagJr, DogTag, and SdyTag (all as defined above in the definition section); more preferably the peptide tag is SpyTag. When more than one peptide tag is inserted in the adenovirus penton base protomer, the adenovirus penton base protomer preferably comprises two peptide tags. A particularly preferred adenovirus penton base promoter comprises the two peptide tags SpyTag and SnoopTag. The adenovirus penton base protomer of the invention is as the penton base protomer of the engineered protein ad defined in the preceding section. In particular, preferred penton base proteins (where the peptide tag is to be inserted) are those having at least 85%, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity, with any of the sequences indicated in SEQ ID NOs: 1 to 11. Most preferred penton base proteins are those indicated in SEQ ID NOs: 1 to 6 and 11, more preferably those indicated in SEQ ID NOs: 1 and 11.

Advantageously, the penton base protomer (used for and/or present in the engineered protein of the invention), can spontaneously pentamerize to form a pentamer. The pentamers can advantageously auto- assemble in a dodecahedron (i.e. 12 pentamers). In a preferred embodiment, the engineered protein of the invention is capable of assembling into a VLP.

In one embodiment, the peptide tag is inserted upstream the RGD motif of the RGD loop, or downstream the RGD motif of the RGD loop, in the RGD motif, or instead of the RGD motif, or any combination thereof. In one embodiment, the peptide tag is inserted in the first RGD-subloop, or in the RGD motif, or in the second RGD-subloop, or any combination thereof. In preferred embodiments, the peptide tag is inserted in the insertion sites as defined above in relation to the penton base protomer of the engineered protein of the invention.

In the context of the present invention, it is also described a post-translationally-modified antigen or a post-translationally-modified domain of an antigen, fused to a binding partner of a peptide tag; wherein the post-translationally modified antigen is preferably a post-translationally-modified antigen of an enveloped virus, more preferably a receptor binding protein of an enveloped virus; or wherein the post- translationally-modified domain of an antigen is preferably a post-translationally-modified domain of an antigen of an enveloped virus, more preferably a post-translationally-modified domain of a receptor binding protein of an enveloped virus; preferably wherein the post-translational modification is selected from glycosylation, phosphorylation, acylation, carboxylation, acetylation, biotinylation, hydroxylation, lipoylation, amidation, ubiquitination, sumoylation, deamination, and any combination thereof; wherein the post-translational modification is more preferably selected from glycosylation, phosphorylation, acylation, carboxylation, and any combination thereof; most preferably glycosylation.

The enveloped virus is preferably selected from the group consisting of Coronaviridae viruses, Flaviviridae viruses, Alphaviruses, Orthomyxoviridae (in particular Alphainfluenzaviruses), Filoviridae, Bunyaviridae, Arenaviridae, Retroviridae, more preferably from the group consisting of Coronaviridae viruses, more preferably from the group consisting of SARS-CoV viruses, more preferably from the group consisting of SARS-CoV2 and variants thereof.

Most preferably, the post-translationally-modified antigen is the glycosylated Spike protein of SARS-CoV2 or any variant thereof; and/or the post-translationally-modified domain of an antigen is a glycosylated domain of Spike protein of SARS-CoV2 or any variant thereof, and preferably the glycosylated RBD domain of the Spike protein of SARS-CoV2 or any variant thereof.

In addition, the post-translationally-modified antigen and/or the post-translationally-modified antigen fragment may be fused to N-terminal position (i.e. to the N-terminus) or the C-terminal position (i.e. to the C-terminus) of the binding partner of the peptide tag (preferably to the N-terminal position (i.e. to the N-terminus) of the binding partner of the peptide tag). Indeed, the Inventors have shown that fusing a cargo to the N-ter of the binding partner is more efficient than fusing a cargo in C-ter of the binding partner.

Advantageously, the post-translationally-modified antigen and/or the post-translationally-modified antigen fragment has two or more post-translational modifications, preferably selected from glycosylation, phosphorylation, acylation, carboxylation, acetylation, biotinylation, hydroxylation, lipoylation, amidation, ubiquitination, sumoylation, deamination, and any combination thereof; more preferably selected from glycosylation, phosphorylation, acylation, carboxylation, and any combination thereof; most preferably glycosylation.

The present invention further concerns a composition comprising, or consisting essentially of, the adenovirus penton base protomer of the invention, the post-translationally-modified antigen and/or the post-translationally-modified domain of an antigen of the invention, the engineered protein of the invention, or any combination thereof. The composition is preferably an immunogenic composition and/or a vaccine. Advantageously, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition may further comprise additional therapeutic/vaccine compounds.

Uses

The original and efficiently adaptable system combining the ADDomer protein presenting capacity with a highly modulable binary tag-tag partner as developed by the Inventors can be used to present a wide variety of cargos, including peptide epitope (as herein demonstrated with A2L tumour epitope), large well- folded functional protein (as herein exemplified using a fluorescent protein, mCherry), large antigens (as herein demonstrated with "melan A" tumour antigen), large well-folded post-translationally modified proteins such as viral antigens (herein illustrated with SARS-CoV2 glycosylated spike protein and fragments thereof, including the glycosylated Spike Receptor Binding Domain (RBD).

Importantly, the data demonstrate that it is possible to bind multiple distinct cargos on the same ADDomer particle, allowing for instance the display of several antigen variants, highly useful for e.g. multivalent vaccines, cancer vaccine or medicaments.

In addition, the data obtained by the Inventors confirmed that the cargos displayed by the ADDomer are fully functional and immunogen. Notably, the large viral SARS-CoV2-RBD displayed on the ADDomer (ADD- RBD) was capable of specifically recognizing and strongly and quasi-irreversibly binding to its natural cell receptor (the ACE2 receptor) and of being recognized by serum from Covid-19 convalescent patients or anti-RBD monoclonal antibodies, thereby validating uses in active compound screening, and antibody detection. Moreover, the Inventors showed that the antigens displayed on the ADDomer can be efficiently taken up by different subsets of dendritic cells, thus triggering a cellular immune response beneficial in both infectiology and vaccination against cancer. Altogether, the data reveal the biological significance of this novel and adaptable cargo-presenting system and validate crucial applications especially in vaccination, but also in targeted drug-delivery within cells, active compound screening, and antibody detection. In particular SARS-Cov2-RBD displayed on the ADDomer and ACE2 receptor immobilized on a surface can be directly used as a system for active compounds screening (e.g. medicaments).

