Fusion proteins for the treatment of viral infections

Abstract

The present disclosure relates to bifunctional fusion proteins comprising domains which bind to a viral envelope protein and to proteins present in the surface of dendritic cells as well as to methods for the prevention and treatment of disorders caused by viral infections.

Description

[0001] FUSION PROTEINS FOR THE TREATMENT OF VIRAL INFECTIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention pertains to the field of protein engineering and medicine, more specifically, the present disclosure provides fusion proteins which target viruses to dendritic cells.

[0004] BACKGROUND OF THE INVENTION

[0005] Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has given rise to one of the worst pandemics in recent history. As of 21 June 2023, the virus has infected more than 768 million individuals, causing over 6.9 million deaths. Although multiple effective vaccines preventing COVID-19 are being widely administered worldwide, the emergence of multiple SARS- CoV-2 variants causing increased viral dispersion and immune evasion requires the continual development of effective therapeutics against COVID-19. In this context, monoclonal antibodies (mAb) have shown efficacy in animal models of SARS-CoV-2 infection and several mAb-based therapeutics received Emergency Use Authorization.

[0006] SARS-CoV-2 infection requires the spike (S) protein receptor-binding domain (RBD) docking to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) for viral entry into host cells, so most SARS-CoV-2-neutralizing antibodies (nAb) block this interaction by direct binding to the RBD. Accordingly, viral strains with mutations altering the RBD surface can avoid antibody recognition and neutralization. A particularly negative effect has been attributed to the K417N / T, L452R, T478K, and E484K / Q mutations, as seen in the B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta) or B.1.1.529 (omicron) variants of concern (VOCs). The spread of these VOCs has reduced the efficacy of vaccines and mAb-based therapeutics, making it mandatory to update them against present and future SARS-CoV-2 variants.

[0007] Camelid-derived single-domain antibodies, also known as VHHs or nanobodies, combine antigen-binding affinities that are comparable to conventional antibodies with a smaller size (15 kDa), high stability, and engineering simplicity. Their potential for use against SARS-CoV-2 infection has been widely reported both in vitro and in vivo.

[0008] Dendritic cells (DCs) are professional antigen-presenting cells that play a central role in the induction of antigen-specific adaptive immune responses during infection. C-type lectin receptors (CLR), such as DEC-205, DCIR-2 and DC-SIGN, have been increasingly used in preclinical models for in vivo targeting of antigens to DCs. Despite the large number of possible therapies available, therapies which are more robust to the ever-changing coronavirus variants are required,

[0009] SUMMARY OF INVENTION

[0010] The authors of the present invention have observed that an improved immune protection effect is obtained when the complete virus particles are presented directly to dendritic cells through fusion proteins which recognize viral envelope proteins as well as receptors present in the surface of dendritic cells.

[0011] As such, a first aspect relates to a fusion protein comprising:

[0012] (a) a region which binds to a viral envelope protein

[0013] (b) a trimerization domain; and

[0014] (c) a region which binds specifically to a receptor present in the surface of dendritic cells.

[0015] Another aspect relates to a trimer comprising three monomers of the fusion protein according to the invention.

[0016] Yet another aspect relates to a polynucleotide encoding the fusion protein according to the invention.

[0017] Another aspect relates to a vector comprising the polynucleotide according to the invention.

[0018] One more aspect relates to a host cell comprising the polynucleotide according to the invention or the vector according to the invention.

[0019] Yet another aspect relates to an immunogenic composition or a vaccine composition comprising the fusion protein according to the invention, the trimer according to the invention, the polynucleotide according to the invention, the vector according to the invention or the host cell according to the invention and a pharmaceutically suitable carrier or excipient.

[0020] One aspect relates to a fusion protein according to the invention, a trimer according to the invention, a polynucleotide according to the invention, a vector according to the invention, a host cell according to the invention for use in medicine.

[0021] Yet another aspect relates to a fusion protein according to the invention, a polynucleotide according to the invention, a vector according to the invention, a host cell according to the invention or an immunogenic composition or vaccine composition according to the invention for use in the prevention and / or treatment of a disorder caused by and / or associated with an infection by a virus, wherein the virus contains in its envelope the protein to which the region (a) of the fusion protein binds.

[0022] LEGENDS TO FIGURES

[0023] Figure 1 : The TNTtrimerbody efficiently blocks RBD-hACE2 interaction by simultaneous binding to the three RBDs of the SARS-CoV-2 S protein causing broad effective neutralization activity. A) Hypothetical model of the TNTtrimerbody displayed in lateral and top views showing VHHS and TIE trimerization domain disposition. Each monomer was colored differently (blue, red and purple). B) The ability of TN and TNTantibodies to block the interaction of ACE2 with RBD was investigated using BLI. TN (black) or TNT(blue) were associated with immobilized B.1 RBD, after which ACE2 was added. Unblocked control is also shown (red). C) Side and top views of sharpened cryo-EM map corresponding to the trimeric S protein ectodomain after incubation with TNT. The map is shown in pale grey and the S protein / TNTmodelization is included in the map as cartoon representation. Each protomer of the S protein (3-up RBD) is colored differently (yellow, blue and green) and the docked domains, E and V VHHS forming the three-arms of the TNT, bound simultaneously to the three RBDs are shown in purple and magenta, respectively. D) The alignment of the three RBDs bound to E and V VHHS from our model illustrates the similar way of interaction between the three arms of TNTand each RBD. E) Neutralization of SARS-CoV-2 S-pseudotyped rVSV-luc. Two-fold serial dilutions of control nAb (SB-40592 or sotrovimab), TN or TNTwere incubated with pseudoviruses expressing S protein from different variants prior to Vero E6 cells infection. Normalized values from three independent experiments ± SEM are plotted. Overall, there was an excellent correlation between the three neutralization assays (R2= 0.92).

[0024] Figure 2: The bispecific TNTDNGR-1 trimerbody combines potent and broad SARS- CoV-2 neutralization with specific targeting of viral antigens to DNGR-1 -expressing DCs for boosting systemic S-specific effector CD8+T cell responses. A) TNTDNGR-1 hypothetic model showing VnH-tandem, TIE trimerization domain and anti-DNGR-1 7H11 scFv antibody. Each polypeptide chain is represented in one different color (blue, red and purple). B) Bispecificity of TNTDNGR-1 was investigated using BLI by incubating RBD-coated biosensors first with antibody, and then with DNGR-1. Control RBD-coated biosensors with TNTDNGR-1 but without DNGR-1 and just with DNGR-1 were also included. C) Neutralization of SARS-CoV-2 S-pseudotyped rVSV-luc. Two-fold serial dilutions of each trimerbody were incubated with SARS-CoV-2 S-pseudotyped rVSV-luc expressing S protein from beta and delta variants prior to infecting Vero E6 cells. D) B.1 , beta and delta SARS-CoV-2 virus neutralization of infection. Five-fold serial dilutions of sotrovimab, TN, TNTor TNTDNGR-1 were incubated with live viruses prior to infecting Vero E6 / TMPRSS2 cells. E) Flow cytometry analysis of Flt3-L BMDCs after incubation at 4 °C or 37 °C with TNTDNGR-1 or control TNTand subsequent stained with AF647- RBD staining and antibodies to discriminate DC subsets. Upper histograms represent actual staining for the different cell subsets at 1 hour and 4°C. Lower data represent geometric mean fluorescence intensity (gMFI) for the cDC1 , cDC2 and pDC subsets at the different conditions. F) Representative pictures of MuTu-DC cells incubated with TNTDNGR-1 or control TNTand AF647-RBD at 4 °C and then for different times at 37 °C before fixation and visualization using confocal microscopy. G) Immunization of C57BL / 6 mice using a prime-boost scheme with either: 1) PBS; 2) S protein + adjuvants (poly l:C + CpG); 3) S protein + TNT+ adjuvants; or 4) S protein + TNTDNGR-1 + adjuvants. One representative from two independent experiments (n=3-5 mice / group / experiment) is shown. Spleens were harvested on day 14 and stained for S-specific CD8+T cells using specific S protein tetramer, H-2Kb (539-VNFNFNGL-546) (S-Tet). Frequency of CD44+S-Tet+cells within the T CD8 gate (H) and total number of CD44+S-Tet+CD8+T cells (I) are shown. *p<0.05; **p<0.01 by one-way ANOVA followed by Tukey’s multiple comparison test. J) The S-specific lgG2c / lgG1 ratio by mean of serum S-specific subclass IgG determination by ELISA at a 1 / 250 dilution in serum samples obtained on day 14.

[0025] Figure 3: Therapeutic application of TNTDNGR-1 , but not TNT, protects mice of SARS- CoV-2 lethal infection by reducing viral load in the lungs. A) Efficacy experiment in K18- hACE2 mice was done using a single antibody intraperitoneal (i.p.) administration 17 hours after intranasal (i.n.) viral challenge (105PFUs / mouse). Experimental groups (n=8 mice / group, 1 experiment) included: uninfected control group (black) and infected groups treated with PBS (grey), sotrovimab biosimilar (green), TNT(red) and TNTDNGR-1 (blue). On day 5 p.c. 4 mice per group were euthanized to obtained serum samples and lungs (n=3 in the case of the TNTgroup). Serum was collected on day 5 and 20 post-challenge (p.c.) for humoral immune response analysis. Lungs were harvested on day 5 p.c. for viral load determination and on day 20 p.c. for the analysis of S-specific T cell responses. B) Weight change after B.1 (isolate MAD6) SARS-CoV-2 challenge followed by antibody administration (mean ± SEM) expressed as percentage of initial body weight. C) Kaplan- Meyer survival representation of the efficacy experiment. D) Viral mRNA levels at day 5 p.c. in the right lung measured for E (envelope) and RNA-dependent RNA polymerase (RdRP) genes. E) Infective virus in the lungs at day 5 p.c. represented as PFUs / g of lung tissue. All graphs represent individual values plus mean ± SEM for a single experiment. *p<0.05; **p<0.01 ; ***p<0.001 by one-way ANOVA followed by Tukey’s multiple comparison test.

[0026] Figure 4: TNTNDGR-1 mediates an effective humoral and cellular immune response against SARS-CoV-2, specifically by providing enhanced S-specific cytotoxic T CD8+cell responses in the lungs. A) Anti-S IgM levels in serum 5 days p.c. expressed as absorbance summation. B) Quantification of S-, RBD- and N-specific total IgG levels at 5 days p.c. expressed as absorbance summation. C) Quantification of S-, RBD- and N- specific total IgG levels at 20 days p.c. expressed as absorbance summation. D,E) SARS-CoV-2 neutralization. Serum neutralization of live SARS-CoV-2 (B.1 strain), expressed as NT50, at day 5 (D) and 20 (E) p.c. Lungs were harvested on day 20 p.c. and stained for S-specific CD8+T cells using specific S protein tetramer H-2Kb (539- VNFNFNGL-546) (S-Tet). F) Frequency of CD44+S-Tet+cells within the T CD8 gate. G) Frequency of S-specific T CD4+(left panel) and T CD8+cells (right panel) in the lungs at 20 days p.c. and evaluated by an intracellular cytokine staining assay measuring expression of CD107a, and secretion of IFN-y, TNF-a and IL-2. All graphs represent individual values plus mean ± SEM (n=4 mice / group; n=3 mice in the case of TNTat day 5 p.c.) for a single experiment. *p<0.05; **p<0.01 ; ***p<0.001 by one-way ANOVA followed by T ukey’s multiple comparison test. *p<0.05 for S specific CD8+T cells analysis (G) was done by Dunn's multiple comparisons test following Friedman test.

[0027] Figure 5: The bispecific J3TDNGR-1 trimerbody efficiently interacts with their two targeted antigens simultaneously. A) Schematic diagram of the J3TDNGR-1 showing the protein arrangement in lateral view showing J3 HHS (red), TIE trimerization domain (light blue) and anti-DNGR-1 7H11 scFv (yellow) disposition within the molecule. Due to their trimeric conformation in solution, the expected mass values for J3TDNGR-1 is 150 kDa, being 50 kDa for each monomeric chain. B) Western blot analysis of supernatants from conditioned cells for each construct 72 hours after transfection using StrepTagll detection. C) Conditioned media for J3, J3Tand J3TDNGR-1 bind specifically to plate immobilized recombinant gp140. D) Purified J3Tand J3TDNGR-1 (2 pg / lane) SDS-PAGE analysis in reducing conditions. E) Both J3-based trimerbodies bound strongly to recombinant gp140. E) J3TDNGR-1 bispecific simultaneous binding to coated gp140 and soluble DNGR-1.

[0028] Figure 6: HEK-293T cells transfection with mRNA coding for TNTand TNTDNGR-1 produced functional trimerbodies. A) Conditioned media for RNA-TNTand RNA- TNTDNGR-1 showed specific interaction with B.1 RBD in a similar amount to vector transfected cultures. B) Bispecific ELISA detecting TNTDNGR-1 bound to RBD by interaction with soluble DNGR-1. In accordance, mRNA produced TNTDNGR-1 , but not TNT, showed simultaneous binding to both target antigens. C) B.1 SARS-CoV-2 (MAD6 isolate) neutralization performed by serial dilutions of conditioned supernatants of HEK- 293T cell cultures transfected with plasmids and mRNA coding for TNTand TNTDNGR- 1.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] The authors of the present invention have observed that an improved immune protection effect is obtained when the complete virus particles are presented directly to dendritic cells through fusion proteins which recognize viral envelope proteins as well as receptors present in the surface of dendritic cells.

[0031] The inventors of the present invention have developed a fusion protein that comprises a region which binds to a viral envelope protein, a trimerization domain; and a region which binds specifically to a receptor present in the surface of dendritic cells.

[0032] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0033] All the embodiments and definitions disclosed in the context of one aspect of the invention are also applicable to the other aspects of the invention.

