Universal principles of active and passive immunization against viral antigens

Abstract

The present invention pertains to protein oligomers for immunization against respiratory viruses and treatment of diseases caused by respiratory viruses.

Description

[0001] Universal Principles of Active and Passive Immunization Against Viral Antigens

[0002] The present invention pertains to protein oligomers for immunization against respiratory viruses and treatment of diseases caused by respiratory viruses.

[0003] Respiratory infections remain a major public health problem and represent an increased economic burden on healthcare systems, morbidity and mortality worldwide, especially in children, elderly and immunocompromised subjects. For more than four years now, the CO VID-19 pandemic has been ongoing. As of February 2024, there have been more than 774 million confirmed cases of CO VID-19, including over 7 million deaths reported globally (WHO coronavirus dashboard). The emergence of this new pandemic led to a race to develop vaccines to achieve herd immunity and curtail the damaging effects of COVID-19. The efforts to develop a vaccine are paying off as many vaccine candidates have shown major success in mitigating the pandemic. However, new viral strains continue to appear. Each new variant and subvariant has proved more transmissible than the last, and in addition, was capable of evading some of the protection offered by current vaccines, as seen with Delta, Omicron, BA.4, BA.5 and other variants.

[0004] Other respiratory virus infections cause an enormous disease burden both in children and in adults in all parts of the world, too. Influenza A and B viruses and respiratory syncytial virus (RSV) are responsible for the highest numbers of deaths and hospitalizations but other respiratory viruses, including rhinovirus, parainfluenza viruses, adenovirus, human bocavirus and coronaviruses, are also currently known to cause severe diseases in addition to mild upper respiratory tract infections. Furthermore, respiratory viruses of animal origin keep on emerging as novel human pathogens with epidemic or pandemic potential. Such viruses include avian influenza viruses and the Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) coronaviruses. Skariyachan et al. (Front. Microbiol., 26 March 2019 Sec. Virology Volume 10 - 2019 | https: / / doi.org / 10.3389 / fimicb.2019.00569) reviewed the pathogenesis, animal models and therapeutics for MERS coronavirus infections.

[0005] Seasonal influenza A and B virus epidemics globally cause up to 600,000 deaths per year. Influenza A viruses have an animal reservoir in avian species, which forms the basis for a permanent threat of pandemics. Vaccines and specific antiviral drugs against influenza are available but they have limitations including variable vaccine efficacies. McAuley et al. (Front Microbiol. 2019 Jan 29;10:39. doi: 10.3389 / fmicb.2019.00039. eCollection 2019) reviewed the current knowledge on the structure of the influenza virus neuraminidase and link the structural features of this surface glycoprotein with the multiple functions of neuraminidase in virus infection and fitness. Neuraminidase inhibitors are widely used for treatment (and sometimes prevention) of influenza infection, and they have demonstrated efficacy. However, new vaccines and drugs are needed to reach better treatment results in vulnerable patient groups particularly in the scenario of developing antiviral resistance. Several new antiviral drugs against influenza have been developed or are under development, including polymerase inhibitors and monoclonal antibodies. These new drugs are reviewed in the paper by Principi et al. (Front. Med., 28 May 2019, Sec. Infectious Diseases - Surveillance, Prevention and Treatment - Volume 6 - 2019 RSV occurs in yearly epidemics that cause high number of severe lower respiratory tract infections in young infants and also in the elderly. There is variation in the timing of RSV epidemics between geographic areas. Recognition of the onset of the epidemic is important in order to accurately start adequate control measures, particularly passive immunization of high- risk infants by monoclonal antibodies such as palivizumab.

[0006] Rhino viruses (RVs) are highly prevalent and can cause both upper and lower respiratory tract infections (URTIs and LRTIs). The clinical spectrum includes common colds, otitis media, and sinusitis, as well as more severe conditions, such as bronchiolitis and pneumonia. Moreover, RVs have been associated with the development and exacerbation of asthma and wheezing in children (allergic sensitization) and are the main viral cause of exacerbations of chronic obstructive pulmonary disease (COPD) in adults (Neugebauer, Viruses. 2022 Aug; 14(8): 1829. Published online 2022 Aug 20. doi: 10.3390 / vl4081829).

[0007] Despite the high clinical importance of respiratory viruses, the mechanisms of pathogenicity, transmission and evolution are only partially known, and most respiratory virus infections lack effective treatment or prevention modalities.

[0008] Due to the ongoing and fast emergence of new variants of respiratory viruses, vaccines need to be constantly adapted resulting in challenges in mass production and distribution to provide vaccines globally. As such, development of alternative and novel vaccines could circumvent these issues.

[0009] In light of the above, there is still a strong need for the development of novel vaccines and drugs that could meet the constant global demand for effective vaccines against respiratory viruses and pharmaceutical compositions against diseases caused by said respiratory viruses.

[0010] The technical problem underlying the present invention could be seen as the provision of means and methods, which comply with the aforementioned needs.

[0011] This technical problem has been solved by the embodiments characterized in the claims and herein below.

[0012] The present invention relates to a protein oligomer comprising at least a first monomer and a second monomer, said at least first and second monomer comprising, in N- to C-terminal order, at least one first receptor binding protein (RBD), an immunoglobulin Fc (Ig Fc), and at least one second receptor binding protein (RBD), wherein a) the Ig Fc has enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, compared to wildtype Ig Fc; and b) the receptor binding domain (RBD) is selected from the group consisting of i) the receptor binding domain (RBD) from the spike protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), wherein the receptor binding domain comprises amino acid residues 367 to 606 of UniProt accession number K9N5Q8, or SEQ ID NO. 69, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 367 to 606 of UniProt accession number K9N5Q8, or to SEQ ID NO. 69; ii) the receptor binding domain (RBD) from influenza virus hemagglutinin (HA), wherein the receptor binding domain comprises amino acid residues 63 to 286 of GenBank accession number ACQ99608, or an amino acid sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 77, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 63 to 286 of GenBank accession number ACQ99608, or to an amino acid sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 77; iii) the receptor binding domain (RBD) from parainfluenza virus hemagglutinin-neuraminidase (HN), wherein the receptor binding domain comprises amino acid residues 54 to 572 of GenBank accession number AY283063, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 54 to 572 of GenBank accession number AY283063; iv) the receptor binding domain (RBD) from rhinovirus capsid protein VP1, wherein the receptor binding domain is encoded by nucleotides 2305 to 3126 of GenBank accession number EF582385.1, or an nucleotide sequence at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to nucleotides 2305 to 3126 of GenBank accession number EF582385.1 encoding a receptor binding domain (RBD) from rhinovirus capsid protein VP1; v) the receptor binding domain (RBD) from human adenovirus fiber protein, wherein the receptor binding domain comprises amino acid residues 386-581 of UniProt accession number Pl 1818, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 386-581 of UniProt accession number Pl 1818; vi) the receptor binding domain (RBD) from respiratory syncytial virus glycoprotein G, wherein the receptor binding domain comprises amino acid residues 64-298 of UniProt accession number P03423, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 64-298 of UniProt accession number P03423; vii) the receptor binding domain (RBD) from human metapneumovirus fusion (F) protein, wherein the receptor binding domain comprises amino acid residues 66 to 87 contained within the F2 fragment of pre-fusion F protein (LIKTELDLTKSALRELRTVSAD) (SEQ ID NO. 56), or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 56; viii) the receptor binding domain (RBD) from parvovirus B19 VP1 unique region, wherein the receptor binding domain comprises amino acid residues 5 to 68 of UniProt accession number Q9JGS0, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 5 to 68 of UniProt accession number Q9JGS0; ix) the receptor binding domain (RBD) from SARS-CoV (or SARS-CoV-1) spike protein, wherein the receptor binding domain comprises amino acid residues 306 to 527 of GenBank accession number ABF65836.1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 306 to 527 of GenBank accession number ABF65836.1; x) the receptor binding domain (RBD) from SARS-CoV-2 spike protein, wherein the receptor binding domain comprises amino acid residues 320 to 541 of GenBank accession number QHD43416.1, or SEQ ID NO. 13, or SEQ ID NO. 14, or SEQ ID NO. 78, or SEQ ID NO. 79, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 320 to 541 of GenBank accession number QHD43416.1, or to SEQ ID NO. 13, or SEQ ID NO. 14, or SEQ ID NO. 78, or SEQ ID NO. 79; xi) the receptor binding domain (RBD) from hCoV-NL63 spike protein, wherein the receptor binding domain comprises SEQ ID NO. 70, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 70; xii) the receptor binding domain (RBD) from HCoV-OC43 spike protein, wherein the receptor binding domain comprises SEQ ID NO. 75 or 76, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 75 or 76; xiii) the receptor binding domain (RBD) from NeoCoV spike protein, wherein the receptor binding domain comprises SEQ ID NO. 71, the receptor binding domain (RBD) from PDF- 2180 spike protein, wherein the receptor binding domain comprises SEQ ID NO. 72, the receptor binding domain (RBD) from HKU5-CoV-l spike protein, wherein the receptor binding domain comprises SEQ ID NO. 73, or the receptor binding domain (RBD) fromHtHKU5-CoV- 2 or HtHKU5-CoV-2-441 spike protein, wherein the receptor binding domain comprises SEQ ID NO. 74, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 71, 72, 73, or 74.