Accordingly, the present invention relates to a use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above) for screening therapeutic molecules/compounds, preferably antiviral and/or antitumour molecules/compounds. The use is preferably an in vitro use (more preferably an in cellulo use). The use preferably comprises contacting the molecules/compounds to be tested with the engineered protein of the invention. The use preferably further comprises detecting inhibition of the binding of the engineered protein of the invention by the molecules/compounds to be tested with using any technique of molecular interaction detection (such as SPR, BLI, ELISA, Immunofluorescence, FRET, BRET, FACS, radioactivity counting, etc.; when the use is an in cellulo use, preferred techniques are Immunofluorescence, FRET, BRET, FACS, radioactivity counting, etc). In one embodiment, the therapeutic molecules/compounds are screened by competition for binding to a cell receptor.

In a specific example, when using the engineered protein of the invention comprising an antigen or an antigen fragment of a coronavirus, the use may comprise contacting the molecules/compounds to be tested with the engineered protein of the invention, in the presence of a cell receptor of said antigen (e.g. ACE2 receptor). The use may further comprise detecting inhibition of the binding of the engineered protein to the receptor by using any of the molecular interaction detection technique mentioned above. Thus, viral inhibitors that prevent the binding of the antigen or fragment thereof to the receptor will be identified. Similar experiments could be done by in cellulo imaging on cell line expressing said receptor (e.g. ACE2) (preferably by Immunofluorescence, FRET, BRET, FACS or radioactivity counting) to identify the viral inhibitors. The same procedure can be adapted to any other SARS-Cov2 receptor or interacting molecules (i.e not ACE2) or to any molecule interacting with any cargo displayed on the VLP.

The invention also concerns a method for screening therapeutic molecules/compounds, preferably antiviral and/or antitumour molecules/compounds, comprising using the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above). The method preferably comprises a step of contacting the molecules/compounds to be tested with the engineered protein of the invention.

The present invention also concerns a use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above) for detecting, in a biological sample from a subject, the presence of antibodies (directed/specific) to a pathogen and/or to a tumour, preferably to a pathogen and/or to a tumour containing (e.g. expressing) the protein or protein fragment as defined in (ii) above (i.e. the cargo). In preferred embodiments, the VLP of the invention is immobilized on a surface and subsequent binding of antibodies to the antigen is monitored. Any detection method can be used, including, but not limited to, ELISA (Enzyme Linked Immunosorbent Assay), Dot-Blot, Western Blot, SPR (surface plasmon resonance), BLI (BioLayer Interaction).

The invention also concerns a method for detecting, in a biological sample from a subject, the presence of antibodies (directed/specific) to a pathogen and/or to a tumour, preferably to a pathogen and/or to a tumour containing (e.g. expressing) the protein or protein fragment as defined in (ii) above (i.e. the cargo), comprising using the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above). The method preferably comprises a step of contacting the biological sample from a subject with the engineered protein of the invention.

In a preferred embodiment, the detection comprises coating of the engineered protein, or the pentamer, or the dodecahedron, or the VLP, or any combination thereof, on a support and contacting the coated support with the biological sample.

The present invention also relates to a use of the adenovirus penton base protomer of the invention (as described above) for increasing immunogenicity of one or more immunogenic protein(s), as far as said immunogenic protein(s) is(are) fused to the binding partner(s) of the peptide tag(s) which is(are) inserted in the adenovirus penton base protomer. Preferably, said binding partner(s) is(are) inserted in the adenovirus penton base protomer. Preferably, the immunogenic protein is fused to a binding partner of the SpyTag peptide, and more preferably, fused to SpyCatcher. When more than one peptide tag is used in the adenovirus penton base promoter, the immunogenic proteins should be fused to the respective binding partners of the peptide tags. For example, if an adenovirus penton base promoter comprising a SpyTag peptide and a SnoopTag peptide is used for increasing immumnogenicity of two immunogenic proteins, one of these immunogenic proteins has to be fused to SnoopCatcher and the other one to SpyCatcher. The use is preferably an in vitro use, more preferably an in cellulo use.

The present invention also relates to the in vitro use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof, for selective delivery of a cargo (as described above in the section "Engineered protein") to a chosen cell type, such as immune cells (including unstimulated T cells, B cells, hematopoietic cells, etc.), cancer cells, cells infected with a pathogen, etc.

Therapeutic uses and methods

The original and efficiently adaptable system combining the ADDomer protein presenting capacity with a highly modulable binary tag-tag partner as developed by the Inventors can be used to present a wide variety of cargos, including peptide epitope, such as tumour or viral epitopes (as herein demonstrated with A2L tumour epitope), large well-folded functional protein (as herein exemplified using a fluorescent protein, mCherry), large antigens (as herein demonstrated with "melan A" tumour antigen), large well- folded post-translationally modified proteins such as viral antigens (herein illustrated with SARS-CoV2 glycosylated spike protein and fragments thereof, including the glycosylated Spike Receptor Binding Domain (RBD).

Importantly, the data demonstrate that it is possible to bind multiple distinct cargos on the same ADDomer particle, allowing for instance the display of several antigen variants, highly useful for e.g. multivalent vaccines, cancer vaccines or medicaments. In addition, the data obtained by the Inventors confirmed that the cargos displayed by the ADDomer are fully functional and immunogen. Notably, the large viral SARS-CoV2-RBD displayed on the ADDomer (ADD- RBD) was capable of specifically recognizing and strongly and quasi-irreversibly binding to its natural cell receptor (the ACE2 receptor) and of being recognized by serum from Covid-19 convalescent patients or anti-RBD monoclonal antibodies, thereby validating uses in active compound screening, and antibody detection. In addition, vaccination of animals with ADD-RBD induced a potent and specific anti-SARS- CoV2-RBD response. Importantly, the data demonstrate that the immunogenic and protective effect is even more potent when the animals have been previously pre-immunized with ADD alone (to mimic an Adenovirus type 3 pre-existing immunity). Therefore, the ADD vector has a beneficial effect to elicit a better immune response after only one dose of vaccine. Altogether, the data reveal the biological significance of this novel and adaptable cargo-presenting system and validate crucial applications in vaccination, targeted drug-delivery within cells, active compound screening, and antibody detection. In particular SARS-Cov2-RBD displayed on the ADDomer and ACE2 receptor immobilized on a surface can be directly used as a system for active compounds screening (e.g. medicaments).