[0034] Fusion protein

[0035] Therefore, a first aspect relates to a fusion protein, from here onwards the fusion protein of the invention, comprising:

[0036] (a) a region which binds to a viral envelope protein

[0037] (b) a trimerization domain; and

[0038] (c) a region which binds specifically to a receptor present in the surface of dendritic cells.

[0039] The term “fusion protein” as used herein, relates to a single polypeptide chain artificially designed and generated by genetic engineering which consist of two or more functional domains derived from different origins. The fusion protein, per definition, is never found in nature as such. A fusion protein may be obtained by conventional methods known by the person skilled in the art (e.g. by means of gene expression of the nucleotide sequence encoding for said fusion protein in a suitable cell) [Sambrook et al., 2001 , “Molecular cloning, to Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3 a]. The term “peptide” or “polypeptide”, as used herein, generally refers to a linear chain of around 2 to 40 amino acid residues joined together with peptide bonds. It will be understood that the terms “peptide bond”, “peptide”, “polypeptide” and protein are known to the person skilled in the art. From here on, “peptide” and “polypeptide” will be used indistinctly.

[0040] As used herein, an "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative or any amino acid mimic known in the art. In certain embodiments, the residues of the protein or peptide are sequential, without any non-amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may comprise one or more non-amino acid moieties. In particular embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non- amino acid moieties.

[0041] In one embodiment, element (a) of the fusion protein (the region which binds to a viral envelope protein) and element (c) (the region which binds specifically to a receptor present in the surface of dendritic cells) comprise, consist or consist essentially of an antibody.

[0042] The term “antibody” (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments which have been previously defined. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique.

[0043] As used herein, the antibodies suitable for the invention encompass not only full length antibodies (e.g., IgG), but also antigen-binding fragments thereof, for example, Fab, Fab’, F(ab')2, Fv fragments, human antibodies, humanized antibodies, chimeric antibodies, antibodies of a non-human origin, recombinant antibodies, and polypeptides derived from immunoglobulins produced by means of genetic engineering techniques, for example, single chain Fv (scFv), diabodies, heavy chain or fragments thereof, light chain or fragment thereof, VH or dimers thereof, VL or dimers thereof, Fv fragments stabilized by means of disulfide bridges (dsFv), molecules with single chain variable region domains (Abs), minibodies, scFv-Fc, and fusion proteins comprising an antibody, or any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of a desired specificity. The antibody of the invention may also be a bispecific antibody. An antibody fragment may refer to an antigen binding fragment. An antibody includes an antibody of any class, namely IgA, IgD, IgE, IgG (or sub-classes thereof), and IgM, and the antibody need not be of any particular class. An antibody mimetic, an organic compound that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.

[0044] In a particular embodiment of the fusion protein of the invention the antibody that binds to a viral envelope protein is a VHH.

[0045] The term ‘VHH’ refers to the single heavy chain variable domain antibodies devoid of light chains. Preferably a HH is an antibody of the type that can be found in Camelidae or cartilaginous fish which are naturally devoid of light chains or to a synthetic and nonimmunized VHH which can be constructed accordingly. Said VHH may be a natural VHH antibody, preferably a Camelid antibody, or a recombinant protein comprising a heavy chain variable domain.

[0046] The term “viral envelope” or “envelope”, as used herein, relates to the viral structure covering the viral capsid that typically is derived from portions of the host cell membranes. The viral envelope comprises phospholipids and proteins from the membrane of the host cell, and may include as well viral glycoproteins. The term “viral envelope protein”, as used herein, relates to a protein constituent of the viral envelope.

[0047] In some embodiments, a viral envelope protein (e.g., a pseudotyping element and / or a fusogenic envelope protein) herein is a full-length polypeptide(s), functional fragment(s), homolog(s), or functional variant(s) of a syncytium-inducing polypeptide. In some embodiments, a viral envelope protein herein is a full-length polypeptide, functional fragment(s), homolog(s), or functional variant(s) of Human immunodeficiency virus (HIV) gpl60, Murine leukemia virus (MLV) gp70, Gibbon ape leukemia virus (GALV) gp70, Feline leukemia virus (RD114) gp70, Amphotropic retrovirus (Ampho) gp70, 10A1 MLV (10A1) gp70, Ecotropic retrovirus (Eco) gp70, Baboon ape leukemia virus (BaEV) gp70, Measles virus (MV) H and F, Nipah virus (NiV) H and F, Rabies virus (RabV) G, Mokola virus (MOKV) G, Ebola Zaire virus (EboZ) G, Lymphocytic choriomeningitis virus (LCMV) GP1 and GP2, Baculovirus GP64, Chikungunya virus (CH1 KV) El and E2, Ross River virus (RRV) El and E2, Semliki Forest virus (SFV) El and E2, Sindbis virus (SV) El and E2, Venezuelan equine encephalitis virus (VEEV) El and E2, Western equine encephalitis virus (WEEV) El and E2, Influenza A, B, C, or D HA, Fowl Plague Virus (FPV) HA, Vesicular stomatitis virus VSV-G, or Chandipura virus and Piry virus CNV-G and PRV-G. In some embodiments, the fusion glycoprotein or functional variant thereof is a full-length polypeptide, functional fragment, homolog, or functional variant of the G protein of Vesicular Stomatitis Alagoas Virus (VSAV), Carajas Vesiculovirus (CJSV), Chandipura Vesiculovirus (CHPV), Cocal Vesiculovirus (COCV), Vesicular Stomatitis Indiana Virus (VSIV), Isfahan Vesiculovirus (ISFV), Maraba Vesiculovirus (MARAV), Vesicular Stomatitis New Jersey virus (VSNJV), Bas-Congo Virus (BASV). In some embodiments, the fusion glycoprotein or functional variant thereof is the Cocal vims G protein. In some embodiments, a viral envelope protein, (e.g., a pseudotyping element and / or a fusogenic envelope protein) herein is a full-length polypeptide(s), functional fragment(s), homolog(s), or functional variant(s) of type G membrane glycoprotein of rabies virus, type G membrane glycoprotein of Mokola virus, type G membrane glycoprotein of vesicular stomatitis virus, type G membrane glycoprotein of Togaviruses, murine hepatitis virus JHM surface projection protein, porcine respiratory coronavirus spike glycoprotein, porcine respiratory coronavirus membrane glycoprotein, avian infectious bronchitis spike glycoprotein and its precursor, bovine enteric coronavirus spike protein, paramyxovirus SV5 F protein, Measles virus F protein, canine distemper virus F protein, Newcastle disease virus F protein, human parainfluenza virus 3 F protein, simian virus 41 F protein, Sendai virus F protein, human respiratory syncytial virus F protein, Measles virus hemagglutinin, simian virus 41 hemagglutinin neuraminidase proteins, human parainfluenza virus type 3 hemagglutinin neuraminidase, Newcastle disease virus hemagglutinin neuraminidase, human herpesvirus 1 gH, simian varicella virus gH, human herpesvirus gB proteins, bovine herpesvirus gB proteins, cercopithecine herpesvirus gB proteins, Friend murine leukemia virus envelope glycoprotein, Mason Pfizer monkey virus envelope glycoprotein, HIV envelope glycoprotein, influenza virus hemagglutinin, poxvirus membrane glycoproteins, mumps virus hemagglutinin neuraminidase, mumps virus glycoproteins Fl and F2, West Nile virus membrane glycoprotein, herpes simplex virus membrane glycoprotein, Russian Far East encephalitis virus membrane glycoprotein, Venezuelan equine encephalitis virus membrane glycoprotein, and varicella virus membrane glycoprotein.

[0048] In some embodiments, region (a) of the fusion protein according to the invention is capable of specifically binding to a viral envelope protein derived from MLV, 10A1 , BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (Fowl plague virus), and influenza virus. Representative examples include, FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV.

[0049] In some embodiments the viral envelop protein is a lentivirus viral envelop protein or a coronavirus envelop protein.

[0050] The term “lentivirus” as used herein refers a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. Lentiviruses are distributed worldwide, and are known to be hosted in apes, cows, goats, horses, cats, and sheep as well as several other mammals.

[0051] The term “HIV”, as used herein, refers to the human immunodeficiency virus. It includes HIV-1 , HIV-2 and SIV; preferably, it relates to HIV-1 and / or HIV-2. “HIV-1” means the human immunodeficiency virus type-1. HIV-1 includes but is not limited to extracellular virus particles and the forms of HIV-1 associated with HIV-1 infected cells. The HIV-1 virus may represent any of the known major subtypes (Classes A, B, C, D E, F, G and H) or outlying subtype (Group O) including laboratory strains and primary isolates. “HIV- 2” means the human immunodeficiency virus type-2. HIV-2 includes but is not limited to extracellular virus particles and the forms of HIV-2 associated with HIV-2 infected cells. The term “SIV” refers to simian immunodeficiency virus which is an HIV-like virus that infects monkeys, chimpanzees, and other nonhuman primates. SIV includes but is not limited to extracellular virus particles and the forms of SIV associated with SIV infected cells.

[0052] The HIV viral envelop comprises the trimeric envelope glycoprotein spike of HIV-1 mediates virus entry into human cells. The exposed part of the trimer, gp140, consists of two noncovalently associated subunits, gp120 and gp41 ectodomain. The surface of an HIV-1 virus is covered with 20-50 trimeric envelope spikes that are embedded in the viral envelope.

[0053] In some embodiments the viral envelop protein is an HIV viral envelop protein. In some embodiments the viral envelop protein is HIV gp140.

[0054] The term “coronavirus” as used herein refers to a group of related RNA viruses that cause diseases in mammals and birds. In humans and birds, they cause respiratory tract infections that can range from mild to lethal. Mild illnesses in humans include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause SARS, MERS and COVID-19, which is causing the ongoing pandemic. In cows and pigs they cause diarrhea, while in mice they cause hepatitis and encephalomyelitis. Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales and realm Riboviria.

[0055] In some embodiments the viral envelop protein is a SARS-CoV-2 viral envelop protein.

[0056] The term “SARS-CoV-2” as used herein refers to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a beta coronavirus that was earlier identified in late December 2019 in the city of Wuhan, China. SARS-CoV- 2 is responsible for the contagious respiratory illness termed Coronavirus disease 2019 or COVID-19.

[0057] Preferably, the term "SARS-CoV-2" when used herein further, i.e., in addition to wildtype SARS-CoV-2, includes SARS-CoV-2 variants, such as Alpha, Beta and / or Delta SARS-CoV-2 variants. There are multiple ways in which SARS-CoV-2 viruses are classified. SARS-CoV-2 is often discussed in the context of lineages (and sublineages). The most commonly used classification system for lineages is Pango. In a larger context, lineages or groups of related lineages may be classified using Greek letters (such as Omicron) by the World Health Organization (WHO).

[0058] The Pango lineage system is hierarchical, like a family tree. Lineages are evolutionarily descendants of a “parent” lineage. A lineage may be described as a “sublineage” when it is being discussed in relation to its parent lineage. Lineages are named using an alphabetical prefix (such as B or BA) and numerical suffix (such as “.1” or “.1.1.5”). When a new lineage is defined, the Pango system assigns an additional number to the name of its parent lineage (e.g., BA.2.75 is a sublineage of BA.2). As the virus continues to change, the Pango lineage names can become very long. Lineages with longer names may be given alphabetic aliases and numbering continues (e.g., “BA” stands for “B.1.1.529,” thus BA.2 is the same as B.1.1 .529.2).

[0059] In a particular embodiment viral envelop protein is a SARS-CoV-2 variant viral envelop protein wherein the SARS-CoV-2 variant is selected from the variants described in Table 1 or a descendant thereof.

[0060] Table 1 : List of SARS-CoV-2 variants.

[0061] SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The term “spike (S) protein” or “Spike glycoprotein” as used herein refers to the largest of the structural proteins of SARS-CoV-2. The spike protein assembles into trimers that form large structures, called spikes or peplomers, that project from the surface of the viral envelope. The function of the spike glycoprotein is to mediate viral entry into the host cell by first interacting with molecules on the exterior cell surface and then fusing the viral and cellular membranes. The Spike glycoprotein is a class I fusion protein that contains two regions, known as S1 and S2, responsible for these two functions. The S1 region contains the “receptor-binding domain” or “RBD” that binds to receptors on the cell surface of the host cell, in SARS- CoV-2 (which causes COVID-19) the RBD binds to the angiotensin-converting enzyme 2 (ACE2). The S2 region contains the fusion peptide and other fusion infrastructure necessary for membrane fusion with the host cell, a required step for infection and viral replication.

[0062] In some embodiments according to the invention, region (a) of the fusion protein according to the invention binds to the SARS-CoV-2 spike protein. In another embodiment the region (a) binds to the SARS-CoV-2 spike protein through the receptor binding domain (RBD).

[0063] In yet another embodiment, region (a) of the fusion protein according to the invention specifically binds the HIV gp140 protein.

[0064] In a particular embodiment, region (a) of the fusion protein according to the invention comprises a VHH according to the sequence of SEQ ID NO: 3 which specifically binds the HIV gp140 protein. In a particular embodiment, the fusion protein according to the invention wherein the first region comprises a first VHH and a second VHH in tandem, wherein each VHH binds to a different epitope in the viral envelope protein.

[0065] The term “in tandem” as used herein refers to one behind another. A sequence in tandem with another is adjacent sequences in continuation.

[0066] As used herein, the term “epitope” includes any protein determinant capable of specific binding to / by an immunoglobulin or fragment thereof, in the present invention, any protein determinant capable of specific binding by the VHH of the fusion protein of the invention. The expression “each HH binds to a different epitope in the viral envelope protein” as used herein refers to each VHH of the fusion protein binding a different determinant of the viral envelope protein. For example, each VHH binds to a different epitope in the RBD of the spike protein of the SARS-CoV-2 viral envelope.