[0013] Accordingly, particularly preferred RBD or RBM of respiratory viruses that can be used in the protein oligomer of the invention are shown in any one of SEQ ID Nos. 13, 14, and 56-79 and in Figures 12 to 15. Encompassed by preferred RBD are also minimized RBD as shown, e.g., in Figures 12 and 14 (named ’’min.RBD region”). The present inventors have developed novel protein oligomers that can advantageously be used for immunization of subjects against infections by respiratory viruses and for therapy or treatment of symptoms and diseases caused by said respiratory viruses in subjects, such as human. Said protein oligomers of the invention are particularly suitable for mucosal administration.

[0014] Preferably, mucosal pH as referred to herein is nasal or nasopharyngeal pH, such as a pH of about 6.0 to 6.7, more preferably of about 6.17 to 6.65, and even more preferably of about 6.5.

[0015] Specifically, the protein oligomer of the invention works with a tetrameric RBD both at N- and C -terminus of an IgGl Fc domain compared to, for instance, mRNA vaccine known superior toxicity profile of a recombinant protein-based vaccine. While there are rationales favoring C- (Genentech) or N-terminal (Lexigen / Merck) protein Fc-fiisions for different candidate proteins, it was not evident that combined N- and C-terminal fusion of RBD to the Fc molecule will result in efficient expression of a highly functional and stable protein - the protein oligomer of the invention. Moreover, the protein oligomer of the invention was a priori designed for mucosal administration. Therefore, the glycine linkers (G4S) both at N- and C-termini were designed to be short (only two repeats) to enhance stability and avoid degradation at the potential cost of RBD protein-linker-Fc flexibility. While the C-terminal RBD was linked with the two G4S linker amino acid sequence GGGGSGGGGSDKTHT (SEQ ID NO. 52) to the hinge-region of IgGl -Fc, the C-terminal Lysine of the Fc (SPGK) was substituted by Glycine forming a G5S-G4S linker SPGGGGGSGGGGS (SEQ ID NO. 53) fused to the C-terminal RBD. In addition, two key modifications of the Fc-domain were utilized. First, enhanced mucosal uptake at nasal pH levels of about 6.5 was engineered by using L309D / Q311H / N434S (DHS) substitutions at the Fc-Rn domain. Fc-Rn may also play an important role for in-vivo persistence and therefore improved stability of the protein oligomer of the invention, as it was originally designed for improved circulation half-life of antibodies via high affinity binding to FcRn at low lysosomal pH (Lee et al., Nat Commun, 2019). Therefore, this pH-toggle function of DHS Fc substitutions was hijacked for mucosal adaptation. This resulted in a successful combination of DHS Fc within tetrameric Fc fusion proteins, the BioVac constructs (tetrameric RBD-Fc-RBD fusion proteins containing the receptor binding domains (RBD) with amino acid residues 320 to 541 of the SI subunit of the spike protein of SARS-CoV-2 (SEQ ID NO. 13), and overall mucosal and intramuscular (i.m.) application of a DHS mutation based FcRn enhanced molecule in mouse and human. The second engineering element was the silencing of the FcR preferably via LALAPG or STR mutations, respectively. The original intention for this modification was that in contrast to prime immunization, an FcR silent and less immunogenic boost immunization should select for high-affinity adaptive immune cells that very specifically bind to the RBD region. Therefore, FcR silencing under boost immunization conditions would preclude non-specific Fc mediated uptake by antigen presenting cells (APC) and restrict the immune response to high affinity RBD recognizing adaptive cells. Overall, the rationale design of the Fc modifications was to have an improved mucosal uptake and persistence combined with an enhanced immunogenic FcR (wt) mediated prime immunization versus (vs.) highly specific RBD centric boost immunization avoiding formation of low-affinity non-neutralizing antibodies that may elicit undesired antibody directed enhancement (ADE) effects. Further, this selection processes for the highly RBD specific B- and T-cell responses vs. anergy for low- affinity less specific binder in a booster strategy might be of relevance for correction of outranged “autoimmune” like humoral and cellular responses induced by infection or vaccination. The latter is currently discussed as a potential underlying mechanism behind the development of postVacs and post / long-covid diseases with respect to Covid- 19. In analogy to natural infection, the prevailing vaccination principles today rely on immunization using the entire highly glycosylated SARS-CoV-2 spike protein as antigen, in case of mRNA membrane anchored, in case of recombinant ectodomain protein expressed in insect cells even with formation of higher order micelles like structures (Bangaru et al., Science, 2020), all rising the possibility of molecular mimicry with human proteins. Therefore, restricting the immunogen to a 3D well folded recombinantly expressed immunodominant RBD subunit region might reduce these adverse effects. At the same time, restricting the immunogen to this subunit region requires additional design steps e.g., multivalent display increasing the immunogenicity. However, engineering a tetrameric RBD-Fc-RBD fusion protein with disulfide bounds and N- glycosylation sites must be considered a challenging approach, the compatibility of different protein design elements on top was even less foreseeable and required extensive experimental iteration, optimization and validation studies. Intriguingly, in proof-of-concept experiments, high expression titers were achieved by the present inventors for the BioVac constructs (tetrameric RBD-Fc-RBD fusion proteins containing the receptor binding domains (RBD) with amino acid residues 320 to 541 of the SI subunit of the spike protein of SARS-CoV-2) in experimental HEK cells and later in standard industrial CHO cells with most recent cell line optimization and selection resulting into ~ lOg / L protein expression rates of a highly pure protein (>99%) using standard protein A / G purification and size exclusion (SEC) methods with exceptional long-term stability despite varying pH and temperature conditions evaluated. These characteristics combined with high functionality (picomolar binding efficacy to the viral receptor on the target cell of the subject) and high immunogenicity due to multivalent display underscore the relevance of protein oligomer of the invention as a novel principle for active mucosal and systemic immunization.

[0016] Accordingly, the protein oligomers of the invention advantageously induce protective immune responses at the relevant mucosal sites of pathogen entry by mucosal delivery of said protein oligomers, in active immunization approaches.

[0017] The protein oligomers of the invention are able to induce both a systemic immune response, dominated by IgGl, as well as a mucosal immune response in the upper respiratory tract that is dominated by slgAl . In light of this, the administration of the protein oligomers of the invention can lead to immunity for many respiratory viruses, thereby providing optimal protection from respiratory viral infection.

[0018] The protein oligomers of the invention can also be used for blocking the interaction between the respiratory virus and the viral receptor on the target cell of a subject, due to the high avidity and affinity of the protein oligomers of the invention to the viral receptor on the viral target cell of a subject. Thus, it is a further advantage that the protein oligomers of the invention can be used for prophylactic administration for avoiding or preventing respiratory viral infection, i.e. for passive immunization, in (a) subject(s) because they prevents efficiently the respiratory virus from binding to its receptor on viral target cells of a subject, thereby blocking viral entry into said target cells in the subject. Specifically, the protein oligomer of the invention advantageously induces protective immune responses at the relevant mucosal sites of pathogen entry by mucosal delivery of said protein oligomer.

[0019] What is more, the protein oligomer of the invention can advantageously be used for treating subjects suffering from infections by respiratory viruses and to ameloriate or even cure symptoms and diseases causes by said respiratory viruses in said subjects.

[0020] The novel protein oligomers of the invention are superior in comparison to known vaccines, in different aspects, as set forth elsewhere herein.