Accordingly, the present invention relates to the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), for use as a medicament, in particular in a subject having already been exposed to an adenovirus. The invention also concerns the use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or of the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), for manufacturing a medicament, in particular for a subject having already been exposed to an adenovirus. The invention also concerns the use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or of the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), as a medicament, in particular in a subject having already been exposed to an adenovirus.

The invention also relates to the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), for use for preventing and/or treating a disease, preferably a cancer or an infectious disease, in particular in a subject having already been exposed to an adenovirus.

The invention also relates to a method for preventing and/or treating a disease, preferably a cancer or an infectious disease, comprising administering to a subject in need thereof the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), in particular in a subject having already been exposed to an adenovirus. The invention also concerns the use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or of the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), for manufacturing a medicament for preventing and/or treating a disease, preferably a cancer or an infectious disease, in particular in a subject having already been exposed to an adenovirus.

The invention also concerns the use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or of the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), for preventing and/or treating a disease, preferably a cancer or an infectious disease, in particular in a subject having already been exposed to an adenovirus.

The invention also concerns the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), for use as a vaccine, preferably as a vaccine against a cancer or an infectious disease, in particular in a subject having already been exposed to an adenovirus. The invention also concerns the use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), as a vaccine, preferably as a vaccine against a cancer or an infectious disease, in particular in a subject having already been exposed to an adenovirus.

The invention also concerns the use of the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), for manufacturing a vaccine, preferably a vaccine against a cancer or an infectious disease, in particular for a subject having already been exposed to an adenovirus.

The invention also concerns a method of vaccinating a subject in need thereof, comprising administering to the subject the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), preferably for vaccinating the subject against a cancer or an infectious disease, in particular in a subject having already been exposed to an adenovirus.

In a further aspect, said methods or uses are for eliciting or stimulating and/or re-orienting an immune response. According to this aspect, said methods or uses preferably comprise administering the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention (as described above; preferably an immunogenic composition), to a subject in need thereof, in an amount sufficient to activate the subject's immunity. In a further embodiment, said method or use is carried out according to a prime boost approach which comprises sequential administrations of a priming composition(s) and a boosting composition(s). Typically, the priming and the boosting compositions may use the same active agent (i.e. the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above), or the composition comprising the engineered protein of the invention)) or may use different active agent (i.e. the engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof (as described above)). Moreover, the priming and boosting compositions can be administered at the same site or at alternative sites by the same route or by different routes of administration. A preferred prime boost approach involves a first injection (eg. subcutaneous, intramuscular, intradermal, intratumoral, or intravenous) (prime) followed by a second injection (eg. subcutaneous, intramuscular, intradermal, intratumoral, or intravenous) after an optimal period of time. The present invention encompasses one or several administration(s) of the priming and/or the boosting composition(s) with a preference for subcutaneous, intramuscular, intradermal, intratumoral, intranasal and intravenous routes. The period of time separating the administrations of the priming and the boosting varies from one week to 6 months, with a preference for one week to one month and even more preferably for a period of one to two weeks.

The engineered protein of the invention, or the pentamer of the invention, or the dodecahedron of the invention, or the VLP of the invention, or any combination thereof, or the composition, is formulated for administration once or several times via the same or different routes. Any of the conventional administration routes is applicable in the context of the invention including oral, parenteral, topical and mucosal routes. Parenteral routes are intended for administration as an injection or infusion and encompass systemic as well as local routes. Preferably, the composition is formulated for one or more parenteral administration(s), and preferably intravenous (into a vein), intravascular (into a blood vessel), intra-arterial (into an artery), intradermal (into the dermis), subcutaneous (under the skin), intramuscular (into muscle) or intraperitoneal (into the peritoneum) route. Administration can be in the form of a single bolus dose or may also be by a continuous perfusion pump. Mucosal administrations include without limitation oral/alimentary, intranasal, intratracheal, intrapulmonary, intravaginal or intra-rectal route. Topical administration can also be performed using transdermal means (e.g. patch and the like). Preferably, the M2-based composition is formulated for administration by intravenous infusion.

Administrations may use conventional syringes and needles (e.g. Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or improving delivery of a virus in the subject (e.g. electroporation for facilitating intramuscular administration). An alternative is the use of a needleless injection device (e.g. BiojectorTM device). Transdermal patches may also be envisaged. Several doses within the indicated ranges may be administered to the patient. For repeated administrations over several days or longer, the treatment would generally be sustained until an observable clinical benefit occurs. Such doses may be administered intermittently, e.g. every day, every 2 or 3 days, every week, every 2 weeks, every three weeks or every month (e.g. such that the subject receives from about two to about twenty doses of the composition). Doses may also be adapted at each administration (e.g., one or more initial higher dose(s) followed by one or more lower dose(s)). DESCRIPTION OF THE FIGURES

Figure 1: Sticky particle principle. The insect cell codon-optimized SpyTag nucleotide sequence (italic) is inserted in the gene encoding the nanoparticle building block (the amino acid sequence SEQ ID NO: 53 is shown below the nucleotide sequence SEQ ID NO: 54). Upon expression, it results in a sticky nanoparticle (ADD-ST) displaying 60 SpyTag (sequences depicted by either circles (variable loop) or stars (RGD loop)).