[0067] In a preferred embodiment, the fusion protein according to the invention wherein the first VHH comprises the sequence of SEQ ID NO: 1.

[0068] In a preferred embodiment, the fusion protein according to the invention wherein the second VHH comprises the sequence of SEQ ID NO: 2.

[0069] The fusion protein of the invention comprises a trimerization domain. The term "trimerization domain" is understood as an amino acid sequence whose function is to promote folding and trimerization of monomer polypeptides. Examples of suitable trimerization domains include without restriction, the T4 bacteriophage foldon trimerization domain, the trimerization domain of collagen XV or the trimerization domain of collagen XVIII. In a particular embodiment of the fusion protein according to the invention, the trimerization domain is the human collagen XVIII trimerization domain.

[0070] In another embodiment of the fusion protein according to the invention the human collagen XVIII trimerization domain comprises a sequence according to sequence SEQ ID NO: 4.

[0071] In a preferred embodiment, the fusion protein according to the invention wherein the region comprising at least one antibody is connected to the trimerization domain by a flexible linker.

[0072] In another particular embodiment of the fusion protein according to the invention the first and second VHH are connected by a flexible linker.

[0073] According to the invention, the “flexible linker” or “spacer” amino acid sequences can act as a hinge region between two polypeptide components, such as the antibody and the trimerization domain and / or the first and second VHHS, allowing them to move independently from one another while maintaining the three-dimensional form of the individual domains, such that the presence of peptide spacers or linkers does not alter the functionality of any of the polypeptide components. In this sense, a preferred intermediate amino acid sequence according to the invention would be a hinge region characterized by a structural ductility allowing this movement. In a particular embodiment, said intermediate amino acid sequence is a flexible linker. The effect of the linker region is to provide space between the polypeptide sequences. It is thus assured that the secondary and tertiary structure of polypeptide is not affected by the presence of the other polypeptide. The spacer is of a polypeptide nature. The linker peptide preferably comprises at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids or approximately 100 amino acids. In a particular embodiment the spacer comprises at least 15 amino acids.

[0074] The spacer or linker can be bound to the two polypeptide components by means of covalent bonds, preferably by peptide bonds; and also preferably the spacer is essentially afunctional, and / or is not prone to proteolytic cleavage, and / or does not comprise any cysteine residue. Similarly, the three-dimensional structure of the spacer is preferably linear or substantially linear.

[0075] The preferred examples of spacer or linker peptides include those that have been used to bind proteins without substantially deteriorating the function of the bound peptides or at least without substantially deteriorating the function of one of the bound peptides. More preferably the spacers or linkers used to bind peptides comprise coiled coil structures.

[0076] In an embodiment of the fusion protein according to the invention the flexible linker comprises the sequence according to SEQ ID NO: 6 ((048)3).

[0077] The fusion protein of the invention comprises a region which binds specifically to a receptor present in the surface of dendritic cells.

[0078] The term "dendritic cell" (DC) refers to a subtype of phagocytic cells belonging to the class of antigen presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Recognition of pathogens is enabled by the expression of pattern recognition receptors (PRRs) present in the surface of the DCs that bind conserved pathogenic structures associated with various microorganisms including bacteria, viruses, parasites and fungi. The DCs are equipped with an array of PRRs, including several Toll-like receptors (TLRs) and C-type lectin receptors (CLRs).

[0079] Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as coreceptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. Thus, dendritic cells can actively induce a T cell- or B cell- related immune response.

[0080] The term "antigen presenting cell" (APC) is a cell of a variety of cells capable of displaying, acquiring, and / or presenting at least one antigen or antigenic fragment on (or at) its cell surface. Antigen-presenting cells can be distinguished in professional antigen presenting cells and non-professional antigen presenting cells. The term "professional antigen presenting cells" relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a co-stimulatory molecule inducing activation of the T cell. Professional antigen presenting cells comprise dendritic cells and macrophages. The term "non-professional antigen presenting cells" relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferongamma. Exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.

[0081] In another embodiment, the region which binds specifically to a receptor present in the surface of dendritic cells is an antibody. In another embodiment the antibody binds specifically to DNGR-1.

[0082] The term “DNGR-1”, also known as CLEC9A, DNGR1 , UNQ9341, CD370, DNGR-1 , C- type lectin domain family 9 member A and C-type lectin domain containing 9A, as used herein, refers to a group V C-type lectin-like receptor (CLR) that is expressed on dendritic cells. CLEC9A can function as an endocytic receptor on a small subset of dendritic cells specialized for the uptake and processing of material from dead cells. CLEC9A recognizes filamentous form of actin, that can be associated with cytoskeletal actin- binding proteins, can be exposed when cell membranes are damaged, and can mediate the cross-presentation of dead-cell associated antigens.

[0083] In another embodiment of the fusion protein according to the invention the domain which binds specifically to a receptor present in the surface of dendritic cells is a ScFv.

[0084] As used herein, a "single chain variable fragment (ScFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the ScFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which variable (Fv) regions of immunoglobulin heavy chain (VH chain) and light chain (VL chain) fragments are linked via a spacer sequence. Various methods for preparing a ScFv are known, and include methods described in US Patent No. 4694778, Nature, vol. 334, p. 54454 (1989), and Science, vol. 242, pp. 1038-1041 (1988).

[0085] In another embodiment, the fusion protein according to the invention wherein the domain which binds specifically to a receptor present in the surface of dendritic cells has a sequence according to SEQ ID NO: 5.

[0086] In another embodiment the fusion protein of the invention has a sequence according to SEQ ID NO: 7 or SEQ ID NO: 8.

[0087] The fusion protein of the invention comprises three region / domains, (a), (b) and (c). This domains may be arranged (or fused) in the fusion protein in several arrangements, wherein this arrangements do not affect the ability of the fusion protein to function as described herein. Therefore, in a particular embodiment of the fusion protein of the invention the region / domains are arranged in the following order from N-terminal to C- terminal:

[0088] - (a) - (b) - (c);

[0089] - (c) - (b) - (a);

[0090] - (a) - (c) - (b);

[0091] - (b) - (c) - (a);

[0092] (c) - (a) - (b); and

[0093] - (b) - (a) - (c).

[0094] The hyphen in between the region / domains of the fusion protein as indicated above is for clarity and representation only not indicating the presence or absence of further region / domains or linkers / spacers. T rimer

[0095] Another aspect of the invention relates to a trimer which comprises three monomers of the fusion protein according to the invention.

[0096] The term “trimer” refers to a structure comprising three monomers, for example, three monomer polypeptides. The term “homotrimer” refers to a structure comprising three identical monomers, for example, three identical monomer polypeptides according to the invention. The ability to trimerize can be assessed experimentally, for example, by SDS- PAGE in non-reducing conditions followed by Coomassie-blue staining, by which the relative abundance and molecular masses of trimeric forms with respect to monomeric forms can be visualized and compared.

[0097] Polynucleotide

[0098] Another aspect of the invention relates to a polynucleotide which encodes the fusion protein according to the invention.

[0099] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and their polymers in either single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding capabilities similar to those of a reference nucleic acid and which are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implies conservatively modified variants (e.g., substitutions with degenerate codons), alleles, orthologs, SNPs and complementary sequences, as well as sequences indicated in direct form. In particular, substitutions with degenerate codons can be obtained by creating sequences in which the third position of one or more selected (or all) codons is replaced by residues with mixed bases and / or deoxyinosine residues. Exogeneous mRNA can be sensed by TLRs in the endosomes as well as by receptors such as RIG-I and MDA5. Innate immune sensing can create an unfavorable environment for the translation of mRNA and thus limit protein expression. The incorporation of modified nucleosides, such as pseudouridine and 5-methylcytidine, in engineered mRNA prevents immune activation and increases the stability and translation of mRNA. These chemical modifications, together with efficient delivery methods, such as the use of lipid nanoparticles, have enormously simplified and expanded the use of mRNA for therapeutic applications.

[0100] In a particular embodiment the polynucleotide which encodes the fusion protein according to the invention is a RNA polynucleotide. In another particular embodiment the polynucleotide which encodes de fusion protein according to the invention has a sequence according to SEQ ID NO: 9.

[0101] Vector

[0102] In another aspect, the invention refers to a vector which comprises the polynucleotide according to the invention.

[0103] The vector of the invention can be used to transform, transfect, or infect cells which can be transformed, transfected or infected by said vector. Said cells can be prokaryotic or eukaryotic. By way of example, the vector wherein the polynucleotide according to the invention is introduced can be a plasmid or a vector which, when it is introduced in a host cell, is integrated in the genome of said cell and replicates together with the chromosome (or chromosomes) in which it has been integrated. The term “vector” is also meant to include plasmids, phages, viruses and the like. Said vector can be obtained by conventional methods known by the persons skilled in the art [Sambrook et al., 2001 , “Molecular cloning, to Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press, N.Y. Vol 1-3 a].

[0104] Host cell

[0105] In another aspect, the invention relates to a host cell which comprises the polynucleotide according to the invention or the vector according to the invention.

[0106] The term “host cell” as used herein refers to a cells which has been able to be transformed, transfected or infected with a polynucleotide or vector provided by this invention. The transformed, transfected or infected cells can be obtained by conventional methods known by persons skilled in the art [Sambrook et al., 2001 , mentioned above].

[0107] Host cells suitable for the expression of the conjugates of the invention include, without being limited to, mammal, plant, insect, fungal and bacterial cells.

[0108] Immunogenic composition

[0109] In another aspect, the invention relates to an immunogenic composition or a vaccine composition which comprises the fusion protein according to the invention, the trimer according to the invention, the polynucleotide according to the invention, the vector according to the invention, the host cell according to the invention and a pharmaceutically suitable carrier or excipient.

[0110] In the context of the present invention, the term “immunogenic composition" refers to a composition that can elicit a cellular and / or humoral immune response but does not necessarily confer full or partial immune protection against the virus which comprises the viral envelope protein to which the antibody of the fusion protein binds to. For the avoidance of doubt, however, such immunogenic composition may confer full or partial protection against the virus which comprises the viral envelope protein to which the antibody of the fusion protein binds to and this is also preferred. In contrast, a “vaccine" in the context of the present invention does confer full or partial, but at least partial immune protection against the virus which comprises the viral envelope protein to which the antibody of the fusion protein binds to.

[0111] As used herein, the term "pharmaceutically acceptable carrier" means a non- toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Remington's Pharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0112] Accordingly, the compositions comprising the fusion protein, polynucleotide, vector, host cell, or nanoparticle of the invention and a pharmaceutically acceptable carrier are pharmaceutical compositions.

[0113] The pharmaceutical compositions of this invention can be administered to a patient by any means known in the art including oral and parenteral routes. According to such embodiments, inventive compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).

[0114] Uses and methods of the invention

[0115] All the elements of the invention previously describe find uses in medicine. Therefore, an aspect of the present invention is the fusion protein according to the invention, the trimer according to the invention, the polynucleotide according to the invention, the vector according to the invention, the host cell according to the invention for use in medicine.

[0116] Another aspect of the present invention is the fusion protein according to the invention, the trimer according to the invention, the polynucleotide according to the invention, the vector according to the invention, the host cell according to the invention and the immunogenic composition or vaccine composition according to the invention for use in the prevention and / or treatment of a disorder caused by and / or associated with an infection caused by a virus containing in its envelope the fusion protein to which region (a) of the fusion protein specifically binds. In some embodiments, the disorder caused by and / or associated with an infection by a virus is an infection caused by HIV or by SARS-CoV-2. The term “prevention”, as used herein, relates to the capacity to prevent, minimize, or hinder the onset or development of a disease or condition before its onset. When referred to infection by HIV or SARS-Cov-2 which results in AIDS and COVID-19 respectively, the term “prevention” also encompasses the administration of the fusion protein of the invention prior to the onset of the infection as well as the administration of the protein of the invention after the onset of infection but before the symptoms and or damages caused by infection appear.

[0117] As used herein, the terms "treat", "treatment", "treatment", or "amelioration". The term refers to therapeutic treatment, the purpose of which is to reverse, reduce, suppress, delay or stop the progression or severity of the condition associated with the disease or disorder. The term "treatment" includes reducing or alleviating at least one adverse effect or condition of a condition, such as AIDS or COVID-19, a disease or disorder. Treatment is usually "effective" when one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if disease progression is delayed or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the interruption of at least a condition that indicates the progression or worsening of symptoms that would be expected in the absence of treatment. The beneficial or desirable clinical outcome, whether detectable or not, is a reduction in one or more symptoms, a reduction in the extent of the disease, a stable (ie, not aggravated) condition of the disease, a disease These include, but are not limited to, delayed or slowed progression, amelioration or alleviation of the disease state, and remission (partial or total). The term "treatment" of a disease also includes providing relief from symptoms or side effects of the disease (including symptomatic treatment). The term “treatment” also involves amelioration of the various physiological symptoms involved.

[0118] Another aspect of the present invention relates to a method for preventing or treating a disorder caused by and / or associated with an infection by HIV or by SARS-CoV-2 comprising the administration of an effective amount of the fusion protein according to the invention, the polynucleotide according to the invention, the vector according to the invention, the host cell according to the invention or the immunogenic or vaccine composition according to the invention to a subject

[0119] The expressions “effective amount”, “therapeutic effective amount”, “effective dose” or “therapeutic effective dose” as used herein refer to an amount of an active agent (i.e. , an ingredient such as the fusion protein of the invention) high enough to deliver the desired benefit, either the treatment or prevention of the disease, but low enough to avoid serious side effects within the scope of medical judgment. The particular dose administered according to this invention will of course be determined by the particular circumstances surrounding the case, such as the route of administration, the age of the subject, and the similar considerations.

[0120] The term “subject” as used herein refers to a mammal, preferably a human of any age, race or gender.

[0121] ***

[0122] The invention is described below by way of the following examples which are to be taken as merely illustrative and not limiting the scope of the invention.