[0021] In addition to protein oligomers comprising the receptor binding domain of the spike protein of SARS-CoV-2, protein oligomer constructs comprising the receptor binding domain of spike proteins of other coronaviruses or the receptor binding domain of hemagglutinin (HA) of influenza viruses have been produced, by the present inventors; see e.g. Figure 15. These constructs are well-expressed in the CHO system and demonstrate high-affinity binding to their cognate receptors and high immunogenicity in the classical prime / boost immunization scheme.

[0022] In approaches similar to those described for protein oligomers comprising the receptor binding domain of the spike protein of SARS-CoV-2, mucosal and intramuscular (i.m.) application of these further DHS mutation based FcRn enhanced constructs will be tested for non-SARS-CoV- 2 protein oligomers of the invention, in mouse and human.

[0023] In a preferred embodiment of the protein oligomer of the invention, i) the receptor binding domain (RBD) from the spike protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) binds to Dipeptidyl peptidase IV (DPP4), or CD26 (UniProt P27487); ii) the receptor binding domain (RBD) from influenza virus hemagglutinin binds to sialic acidcontaining proteins or sialylated glycoconjugates; iii) the receptor binding domain (RBD) from parainfluenza virus hemagglutininneuraminidase (HN) binds to sialic acid-linked receptors or sialylated glycoconjugates; iv) the receptor binding domain (RBD) from rhinovirus capsid protein VP1 binds to intercellular adhesion molecule 1 (ICAM-1) receptor (UniProt P05362), very low density lipoprotein (VLDL)-receptor (VLDL-R) (UniProt Q9Y679) or Cadherin Related Family Member 3 (CDHR3) (UniProt Q6ZTQ4); v) the receptor binding domain (RBD) from human adenovirus fiber protein binds to coxsackie and adenovirus receptor (CAR) (UniProt P78310), membrane cofactor protein (MCP) or CD46 (UniProt P15529), GDla glycan, polysialic acid, or desmoglein-2 (DSG-2) (UniProt Q 14126); vi) the receptor binding domain (RBD) from respiratory syncytial virus glycoprotein G binds to CX3C motif chemokine receptor 1 (CX3CR1) (UniProt P49238), heparan sulfate proteoglycans (HSPG), or Nucleolin (UniProt Pl 9338); vii) the receptor binding domain (RBD) from human metapneumovirus fusion (F) protein binds to heparan sulfate, glucosaminoglycan, or integrin a5pi (UniProt P08648; P05556); viii) the receptor binding domain (RBD) from parvovirus B19 VP1 unique region binds to glycosphingolipid (GSL) globoside (Gb4), or tyrosine protein kinase receptor UFO (AXL) (UniProt P30530); ix) the receptor binding domain (RBD) from SARS-CoV spike protein binds to angiotensinconverting enzyme 2 (ACE2) (UniProt Q9BYF1); x) the receptor binding domain (RBD) from SARS-CoV-2 spike protein binds to angiotensinconverting enzyme 2 (ACE2) (UniProt Q9BYF1); xi) the receptor binding domain (RBD) from from hCoV-NL63 spike protein binds to angiotensin-converting enzyme 2 (ACE2) (UniProt Q9BYF1); xii) the receptor binding domain (RBD) from HCoV-OC43 spike protein binds to sialoside receptor; xiii) the receptor binding domain (RBD) from NeoCoV spike protein, PDF-2180 spike protein, HKU5-CoV-l spike protein, HtHKU5-CoV-2 spike protein or HtHKU5-CoV-2-441 spike protein binds to angiotensin-converting enzyme 2 (ACE2) (UniProt Q9BYF1) or bat orthologues thereof, or to Dipeptidyl peptidase IV (DPP4), or CD26 (UniProt P27487).

[0024] Figure 1 contains information with respect to human respiratory viruses, their entry proteins and receptor binding domains (RBD), and human cell surface receptors used by said viruses for human target cell entry, referred to herein.

[0025] In another preferred embodiment of the protein oligomer of the invention, Ig Fc is selected from the group consisting of IgG Fc, IgA Fc, and IgM Fc, preferably IgGl Fc or IgG3 Fc or IgG4 Fc, more preferably IgGl Fc.

[0026] In an even more preferred embodiment of the protein oligomer of the invention, said Ig Fc is human Ig Fc, preferably selected from the group consisting of human IgG Fc, human IgA Fc, and human IgM Fc, more preferably human IgGl Fc or human IgG3 Fc or human IgG4 Fc, most preferably human IgGl Fc.

[0027] In a further preferred embodiment of the protein oligomer of the invention, Ig Fc is a homodimer or a heterodimer, preferably wherein the heterodimer comprises Fc domains from knobs-into-holes (KiH)-engineered IgG, more preferably from knobs-into-holes (KiH)- engineered IgGl, most preferably from knobs-into-holes (KiH)-engineered human IgGl, or heterodimeric Fc variants selected from the group consisting of HA-TF, ZW1, DD-KK, 7.8.60, SEED, EW-RVT, and Al 07.

[0028] In a still further preferred embodiment of the protein oligomer of the invention, enhanced affinity of Ig Fc for the neonatal Fc receptor (FcRn) at mucosal pH, compared to wildtype Ig Fc, is mediated by (i) DHS mutations (L309D / Q311H / N434S in human IgGl Fc), (ii) YTE (M252Y / S254T / T256E in human IgGl Fc), (iii) LS mutations (N428L / N434S in human IgGl Fc), (iv) KF mutations (H433K / N434F in human IgGl Fc), or (v) DE mutations (S239D / I332E in human IgGl Fc), in the Ig Fc.

[0029] In yet another preferred embodiment of the protein oligomer of the invention, the Ig Fc further comprises LALAPG mutations (L234A / L235A / P329G in human IgGl Fc), LALA mutations (L234A / L235A in human IgGl Fc), or STR mutations (L234S / L235T / G236R in human IgGl Fc), for ablating Fc-Fc gamma receptor-mediated effector functions, without essentially affecting affinity for Fc gamma receptor, preferably wherein the Fc gamma receptor is selected from the group consisting of FcgammaRI, FcgammaRIIa, FcgammaRIIc, FcgammaRIIIa, and FcgammaRIIIb.

[0030] Preferably, the Fc-Fc gamma receptor-mediated effector function is antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and / or inflammation via the induction of cytokine secretion.

[0031] In a further preferred embodiment of the protein oligomer of the invention, the RBD or fragment thereof is the same or different, (i) in the at least first monomer of the protein oligomer, or (ii) in the at least second monomer of the protein oligomer, or (iii) both in the at least first monomer and in the at least second monomer of the protein oligomer.

[0032] Preferably, the linker is an independently selected variable linker amino acid sequence, preferably wherein the linker has reduced sensitivity to protease cleavage, more preferably wherein the linker comprises or is the linker (GGGGS)i (SEQ ID NO. 33) or (GGGGS)? (SEQ ID NO. 49) or GGGGGSGGGGS (SEQ ID NO. 54), and the reduced sensitivity to protease cleavage is in comparison to (GGGGS)s (SEQ ID NO. 50) or the linker comprises a Fc hinge region-derived linker (see, e.g., US patent number 6,165,476).

[0033] Preferably, the oligomerization domain is selected from the group consisting of: The non-triple helical trimerization domain of human collagen 18, the C-terminal oligomerization domain of human C4b-binding protein, coiled coils, oligomeric mini-proteins, short peptides with discrete protein-like structures, trimerization domain of the bacteriophage T4 fibritin (foldon), TNF alpha trimerization domain, zinc finger or p53 tetramerization domain.

[0034] The invention further relates to a vaccine comprising the protein oligomer of the invention. In a preferred embodiment of the vaccine of the invention, the vaccine or protein oligomer of the invention is for (i) passive vaccination, or (ii) active vaccination, or (iii) passive vaccination and active vaccination, in a subject, or the vaccine or protein oligomer of the invention is for use in (i) passive vaccination, or (ii) active vaccination, or (iii) passive vaccination and active vaccination, in a subject. Preferably, the subject is human.

[0035] The invention also pertains to a pharmaceutical composition comprising the protein oligomer of the invention.

[0036] In a preferred embodiment of the pharmaceutical composition or vaccine of the invention, the pharmaceutical composition or vaccine further comprises one or more of the following: a pharmaceutically acceptable buffer, a pharmaceutically acceptable carrier, a surfactant, a preservative, a stabilizer, a mucosal drug delivery system, an adjuvant, or combinations thereof.