Figure 2: SpyCatcher (SC) binding to ADD-ST is functional and can result in full decoration of the nanoparticle. (A) Time course analysis of SC binding to ADD-ST by SDS-PAGE. The shift toward higher molecular weight depicted by asterisk * corresponds to the covalent binding of SC to ADD-ST. Negative control (ADD-0 without SpyTag) cannot result in SC binding as expected. (B) Negative staining electron microscopy shows that the overall structure of ADD-ST is conserved upon SC binding and black 'halo' of SC is visible around the particles. (C) mass spectroscopy analysis of undecorated and fully-decorated ADD- ST by SC shows the shift of ADD-ST of 13kDa corresponding to the SC molecular weight. There is no remaining signal for undecorated ADD-ST after SC addition thus reflecting the full decoration of the nanoparticle.

Figure 3: SnoopCatcher binding to ADD-Snoop Tag is functional and the couple SnoopTag/Snoop Catcher can be combined with the couple SpyTag/SpyCatcher. The SnoopTag (SnT) was inserted in the V loop of the ADD to generate a construct ADD-SnoopTag (ADD-SnT) (A). Another construct (ADD-ortho) was made by inserting the SnoopTag in the V loop and the SpyTag in the RGD loop.(B). Specific orthogonal binding of SnoopCatcher (SnoopC) to SnT and SpyCatcher (SpyC) to SpyTag as well as the possibility of simultaneous binding to the two tags was analyzed by the shift of the ADD molecular weight as indicated.

Figure 4: Influence of fusing the Cargo to either the N-ter or the C-ter of SC. (A) Structure of SC (in front, grey ribbons) bound to SpyTag (in the background, white sticks). Note that the N-ter contrary to C-ter is far from the interaction with SpyTag and then cargo fused to the N-ter of SC part would not result in steric hindrance for the interaction with SpyTag. (B) Gel showing two cargos (mCherry and Melan A) expressed in either the C-ter (SC-mCh and SC-Melan) or N-ter (mCh-SC and Melan-SC) of SC. When these constructs are incubated with ADD-ST, cargos fused to the N-ter efficiently bind to ADD-ST (depicted by white asterisks) whereas their counterpart fused to C-ter are less visible.

Figure 5: Glycosylated RBD-SC can bind ADD-ST with a controlled stoichiometry. (A) A shift in migration is visible for RBD-SC treated with N-Glycosydase showing that it was glycosylated. (B) A fixed amount of ADD-ST was incubated with increasing amount of glycosylated RBD-SC (from left to right). After denaturation on SDS-PAGE the number of ADD-ST monomers bound to RBD-SC (decorated) increases while, as expected, the number of non-decorated ADD-ST monomers decreases showing that particle can accommodate from low to high number of antigens (1 to 60 copies). (C) RBD-SC can also form covalent complex with ADD-ST when ST is inserted in the RGD loop having the sequence as shown in SEQ ID NO: 27.

Figure 6: Combo cancer vaccine and panCoV2 vaccine. ADD-ST enables to bind different cargos on the same particle. Proof of concept has been made with two cargos, depicted by different arrows in (A) (cancer related antigens A2L and MelanA) and three cargos (of different molecular weight for clarity), see different arrows in (B). This strategy has also been validated with wt-RBD combined to two different Cov2 RBD variants (B1.351 and P.l variants) also known as South African and Brazilian variants (shown in SEQ ID NOs: 60 and 61 (C). a, b, c, d in (A): ADD-ST alone, RBC-SC alone, MelanA-SC alone and the three combined together; a and b in (B): ADD-ST alone and ADD-ST combined to the three depicted cargos.

Figure 7: Antibody detection by ELISA using (A) coated ADDomer-RBD and serum from patients who have been infected with SARS-CoV2 or not (weak (*) and strong (***) recognition for COVID-19 positive patients are distinguished from negative (#) patients).

Figure 8: ELISA using anti-RBD neutralizing monoclonal antibodies. The two used antibodies (CC12.1 and CC12.3) recognize RBD-SC displayed on the ADD-ST surface (see the two lines at the top of the graph) even better than RBD-SC alone (the two middle lines) while as expected ADD alone is not recognized (the two lines at the bottom of the graph) showing the specificity of RBD recognition.

Figure 9: Vaccination against SARS-CoV2 in mice. Four groups of ten mice were designed as shown in (A) and injected subcutaneously following the calendar described in (B). Pre-immunization with ADD alone was done in one group to mimic the effect of adenovirus pre-immunity. Each mouse received the same amount of RBD-SC. (C) As expected RBD-SC was not bound to empty ADDomer in group II whereas its display on ADD-ST was visible for groups III and IV as shown by SDS-PAGET

Figure 10: Individual anti-RBD response of the ten mice from the different groups after one and two injections. Dilution of serum was performed from 1/20 to 1/327.680 using 4 time serial dilutions. Two weeks after the first injection ('Prime', upper panel) only mice injected with RBD-SC displayed on the ADD particle (ADD-RBD) showed significant anti-RBD responses. Pre-immunity against adenovirus (mimicked by injection of ADD prior to injection of ADD-RBD - group 4) is beneficial since the response is slightly higher in preimmunized mice than in naive mice (group 3). After the second injection ('Boost', lower panel), a very high signal is observed for groups 3 and 4 injected with ADD-RBD (up to dilution 1/327.680) whereas group 1 and 2 after two injections barely reach signal obtained for group 3 and 4 after only one injection.

Figure 11: SPR assay of ADD-RBD binding to ACE2 receptor. Different concentrations of either RBD-SC alone or decorated ADD-RBD were injected onto immobilized ACE2 receptor. Signals obtained for ADD- RBD were significantly higher and more stable than for RBD-SC (ex: 688RU versus 11RU at lOnM). Signal at InM for ADD-RBD (186RU) is 6 time more important than RBD-SC at lOOnm (31 RU).

Figure 12: Cellular assay of binding of ADD-RBD to HeLa cell expressing ACE2 receptor. Non-decorated (ADD-ST), RBD decorated (ADD-RBD), NTD decorated (ADD-NTD) particles were incubated for 1H at either 4°C or 37°C on HeLa-ACE2 cells. Particles were detected using an anti-ADD serum and an Alexa488 labelled secondary antibody. Nuclei are counterstained with DAPI (Dark grey). (NTD: N ter Domain of SARS-CoV2 spike protein, aminoacids 13-320 of SEQ ID NO: 59; i.e. not containing RBD).