[0123] EXAMPLES

[0124] Materials and Methods

[0125] Cell lines

[0126] HEK-293 (# ACC-305, DSMZ) and 293T (# CRL-3216, ATCC) cells were obtained from the ATCC and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Lonza) supplemented with 2 mmol / L L-glutamine (Gibco), 10% (v / v) heat-inactivated fetal bovine serum (FBS) (Merck Life Science), and antibiotics (100 units / mL penicillin, 100 mg / mL streptomycin; Life Technologies) referred as to DMEM complete medium (DCM) As previously described, the parental B3Z cell line (Kurts et al., 1996, The Journal of Experimental Medicine, 184(3), 923-930) and its derived mouse cell line expressing CLEC9A / DNGR-1 (B3ZDNGR-1) werecultured in Roswell Park Memorial Institute (RPMI) medium (Gibco) supplemented with 2 mmol / L L-glutamine, 10% (v / v) heat- inactivated FBS, antibiotics (100 units / mL penicillin, 100 mg / mL streptomycin), herein referred as RCM, and 1 mmol / L sodium pyruvate (Gibco), 0.1 mmol / L MEM non- essential amino acids (NEAA) (Gibco), 55 pmol / L p-mercaptoethanol (Gibco), and. Generation and characterization of the B3ZDNGR-1 cell line was previously described (Poulin et al., 2012, Blood, 119(25), 6052-6062; Sancho et al 2009, Nature, 458(7240), 899-903). BHK-21 / WI-2 (# EH1011 , Kerafast) cells, used for recombinant VSV generation, were grown in DCM. Vero E6 (# 60476, BCRC) cells used in the S- pseudotyped neutralization experiments were grown in DCM supplemented with 0.1 mmol / L NEAA and 12.5 units / mL Nystatin (penicillin-streptomycin-nystatin) (Biological Industries). Vero E6 cells expressing the transmembrane serine protease TMPRSS2 (VeroE6 / TMPRSS2) used in the live SARS-CoV-2 neutralization experiments were maintained in DCM, supplemented with 10 mM HEPES (Gibco), 100 mg / mL geneticin (G 418 disulfate salt, Sigma-Aldrich) and 0.1 mmol / L NEAA. Mouse BMDCs were obtained by using Flt3-Ligand (Flt3-L BMDCs) as previously described (Sancho et al., 2008, The Journal of Clinical Investigation, 118(6), 2098-2110) with slight modifications. Briefly, femur and tibia from C57BL / 6J mice were collected, bone marrow was flushed, red blood cells were lysed and cells were cultured in RCM 1000 units / mL penicillin, 100 pg / mL streptomycin (both from Sigma-Aldrich), 0.1 mmol / L NEAA, 1 mmol / L sodium pyruvate, 2 mmol / L L-glutamine, 10 mmol / L HEPES (all from HyClone) and 50 pmol / L p- mercaptoethanol (Merck), herein called R10, plus 200 ng / mL human Flt3-L (Miltenyi). Media was refreshed on day 7 and cells were harvested on day 9. Mutu-DC cells (kindly provided by Hans Acha-Orbea, Laussane, Switzerland) (Fuertes Marraco et al., 2012, Frontiers in Immunology, 3(NOV)) were cultured in R10 and passaged when confluent using 5 mmol / L EDTA. All 477 adherent cell lines were cultured at 37 °C in 5% (v / v) CO2 at 95% air in a humidified atmosphere. Freestyle 293F and Expi293F (both from Gibco;) cells were respectively cultured in Freestyle 293 and Expi293 Expression Medium at 37 °C in a humidified 8% CO2 incubator rotating at 95 rpm. All cell lines were used within 3 months of thawing and checked for Mycoplasma by PCR every month using the Mycoplasma Gel Detection Kit (Biotools B&M Labs).

[0127] SARS-CoV-2 viruses

[0128] Several SARS-CoV-2 viruses were used in the live SARS-CoV-2 neutralization assays. SARS-CoV-2 MAD6 isolate, a prototypic strain B.1 containing the D614G mutation in the S protein, is a virus collected from a nasopharyngeal swab from a 69-year-old male COVID-19 patient from Hospital 12 de Octubre in Madrid, Spain (Diez et al., 2020, Immunotherapy, 12(17), 1247-1255). The VOCs B.1.351 (beta; hCoV-19 / France / PDL- IPP01065 / 2021 , clade 10H / 501Y.V2) and B.1.617 (delta; SARS-CoV-2, Human, 2021 , Germany ex India, 20A / 452R) were supplied through the European Virus Archive Global (EVAg) platform, by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France) and the Robert Koch Institute (German Federal Institute for Infectious and Non-Communicable Diseases, Berlin, Germany), respectively. All SARS-CoV-2 virus stocks were grown on Vero E6 cells and virus infectivity titers were determined by standard plaque or median tissue culture infectious dose (TCID50) assays as previously described (Perez et al., 2022, Frontiers in Immunology, 12, 824728).

[0129] Mouse strains

[0130] C57BL / 6 mice (Charles River Laboratories) were bred at CNIC under specific pathogen- free conditions. Age-matched female 6- to 8-week-old mice were used and randomized before treatment. Immunogenicity experiments in C57BL / 6 mice were approved by the animal ethics committee at CNIC and by the Division of Animal Protection of the Comunidad de Madrid (PROEX 240 / 16). Transgenic K18-hACE2 mice (The Jackson Laboratory), expressing hACE2 under control of the cytokeratin 18 promoter on the C57BL / 6 background, were used in the protective efficacy studies. 9-week-old female K18-hACE2 mice were used and randomized between treatment groups after SARS- CoV-2 challenge. Protective efficacy experiments with K18-hACE2 mice were performed in the biosafety level 3 (BSL-3) facilities at the Centro de Investigation en Sanidad Animal (CISA)-lnstituto National de Investigaciones Agrarias (INIA)-CSIC (Valdeolmos, Madrid, Spain). Efficacy experiments were approved by the animal ethics committee at I NIA and by the Division of Animal Protection of the Comunidad de Madrid (PROEX 161.5 / 20). All animal procedures were conformed to Spanish law 510 under the Royal Decree (RD 53 / 2013) and in accordance with Ell Directive 2010 / 63EU and Recommendation 2007 / 526 / EC.

[0131] Proteins

[0132] Production and purification of the SARS-CoV-2 S and RBD proteins

[0133] The SARS-CoV-2 HexaPro S protein cDNA (Hsieh et al., 2020), corresponding to Wuhan-Hu-1 strain, was codon-optimized and synthesized (GenScript). The gene was cloned into mammalian expression vector pcDNA3.1(-) carrying a C-terminal Twin-Strep tag. Four days following the transfection in Freestyle 293F cells with 293fectin transfection reagent (Life Technologies), the culture supernatants were collected and purified with Strep-Tactin affinity chromatography resin (I BA LifeSciences) followed by SEC using a Superose 6 Increase 10 / 300 GL column (Cytiva). Concentrated purified fractions were analyzed by 0.1 % (w / v) sodium dodecyl sulfate (SDS)-4-20% (w / v) polyacrylamide gel electrophoresis (PAGE). Proper protein folding and conformation was certified by antibody recognition in ELISA using anti-RBD (# A2286, Biovision), anti-N- terminal domain (NTD) (# A2269, Biovision) and anti-S2 (# 40590-D001 , SinoBiological) mAbs. The SARS-CoV-2 Wuhan-Hu-1 / B.1 strain S protein RBD, defined as amino acids R328-L533, was expressed in pcDNA3.1 (+) with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag (BEI Resources NR-52422) (Walls et al., 2020, In Cell 183, 6, p. 1735). The RBD was produced using Expi293F transfection, supernatant was harvested after 3 days, and protein was purified using a 10 mL bed volume of Talon Superflow Metal Affinity Cobalt Resin (Takara Bio). Purified protein was filtered and concentrated into buffer containing 20 mM Tris, 300 mM NaCI and 200 mM imidazole, pH 8.0. SDS-was run to check purity and a modified competition ELISA as control for binding to hACE2 (Aero BioSystems). Generation of antibody expression vectors

[0134] DNA fragments encoding for E, V and TN (EV-tandem) nanobodies were synthesized by GeneArt (Thermo Fisher Scientific) including them within the following vectors: pcDNA3.1 (+)-E VHH537 Myc-His, pcDNA3.1 (+)-V VHH-Flag-His and pcDNA3.1 (+)-TN- StrepTagll . All constructs included an in frame fused wild type N-terminal interleukin-2 (IL-2) signal peptide for effective protein secretion to the medium. The expression vector for the TNTtrimerbody was generated by subcloning L23-TIE cDNA fragment from vector pCR3.1 (+)-MFE23scFv-L23-TIE-Myc-His (previously described in Blanco-Toribio et al., 2013) into pCR3.1-TN-StrepTagll by mean of Notl / BamHI restriction enzymes. To generate the TNTDNGR-1 construct, the cDNA for L15-7H11scFv-StrepTagll fragment (GeneArt) was subcloned using Bgll l / Xbal cloning sites into the TNTtrimerbody parental vector resulting in pcDNA3.1 (+)-544 TN-L23-TIE-L15-7H11-StrepTagll.AII the sequences were verified using primers FwCMV and RvBGH.

[0135] Also, DNA fragment encoding for J3 VHH was synthesized by GeneArt (Thermo Fisher Scientific) including it within the following vector: pcDNA3.1 (+)-J3 VnH-His. All engineered constructs included an in frame fused wild type N-terminal oncostatin M (OM) signal peptide for effective protein secretion to the medium. First, a C-terminal StrepTagll tagged monomeric J3 nanobody expression vector was created as a control, pCDNA3.1 J3 VHH-StrepTagll. The expression vector for the J3 VHH N-terminal trimerbody, J3T, was generated by subcloning J3 HH CDNA fragment into previously described trimerbody scaffold by mean of Not\ / BamH\ restriction enzymes, creating pCDNA3.1(+)-J3 VHH-L23- TIE-StrepTagll. To generate the TNTDNGR-1 construct, the cDNA for L15-7H11scFv- StrepTagll fragment (GeneArt) was subcloned using Bgl\\ / Xba\ cloning sites into the J3Ttrimerbody parental vector resulting in pcDNA3.1 (+)-J3 VHH-L23-TIE-L15-7H 1 1 - StrepTagll.

[0136] Production and purification of recombinant engineered antibodies

[0137] The generation of H EK-293 stable transfectants was done in DCM by selection with 500 mg / mL G418 (Life Technologies) after Lipofectamine 3000 (Invitrogen) transfection. Large-scale protein production was done by collecting periodically the media of stable transfectants. TN, TNTand TNTDNGR-1 were purified from conditioned culture media by Strep-Tactin affinity chromatography in an AKTA Prime Plus FPLC System (Amersham Biosciences). Finally, the elution fractions corresponding to purified antibodies were dialyzed against PBS. Protein concentration calculation was carried out by UV- absorbance measurements on an Uvikon 930 spectrophotometer (Kontron Instruments). Purified antibodies were analyzed by 0.1 % (w / v) SDS-15% (w / v) in reducing conditions followed by Coomassie brilliant blue R-250 (BioRad Laboratories) staining, for optimal band visualization. Gel image acquisition was performed with a ChemiDoc MP Imaging System (BioRad Laboratories).

[0138] Furthermore, mid-scale protein production was done by Expi293 cells transfection using Expifectamine reagent, collecting the culture supernatants after 120 hours of incubation at 32°C. J3Tand J3TDNGR-1 were purified from conditioned culture media by Strep- Tactin affinity chromatography in an AKTA Prime Plus FPLC System (Amersham Biosciences). The elution fractions corresponding to purified antibodies were dialyzed against PBS. Protein concentration calculation was carried out by UV-absorbance measurements on an Uvikon 930 spectrophotometer (Kontron Instruments). Purified antibodies were analyzed by 0.1% (w / v) sodium dodecyl sulfate (SDS)-4-20% (w / v) polyacrylamide gel electrophoresis (PAGE) in reducing conditions followed by Coomassie brilliant blue R-250 (BioRad Laboratories) staining, for optimal band visualization. Gel image acquisition was performed with a ChemiDoc MP Imaging System (BioRad Laboratories).

[0139] ELISA assays

[0140] The ability of TN based constructs to bind purified SARS-CoV-2 RBD was analyzed by ELISA. Briefly, Nunc MaxiSorp flat-bottom 96-well plates (Thermo Fischer Scientific) were coated (0.2 pg / well) with recombinant B.1 RBD. After washing and blocking with 300 pl PBS-BSA, 100 pl of conditioned media from transfected H EK-293 cells were added to the wells and incubated for 1 hour at room temperature. After three washes, 100 pl of the corresponding anti-Strep mAb (# 2-1507-001 , 1 BA Lifesciences) were added for 1 hour at room temperature. Finally, HRP-conjugated GAM IgG (# 115-035-003, Jackson ImmunoResearch Labs) was added, after which the plate was washed again and developed. Purified TN and TNT(50, 10, and 1 nM) ability to bind different strain RBDs was performed in the same way. RBD interaction with biotinylated hACE2 (#Q9BYF1-1 , AcroBiosystems) was used as control by adding 100 pL at 0.1 pg / mL for 1 hour, followed by HRP-conjugated streptavidin (# 554066, BD Biosciences) in the same conditions.