[0037] Detailed description of the protein oligomer of the invention

[0038] The monomer, such as the first and second monomer included in the protein oligomer of the invention, comprises, at least one first receptor binding domain (RBD), an immunoglobulin Fc (Ig Fc), and at least one second receptor binding domain (RBD), in N- to C-terminal order.

[0039] Immunoglobulin Fc (Ig Fc)

[0040] The center is formed by an immunoglobulin (Ig) Fc (crystallizable fragment) domain, in the monomer of the protein oligomer of the invention.

[0041] The Ig Fc domain can be an IgG Fc, IgA Fc, and IgM Fc. The IgG Fc can be, for example, an IgGl Fc or IgG3 Fc.

[0042] As appreciated by those skilled in the art, the Ig Fc is human Ig Fc, such as human IgG Fc, human IgA Fc, or human IgM Fc, if the protein oligomer of the invention is adminsistered to a human subject for vaccination against the SARS-CoV-2 coronavirus, or against another respiratory virus as referred to or defined herein. For instance, the IgG Fc can be human IgGl Fc or human IgG3 Fc, in this case.

[0043] The Ig Fc domain can be a monomeric Ig Fc domain, or the Ig Fc domain can be capable of dimerization.

[0044] For instance, the Ig Fc can comprise one or more monomeric mutation(s), which means that the Ig Fc domain is engineered to a monomeric Fc in that one, two, three, four, five, six, seven or even more critical amino acid residues at positions located on the Ig Fc dimerization interface are mutated. An Ig Fc carrying one or more of such monomeric mutation(s) is no longer able to dimerize with another Ig Fc domain but only forms a monomer. Such monomeric mutations are known in the art; see, e.g., Ying et al., MAbs. 2014 Sep-Oct; 6(5): 1201-1210. Published online 2014 Oct 30. doi: 10.4161 / mabs.29835; Shan, L., Dyk, N.V., Haskins, N. et al. In vivo pharmacokinetic enhancement of monomeric Fc and monovalent bispecific designs through structural guidance. Commun Biol 4, 1048 (2021). https: / / doi.org / 10.1038 / s42003-021-Q2565- 5; Shan L, Colazet M, Rosenthal KL, Yu X-Q, Bee JS, Ferguson A, et al. (2016) Generation and Characterization of an IgG4 Monomeric Fc Platform. PLoS ONE 11(8): e0160345. https: / / doi.org / 10.1371 / journal.pone.0160345). An example for such a mutation is the monomeric mutation F405R in human IgGl Fc. If the at least first and second monomer in the protein oligomer of the invention comprise monomeric Ig Fc, dimerization will not occur via the Ig Fc but via an oligomerization domain, as explained elsewhere herein. Such an embodiment using a monomeric Ig Fc domain in the at least first and second monomer of the protein oligomer of the invention can be advantageous, for instance, in combination with the usage of a trimerization domain of NC-1, T4 bacteriophage T4 fibritin (foldon) or the p53 tetramerization domain to avoid problems with oligomerization forces working against each other (e.g. trimerization mediated by NC-1 trimerization domain which would work against dimerization mediated by dimeric Fc which is circumvented by the usage of monomeric Ig Fc).

[0045] The Ig Fc domain can form a homodimer or a heterodimer, in the protein oligomer of the invention.

[0046] Dimerization in the Ig Fc can be, for instance, via homodimerization.

[0047] Conventional IgG antibodies are bivalent and monospecific, the assembly of which depends upon in vivo homodimerization of two identical heavy chains (HCs), which is mediated by homodimeric associations between CH3 domains, and subsequently disulfide linkages between each HC and each light chain (LC), in B cells.

[0048] Zheng et al. elucidated the structural and functional roles of engineered disulfide bonds in antibody Fc fragments; see Zheng et al., J Biol Chem. 2018 Dec 7; 293(49): 19127-19135. The Fc fragment of an immunoglobulin (Ig) is dimeric, composed of two copies of CH2 domains and two copies of CH3 domains. In each domain, there is a native disulfide bond that is important for the structural stability. It has been shown that the native disulfide bond between Cys367 and Cys425 in human IgGl Fc (with the Fc residues being numbered according to EU numbering) can support the folding of single CH3 domain, as well as the dimerization process between two CH3 domains, and prevent aggregation formation. Because of the important roles of native disulfide bonds, the introduction of additional disulfide bonds can be used to stabilize the Fc molecule to make it better toward clinical use, as appreciated by the skilled person.

[0049] Accordingly, dimerization of the Ig Fc domain in the protein oligomer of the invention can be achieved, for instance, by native disulfide bridges in the CH2 and CH3 domains, or by genetically engineered (artificial) disulfide bridges in the CH2 and / or CH3 domains; see, e.g., Zheng et al., J Biol Chem. 2018 Dec 7; 293(49): 19127-19135.

[0050] Approaches for enhancing antibody Fc homodimer formation are well described in the literature; see, e.g., Yu et al., J. Biol. Chem. 2017 Oct 27; 292(43): 17885-17896.

[0051] Dimerization in the Ig Fc can also be via heterodimerization.

[0052] Heterodimeric Fc variants have been mainly engineered through the replacement of homodimer-favoring interactions at the CH3 domain interface with heterodimer-favoring interactions. This is achieved by introducing asymmetric mutations in each CH3 domain, which promotes the assembly of HCs from two different antibodies. This heterodimeric Fc engineering, using CH3 variant pairs, has been approached using two strategies: (1) structurebased rational design and (2) directed evolution; see, e.g., Ha et al., Frontiers in Immunology, Volume 7, Article 394, p. 1-16 (2016).

[0053] For instance, heterodimerization of the human Ig Fc domain in the protein oligomer of the invention can also be mediated by knob-into-holes (KiH) mutations, in the Ig Fc domain. Knobs-into-holes is a well-validated heterodimerization technology for the third constant domain of an antibody. Basically, the concept relies on modifications of the interface between the two CH3 domains where most interactions occur. A bulky residue is introduced into the CH3 domain of one antibody heavy chain and acts similarly to a key. In the other heavy chain, a “hole” is formed that is able to accommodate this bulky residue, mimicking a lock. The resulting heterodimeric Fc domain can be further stabilized by artificial disulfide bridges. During the process of optimizing the heterodimerization interface, various rational designs, including steric complementarity, KiH, disulfide bonds and salt bridges juxtaposing oppositely charged residues on either side of the CH3 domain, can be evaluated and ultimately optimized using, e.g., a phage display library. Correct heavy chain association with heterodimerization yields above 97% can be achieved by introducing six mutations: S354C, T366W in the “knob” heavy chain and Y349C, T366S, L368A, Y407V in the “hole” heavy chain of human IgGl (see e.g. Klein et al., MAbs. 2012 Nov 1; 4(6): 653-663; Ridgway et., Protein Eng. 1996 Jul;9(7):617-21); Xu et al., MAbs. 2015 Jan-Feb; 7(1): 231-242; Shatz et al., MAbs. 2013 Nov- Dec;5(6):872-81. doi: 10.4161 / mabs.26307. Epub 2013 Aug 29. In addition, properties of antibodies with KiH mutations such as (thermal) stability, FcyR binding and effector functions (e.g., ADCC, FcRn binding) and pharmacokinetic (PK) behavior are not affected. The noncovalent interactions, along with disulfide bridges in the hinge region, drive assembly toward heterodimer formation and minimize combinatorial heterogeneity. Suitable KiH- engineered Fc domains are depicted, e.g., in SEQ ID NOs. 25, 26, 28 and 30 of WO 2017 / 093569. For example, SEQ ID NO: 25 of WO 2017 / 093569 shows the amino acid sequence of the human IgGl Fc with the “knob” mutations S354C / T366W, and SEQ ID NO: 26 depicts the amino acid sequence of the human IgGl Fc with the “hole” mutations Y349C / T366S / L368A / Y407V.

[0054] An Ig Fc heterodimer can comprise, for instance, Fc domains from knobs-into-holes (KiH)- engineered IgG, such as knobs-into-holes (KiH)-engineered IgGl or IgG3, in the protein oligomer of the invention.