Figure 13: Analysis of ADDomer mediated targeting of melanoma antigens to dendritic cells and activation of CD8+ specific T lymphocytes. (A) Percentage of the different subsets of DC cells positive by FACS for ADDomer staining according to the decoration (empty, decorated with the melanoma peptide A2L or decorated with the melanoma antigen MelanA). (B) ADDomer internalization by DC cells was monitored by confocal microscopy. ADDomer internalization is reflected by dots inside the cells. (C) A2L specific activation of CD8+ T lymphocytes (cross-presentation). Activation of peripheral blood mononuclear cells (PBMCs) from a healthy donor activated for 20 days with corresponding ADDomer was compared with activation by A2L peptide alone at 66 time higher concentration. Activated lymphocytes were recognized by A2L specific dextramers and analyzed by flow cytometry.

EXAMPLES

Although the present invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

EXAMPLE 1: Insertion of SpyTag in the nanoparticle

The ADDomer is a non-infectious 30 nm nanoparticle, formed from 60 copies of a single adenovirus protein, the penton base (i.e. the nanoparticle monomer; Vragniau, C. et al. Synthetic Self-assembling ADDomer Platform for Highly Efficient Vaccination by Genetically-encoded Multi-epitope Display. Science Advances Sep 25;5(9):eaaw2853. doi: 10.1126/sciadv.aaw2853 (2019). Two loops (also called flexible loops), the variable loop and the RGD loop (the latter divided in three subloops), are exposed to the outside and allow the insertion of foreign proteins including antigens, and to present them in 60 copies on the surface of the Addomer.

The SpyTag sequence was inserted in the nanoparticle monomer (i.e. in the penton base protomer) gene in a region coding for the variable loop. This SpyTag was flanked with two linker sequences. This sequence was codon-optimized for expression in insect cells. Expression of the insect cell codon-optimized SpyTag in the baculovirus/insect cell system led to the production of the first component of the system called "ADenovirus Dodecamer - Sticky" (or ADD-ST). The ADD-ST thus obtained is a sticky nanoparticle displaying 60 SpyTag (Figure 1).

Materials and Methods pACEBAC plasmid containing the gene coding the nanoparticle monomer (SEQ ID NO: 12; coding for the amino acid sequence SEQ ID NO: 11) was used for SpyTag insertion. A synthetic DNA coding the SpyTag sequence flanked by linkers at both side (Figure 1, the amino acid sequence SEQ ID NO: 50 is shown below the nucleotide sequence SEQ ID NO: 51) was inserted by synthetic biology instead of amino acids located from positions 159 to 163 of SEQ ID NO: 11. The resulting plasmid was then transformed in Escherichia coli containing the baculovirus genome and a recombinase. Using appropriate antibiotics (Kanamycin, Tetracyclin and Gentamycin) and a blue white screening on LB agar plate, white colonies were selected and used for DNA mini preparation. After transfection with XtremGene in SF21 insect cells, baculovirus coding the nanoparticle (ADD-ST) were amplified and used for batch production in SF21 cells. The resulting nanoparticle (ADD-ST, having the SEQ ID NO: 13) was then purified in two steps :1) sucrose density gradient and 2) ion exchange chromatography. Quality control of the sample was performed by negative staining electron microscopy (Figure 2B). EXAMPLE 2: SpyCatcher makes spontaneous covalent bonds with ADD-ST particle resulting in full decoration

To assess whether the SpyTag sequence is accessible and functional on ADD-ST, the nanoparticle was incubated for different periods of time with the SpyCatcher "SC" (Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690-E697 (2012)). Empty particle (i.e. ADD without SpyTag) was used as negative control. Binding of SC to ADD-ST was controlled by SDS-PAGE analysis, mass spectroscopy and electron microscopy. Results clearly show that SC is covalently bound to ADD-ST and that a full decoration of ADD-ST can be achieved as shown by the disappearance of ADD-ST monomer on both PAGE-SDS (Figure 2A) and mass spectroscopy (Figure 2C). The doublet seen on both SDS-PAGE and mass spectroscopy is due to a second initiation codon in ADD-ST and SC can bind to each form indifferently. Importantly, there is no remaining signal for undecorated ADD-ST after SC addition thus reflecting the full decoration of the nanoparticle. Moreover Electron microscopy (Figure 2B) shows that the dodecameric structure of the particle is conserved.

These data show that SpyCatcher (SC) binding to ADD-ST is functional and results in full decoration of the nanoparticle.

Materials and Methods

ADD-ST (SEQ ID NO: 13) and SC (SEQ ID NO: 48) were incubated in a reactor and samples were taken for analysis at different times after addition (5 minutes to 18H). Negative control consists of empty ADD (i.e. without SpyTag) incubated for 18H with the same amount of SC. SDS-PAGE shows the gradual appearance of a higher molecular weight band (depicted by asterisk) consisting of SC covalently bound to ADD-ST. This sample was further analysed by electron microscopy and electrospray mass spectroscopy.

EXAMPLE 3: SnoopCatcher binds SnoopTag specifically and this process is orthogonal to SpyCatcher binding to SpyTag

To assess whether SnoopTag/SnoopCatcher could be combined with the ADD technology, SnoopTag was inserted in the Variable loop of the ADD, resulting in SEQ ID NO: 96 using the nucleotide sequence SEQ ID NO: 97). Upon addition of SnoopCatcher (SnoopC) for 18h at 25°C, a covalent decoration of ADD-SnoopTag was observed, as indicated by the shift of the ADD-SnoopTag (ADD-SnT) band (indicated by an arrow) from 72 to 95 kDa (1). There was no binding of SnoopC to ADD-SpyTag (2) and no binding of SpyC to ADD- SnoopTag (3) (Figure 3A). Another construct (ADD-ortho, resulting in SEQ ID NO: 98, using the nucleotide sequence SEQ ID NO: 99) was made with SnoopTag in Variable loop and SpyTag in the RGD loop. As shown in Figure 3B, there is about 80% decoration of ADD-ortho by covalent binding of SnoopCatcher(4) and almost total decoration by covalent binding of SpyCatcher (5). Double decoration was observed by sequential binding of SnoopCatcher then SpyCatcher (6) or SpyCatcher followed by SnoopCatcher (7) at half the maximal concentration each, or simultaneous binding (8). Simple and double decorations are indicated by arrows on the right.