[0141] Also, the ability of J3 VHH based trimerbodiess to bind HIV-1 gp140 (M.CON-S gp140CFI) (#12577, NIH-ARP) was analyzed by ELISA. Briefly, Nunc MaxiSorp flat-bottom 96-well plates (Thermo Fischer Scientific) were coated (0.1 pg / well) with recombinant HIV gp140. After washing and blocking with 300 pl 5% (w / v) in PBS, 100 pl of conditioned media from transfected Expi293 cells or purified protein solutions were added to the wells and incubated for 1 hour at room temperature. After three washes, 100 pl of the corresponding anti-Strep mAb (# 2-1507-001 , IBA Lifesciences) were added for 1 hour at room temperature. Finally, HRP-conjugated GAM IgG (# 115-035-003, Jackson ImmunoResearch Labs) was added, after which the plate was washed again and developed. To investigate the bispecific interactions of the J3TDNGR-1 , after incubating immobilized gp140 protein with the purified antibodies (1 pg / ml), we added murine Refused DNGR-1 (1 pg / ml). After washing again, the bispecific binding by J3TDNGR-1 was detected by HRP-conjugated GAM IgG (# 115-035-003, Jackson ImmunoResearch Labs).

[0142] The ability of in vitro mRNA produced TN-based trimerbodiess to bind SARS-CoV-2 B.1 RBD was analyzed by ELISA. Briefly, Nunc MaxiSorp flat-bottom 96-well plates (Thermo Fischer Scientific) were coated (0.2 pg / well) with recombinant B.1 RBD. After washing and blocking with 300 pl 5% (w / v) in PBS, 100 pl of supernatant from HEK-293T transfected cells were added to the wells and incubated for 1 hour at room temperature. After three washes, 100 pl of the corresponding anti-Strep mAb (# 2-1507-001 , IBA Lifesciences) were added for 1 hour at room temperature. Finally, HRP-conjugated GAM IgG (# 115-035-003, Jackson ImmunoResearch Labs) was added, after which the plate was washed again and developed. Bispecific binding of the TNTDNGR-1 produced antibody was probed by adding murine Fc-fused DNGR-1 after antibody binding to coated RBD. Finally, the DNGR-1 chimera was detected by HRP-conjugated GAM IgG (# 115-035-003, Jackson ImmunoResearch Labs).

[0143] All optical density measurements were done using a Multiskan FC apparatus (Thermo Scientific Scientific).

[0144] SDS-PAGE analysis and western blotting

[0145] Recombinant protein secretion to culture media was analyzed by 0.1% (w / v) SDS- 15% (w / v) PAGE followed by western blot. After electrophoresis in reducing conditions, the gel contained proteins were transferred to immunoblot PVDF membrane (Millipore). Prior to antibody detection, membrane was completely blocked by incubation with PBS containing 5% (w / v) of BSA (PBS-BSA). J3, J3Tand J3TDNGR-1 detection was done using anti-Strep mAb (# 2-1507-001 , IBA Lifesciences) according to the manufacturer recommendations, followed by HRP-conjugated GAM IgG (#A2554, SIGMA) incubated in 10% (w / v) non-fat milk supplemented PBS.

[0146] Biolayer Interferometry

[0147] The binding of TN and TNTto immobilized B.1 or B.1.351 (beta) RBDs was measured using biolayer interferometry (BLI) on an Octet RED96 system (Fortebio). The RBD was immobilized onto AR2G biosensors (Fortebio) at pH 5.0 using a standard amine 578 reactive coupling protocol (activation with EDC / s-NHS and quenching with ethanolamine). The antibodies in HEPES buffered saline (HBS; 20 mM HEPES, 150 mM NaCI, pH 7.4) at 50 and 10 nM were associated with the RBD for 30 min, after which the dissociation of antibody from the biosensor was measured for 1 hour, to determine their binding kinetics. The experimental data was then fitted to a 1 :1 binding model using the Octet Data Analysis software (Fortebio). To determine the blocking of ACE2’s RBD binding, 150 nM of TN monomer or 50 nM of TNTwere associated with immobilized B.1 RBD for 10 min, after which 100 nM of soluble hACE2 (Aero Biosystems) was added and associated with RBD for 30 min. To investigate bispecific interactions between the TNTDNGR-1 , immobilized RBD and DNGR-1 in solution, RBD-coated biosensors were first prepared as described above. Then, 50 nM of the TNTDNGR-1 in HBS was then incubated with the biosensors for 30 min, followed by 5 min of dissociation in HBS. The biosensors were then moved into 50 Nm of mouse DNGR-1 Fc chimera in HBS and incubated for 30 min, followed by 5 min of dissociation.

[0148] SEC-MALS experiments

[0149] Static light scattering experiments were performed on a Superdex 200 Increase 10 / 300 GL column (Cytiva) attached in-line to a DAWN EOS light scattering photometer and an Optilab rEX differential refractive index detector (both from Wyatt Technology). The chromatography was run at room temperature and the scattering detector was thermostated at 23 °C. The column was equilibrated with running buffer (PBS pH 7.4 plus 150 mM NaCI, 0.1 pm filtered) and the SEC-MALS system was calibrated with a sample of BSA at 2 mg / mL in the same buffer. Approximately 150 pL of the solutions at 0.5 mg / mL were injected into the column at a flow rate of 0.5 mL / min. Data acquisition and analysis were performed using ASTRA software (Wyatt Technology). The reported molar masses correspond to the center of the chromatography peaks. Based on numerous measurements on BSA samples under similar conditions we estimate that the experimental error in the molar mass is around 5%.

[0150] Circular Dichroism

[0151] Circular dichroism (CD) measurements were performed with a Jasco J-715 spectropolarimeter (JASCO). The spectra were recorded on protein samples at 0.02 mg / mL in PBS pH 7.4 plus 150 mM NaCI using a 0.2 cm path length quartz cuvette at 25 °C. Thermal denaturation curves from 10 to 95 °C were recorded on the same protein samples and cuvette by increasing temperature at a rate of 1 °C / min and measuring the change in ellipticity at 218 nm. Microfluidic diffusional sizing

[0152] The change in hydrodynamic radius (Rh) of TNTtrimerbody and its subsequent complex with SARS-CoV-2 S protein was measured by microfluidic diffusional sizing (MDS) (Zhang et al., 2018). Then, Rh values of fluorescently labeled species in their native state in solution are included into a plot of the hydrodynamic radii of a range of native and denatured standard proteins obtained using dynamic light scattering and pulsed field gradient nuclear magnetic resonance (Wilkins et al., 1999, Biochemistry, 38(50), 16424- 16431). TNT(1 pM) was labelled with Fluidiphore rapid amine 503 through amine coupling. The sample was incubated at 4 °C overnight and the size of the conjugated protein was determined through measuring the hydrodynamic radius, Rh, using the Fluidity One-W platform. To determine the Rh upon complex formation, the unlabeled HexaPro S protein (1 pM) was incubated 1 :1 with labeled TNTfor 5 min. The change in Rh was monitored by Fluidity One-W instrument.

[0153] Cryo-EM

[0154] A sample of 0.5 mg / mL HexaPro S protein was prepared in the protein production core (C1) of the Center for Biomolecular Therapeutics (Rockville, Maryland, USA), as previously described (Schaub et al., 2021 , Nature Protocols, 16(11), 5339-5356). It was mixed in 1 : 1 molar ratio with TNTtrimerbody and vitrified on cryo-EM grids using standard protocols with an FEI Vitrobot Mark IV. Particles were selected using blob picking and these were used to produce an ab initio model that was quickly refined and used to generate particle templates for re-picking and 2D classification / filtering. Multiple cycles of 3D refinement in cryoSPARC (Punjani et al., 2017, Nature Methods, 14(3), 290-296) resulted in electron density maps at 3.80 A resolution as determined from gold-standard Fourier shell correlation (GSFSC) curves. This model was in excellent agreement with overall structure of the S protein; however, density regions corresponding to bound TNTelements were difficult to resolve likely due to heterogeneity and dynamic nature of the protein complex. To improve the quality and interpretability of the electron density, particle stacks were exported into Relion (Afonine et al., 2018, Acta Crystallographica. Section D, Structural Biology, 74(Pt 6), 531-544) and subjected to 3D refinement with imposed C3 symmetry followed by 3D classification without any symmetry to allow determination of subconformations not compliant with C3 symmetry. This produced 3 distinct subconformations that were subjected to a single final round of non-uniform refinement in cryoSPARC and local map sharpening in PHENIX (Afonine et al., 2018, supra).

[0155] Cryo-EM model building and refinement Model building on the map resulting after the incubation of TNTwith 645 the HexaPro S protein was done in Coot 0.9.6 (Emsley & Cowtan, 2004, Acta Crystallographica. Section D, Biological Crystallography, 60(Pt 12 Pt 1), 2126-2132). The coordinates corresponding to PDB 7B18 (Koenig et al., 2021 , Science (New York, N.Y.), 371 (6530) were used as a starting model, placed into it after performing a molecular replacement step and posterior refinement in PHENIX (Adams et al., 2010), that included rigid body, simulated annealing and morphing. The model was manually fitted into the density sharpened map and several runs of refinement done in PHENIX until a final reliable model were obtained. Figures were generated using ChimeraX (Pettersen et al., 2021 , Protein Science : A Publication of the Protein Society, 30(1), 70-82) and Pymol.

[0156] Flow cytometry studies

[0157] The ability of both TN-based trimerbodies to bind SARS-CoV-2 B.1 S protein on cell surface was studied by flow cytometry. H EK-293 or HEK-293S cells were incubated with supernatants or purified trimerbodies (1 pg / mL) followed by anti-Strep mAb and PE-GAM IgG antibody (# 115-116-071 , Jackson ImmunoResearch Labs) for 30 min at 4 °C, each of them. Between incubations and after the final one, the cells were sedimented by centrifugation (1200 g, 4 °C, 5 min) and washed with PBS supplemented with 0.05% (v / v) FBS (PBS-FBS) for three times. As a control, both cell types were incubated with rabbit anti-SARS-CoV-2 IgG SB-40592 (# 40592-R001 , Sino Biological) followed by AF488-conjugated donkey anti-rabbit IgG (# 711-545-152, Jackson ImmunoResearch Labs). The binding of TNTDNGR-1 to cell surface mouse CLEC9A / DNGR-1 was assayed using parental B3Z and transfected B3ZDNGR-1 cell lines following the same protocol and staining as explained above for S protein labelling. The PE-conjugated rat antihuman Clec9A (CD370) antibody (clone 3A4 / Clec9A) (# 563488, BD Biosciences) was used as control. Furthermore, the capacity of TNTDNGR-1 for targeting soluble RBD specifically to B3ZDNGR-1 cells was analyzed by preincubating AF647-conjugated RBD (1 pg / mL) with TNTDNGR-1 or TNT(1 pg / mL) for 30 min at room temperature. Then, the mixtures were added to the cells and followed by incubation of 30 min at 4 °C. All the samples were processed on a FACScan apparatus (BD Biosciences) while data analysis was done using the FlowJo Software (BD Biosciences). For labeling and endocytosis studies in BMDCs, cells were incubated with culture media containing TNTor TNTDNGR- 1 (1X supernatant and 10X, respectively) for 30 min at 4 °C. Cells were then washed and kept for different times at 37 °C or 4 °C before being incubated with AF647-conjugated RBD (1 pg / mL) for 1 hour at 4 °C and further stained with antibodies to distinguish the main DC subpopulations: type 1 DCs (cDC1s, CD11c+B220-XCR1+), type 2 DCs (cDC2s, CD11c+B220- SIRPa+) and pDCs (CD11c+B220+). Stained cells were analyzed on BD FACSCanto™ flow cytometer (BD Biosciences).

[0158] Confocal microscopy

[0159] MuTuDC cells were incubated with culture media containing TNTor TNTDNGR-1 (1X supernatantmand 10X, respectively) for 30 min at 4 °C, washed and stained with AF647- conjugated RBD (1 pg / mL) for 1 hour at 4 °C. Cells were then thoroughly washed and incubated in complete media for different times at 4 °C or 37 °C. Then, stained cells were resuspended in PBS, plated in Cell-Tak (Corning) coated coverslips, fixed using 4% paraformaldehyde (PFA) and mounted for visualization in a Zeiss LSM 700 confocal microscope.

[0160] Production of SARS-CoV-2 S-pseudotyped VSV particles

[0161] Vesicular stomatitis virus-G (VSV-G) pseudotyped rVSV-luc recombinant viruses were produced according to previously published protocol by co-transfection of BHK-21 cells (BHK-21 / WI-2, Kerafast) with expression vector coding for VSV-G and rVSVAG- luciferase vector (G*AG692 luciferase; Kerafast) that contains firefly luciferase instead of the VSV-G open reading frame (Whitt, 2010). Briefly, BHK-21 cells were transfected using Lipofectamine 3000 protocol to express the SARS-CoV-2 S protein and after 24 hours, the transfected cells were inoculated with a replication-deficient VSV-G-rVSV-luc pseudotype (multiplicity of infection (MOI) of 3). Following 1 hour incubation at 37 °C, the inoculum was removed, cells were washed extensively with PBS and fresh DCM was added. SARS-CoV-2 S-pseudotyped rVSV-luc particles were harvested 20 hours postinoculation, clarified from cellular debris by centrifugation and stored at - 80 °C. Infectious titers were estimated as TCID50 / mL by limiting dilution of the SARS-CoV-2 S rVSV-luc- containing supernatants on Vero E6 cells. Luciferase activity was determined by Steady- Glo® Luciferase Assay System in a GloMax® Navigator Microplate Luminometer (both from Promega). The SARS-CoV-2-pseudotyped rVSV-luc variants used were SARS- CoV-2 S protein B.1 , alpha, beta, gamma, delta and kappa. The SARS-CoV-2 S protein mutant D614G (B.1) was generated by site-directed mutagenesis using as an input DNA the expression vector encoding SARS-CoV-2 S 614D by Q5 Site Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s instructions. SARS-CoV-2 alpha (B.1.1.7; GISAID: EPI_ISL_608430), beta (B.1.351 ; GISAID: EPI_ISL_712096), gamma (P.1 ; GISAID: EPI_ISL_833140), delta (B.1.617.2; GISAID: EPI_ISL_1970335) and kappa (B.1.617.1 ; GISAID: EPI_ISL_1970331) were optimized, synthesized and cloned into pcDNA3.1 (GeneArt).