[0055] Heterodimerization of the Ig Fc domain in the protein oligomer of the invention can also be mediated by heterodimeric Fc variants selected from the group consisting of HA-TF, ZW1, DD-KK, 7.8.60, SEED, EW-RVT, and A107, in the human Ig Fc domain (see e.g., Table 1 of Ha et al., Front Immunol. 2016; 7: 394. Published online 2016 Oct 6. doi: 10.3389 / fimmu.2016.00394).

[0056] The protein oligomer of the invention can also comprise combinations of two different Ig Fc domains. Such a combination can be - without limitation - a dimer formed by an IgGl Fc domain and an IgG3 Fc domain, or even combinations of Fc domains from different immunoglobulin isotypes, such as a dimer of a human IgGl Fc domain and a human IgA Fc domain.

[0057] Further defined mutations can be incorporated in the Ig Fc domains of the protein oligomer of the invention to increase half-lives, extend circulation time and / or prolonged duration of protection, and / or to improve binding of said protein oligomer to the neonatal Fc receptor (FcRn) on cells at the apical site of the mucosa of a subject as defined herein and to enhance mucosal uptake and transport across polarized epithelia cells. At mucosal sites, FcRn transports IgG across polarized epithelial cells where it retrieves IgG in complex with luminal antigens that is delivered to tissue-localized immune cells. The Ig Fc advantageously has enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, preferably nasal or nasopharyngeal pH, such as a pH of about 6.0 to 6.7, more preferably of about 6.17 to 6.65, and even more preferably of about 6.5, compared to wildtype Ig Fc, in the protein oligomer of the invention. Such enhanced affinity enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH can be mediated, for instance, by (i) DHS mutations (L309D / Q311H / N434S in human IgGl Fc) [Lee CH, Kang TH, Godon O et al. “An engineered human Fc domain tha behaves like a pH-toggle switch for ultra-long circulation persistence”; Nat Commun. 2019; 10:5031], (ii) YTE (M252Y / S254T / T256E in human IgGl Fc) [Rosenberg YJ, Lewis GK, LaBranche CC et al. “Introduction of the YTE mutation into the non-immunogenic HIV bnAb PGT121 induces anti-drug antibodies in macaques”; PLoS One 2019; 14(2):e0212649], (iii) LS mutations (N428L / N434S in human IgGl Fc) Rosenberg YJ, Lewis GK, LaBranche CC et al. “Introduction of the YTE mutation into the non-immunogenic HIV bnAb PGT121 induces antidrug antibodies in macaques”; PLoS One 2019; 14(2):e0212649], (iv) KF mutations (H433K / N434F in human IgGl Fc) [Grevys AG, Bern M, Foss S et al. “Fc Engineering of Human IgGl for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions”; J Immunol. 2015 Jun 1; 194(11): 5497-5508], or (v) DE mutations (S239D / I332E in human IgGl Fc) [Ilieva et al., Front Immunol. 2017 Sep 11;8: 1112. doi: 10.3389 / fimmu.2017.01112. eCollection 2017.], in the Ig Fc, as disclosed elsewhere herein.

[0058] As evident from the cited references, such mutations in the Ig Fc domain mediating enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, compared to wildtype or nonmodified Ig Fc are well known in the art; see also Kisalu et al., JCI Insight. 2021 Feb 8; 6(3): el43958. Published online 2021 Feb 8. doi: 10.1172 / jci. insight.143958; Jebamani et al., Biotechnology and Bioprocess Engineering volume 26, pages985-992 (2021).

[0059] For example, if the protein oligomer of the invention is administered to a human subject for active immunization or vaccination against the SARS-CoV-2 coronavirus, or against another respiratory virus as referred to or defined herein, the human Ig Fc domain of the protein oligomer of the invention can carry one, two, three, four or all of the aforementioned DHS, YTE, LS, KF, or DE mutations, mediating enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, and showing no or essentially no detectable binding to FcRn at serum pH (about pH 7.35 to 7.40; preferably about pH 7.4), compared to wildtype or non-modified Ig Fc. Advantageously, the Ig Fc domain of the protein oligomer of the invention can further comprise mutations for ablating Fc-Fc gamma receptor-mediated effector functions, without essentially affecting affinity for Fc gamma receptor. Such mutations are well described in the literature and comprise, e.g., (i) LALAPG mutations (L234A / L235A / P329G in human IgGl Fc) [Wilkinson I, Anderson S, Fry J et al. “Fc-engineered antibodies with immune effector functions completely abolished”; PLoS One 2021; 16(12): e0260954], (ii) LALA mutations (L234A / L235A in human IgGl Fc) [Wilkinson I, Anderson S, Fry J et al. “Fc-engineered antibodies with immune effector functions completely abolished”; PLoS One 2021; 16(12): e0260954] or (iii) STR mutations (L234S / L235T / G236R in human IgGl Fc) [Wilkinson I, Anderson S, Fry J et al. “Fc-engineered antibodies with immune effector functions completely abolished”; PLoS One 2021; 16(12): e0260954], as disclosed elsewhere herein.

[0060] The Fc gamma receptor can be selected from the group consisting of FcgammaRI, FcgammaRIIa, FcgammaRIIc, FcgammaRIIIa, and FcgammaRIIIb.

[0061] The Fc-Fc gamma receptor-mediated effector function as used herein encompasses antibodydependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and / or inflammation via the induction of cytokine secretion; see, e.g., Schlothauer et al., Protein Eng Des Sei. 2016 Oct;29(10):457-466. doi: 10.1093 / protein / gzw040. Epub 2016 Aug 29).

[0062] As appreciated by the skilled person, Ig Fc domains completely devoid of binding to Fey receptors (FcyRs) and complement protein Clq, and thus with abolished immune effector functions, are of use for various therapeutic or medical applications in order to reduce FcyR activation and Fc-mediated toxicity. Such engineered Fc domains are also known as 'effector- silent' Fc variants, in the art. For instance, HD-BioVac002 (SEQ ID NO. 2) contains LALAPG mutations, in human IgGl Fc. Said LALAPG mutations are for ablating Fc-Fc gamma receptor- mediated effector functions without essentially affecting affinity for the Fc gamma receptor. Ablation of the paracrine immunostimulatory signals by Fc-silencing (e.g., LALAPG and STR mutations) could further restrict B-Cell stimulation to those cells with high-affinity RBD- binding only, hence restraining overt and less / or non-specific adaptive immune responses. In addition, this could help to fine tune or ablate potential undesired immune responses as reported, for example, in patients with post vaccine or infection (post / long-Covid) syndromes.

[0063] Respiratory viruses as referred to herein

[0064] The respiratory viruses referred to herein are characterized in that they comprise a specific viral protein with a receptor binding domain or even receptor binding motif which binds to a receptor on the target cell of a subject, thereby mediating entry of the respiratory virus into the target cell of the subject, such as a human subject.

[0065] Middle East respiratory syndrome coronavirus (MERS-CoV)

[0066] Coronaviruses can be categorized into three main genera or groups, with group 1 - alpha coronaviruses, group 2 - beta coronaviruses, and group 3 - gamma coronaviruses. Both MERS- CoV and SARS-CoV belong to the betacoronavirus genus, but are classified into different lineage subgroups (subgroup 2b for SARS-CoV and SARS-CoV-2, and subgroup 2c for MERS- CoV) (Lu G, Liu D. SARS-like virus in the Middle East: a truly bat-related coronavirus causing human diseases. Protein Cell. 2012;3:803-805. doi: 10.1007 / sl3238-012-2811-1).

[0067] The Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans, representing a further example of a highly pathogenic coronavirus, the first being SARS-CoV and SARS-CoV-2. CD26 (also known as dipeptidyl peptidase 4, DPP4) was identified as the cellular receptor for MERS-CoV (Raj VS, et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature. 2013;495:251-254. doi: 10.1038 / naturel2005). CD26 is a type II transmembrane protein. It is present as a homodimer on the cell surface. The dimerization of the peptidase relies on broad intermolecule contacts contributed by the hydrolase domain and the extended strands in blade IV of the P- propeller. The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus-cell fusion, thereby initiating infection. The study by Lu et al. (Nature. 2013; 500(7461): 227-231.Published online 2013 Jul 7. doi: 10.1038 / naturel2328) delineates the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 P-propeller. The atomic details at the interface between the two binding entities reveal a surprising protein-protein contact mediated mainly by hydrophilic residues.