These data show that decoration of ADD by SnoopCatcher and SpyCatcher are independent phenomena. This feature shows that it is possible to display two different proteins or protein fragments, in a controlled manner on the Addomer. It allows a higher complexity of protein cocktails that can be displayed, a number of different proteins or protein fragments can be displayed with a single tag, and this is multiplied by two using two peptide tags (ADD-ortho). The system of the invention is thus particularly advantageous to broaden the spectrum of the vaccine for example against a family of viruses and/or a series of serotypes, or different tumour antigens.

EXAMPLE 4: Expression of antigens in fusion to SpyCatcher

Spycatcher (SC) was previously described as capable of making a spontaneous isopeptidic bond with SpyTag. A novel system enabling to fuse SC to a cargo of interest was then designed.

Figure 4 shows successful fusion of two distinct cargos to SC: a fluorescent protein (mCherry; SEQ ID NO: 55) and a large tumor antigen ("Melan A" tumour antigen, SEQ ID NO: 56). When these constructs are incubated with ADD-ST, cargos fused to the N-ter efficiently bind to ADD-ST (depicted by white asterisks) whereas their counterparts fused to C-ter were less visible. These results show that fusing these specific cargos to the N-ter of the SC was more efficient than fusing the cargo in C-ter of the SC (Figure 4).These data demonstrate for the first time that any cargo can be used in this novel system, including (i) "small peptides" such as tumour or viral epitopes, (ii) large well-folded functional protein (as herein exemplified using a fluorescent protein, mCherry), (iii) large antigens (as herein demonstrated with "MelanA" tumour antigen) or even (iv) large well-folded glycoproteins such as viral antigens from different emergent viruses (e.g. part of SARS-CoV2 spike protein including the receptor binding domain (RBD) (Hsieh, C.-L. et al. Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes. Science. Sep 18;369(6510):1501-1505. doi: 10.1126/science.abd0826 (2020)); see example 5 below).

Whatever the nature of the cargo, it is possible to fuse it to either the N-ter or C-ter part of SC; although fusion to the N-Ter was more efficient for these specific examples.

Materials and Methods

Cargos were fused to either the N-ter or the C-ter of SC and expressed in baculovirus/insect cell system. Proteins were purified by IMAC (Immobilized Metal Affinity Chromatography) using a hexa-his sequence added to SC. To assess their respective binding capacity to ADD-ST, a fixed amount of each construct was incubated for 18H at room temperature with a fixed amount of ADD-ST. SDS-PAGE was then used to see whether a covalent adduct of each different cargo to ADD-ST was made.

EXAMPLE 5: Creation of a mime of SARS-CoV2 with glycosylated Spike-RBD

The results presented in example 3 above show that large epitopes can be efficiently attached to the nanoparticle. This novel technology was thus adapted to bind large glycosylated antigen from emergent viruses in order to mimic their surface. The pandemic SARS-CoV2 was the first target. This coronavirus enters cell through the interaction of the viral Spike protein and more particularly the Receptor Binding Domain (RBD) of the protein Spike. SARS-CoV2 RBD (amino acid residues 321-554 of SEQ ID NO: 58) was fused to the N-ter of SpyCatcher (RBD-SC) and expressed in insect cells with a signal peptide, to allow the protein to follow the glycosylation route. After purification from the cell supernatant, RBD-SC was incubated with ADD-ST at different ratio. Results clearly show that the nanoparticle can be decorated by different amounts of glycosylated RBD (Figure 5). Figure 5B shows that the number of ADD-ST monomers bound to RBD-SC (decorated) increases while, as expected, the number of non-decorated ADD-ST monomers decreases showing that particle can accommodates from low to high number of antigens (1 to 60 copies), as desired. These data confirm that glycosylated RBD-SC can bind ADD-ST with a controlled stoichiometry.

A second ADD-ST particle was constructed by synthetic biology with SpyTag (ST) inserted in the RGD loop, instead of amino acid residues located from positions 335 to 348 of SEQ ID NO: 11. The resulting nanoparticle (ADD-ST in RGD loop) has the SEQ ID NO: 15.

Figure 5C shows that similar decoration can be achieved when ST is inserted in the RGD loop (SEQ ID NO: 15) instead of the variable loop (SEQ ID NO: 13).

EXAMPLE 6: Combo cancer vaccine and panSARS-CoV2 vaccine including multiple antigens or viral variants

Since ADD-ST is composed of 60 identical monomers displaying SpyTag, it can be envisaged to bind different cargos on the same particle. This strategy is highly desirable to target emergent viruses such as SARS-CoV2 and its different variants. Since RBD-SC and variants thereof (RBDvar-SC) have the same molecular weight, proof of concept has been made using unrelated cargos with different molecular weights (Figure 6). Figure 6 shows that ADD-ST enables to bind multiple distinct cargos on the same particle (Fig 6A: two cargos; Fig 6B: three distinct cargos: mCherry (SEQ ID NO: 55), MelanA (SEQ ID NO: 56). and A2L (SEQ ID NO: 57).

Of note, MelanA (also known as MART-1: melanoma antigen recognized by T cell 1) is a tumour antigen found specifically at the surface of melanocytes. A decameric peptide encompassed in Melan A (26-35) is recognized by MFIC class I complexes and the mutation A27L (named A2L) was reported to enhance binding. The simultaneous display of both tumour antigen Melan A and engineered tumour epitope A2L on the same vaccine particle paves the way to new anti-tumour therapies.

The data illustrate that a similar strategy can be transposed to wild-type RBD (wt-RBD; SEQ ID NO: 59) combined with two or more different Cov2 RBD variants such as B1.351 (SEQ ID NO: 60) and P.l (SEQ ID NO: 61) variants, also known as South African and Brazilian variants (Fig 6C), or any other variants.