[0162] SARS-CoV-2 S protein-pseudotyped VSV neutralization assay TN and TNTneutralization activity were examined in triplicates at 712 concentrations in the range 0.64 pM - 10 nM using a SARS-CoV-2-pseudotyped rVSV-luc system. As controls, the SB-40592 anti-RBD neutralizing mAb and sotrovimab biosimilar (anti-S glycoprotein mAb) were used in the range 0.88 pM - 13.86 nM. For neutralization experiments, viruses-containing transfection supernatants were normalized for infectivity to a MOI of 0.5-1 and incubated with the antibodies at 37° C for 1 hour in 96-well plates. After the incubation time, 2 x 104 Vero E6 cells were seeded onto the virus-antibody mixture and incubated at 37 °C for 24 hours. Cells were then lysed and assayed for luciferase expression. Half maximal effective concentration (EC50) and 95% confidence intervals (95% Cl) were calculated using a nonlinear regression model fit with settings for log agonist versus normalized response curve using GraphPad Prism v8 Software.

[0163] Live SARS-CoV-2 neutralization assay

[0164] The neutralization activity of TN-based trimerbodies was also measured by a microneutralization test assay by using live SARS-CoV-2 virus, as previously described (Perez et al., 2022, Frontiers in Immunology, 12, 824728). Serially five-fold diluted antibodies (in the range 0.3 pM - 5 nM) in DMEM-2% (v / v) FBS medium were incubated at a 1 :1 ratio with 200 TCID50 of live SARS-CoV-2 B.1 (MAD6 isolate, containing the D614G mutation), B.1.351 (beta) and B.1.617.2 (delta) VOCs in Il-bottom 96-well tissue culture plates for 1 hour at 37 °C. Then, mixtures of antibodies and live SARS-CoV-2 virus were added in triplicates to Vero E6 / TMPRSS2 cell monolayers seeded in 96-well plates at 2 x 104 cells / well, and plates were incubated at 37°C, in a 5% CO2 incubator for 3 days. Then, cells were fixed with 10% formaldehyde for 1 hour and stained with crystal violet. When plates were dried, crystal violet was diluted in H2O-10% (w / v) SDS and optical density was measured in a luminometer at 570 nm. Fifty percent neutralization titer (NT50) and 95% Cl were calculated using a nonlinear regression model fit with settings for log agonist versus normalized response curve using GraphPad Prism v8 Software.

[0165] Immunization of immunocompetent C57BL / 6 mice

[0166] Trimerbodies in combination with SARS-CoV-2 S protein super stable trimer (Aero Biosystems) were injected subcutaneously (s.c.) into both hind paws of C57BL / 6 mice. The immunization followed a prime-boost scheme 7 days apart. Mice (n=5 / group, but PBS n=3 / group) were primed either with 1) PBS as control; 2) 2 pg of S protein alone; 3) 3 pg of TNT+ 2 pg of S protein; or 4) 3 pg of TNTDNGR-1 + 2 pg of S protein. For the boost, mice were injected s.c. respectively with 1) PBS as control; 2) 1 pg of S protein alone; 3) 1.5 pg of TNT+ 1 pg of S protein; or 4) 1.5 pg of TNTDNGR-1 + 1 pg of S protein. These amounts were divided equally between the two hind paws. All conditions except PBS included 5 pg CpG ODN 1826 746 and 5 pg Poly (l:C) LMW (both from InvivoGen) per mouse as adjuvants. The injection volume was 25 pL per paw. Mice were euthanized on day 14 (7 days post-boost) and spleens and popliteal lymph nodes (pLNs) were harvested to assess cellular responses by flow cytometry analysis, and serum samples were collected to evaluate humoral responses.

[0167] Cellular response analysis of immunized C57BL / 6 mice

[0168] Spleens and pLNs were smashed through a 70 pm filter in R10 medium. In spleen suspensions, erythrocytes were lysed using 1 mL of red blood cell lysing buffer Hybri- Max (Sigma-Aldrich). Cells were stained with fluorochrome labelled antibodies directed against B220-FITC (# MABF608, Invitrogen), CD11c-BV421 (# 566877, BD Biosciences), CD11c-APC / FireTM (# 117351 , BioLegend), CD19-BV421(# 302233, BioLegend), CD8-APC / FireTM (# 100765, BioLegend), CD4-APC (# 553051 , BD Biosciences), CD44-FITC (#11-0441-82, Invitrogen ), Sirpa-PE / Cy7 (# 25-1721-82, Invitrogen) and XCR1-PE (# 148203, Biolegend) . CD16 / CD32 Fc Shield (# 70-0161 , Tonbo Biosciences) was used to reduce nonspecific binding. All antibodies were used at a 1 :200 dilution. Tetramer specific for SARS-CoV-2 S protein, H-2Kb (539VNFNFNGL546), was provided by the NIH Tetramer Facility at Emory University (1 :100 dilution). Samples were stained in cold PBS supplemented with 2.5% (v / v) FBS and 2 mM EDTA. Tetramer staining was performed at room temperature for 15 min, followed by sequential surface staining with the appropriate antibody cocktail at 4 °C for 15 min. Dead cells were excluded using DAPI. Data was acquired on a LSRFortessaTM Cell Analyzer (BD Biosciences), using FACSDiva software, and analyzed using FlowJo v10.8 Software (BD Biosciences).

[0169] Humoral response analysis of immunized C57BL / 6 mice

[0170] Serum samples were prepared by incubating blood samples in collection tubes without anticoagulant for 15 min at room temperature to allow clotting to occur. Then samples were centrifuged at 15,000 rpm for 15 min, and serum was moved to a fresh collection tube. S- and RBD773 specific immunoglobulin (Ig) levels were determined by ELISA. Briefly, SARS-CoV-2 S protein or RBD were plated in Nunc MaxiSorpTM flat-bottom 96- well plates (Thermo Fischer Scientific) in carbonate buffer or PBS, respectively, at 1 pg / mL overnight at 4°C. Then, the plates were washed three times with PBS and blocked with BSA-PBS at least 1 hour at 37 °C. After washing again, serum was plated by duplicate at 1 / 50, 1 / 250, 1 / 1 ,250, and 1 / 6,250 serial dilutions in PBS containing 1% (w / v) BSA to visualize dose-response signals. Samples were incubated 2 hours at room temperature and washed three times with PBS-0.05% (v / v) Tween-20. Plates containing serum samples were incubated with biotinylated anti-mouse IgM, 780 total IgG, lgG1 , and lgG2c (all from BD) at 2 pg / mL for 1 hour at room temperature followed by three washes with PBS-0.05% (v / v) Tween-20 and incubation with HRP-conjugated streptavidin at 1 pg / mL for 30 min at room temperature. All plates were then washed again and developed in the presence of 3, 3', 5, 5'- Tetramethylbenzidine (TMB) substrate (Sigma-Aldrich) and stopped by 1M H2SO4 solution. Absorbance was read at 450 nm subtracting background at 620 nm.

[0171] Protective therapeutic efficacy study in transgenic K18-hACE2 mice

[0172] Female K18-hACE2 mice (9 weeks old) were used to evaluate the capacity of TNTand TNTDNGR-1 antibodies to protect against SARS-CoV-2 infection in a therapeutic administration schedule. After a lethal dose challenge (1 x 105 PFUs / mouse) with SARS- CoV-2 (MAD6 strain) by intranasal route in 50 pl of PBS, mice were divided randomly in four groups (n=8 / group) and 17 hours laterwere inoculated i.p. with 100 pg of TNTDNGR- 1 , 65 pg of TNT(equimolar to TNTDNGR-1), 100 pg of sotrovimab or PBS (as a control of infection) in a total volume of 200 pl. In addition, non794 infected and non-treated mice (n=8) were used as a control group of healthy animals. Mice were monitored for body weight change and mortality for 20 days p.c. Animals with more than 25% of body weight loss were euthanized. At days 5 and 20 p.c., four mice per group were euthanized, and lungs and serum samples were collected. Right lung lobes were divided longitudinally in two, with one part placed in RNALater stabilization reagent (Sigma-Aldrich) and stored at -80 °C for RNA extraction, and the other part stored also at -80 °C for the analysis of infective virus yields. At day 20 p.c. left lung was freshly processed for cellular immune response analysis. At both end point times blood was collected by submandibular bleeding, maintained at 37°C for 1 hour, kept at 4 °C overnight, and centrifuged at 3,600 rpm for 20 min at 4 °C to obtain the serum samples, which was then inactivated at 56 °C for 30 min and kept at -20 °C until use.

[0173] Analysis of SARS-CoV-2 RNA by RT-qPCR

[0174] Lungs from K18-hACE2 mice were harvested at day 5 p.c. and stored in RNALater (Sigma-Aldrich) at -80°C until homogenized with a gentleMACS dissociator (Miltenyi Biotec) in 2 ml of RLT buffer (Qiagen) plus p-mercaptoethanol (Sigma-Aldrich) and aliquoted. Then, 600 pl of homogenized lung tissue was used to isolate total RNA using the RNeasy minikit (Qiagen), according to the manufacturer’s specifications. First strand cDNA synthesis and subsequent real time PCR were performed in one step using NZYSpeedy One-step RT-qPCR Master Mix (NZYTech), according to the manufacturer’s specifications using ROX as reference dye. SARS CoV-2 viral RNA content was determined using previously validated set of primers and probes specific for the SARS-CoV-2 subgenomic RNA for the protein E and the genomic virus RNA dependent RNA polymerase (RdRp) gene (Lazaro-Frias et al., 2022, NPJ Vaccines, 7(1), 17). Data were acquired with a 7500 real-time PCR system (Applied Biosystems) and analyzed with 7500 software v2.0.6. Relative RNA arbitrary units (A.U.) were quantified relative to a negative group (non-infected mice) and were performed using the 2-AACt method. All samples were tested in duplicate.

[0175] Analysis of SARS-CoV-2 virus yields by plaque assay

[0176] Lungs from K18-hACE2 mice were harvested at day 5 p.c., weighted, and stored directly at -80 °C until homogenized with a gentleMACS dissociator (Miltenyi Biotec) in 2 ml of PBS and aliquoted. Then, undiluted and serial ten-fold dilutions of homogenized lung tissue were added in triplicate to Vero E6 / TMPRSS2 cell monolayers seeded in 12-well plates at 5 x 105 cells / well. After 1 hour of adsorption the inoculums were removed and plates were incubated at 37 °C, 5% (v / v) CO2 in 2:1 DM EM 2X containing 4% FBS and Avicel® RC-591 (DuPont Nutrition Biosciences ApS). After 4 days, cells were fixed for 1 hour with 10% formaldehyde (Sigma-Aldrich), then the supernatant was removed, and plaques were visualized by adding 0.5% (w / v) crystal violet solution (Sigma-Aldrich). SARS-CoV-2 titers were determined in PFUs per gram of lung tissue.

[0177] Humoral response analysis in the protective therapeutic efficacy experiment in transgenic K18-hACE2 mice

[0178] S-specific IgM measurements in individual sera samples from SARS-CoV-2 infected and treated K18-hACE2 mice were done by ELISA as described above in prime-boost immunization assays. S-, RBD- and N-specific IgG antibody levels were determined by ELISA, as previously described (Garcia-Arriaza et al., 2021 , Journal of Virology, 95(7); Lazaro-Frias et al., 2022, NPJ Vaccines, 7(1), 17; Perez et al., 2022, Frontiers in Immunology, 12, 824728). Briefly, 96-well Nunc MaxiSorp plates were coated with 50 pl of purified recombinant SARS-CoV-2 S or RBD proteins (2 pg / ml) in PBS overnight at 4°C. The SARS-CoV-2 S and RBD proteins used to coat the plates derived from the Wuhan strain (GenBank accession number MN908947.3) and were previously described (Garcia-Arriaza et al., 2021 , Journal of Virology, 95(7); Lazaro-Frias et al., 2022, NPJ Vaccines, 7(1), 17; Perez et al., 2022, Frontiers in Immunology, 12, 824728). Plates were washed with PBS-0.05% (v / v) Tween-20 and blocked with 5% milk in PBS for 2 hours at room temperature. Individual serum samples were diluted in duplicate in PBS-0.05% Tween-1 % milk, added to plates, and incubated for 1.5 hours at room temperature. Plates were then washed, and secondary HRP-conjugated GAM IgG mAb (Southern Biotech) diluted 1 :1 ,000 in PBS-0.05% Tween-1 % milk was added and incubated for 1 hour at room temperature. Plates were washed, the TMB substrate (Sigma-Aldrich) was added, and the reaction was stopped by adding 1M H2SO4. Absorbance was read at 450 nm. Antigen-specific IgG and IgM serum levels were evaluated by absorbance summation method, which sums the observed absorbance 848 values from all dilutions employed to obtain one data point for each sample to be used for comparison (Hartman et al., 2018, PloS One, 13(6)).

[0179] Determination of administrated antibody concentration in serum

[0180] The concentration of TNT, TNTDNGR-1 or sotrovimab in serum samples at day 5 and 20 p.c. was determined by ELISA using 1 :5 serial dilutions of the corresponding purified antibodies for standard. Briefly, Nunc MaxiSorp flat-bottom 96-well plates (Thermo Fischer Scientific) were coated with recombinant B.1 RBD (0.2 pg / well). After washing and blocking with 300 pl PBS BSA, 50 pl of individual sera, 1 / 20 and 1 / 100 times diluted, were incubated for 1 hour at room temperature. For TNTand TNTDNGR-1 or sotrovimab, after three washes, 100 pl of the corresponding HRP-conjugated rabbit anti-camelid VHH cocktail (# A02016, Genescript) or HRP conjugated goat anti-human IgG mAb (# A0170, Sigma-Aldrich) were added, respectively, and incubated 1 hour at room temperature. Finally, the plate was washed again and developed. All samples were analyzed by duplicate per each dilution factor.