[0068] The recognition of CD26 by MERS-CoV is mediated by virus surface spike (S) protein. As with other coronaviruses, the MERS-CoV S protein is cleaved in host cells into SI and S2 subunits. SI engages the receptor whereas S2, with typical sequence motifs homologous to those identified as the heptad repeats in class I enveloped viruses mediates membrane fusion.

[0069] The coding sequences for MERS-CoV RBD can be derived from e.g. GenBank accession number JX869059 (spike protein amino acid residues 367-606). SEQ ID NO. 69 shows the receptor binding domain of the MERS-CoV spike protein; see also amino acid residues 367 to 606 of UniProt accession number K9N5Q8. The coding sequences for human CD26 are shown in accession number NP 001926 (amin acid residues 39-766). The coding sequences for human ACE2 are shown in accession number BAJ21180 (residues 19-615). See, e.g., Lu et al., Nature. 2013; 500(7461): 227-231; Tai et al., Volume 499, December 2016, Pages 375-382.

[0070] Interestingly, it has been found that close relatives of MERS-CoV in bats use ACE2 as their functional receptors, recently. Using a pseudotype virus entry assay, the authors of Xiong Q. et al. found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins; see Xiong Q, Cao L, Ma C, Tortorici MA, Liu C, Si J, Liu P, Gu M, Walls AC, Wang C, Shi L, Tong F, Huang M, Li J, Zhao C, Shen C, Chen Y, Zhao H, Lan K, Corti D, Veesler D, Wang X, Yan H. Close relatives of MERS-CoV in bats use ACE2 as their functional receptors. Nature. 2022 Dec;612(7941):748-757. doi: 10.1038 / s41586-022-05513-3. Epub 2022 Dec 7.

[0071] Influenza virus

[0072] Four types of influenza virus, A, B, C, and D, are known. Influenza A and B viruses can cause severe symptoms and mortality in the human population, whereas influenza C virus only manifests itself in mild disease and influenza D virus does not circulate in humans. A major difference between influenza A and B viruses is that influenza B virus is almost exclusively observed in humans, whereas influenza A virus has a diverse and extensive reservoir in aquatic birds that occasionally spills over to humans directly or via domestic animals, such as pigs, as new pandemics or emerging viruses. As a result, influenza A viruses receive much more attention than other influenza types even though influenza A and B both co-circulate in the human population as seasonal viruses. Influenza A virus can be further divided into subtypes based on the antigenicity of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), with 18 known subtypes of HA (H1-H18) and 11 subtypes of NA (Nl-Nl 1). Similar to influenza A virus, influenza B virus also has two surface glycoproteins HA and NA, which diverged into two lineages, Victoria and Yamagata, during the 1980s. In contrast, influenza C and D viruses only have one surface glycoprotein hemagglutinin-esterase fusion (HEF) that encompasses both HA and NA activities. Four known influenza A pandemics have been documented in human history, namely 1918 Spanish flu (H1N1), 1957 Asian flu (H2N2), 1968 Hong Kong flu (H3N2), and 2009 swine flu (H1N1), although others undoubtedly have occurred prior to these. Occasionally, other influenza A subtypes, such as H5N1, H5N6, H6N1, H7N7, H7N9, H9N2, and H10N8, also infect humans through cross-species transmission but so far lack the ability for human-human transmission (see e.g. https: / / www.nejm.org / doi / full / 10.1056 / NEJMc2405495). Nevertheless, zoonotic influenza subtypes can be highly pathogenic, with a mortality rate of up to 60% in hospitalized patients. Currently, vaccines (trivalent and quadrivalent) are available against seasonal influenza viruses, including subtypes H1N1 and H3N2 of influenza A virus and for the two lineages of influenza B virus. However, the effectiveness of seasonal influenza vaccine is often quite low, especially against H3N2 viruses, despite the vaccine components being updated annually. Therefore, influenza vaccine development remains an active research area.

[0073] As set forth above, Haemagglutinin (HA) is one of two main surface fusion glycoproteins embedded in the envelope of influenza viruses, the other being neuraminidase (NA). There are sixteen known HA subtypes (H1-H16) and nine NA subtypes (N1-N9), which together are used to classify influenza viruses (e.g. H5N1). The antigenic variations in HA and NA enable the virus to evade host antibodies made to previous influenza strains, accounting for recurrent influenza epidemics. The HA glycoprotein is present in the viral membrane as a single polypeptide (HAO), which must be cleaved by the host's trypsin-like proteases to produce two peptides (HA1 and HA2) in order for the virus to be infectious. Once HAO is cleaved, the newly exposed N-terminal of the HA2 peptide then acts to fuse the viral envelope to the cellular membrane of the host cell, which allows the viral negative- stranded RNA to infect the host cell. The type of host protease can influence the infectivity and pathogenicity of the virus. The haemagglutinin glycoprotein is a trimer containing three structurally distinct regions: a globular head consisting of anti-parallel P-sheets that form a P-sandwich with a jelly-roll fold (contains the receptor binding site and the HA1 / HA2 cleavage site); a triple- stranded, coiled- coil, a-helical stalk; and a globular foot composed of anti-parallel P-sheets.

[0074] Each monomer consists of an intact HAO polypeptide with the HA1 and HA2 regions linked by disulphide bonds. The N terminus of HA1 provides the central strand in the 5-stranded globular foot, while the rest of the HA1 chain makes its way to the 8-stranded globular head. HA2 provides two a helices, which form part of the triple-stranded coiled-coil that stabilises the trimer, its C terminus providing the remaining strands of the 5-stranded globular foot.

[0075] The RBD of influenza A HA is composed of four structural elements, 130-loop, 150-loop, 190- helix, and 220-loop, which are named after their positions on the primary amino acid sequence. Similarly, RBD of influenza B HA is composed of the 140-loop, 190-helix, and 240-loop, which are structurally equivalent to the 130-loop, 150-loop, and 190-helix in influenza A HA. Four residues in the RBD are highly conserved across influenza A and B HAs: Trpl53, Hisl83, Leul94, and Tyrl95 (H3 numbering, i.e., Trpl58, Hisl91, Leu201, and Tyr202 in influenza B numbering). However, the RBD of influenza and B also have important differences. For example, while Phe98 (H3 numbering, i.e., residue 95 in influenza B numbering) is highly conserved in influenza B virus, influenza A virus has a highly conserved Tyr at residue 98. In influenza A virus, the Y98F mutant has very poor receptor binding. Although some animal influenza A viruses use N-glycolyl-neuraminic acid (NeuGc), most influenza A and B viruses use N-acetyl-neuraminic acid (Neu5Ac) as a receptor [Wasik B.R., Barnard K.N., Parrish C.R. Effects of sialic acid modifications on virus binding and infection. Trends Microbiol. 2016;24:991-1001. doi: 10.1016 / j.tim.2016.07.005], whereas influenza C virus mainly uses N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). In comparison, influenza D virus seems to be able to tolerate the broadest range of sialic acid modifications on the host receptor, likely due to its more open receptor-binding cavity. See Wu and Wilson, Viruses. 2020 Sep; 12(9): 1053. Published online 2020 Sep 22. doi: 10.3390 / vl2091053.

[0076] The receptor binding domain (RBD) of influenza virus hemagglutinin (HA) corresponds, e.g., to amino acid residues 63 to 286 of GenBank accession number ACQ99608 (residues 55 to 271 in H3 numbering); see e.g. DuBois et al., J Virol. 2011 Jan; 85(2): 865-872. Published online 2010 Nov 10. doi: 10.1128 / JVI.01412-10; Yen et al., Proc Natl Acad Sci U S A. 2009 Jan 6; 106(l):286-91. doi: 10.1073 / pnas.0811052106. Epub 2008 Dec 30.

[0077] Figures 12 and 14 shows the receptor binding domain (RBD) of hemagglutinin (HA) from relevant influenza strains; see also SEQ ID Nos. 57 to 68 and 77.

[0078] For instance, SEQ ID NO. 57 shows the receptor binding domain for Influenza A virus hemagglutinin (A / Mexico / 4603 / 2009(H1N1), as set forth elsewhere herein. SEQ ID NO. 58 represents HA-RBD amino acid sequence derived from COBRA Pl and is further described elsewhere herein. The amino acid sequence of the HA1 chain of hemagglutinin is shown under 2VIU in RCSB Protein Data Bank for influenza A, and in UniProt P04664 for influenza A and Uniprot P03460 for influenza B.