Altogether, these data showed that mosaic vaccine harbouring different antigens at once can be produced using the vaccine system of the present invention. The number of possible antigens bound to the vaccine system can be multiplied by two when two peptide tags are combined, as in example 3. The system of the invention is thus particularly advantageous to broaden the spectrum of the vaccine for example against a family of viruses and/or a series of serotypes, or different tumour antigens.

EXAMPLE 7: The SARS-CoV2-RBD displayed on ADD-ST can be used to detect antibodies in serum from Covid-19 convalescent patients or anti-RBD monoclonal antibodies

To assess whether RBD is functional when displayed on ADD-ST, serum from SARS-CoV2 convalescent patients was used. RBD-decorated ADD was coated in 96-well ELISA plate then blocked by BSA. Serums from COVID-19 convalescent patients or non-infected patients were then incubated. After washes, anti human antibody conjugated to H RP was used to detect the presence of anti-SARS CoV2 bound to RBD- decorated particles (Figure 7). Figure 7 demonstrates that recognition of antibodies for COVID-19 positive patients can be clearly distinguished from negative patients.

Of note, ADD alone is recognized by serums from COVID-19 convalescent patients and by some of the COVID-19 uninfected patients. This pre-existing immunity, which is known to exist in humans who had been previously exposed to adenovirus, accurately correlates with the positive effect observed in mice in Figure 10.

A complementary experiment was performed with two monoclonal antibodies described to inhibit SARS- CoV2 infection. These antibodies (CC12.1 and CC12.3) were tested by ELISA on either empty ADD, RBD alone or RBD-decorated ADD-ST. The experiment clearly shows that RBD displayed by ADD is well- recognized by those antibodies showing that RBD is immunologically functional (Figure 8).

Altogether, these data demonstrate that the particle displaying the antigen of interest can be used for detection of specific antibodies in patient serums.

EXAMPLE 8: Vaccination in mice

Vaccination was assessed on mice. Four groups corresponding to RBD-SC alone, RBD-SC with empty ADD, RBD-decorated ADD-ST on naive mice and RBD-decorated ADD-ST on adenovirus preimmunized mice were designed (Figure 9A). Each group was composed of 10 mice receiving the same dose (5pg per mouse) of RBD-SC (either alone, in addition to empty ADDomer or displayed on ADD-ST) in presence of Addavax (Invivogen). A prime injection was done at day 0 followed by a boost injection at day 14. Serums were taken one day before each injection then at day 27 and 41 (Figure 9B). As expected RBD-SC was not bound to the nanoparticle in group II whereas its display on ADD-ST was visible for groups III and IV as shown by SDS-PAGE in Figure 9C.

Flumoral response against SARS-Cov2 RBD investigated by ELISA

In order to assess if an anti-SARS-CoV2-RBD response was triggered in animals, an analysis was performed with the 10 individual mice of each group at 2 weeks after prime and 4 weeks after the boost. RBD was coated on 96 well-plate overnight, blocked with BSA and then incubated with different dilutions of serum from all the mice of each group. Sequential four-time dilutions were performed starting from 1/20 and ending at 1/327.680. After washes, wells were incubated with an anti-mouse secondary antibody linked to H RP then washed again. The TMB (3,3',5,5'-tetramethylbenzidine) substrate was then added and the reaction was stopped by sulfuric acid after 1 minute. OD450nm was then read.

Figure 10 shows the results of detection of the anti-RBD response in all individual mice from the different groups. Clearly, the display of RBD on the nanoparticle is beneficial. Indeed, significantly increased responses were obtained after the prime injection for group 3 and 4 whereas no response was observed when RBD was not displayed on ADDomer in group 1 and 2 (Figure 10 upper panel). Moreover, preimmunity against adenovirus, mimicked here by pre-injection of ADD alone (group 4) is not an obstacle, and on the contrary the anti-coronavirus response was slightly higher in preimmunized mice (group 4) than in naive mice (group 3). After the second injection, a very high signal (up to dilution 1/327.680) was observed for groups 3 and 4 injected with ADD-RBD (Figure 10, lower panel) whereas group 1 and 2 after two injections barely reached signal obtained for groups 3 and 4 after only one injection.

Altogether these data showed that the system of antigen display of the invention enables to get a rapid humoral response from the prime immunization reinforced by high titers of antibody against the antigen of interest after the boost immunization, and this, independently from adenovirus pre-existing immunity. In other terms adenovirus pre-immunity is not an obstacle and even has slightly favourable effect.

EXAMPLE 9: A highly sensitive SPR detection method showing the interaction of RBD displayed by ADDomer with immobilized ACE2 receptor.

Angiotensin Converting Enzyme 2 (ACE2) is the receptor for SARS-CoV2. The RBD is the part of the spike known to bind ACE2 receptor. The hypothesis where the display of RBD-SC on ADD-ST particle can be considered as a non-infectious mime of SARS-CoV2 for attachment to ACE2 was assessed. Surface Plasmon Resonance (SPR) is a gold standard method to investigate real-time molecular interactions. In this experiment, the ACE2 receptor (aa 18 to 615 of SEQ ID NO: 62). fused to Fc (Fc-ACE2) was immobilized on the sensorship in an oriented manner using anti-Fc antibody. As a control, a FlowCell with anti-Fc antibody without Fc-ACE2 was used. Specific SPR signal was then calculated by subtracting the signal of the control FlowCell to the one of the Fc-ACE2 containing FlowCell. Different amounts of SC-RBD alone or ADD-RBD (RBD displayed on ADD-ST) were injected (amounts shown in Table 1 below) and SPR signal was recorded (Figure 11 and Table 1).

Table 1 : Amounts of SC-RBD alone or ADD-RBD injected, and SPR signals recorded:

Signals obtained for ADD-RBD were significantly higher and more stable than for RBD-SC (ex: 688RU versus 11RU at lOnM). Note that signal at InM for ADD-RBD (186RU) is 6 time more important than RBD-SC at lOOnm (31 RU). Overall, sensitivity of detection is increased up to 600 times when using RBD-decorated ADD and no dissociation is observed at the end of injection (depicted by arrow) as reflected by the flat curves.

These results show that the display of a protein binding domain on the ADDomer can induce a high affinity binding of its corresponding target (e.g. receptor), with virtually no dissociation, which is of great interest to set up highly sensitive screening tests.