[0181] Cellular response analysis in the protective therapeutic efficacy experiment in transgenic K18-hACE2 mice

[0182] Lung cellular suspensions were obtained from the left lung of K18-hACE2 mice harvested at day 20 p.c., homogenized with a gentleMACS dissociator (Miltenyi Biotec) in R10 medium and then erythrocytes were lysed by using ACK lysis buffer (Gibco). S- specific H-2Kb tetramer staining was performed, acquired and analyzed as previously described for the C57BL / 6 prime-boost immunization assay. The magnitude, breadth and polyfunctionality of SARS-CoV-2 S-specific CD4+ and CD8+ T cells expressing CD107a, and / or secreting IFNy, and / or TNFa, and / or IL-2 were analyzed by ICS as previously described (Garcia-Arriaza et al., 2021 , Journal of Virology, 95(7); Lazaro- Frias et al. , 2022, NPJ Vaccines, 7(1)) in lung cell samples stimulated with a SARS-CoV- 2 S peptide pool (1 pg / ml) (JPT Peptide Technologies), spanning the S1 and S2 regions of the S protein from the Wuhan strain, and containing 158 (S1) and 157 peptides (S2) as consecutive 15-mers overlapping by 11 amino acids. After left lung processing, 4 x 106 fresh isolated cells were seeded on M96 plates and stimulated overnight in complete RPMI 1640 medium supplemented with 10% FBS containing 1 pl / ml Golgiplug (BD Biosciences) to inhibit cytokine secretion, 1X monensin (eBioscience, Thermo Fisher Scientific), and anti-CD107a-FITC (BD Biosciences). Cells were then washed, stained for surface markers, fixed, permeabilized (Cytofix / Cytoperm kit; BD Biosciences), and stained intracellularly with the appropriate antibodies. Dead cells were excluded using the violet LIVE / DEAD stain kit (Invitrogen). The fluorochrome-conjugated antibodies used for functional analyses were CD3-PE-CF594, CD4-APC-Cy7, CD8-V500, IFN-882 y-PE-Cy7, TNF-a-PE, and IL-2-APC. All antibodies were from BD Biosciences. Cells were acquired with a Gallios flow cytometer (Beckman Coulter), and data analyzed with the FlowJo software version 10.4.2 (Tree Star), as previously described (Garcia-Arriaza et al., 2021 , Journal of Virology, 95(7); Lazaro-Frias et al., 2022, NPJ Vaccines, 7(1)).

[0183] Generation of antibody codifying mRNA mRNA polynucleotides encoding for TNTand TNTDNGR-1 were synthesized by TriLink BioTechnologies. The engineered mRNA sequences, mRNA-TNTand mRNA- TNTDNGR-1 , codified for exactly the same amino acid sequences as pcDNA3.1 (+)-TN- L23-TIE-StrepTagll and pcDNA3.1 (+)-TN-L23-TIE-L15-7H11-StrepTagll, respectively. Both constructs included an in frame fused wild type N-terminal interleukin-2 (IL-2) signal peptide for effective protein secretion to the medium and C-terminal StrepTactinll for detection and purification purposes.

[0184] HEK-293T mRNA transfection of the recombinant engineered antibodies

[0185] Both mRNA constructs (1 pg per molecule) were transfected using TransIT (Fisher Scientific) reagent according to manufacturer solutions in seed HEK-293T cells. Parental vectors encoding for TNTand TNTDNGR-1 (2.5 pg per plasmid, respectively) were included as transfection controls. After 48 hours of incubation at 37° C, the supernatants were collected for ulterior analysis.

[0186] Equipment and settings

[0187] If brightness or contrast processing of gel and blot images has been performed, it was applied to the entire image, including controls. No high-contrast gels or blots have been displayed. When necessary, cropped gels and juxtaposing images were displayed to improve the clarity and conciseness of the presentation, being shown in the figure. All optical density measurements were done using a Multiskan FC apparatus (Thermo Fischer Scientific).

[0188] Live SARS-CoV-2 neutralization assay The neutralization activity of mRNA expressed TN-based trimerbodies was studied by a microneutralization test assay by using live SARS-CoV-2 virus. Serially three-fold diluted supernatants in DMEM-2% (v / v) FBS medium were incubated at a 1 :1 ratio with 200 TCID50 of live SARS-CoV-2 B.1 (MAD6 isolate, containing the D614G mutation) in II- bottom 96-well tissue culture plates for 1 hour at 37 °C. Then, mixtures of antibodies and live SARS-CoV-2 virus were added in triplicates to Vero E6 / TMPRSS2 cell monolayers seeded in 96-well plates at 2 x 104 cells / well, and plates were incubated at 37°C, in a 5% CO2 incubator for 3 days. Then, cells were fixed with 10% formaldehyde for 1 hour and stained with crystal violet. When plates were dried, crystal violet was diluted in H2O- 10% (w / v) SDS and optical density was measured in a luminometer at 570 nm.

[0189] Statistical analysis

[0190] Statistical analysis was performed using GraphPad Prism Software version 8.0. In general, the in vitro experiments were done in triplicates and values are presented as mean ± standard error of the mean (SEM) from one of at least two independent experiments. In the immunogenicity experiments in C57BL / 6 mice, significant differences (P value) were discriminated by applying one-way ANOVA followed by Tukey’s multiple comparison test. The multiparametric read out analysis of protective efficacy experiment in K18-hACE2 mice, in general was done by applying one-way ANOVA followed by Tukey’s multiple comparison test for significant differences discrimination.

[0191] Results

[0192] Design of a broadly neutralizing biparatopic trimeric antibody targeting the SARS- CoV-2 S protein RBD

[0193] For the generation of the biparatopic VHH-tandem neutralizing antibody (TN), we used two well characterized neutralizing VHHs (E and V) that recognize two non-overlapping epitopes on the SARS-CoV-2 Wuhan-Hu-1 / B.1 RBD as building blocks (Koenig et al., 2021), fusing them with a 15-amino acid-long (G4S)3 linker. To improve TN’s binding to RBD through multivalency (i.e. the avidity effect) and to increase RBD occupancy within the S protein trimer, TN was integrated into a trimerbody scaffold by fusing it to a human collagen XVIII-derived homo-trimerization (TIE) domain (Compte et al., 2021 , Clinical Cancer Research : An Official Journal of the American Association for Cancer Research, 27(11), 3167-3177) generating a biparatopic VHH-based trimerbody (TNT) (fig. 1A). TN and TNTwere purified from HEK-293 conditioned media, showing migration patterns in SDS under reducing conditions consistent with their theoretical molecular weights (29.6 and 38 kDa, respectively) . In a size exclusion chromatography-multiangle light scattering (SEC MALS) analysis TN eluted as a major peak with a molar mass of 31 kDa and TNTas a single peak with 118 kDa (calculated masses are 29.6 and 114 kDa, respectively) . Circular dichroism (CD) spectra display minima at 217 nm, characteristic of b-sheet secondary structure. The cooperative thermal denaturation curves, with mid-point 171 temperatures > 60 °C, indicate stable three-dimensional structures in both antibodies.

[0194] TN-based antibodies bind SARS-CoV-2 S protein RBD with high affinity and efficiently block the RBD-ACE2 interaction

[0195] Biolayer interferometry (BLI) analysis confirmed the ability of TN and TNTto bind B.1 RBD and block its interaction with human ACE2 (hACE2). Treating the biosensor- immobilized B.1 RBD with any of the two antibodies prevented nearly all (>95%) subsequent binding of hACE2 (fig. 1 B). Moreover, TN and TNTexhibited similar reactivity against B.1.351 (beta), P.1 (gamma), B.1.617.1 (kappa) and B.1.617.2 (delta) RBDs compared to the B.1 RBD, as measured by enzyme-linked immunosorbent assay (ELISA). BLI analysis confirmed that TN and TNTbind with high affinity to immobilized B.1 RBD and demonstrated negligible dissociation over one hour of measurement. This precluded a precise determination of their dissociation rate but was indicative of high- affinity sub-nanomolar dissociation constants (KD). As expected from their respective monomeric and trimeric natures, TNTassociation rate constant was approximately 3-fold greater than for TN (ka values of ~16 and ~6 104*M-1*s-1 , respectively). A similar study was performed where both TN and TNTbound the beta variant RBD essentially identically to the B.1 RBD.

[0196] Biparatopic TNTbinds simultaneously to all three S protein RBDs

[0197] The stoichiometry of the interaction between S protein and TNTwas studied by microfluidic diffusional sizing (MDS) analysis. The hydrodynamic radius (Rh) of TNT(5.06 nm) is consistent with its trimeric form, increasing to 16.6 nm when mixed with equimolar amount of HexaPro S protein. This indicates that TNTbinds to trimeric S protein at a 1 :1 stoichiometry. Cryo-electron microscopy (cryo-EM) was used to determine the structure of the HexaPro S protein / TNTcomplex. An electron density map with 3.8 A resolution was used to build a consistent model of the S protein ectodomain (yellow, blue and green, fig. 1C). While the isolated S protein exists predominantly in a closed RBD conformation (Walls et al. , 2020), the TNT-bound S protein is mainly in the prefusion state with all three RBDs in the open conformation. The TIE domain and the linkers of TNTwere not observed (likely due to their flexibility and lack of direct binding to the S protein), but there were densities in the map that made it possible to model the six TNTVHHs bound to their RBD epitopes. As the length of the 15-amino acid linker connecting the C- terminus of E VHH and the N-terminus of V VHH (53 A in an extended conformation) is compatible with this distance between the E and V VHHs binding to the same RBD (43 A), but not compatible with those bound to different RBDs (>90 A), each tandem VHH must interact with a single RBD. In concert, the three tandem VHHs of TNTcompletely block all of the S protein’s sites of interaction with the hACE2 receptor. The RBD binding by the E and V VHHs within the TNTis very similar to that of the monomeric tandem VHHs, which was previously described (PDB: 7B18) (Koenig et al., 2021), although in the S protein / TNTmodel there is a major shift in the position of one of the RBDs relative to the other two in the complex.

[0198] Trimerization enhances SARS-CoV-2 neutralization by the TN antibody and prevents escape caused by mutations found in most VOCs

[0199] The neutralizing activity of TN and TNTwas assessed using replication-deficient G- luciferase VSV pseudotyped with the SARS-CoV-2 S protein. SB-40592 and sotrovimab biosimilar were used as control mAbs. SB-40592 neutralized S protein-pseudotyped particles with half-maximal effective concentration (EC50) of 37 pM for B.1 , 47 pM for VOC B.1.1.7 (alpha) and 41 pM for VOC delta, but was ineffective against beta, gamma and kappa strains (fig. 1 E). Sotrovimab EC50 values were between 156 and 335 pM for all assayed strains (fig. 1 E). TN neutralized infection strongly in a dose-dependent manner, with EC50 values of 147 and 182 pM for the B.1 and delta pseudoviruses, respectively; however, its efficacy was significantly decreased against beta (~ 10-fold), kappa (~ 8- fold) and gamma (~ 5-fold) variants (fig. 1 E). The trimerization-conveyed avidity of TNTreduced the EC50 concentration against B.1 pseudovirus to 26 pM (~ 6- fold) and it retained this efficacy against all the variants studied. Specifically, TNTwas nearly 70- and 30-fold more effective than TN against beta and gamma VOCs (EC50 of 23 and 24 pM, respectively) (fig. 1 E).

[0200] Design of a SARS-CoV-2-neutralizing trimerbody targeting dendritic cells

[0201] To allow TNTto prime adaptive immune responses in addition to its short-term virusneutralizing capacity, we aimed to deliver the TNT-bound virus to cDC1s, which excel at priming anti-viral responses. With this purpose, we generated a bispecific trimerbody (TNTDNGR-1) by fusing the anti-DNGR-1 7H11 scFv (Sancho et al., 2008) to the C- terminus of TNTthrough a flexible 15-amino acid linker (fig. 2A). SDS-analysis of purified TNTDNGR-1 revealed a single band consistent with its estimated molecular weight (64.7 kDa) and SEC-MALS analysis showed one major symmetric peak with a molar mass of 175 kDa, close to its theoretical trimeric mass of 194 kDa. BLI studies proved that TNTDNGR-1 bound to immobilized B.1 RBD with high affinity (fig. 2B) and could bind soluble DNGR-1 while remaining bound to the immobilized RBD, demonstrating its ability to bind both antigens simultaneously (fig. 2B). TNTDNGR-1 bound B.1 RBD in ELISA assays and HEK-293 cells 239 expressing B.1 S protein as efficiently as TNT. Furthermore, in accordance with its high-affinity binding to immobilized RBD protein, TNTDNGR-1 neutralized pseudovirus infection as efficiently as TNT, with EC50 values of 49.2, 36.9 and 42.6 pM for B.1 , beta and delta variants, respectively (fig. 20). Moreover, TN inhibited SARS-CoV-2 infection of Vero E6 / TMPRSS2 cells in a similar way to sotrovimab for B.1 and delta strains, but its EC50 was 5-fold increased against beta VOC. Comparatively, TNTDNGR-1 and TNTpromoted a higher neutralization effect than monomeric TN and sotrovimab for all assayed variants (fig. 2D). Both trimerbodies avoid viral escape with EC50 of 0.3 and 0.7 pM for B.1 strain, 0.15 and 1 pM for VOC beta, and 2.3 and 0.64 pM for VOC delta, respectively (fig. 2D). Comparatively sotrovimab EC50 values for these strains were 21 , 11 and 16 nM, respectively (fig. 2D).