[0079] Parainfluenza virus

[0080] Acute respiratory infection is the leading cause of mortality in children under age 5 years, accounting for 20% of childhood deaths worldwide and killing between 2 million and 3 million children each year, yet for most of the important viral causes of lower respiratory tract disease, we have no vaccine or drug treatment. The human parainfluenza viruses (HPIVs), along with respiratory syncytial virus (RSV) and human metapneumovirus, cause the majority of cases of childhood croup, bronchiolitis, and pneumonia, the three major manifestations of the acute respiratory infections that affect young infants. No vaccines or drugs exist for the HPIVs, HPIV type 1 (HPIV1), HPIV2, HPIV3, or HPIV4 (4), despite the more than 23,000 hospitalizations for HPIV infection yearly in the United States. Parainfluenza viruses are a significant cause of lower respiratory tract infection in pediatric and adult patients following hematopoietic stem cell transplantation (HSCT), with HPIV3 being the most important and being associated with high rates of mortality. In adults with HPIV infection after HSCT, the mortality rate may be 75%, and no therapies are effective.

[0081] Parainfluenza virus entry is mediated by fusion of the viral and target host cell membranes at the cell surface. Virus-cell fusion results from the coordinated action of the two envelope glycoproteins that comprise the viral entry complex: the receptor binding protein hemagglutinin-neuraminidase (HN) and the fusion protein (F). These two envelope glycoproteins form a fusion complex and work together to mediate virus attachment and entry into target cells. The HPIV3 HN is a type II transmembrane protein that executes both receptor binding during viral entry and receptor cleavage during viral release from an infected cell. HN also has a dual effect on the F protein: before receptor engagement, HN stabilizes the F protein, but upon receptor engagement, HN activates F. F is synthesized as a precursor (F0) that is cleaved within the cell to yield the prefusion F trimer, with Fl and F2 remaining covalently linked via a disulfide bond. This trimeric F structure is present on the surface of an infectious viral particle in a metastable prefusion conformation, with its hydrophobic fusion peptide being buried in the interior of the molecule. After HN engages its cell surface sialic acid receptor, it activates F, and the prefusion F undergoes a conformational transition, extending and inserting its hydrophobic fusion peptide N-terminal domain into the target cell. F proceeds to refold into its energetically stable postfusion structure as the N-terminal and C-terminal complementary heptad repeats meet to form a stable six-helix bundle, and this refolding drives fusion of the viral and cell membranes and release of the viral genetic material into the target cell.

[0082] The sequences of the hemagglutinin-neuraminidase of human parainfluenza 3 virus are shown, e.g. in UniProt P12564 and GenBank accession numbers Z26523 and AY283063.

[0083] Amino acid residues 54 to 572 of GenBank accession number AY283063 contains the receptor binding domain. Rhinovirus

[0084] Rhinoviruses are among the most common viral infectious agents found in humans. Human Rhino viruses (HRVs) account for more then 50% of upper respiratory tract infections and infection rates among young children can be as high as 8-12 times a year. HRV infections are generally associated with an incubation period of 2 days followed by a symptomatic period of 1 to 2 weeks before clearance. Asymptomatic infections of the nasopharynx are quite common for rhinoviruses, especially among young children. Upper respiratory tract infections are associated with common cold-like symptoms including rhinorrhea, sore throat, coughing, sneezing, nasal congestion, and general malaise. In addition to the common cold, rhinovirus infections are often linked to acute otitis media and rhinosinusitis, which also frequently coincide with bacterial coinfection. Despite an optimal temperature for HRV replication in the cooler surfaces (32-33°C) of the upper respiratory tract, rhinoviruses are also implicated with lower respiratory diseases including pneumonia, bronchitis, and bronchiolitis, and exacerbation of asthma. Over half of all asthma exacerbation incidents are known to be associated with HRV infections and early HRV infections resulting in wheezing promote a greater risk of asthma development later in life. Approximately, 90% of children hospitalized with acute asthma attacks were shown to have detectable HRV. Furthermore, a study evaluating 3 to 18-year-old patients admitted with wheezing identified nearly half tested positive for HRV. Collectively, these studies demonstrate that HRV, though not typically considered a pathogen of high mortality, has a high potential for acute respiratory illness and the potential to promote or exacerbate chronic respiratory health conditions.

[0085] Three species of rhinovirus are currently known: HRV-A, HRV-B, and HRV-C. However, sequencing and serologic methods have defined approximately 83 HRV-A types, 32 HRV-B types, and 55 HRV-C types with potentially as many as 150 to 170 serological distinct HRV types in circulation. Rhinovirus strains within a given species share greater than 70% amino acid identity and new types are classified almost exclusively now based on VP1 or VP4 / VP2 sequence alignments. Isolates which share greater than 87% identity may be merged with existing types. A major challenge to the development of an HRV vaccine and subsequent establishment of protective immunity is the phylogenetic breadth of existing HRV serotypes. Infection with one type of HRV is unlikely to afford any immunity to other types resulting in lifelong infections to different HRV type exposures. However, limited cross-serotype protection has been demonstrated for some closely-related types. Among known types of HRV, HRV-A, and HRV-C are generally associated with more severe disease and asthma exacerbations than HRV-B. Structures of all three species exhibit distinct surface pocket differences which may account for their phenotypic differences.

[0086] Human rhinovirus is a non-enveloped virus with a positive-sense single-stranded RNA (+ssRNA) of approximately 7.2 kb that encodes 11 proteins. The viral capsid of HRV is comprised of four viral proteins (VPs): VP1, VP2, VP3, and VP4. The remaining viral proteins are responsible for viral replication and subsequent assembly. Antigenic variation among HRV types is derived from variations in the exposed surface of VP1, VP2, and VP3, while embedded VP4 is responsible for RNA packaging during assembly. Compared to the rest of the HRV genome, the capsid proteins exhibit a high degree of heterogeneity resulting in a wide range of antigenic diversity. Several antigenic sites have been identified for HRV strains through study of binding of neutralizing antibodies. However, the locations of these sites are often not conserved.

[0087] VP1 mediates cell surface attachment through engagement of a variety of cell surface receptors. Traditionally, the majority of HRV types were known to bind the intercellular adhesion molecule 1 (ICAM-1) receptor and a minority of HRV types utilized the low-density lipoprotein receptor (LDLR) for binding. However, with the recent identification of a specific variant of cadherin-related family member 3 (CDHR3) as the primary receptor for HRV C species and the breadth of circulating HRV types, yet-to-be identified receptors may also exist. HRV may enter the cell through several pathways including macropinocytosis and clathrin-dependent and clathrin-independent endocytosis. Upon entry and uncoating, the viral genome is translated and subsequently proteolytically processed by virus-encoded proteases, 2 A and 3C. During virion assembly and genomic packaging, 60 units of each capsid protein associate, with 1 unit of each capsid protein per face, to form the icosahedral structure encapsulating the RNA genome of the virus. See e.g. Stobart et al., Front Microbiol. 2017; 8: 2412. Published online 2017 Dec 5. doi: 10.3389 / fmicb.2017.02412; Palmenberg A. C., Spiro D., Kuzmickas R., Wang S., Djikeng A., Rathe J. A., et al. (2009). Sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution. Science 324 55-59. 10.1126 / science.1165557; Palmenberg A. C. (2017). Rhinovirus C, asthma, and cell surface expression of virus receptor CDHR3. J. Virol. 91:e00072-17. 10.1128 / JVI.00072-17.

[0088] The amino acid sequence for the rhinovirus capsid protein VP1 can be found in Genbank accession number FJ445189.1, D00239.1, NC001490, FJ445151, EF186077, EF582386, and RCSB Protein Data Bank: The Protein Database (PDB) identification codes for the obtained sequence and secondary structures are for HRV1A: 1R1A; for HRV2: 1FPN; for HRV3: 1RHI; for HRV14: 4RHV; and for HRV16: 1AYM. Nucleotides 2305 to 3126 of GenBank accession number EF582385.1 encodes VP1.

[0089] Adenovirus

[0090] Phylogenetically, the human adenoviruses (HAdV’s) are diverse, subdivided across seven species, A-G5, based classically on serological cross-reactivity, receptor usage, haemagglutination properties and, more recently, phylogenetic sequence similarity.