EXAMPLE 10: Cellular immuno-fluorescent detection of ADD displayed RBD binding to ACE2 receptor.

Example 9 above demonstrates that ADD-RBD does bind ACE2 by SPR, paving the way of an effective screening of antiviral compounds targeting the RBD/ACE2 interaction. To go further in the screening process, it was investigated whether those putative antiviral compounds would be also effective in the cellular context, knowing that viruses are ultimate parasites requiring to enter cell to replicate. To do so, FleLa cells overexpressing ACE2 receptor were used. This cell line was then incubated at 4°C (to prevent internalization) or at 37°C (permissive for internalization) with non-decorated ADD, RBD-decorated ADD or ADD decorated with the N-terminal part of the spike of the SARS-CoV2 without the RBD domain (NTD; aa 13-321 of SEQ ID NO :58). After 1H incubation, particles were detected using anti-ADD rabbit serum and anti-rabbit antibody labelled by Alexa488. Similar experiment was also performed at 37°C for 1H to verify whether internalization can occur (Figure 12).

Figure 12 reveals specific binding of ADD-RBD to FleLa cell expressing ACE2 receptor. These results show that antiviral compounds targeting/blocking SARS-CoV2 binding to ACE2 can be efficiently screened using the system of the present invention (for example by combining ADD-RBD concomitantly or sequentially to SPR and cell binding techniques).

EXAMPLE 11: Melanoma antigens displayed on ADDomer are able to enter dendritic cells and trigger cross-presentation to CD8+ B lymphocytes.

Empty ADDomer, ADDomer-A2L and ADDomer-MelanA were made by incubation of ADDomer Spy Tag with A2L and Melan A fused to Spy Catcher. ADDomers Spy Tag was previously labelled by Alexa fluor 647 using the manufacturer's protocol prior to incubation with the different antigens. Peripheral blood mononuclear cells (PBMCs) from healthy HLA-A2+ donors were cultured in RPMI medium with 10% Fetal Calf Serum and treated by the labelled ADDomers (at 1, 3 and 10 pg/ml) carrying the antigens. After 4h, FACS analysis was performed to measure the percentage of labelled cells using both Alexa647 label and specific markers of the different dendritic cells' subsets. The mean fluorescence intensity was plotted.

For cross presentation, there is only A2L specific dextramers, so the experiment was done with A2L only. PBMCs from healthy donors were cultivated for 20 days and stimulated with TLR3 ligand to yield all 3 subsets of dendritic cells. They were treated every 7 days with ADD or ADD-A2L and in parallel with A2L peptide. CD8+ T cells were detected by A2L specific dextramer and an irrelevant dextramer was used as negative control.

Figure 13 shows entry of ADD into dendritic cells, a process required for efficient access to the immune system. All 3 subsets of dendritic cells were targeted and a dose-effect is shown with the use of 3 doses of ADDomer (Figure 13A). The presence of ADD inside the cells was visualized by immunofluorescence (Figure 13B). A cross-presentation experiment was done in which PBMCs were treated for 20 days with ADD-A2L, which resulted in A2L-specific activation of CD8+ T lymphocytes, in a way comparable to that obtained with A2L peptide at higher concentration (Figure 13C).

These data showed that a cellular cytotoxic response against the displayed tumoral antigen is triggered by the vaccine system of the invention, in addition to the humoral response. This is of high importance for cancer purposes as well as for long term protection against pathogens.

Claims

1. Engineered protein comprising:

2. The engineered protein according to claim 1, wherein the peptide tag is SpyTag.

3. The engineered protein according to claim 1 or claim 2, wherein the binding partner is SpyCatcher or any variant thereof.

4. The engineered protein according to any one of the preceding claims, wherein the protein or the protein fragment of (ii) has at least one post-translational modification, preferably selected from glycosylation, phosphorylation, acylation, carboxylation, and any combination thereof, most preferably glycosylation.

5. The engineered protein according to any one of the preceding claims, wherein the protein of (ii) is an antigen, or the protein fragment of (ii) is a fragment of an antigen.

6. The engineered protein according to claim 5, wherein the protein of (ii) is a glycosylated antigen, preferably a glycosylated antigen of an enveloped virus, more preferably a glycosylated receptor binding protein of an enveloped virus; or wherein the protein fragment of (ii) is a glycosylated domain of an antigen, preferably a glycosylated domain of an antigen of an enveloped virus, more preferably a glycosylated domain of a receptor binding protein of an enveloped virus.

7. The engineered protein according to any one of claims 1 to 6, wherein the protein of (ii) is the Spike protein of SARS-CoV2 or any variant thereof, preferably the glycosylated Spike protein of SARS-CoV2 or any variant thereof ; or wherein the protein fragment of (ii) is a fragment of the Spike protein of SARS-CoV2 or any variant thereof, preferably a glycosylated domain of Spike protein of SARS-CoV2, and more preferably the glycosylated RBD domain of the Spike protein of SARS-CoV2 or any variant thereof.

8. An adenovirus penton base protomer comprising a peptide tag in the variable loop and/or in the RGD loop.

9. The adenovirus penton base protomer according to claim 8, which comprise two peptide tags, preferably SpyTag and SnoopTag.

10.In vitro use of the adenovirus penton base protomer according to claim 8 or 9 for increasing immunogenicity of immunogenic protein(s) fused to the binding partner(s) of the peptide tag(s).

11. The engineered protein according to any one of claims 1 to 6 or an immunogenic composition comprising said engineered protein, for use as a vaccine, preferably as a vaccine against cancer or infectious disease, in particular in a subject having already been exposed to an adenovirus.

12. The engineered protein according to any one of claims 1 to 7 or a composition comprising said engineered protein, for use as a medicament.

13. In vitro use of the engineered protein according to any one of claims 1 to 7 for screening therapeutic molecules/compounds, preferably antiviral molecules/compounds. lA.ln vitro use of the engineered protein according to any one of claims 1 to 7 for detecting, in a biological sample from a subject, the presence of antibodies to a pathogen containing the protein or protein fragment as defined in claim l(ii).