[0202] TNTDNGR-1 trimerbody targets RBD to DNGR-1 -expressing dendritic cells promoting its internalization

[0203] To test the specificity of TNTDNGR-1 we stained B3Z and B3ZDNGR-1 cells with AF647- conjugated

[0204] B.1 RBD (AF647-RBD) preincubated with TNTor TNTDNGR-1 and found that B3ZDNGR- 1 cells were labeled by AF647-RBD only in the presence of TNTDNGR-1 but not the TNTcontrol. To address whether TNTDNGR-1 (as well as neutralized RBD or virions) is internalized by DCs, we incubated Flt3L bone marrow-derived dendritic cells (BMDCs) with TNTor TNTDNGR-1 for up to two hours at 4 °C or 37 °C and then added AF647- RBD for detecting cell surface-bound trimerbodies. DNGR1 -expressing DCs, mainly cDC1s but also plasmacytoid (pDCs), were stained with AF647-RBD when DCs were preincubated with TNTDNGR-1 , but not with TNT, at 4 °C (fig. 2E). Notably, after 1- or 2- hour incubation with TNTDNGR-1 at 37 °C, the AF647-RBD signal was reduced 4-fold in these subpopulations, suggesting its internalization (fig. 2E, lower graphs). The ability of TNTDNGR-1 to promote cell internalization of soluble RBD was confirmed in MuTu-DCs (a mouse DC cell line expressing DNGR-1) by confocal microscopy. Only cell preparations treated with TNTDNGR-1 , but not with TNT, followed by immediate AF647- RBD incubation showed specific staining and internalization of the TNTDNGR-1 / AF647- RBD complex after 2 hours of incubation at 4 °C or 37 °C (fig. 2F).

[0205] TNTDNGR-1 trimerbody increases S protein-specific CD8+ T cells in immunocompetent mice We investigated whether TNTDNGR-1 could increase the generation of adaptive immunity by specific targeting of captured SARS-CoV-2 S protein towards cDC1s. We treated immunocompetent C57BL / 6 mice in a prime-boost scheme with the following stimuli: 1) PBS; 2) S protein + adjuvants (poly l:C + CpG); 3) S protein + TNT273 + adjuvants; or 4) S protein + TNTDNGR-1 + adjuvants (fig. 2G). Immunization with S protein, like S protein + TNT, led to a significant increase in the generation of systemic S protein-specific effector CD8+ T cells quantified by S protein-specific tetramer (S-Tet) staining, compared to PBS-treated control mice (fig. 2H, I). Notably, immunization with S protein in the presence of TNTDNGR-1 resulted in a further increase of the frequency and number of S protein-specific effector CD8+ T cells (fig. 2H, I). Moreover, ELISA analysis in serum samples reveals that all the S protein treated mice groups induced potent S protein-specific IgM and total IgG antibody responses compared to PBS treated mice. By contrast, RBD-specific IgM and total IgG antibody responses were only induced in mice immunized with S protein and with S protein + TNTDNGR-1 , but not in animals immunized with S protein + TNTor PBS-treated mice. The measurement of S protein specific lgG1 and lgG2c antibody subclasses showed that S protein + TNTDNGR-1 administration induced a Th1 -polarized antibody profile response marked by an increased ratio of S protein-specific lgG2c to lgG1 (fig. 2J). RBD-specific lgG1 and lgG2c responses were only enhanced compared PBS by the immunization with S protein, but no significant differences we found for TNTand TNTDNGR-1 coadministration groups.

[0206] TNTDNGR-1 trimerbody protects against a lethal SARS-CoV-2 challenge

[0207] To evaluate the capacity of the TNTDNGR-1 trimerbody to protect against SARS-CoV-2 infection, we performed an in vivo protection study in K18-hACE2 transgenic mice, which express the human ACE2, and are therefore susceptible to SARS-CoV-2 infection (Moreau et al., 2020) (fig. 3). K18-hACE2 mice (n= 8 / group) were challenged intranasally (i.n.) with a lethal dose [1 x 105 plaque forming units (PFUs) / mouse] of SARS-CoV-2 (isolate MAD6, a prototypic B.1 strain) and 17 hours later were treated by intraperitoneal (i.p.) injection of TNT, TNTDNGR-1 , sotrovimab or PBS (as a non-treated infected control). In addition, non-infected and non-treated mice were used as a control group of healthy animals (fig. 3A). Mice were monitored for 20 days to determine body weight loss and survival (fig. 3B, C). All treated animals lost approximately 5% to 8% of their initial body weight during the first days post-challenge (p.c.), 10 to 13% in the case of the control PBS-treated group, but by day 6 p.c. mice treated with TNTDNGR-1 or sotrovimab began to recover body weight reaching the baseline body weight by day 9 p.c. (fig. 3B) and all survived (fig. 3C). However, PBS and TNT-treated mice continue to lose body weight (fig. 3B) and succumbed or were euthanized on day 7 p.c. (fig. 3C). Next, to assess the impact of TNTDNGR-1 treatment on the replication of SARS-CoV-2, four mice per group were sacrificed on day 5 p.c., the lungs were harvested and processed, and the presence of SARS-CoV-2 subgenomic E and genomic RdRp RNA (fig. 3D), and infectious virus (fig. 3E) analyzed. Treatment with TNTDNGR-307 1 and sotrovimab significantly reduced the subgenomic and genomic SARS-CoV-2 RNA levels in the lungs compared to PBS- or TNT-treated mice (fig. 3D). Live infectious virus determination showed that TNTDNGR-1 induced a more potent reduction than sotrovimab in virus titers in the lungs, whereas PBS- and TNT-treated mice show similar titers (fig. 3E).

[0208] TNTD / VGR-1 trimerbody induces SARS-CoV-2-specific humoral and cellular immune responses

[0209] Next, to study whether TNTDNGR-1 could promote SARS-CoV-2-specific adaptive immunity, we analyzed the humoral and cellular immune responses induced in K18- hACE2 mice. On day 5 p.c., mice treated with TNTDNGR-1 and sotrovimab induced significantly higher titers of S-specific IgM (fig. 4A) and S-, RBD-, and N-specific IgG antibodies than non-treated or TNTtreated mice (fig. 4B). Furthermore, on day 20 p.c., mice treated with TNTDNGR-1 or sotrovimab both elicited similar high titers of S-, RBD- , and N-specific IgG antibodies (fig. 4C,), which were highly increased when compared to the levels induced on day 5 p.c. Treatment with TNTDNGR-1 and sotrovimab led to higher titers of serum nAb against live SARS CoV-2 on day 5 p.c, than PBS- or TNT- treated mice. (fig. 4D). Consistently and similar to IgG antibody titers at day 20 p.c., neutralizing antibody titers increased highly in TNTDNGR-1- and sotrovimab-treated mice compared to day 5 p.c. levels (fig. 4E). To assess the contribution of the administrated antibodies to this neutralizing activity, we determined the concentration of TNT, TNTDNGR-1 and sotrovimab in mice serum at day 5 and 20 p.c., obtaining significant higher levels for sotrovimab (8.26 pg / ml and 12.72 ng / ml, respectively) than for TNTDNGR-1 (38.73 and 0.11 ng / ml, respectively) and TNT(0.32 ng / ml and undetermined, respectively). The study of T cell responses by S-Tet staining in the lungs obtained on day 20 p.c. showed that TNTDNGR-1 treatment selectively induced a significant local increase of S protein-specific effector CD8+ T cells compared to noninfected mice (fig. 4F). Notably, the magnitude of this effect was superior to that promoted by sotrovimab in the same non-infected mice (fig. 4F). In addition, the presence of S-specific CD4+ and CD8+ T cell immune responses was further assessed by an intracellular cytokine staining (ICS) assay in lungs cells obtained on day 20 p.c. and restimulated in vitro with SARS-CoV-2 S peptide pools. Treatment with TNTDNGR- 1 and sotrovimab induced S-specific CD4+ and CD8+ T cells expressing the cytotoxic marker CD107a and / or secreting IFNy, TNFa, and / or IL-2 cytokines (fig. 4G). While TNTDNGR-1 and sotrovimab elicited similar levels of S-specific CD4+ T cells, TNTDNGR- 1 -treatment specifically triggered a significantly increased number of S-specific CD8+ T cells compared to non-treated 341 group (fig. 4G). In detail, TNTDNGR-1 triggered a trend towards a higher amount of S-specific CD8+ T cells individually secreting I FNy, TN Fa, or expressing CD107a compared to sotrovimab, but did not show any differences in the CD4+ T cell subset. Together, these results suggest that the addition of DNGR-1 specificity to the TNTmoiety redirects the virus to cDC1s and favors cross-priming and a boosted adaptive response against the S protein of SARS-CoV-2.

[0210] J3TDNGR-1

[0211] The broadly HIV-neutralizing antibody J3 VHH was included into a trimerbody scafold to improve its interaction with trimeric HIV-1 Env protein through multivalency (i.e., the avidity effect) generating trimeric J3T. Then, by fusing the anti-DNGR-1 7H11 scFv at C- terminal of the construct we generated the cDC1 targeted J3TDNGR-1 bispecific trimerbody (Fig. 5 A). Both proteins were efficiently secreted to the Expi293 culture supernatant recognizing immobilized gp140 antigen (Fig.5 B, C). J3T and J3TDNGR-1 were purified in high yield to homogeneity by single step affinity chromatography showing migration patterns in SDS-PAGE under reducing conditions consistent with their theoretical molecular weights (19 and 50 kDa, respectively) (Fig. 5 D). Purified J3T and J3TDNGR-1 (1 pg / ml) bound similarly to immobilized recombinant HIV-1 gp140 (M.CON- S gp140CFI) (Fig. 5 E), while just J3TDNGR-1 could bind soluble DNGR-1 while remaining bound to the immobilized RBD, demonstrating its ability to bind both antigens simultaneously (Fig. 5 F).

[0212] TNTand TNTDNGR-1: mRNA

[0213] According to anti-RBD ELISA assays, both trimerbodies were produced efficiently by mRNA transfection of HEK-293T, showing pair by pair similar binding activity to the supernatants from the corresponding plasmid cell transfection (Fig. 6 A). Although both mRNA-TNTand mRNA-TNTDNGR-1 demonstrated to bind specifically to SARS-CoV-2 RBD, in the same way as plasmid expressed TNTDNGR-1 , mRNA-TNTDNGR-1 bound simultaneously to soluble DNGR-1-mFc (Fig. 6 B). Interestingly, both supernatants containing mRNA expressed TNTand TNTDNGR-1 showed a potent neutralizing activity against live B.1 SARS-CoV-2 (MAD6 isolate), similar again to the neutralization profile for plasmid transfected medium cultures (Fig. 6 C).

Claims

CLAIMS1 . A fusion protein comprising:(a) a region which binds to a viral envelope protein(b) a trimerization domain; and(c) a region which binds specifically to a receptor present in the surface of dendritic cells.

2. The fusion protein according to claim 1 wherein the region that binds to a viral envelope protein is an antibody, preferably a VHH.

3. The fusion protein according to claim 2 wherein the viral envelope protein in HIV gp140 or the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

4. The fusion protein according to claim 3 wherein the HH which binds to the HIV gp140 comprises the sequence of SEQ ID NO: 3.

5. The fusion protein according to any of claims 2 or 3 wherein region (a) comprises a first VHH and a second VHH in tandem, wherein each VHH binds to a different epitope in the viral envelope protein.

6. The fusion protein according to claim 5 wherein the first VHH comprises the sequence of SEQ ID NO: 1.

7. The fusion protein according to claim 5 or 6 wherein the second VHH comprises the sequence of SEQ ID NO: 2.

8. The fusion protein according to any of claims 5 to 7 wherein the first and second VHH are connected by a flexible linker.

9. The fusion protein according to claim 8 wherein the flexible linker has at least 15 amino acids.

10. The fusion protein according to claim 8 or 9 wherein the flexible linker comprises the sequence according to SEQ ID NO: 6 ((648)3).

11. The fusion protein according to any of claim 1 to 10 wherein the trimerization domain is the human collagen XVIII trimerization domain.

12. The fusion protein according to claim 11 wherein the human collagen XVIII trimerization domain comprises a sequence according to sequence SEQ ID NO: 4.

13. The fusion protein according to any of claims 1 to 12 wherein region (a) is connected to the trimerization domain by a flexible linker.

14. The fusion protein according to claim 13 wherein the flexible linker has at least 15 amino acids.

15. The fusion protein according to claim 13 or 14 wherein the flexible linker comprises the sequence according to SEQ ID NO: 6 ((048)3).

16. The fusion protein according to any of claims 1 to 15 wherein the region which binds specifically to a receptor present in the surface of dendritic cells binds to DNGR-1.

17. The fusion protein according to claim 16 wherein the region which binds specifically to a receptor present in the surface of dendritic cells is an antibody, preferably a ScFv.

18. The fusion protein according to claim 17 wherein the domain which binds specifically to a receptor present in the surface of dendritic cells has a sequence according to SEQ ID NO: 5.

19. A trimer comprising three monomers of the fusion protein according to any of claims 1 to 18.

20. A polynucleotide encoding the fusion protein according to any of claims 1 to 19.

21. A vector comprising the polynucleotide according to claim 20.

22. A host cell comprising the polynucleotide according to claim 20 or the vector according to claim 21.

23. An immunogenic composition or a vaccine composition comprising the fusion protein according to any of claims 1 to 18, the trimer according to claim 19, the polynucleotide according to claim 20, the vector according to claim 21 or the host cell according to claim 22 and a pharmaceutically suitable carrier or excipient.

24. A fusion protein according to any of claims 1 to 18, a trimer according to claim 19, a polynucleotide according to claim 20, a vector according to claim 21 , a host cell according to claim 22 for use in medicine.

25. A fusion protein according to any of claims 1 to 18, a trimer according to claim19, a polynucleotide according to claim 20, a vector according to claim 21 , a host cell according to claim 22 or an immunogenic composition or vaccine composition according to claim 23 for use in the prevention and / or treatment of a disorder caused by and / or associated with an infection by a virus containing in its envelope the protein to which region (a) of the fusion protein binds.

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