[0091] Human Adenoviruses (HAdVs) are a family of non-enveloped double-stranded deoxyribonucleic acid (dsDNA) viruses with genomes of about 35 kilobases (kb). They are causative agents of a wide range of illnesses, such as conjunctivitis, gastroenteritis and respiratory infections. Over 100 types of HAdVs, classified into seven groups (A-G), have been reported to the HAdV Working Group (http: / / hadvwg.gmu.edu / ). Viruses among these groups vary in pathology and molecular characteristics, for instance receptor specificity and host cell tropism. The HAdV capsid possesses T= 25 icosahedral symmetry, and consists of three major proteins: the hexon, the penton base and the fibre (sometimes also referred to as fiber; both terms are used interchangeably herein). Both the fibre and penton base, forming the penton complex at the vertices of the icosahedron, can engage host cell receptors. The fibre can be divided into a C -terminal, globular structure, the “knob”, which protrudes away from the capsid and mediates the initial interaction with attachment receptors, and an N-terminal, elongated “shaft” that anchors the fibre into the viral capsid. Binding of the knob to attachment receptors is followed by the penton base binding to the integrin entry receptor.

[0092] The fibre is a homotrimeric structure of between 60-80 kDa per monomer and its globular knob can engage different receptors. The monomeric C-terminal fibre knob contains an eight- stranded antiparallel P-sandwich fold, comprised of two P-sheets, with multiple loops. The trimeric propeller-like knob is formed by intertwining P-sheets and has a deep depression at the centre, which thins into narrowing channels, exposing around 65% residues to the solvent. The knob is mounted on the shaft, which consists of repeats of a hydrophobic 15-residue sequence assembled into a trimeric P-spiral. The number of repeats, and hence the length of the shaft, varies between HAdV types. Residues at the very N-terminal region of the fibre knob form a tail that interacts non-covalently with the penton base.

[0093] Multiple HAdV attachment receptors have been identified: the cell surface proteins Coxsackie and Adenovirus Receptor (CAR), CD46, and desmoglein-2 (DSG-2), as well as the glycans GDI a and poly sialic acid. The interactions of all five receptors with HAdV have been established using structural biology techniques. Other adenovirus receptors, such as heparan sulphate glycosaminoglycans, or factors IX and X, have also been described, but we currently lack detailed structural information about their modes of binding to the virus.

[0094] Once HAdV has attached to the cell surface, the fibre starts disassociating from the capsid, exposing the penton base. The penton base forms the vertex pentamer, which binds to the integrin entry receptor and exploits integrin-mediated signalling to enter the cell by endocytosis. HAdVs have been shown to use multiple types of integrins as their receptors, again highlighting their broad tropism.

[0095] The penton monomer consists of two domains: a jelly-roll domain proximal to the virion centre, and a distal insertion domain. The latter contains the variable, highly mobile RGD loop, so named because it contains an arginine-glycine-aspartic acid tripeptide sequence motif. The integrin-binding RGD motif mediates binding between the integrin and the penton base, with the exception of group F HAdV-40 and -41, where the interaction is presumed to take place in another manner due to the lack of this motif. The length of the RGD loop varies significantly between strains, ranging from 36 amino acids for HAdV-12 to 99 amino acids for HAdV-5. See e.g. Stasiak and Stehle, Med Microbiol Immunol. 2020 Jun;209(3):325-333. doi: 10.1007 / s00430-019-00645-2. Xia et al., Structure. 1994 Dec 15;2(12): 1259-70. doi: 10.1016 / s0969-2126(94)00126-x. Baker et al., Nature Communications volume 10, Article number: 741 (2019). The RBD of the fiber protein corresponds to residues 386-581 of the intact fiber protein shown in UniProt Pl 1818; see Xia et al., Structure. 1994 Dec 15;2(12): 1259-70.

[0096] Respiratory syncytial virus

[0097] Respiratory syncytial virus (RSV) is a globally prevalent virus that affects the airways and lungs. Infants and young children are at the highest risk of severe outcomes from RSV infection, with 33.1 million episodes of lower respiratory tract infection and approximately 3.2 million hospital visits and 118,200 deaths per year worldwide in children under the age of 5 years due to RSV. RSV is also a major cause of illness in adults older than 65 years of age and immunocompromised individuals, with an estimated 14,000 deaths per year in the United States. Hospitalization due to RSV is a major economic burden, especially in preterm infants and older adults.

[0098] Currently, only two licensed vaccine exist for the prevention of RSV infection, GSK Arexvy and Pfizer Abrysvo, making RSV one of the highest-burden diseases. In 2023, the U.S. Food and Drug Administration approved Arexvy, the first respiratory syncytial virus (RSV) vaccine approved for use in the United States. Arexvy is approved for the prevention of lower respiratory tract disease caused by RSV in individuals 60 years of age and older. Also in 2023, U.S. FDA approved ABRYSVO™, Pfizer’ s vaccine for the prevention of Respiratory Syncytial Virus (RSV) in infants through active immunization of pregnant individuals 32-36 weeks of gestational age.

[0099] RSV is a negative-sense single- stranded RNA virus with two major glycoproteins on the virion surface: the attachment glycoprotein (G) and the fusion glycoprotein (F). RSV G is responsible for cellular attachment to host cells, and RSV F causes the viral membrane to fuse with the target host cell membrane. While both RSV F and G are immunogenic and are targeted by neutralizing antibodies, the majority of neutralizing antibodies in human sera target RSV F. As such, most RSV vaccine candidates and therapeutic antibodies currently in development focus on RSV F. However, RSV that does not express the G protein is highly attenuated in vivo, and monoclonal antibodies that target RSV G are protective in vivo. In humans, anti-G antibodies are associated with lower clinical disease severity scores, despite an abundance in sera more than 30 times lower than anti-F antibodies. Thus, the RSV G protein is increasingly recognized as an important target for RSV vaccine and therapeutic antibody development (Tripp RA, Power UF, Openshaw PJM, Kauvar LM. 2018. Respiratory syncytial virus: targeting the G protein provides a new approach for an old problem. J Virol 92:e01302-17. doi: 10.1128 / JVI.01302-17).

[0100] RSV G is a type II membrane protein containing two mucin-like regions coated with 30 to 40 O-linked glycans and 3 to 5 N-linked glycans (McLellan JS, Ray WC, Peeples ME. 2013. Structure and function of respiratory syncytial virus surface glycoproteins. Curr Top Microbiol Immunol 372:83-104. doi: 10.1007 / 978-3-642-38919-l_4). There are two forms of RSV G produced during infection. Membrane-bound RSV G is responsible for virus attachment to airway epithelial cells via the human chemokine receptor CX3CR1. A secreted form of RSV G, derived from a second translation initiation site at Met48 and released from the membrane by proteolysis, is expressed early in infection (first ~6 h, prior to the release of virions at ~ 12 h). Secreted RSV G modulates signaling and trafficking of CX3CR1+ immune cells, contributing to airway congestion and pathogenesis. Between the two mucin-like regions of RSV G is a central conserved domain (CCD) of ~40 highly conserved amino acids, including 4 invariant cysteines forming a cysteine noose motif with two disulfide bonds (1-4, 2-3 connectivity) (Langedijk JP, de Groot BL, Berendsen HJ, van Oirschot JT. 1998. Structural homology of the central conserved region of the attachment protein G of respiratory syncytial virus with the fourth subdomain of 55-kDa tumor necrosis factor receptor. Virology 243:293-302. doi: 10.1006 / viro.1998.9066). While the C terminus of the RSV G CCD possesses a heparin binding domain, initial RSV infection is thought to be mediated primarily by interactions between the RSV G CCD and CX3CR1 on ciliated airway cells, which do not have measurable heparan sulfate proteoglycans on their surfaces.

[0101] The amino acid sequence of Glycoprotein G from RSV is shown in UniProt P03423.

[0102] Amino acid residues 64-298 of UniProt P03423 comprise the receptor binding domain of the RSV Glycoprotein G.

[0103] The amino acid sequence from the CX3C motif chemokine receptor 1 (CX3CR1) is shown in UniProt P49238), and the amino acid sequence from Nucleolin is shown in UniProt P19338.

[0104] Metapneumovirus

[0105] Human metapneumovirus (hMPV) was first isolated in 2001 by van den Hoogen et al. (van den Hoogen BG, et al., A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med. 2001;7:719-724. doi: 10.1038 / 89098), from children with respiratory infections from which the etiological agent had not been identified. Compelling evidence accumulated since then indicates that hMPV is a common cause of acute lower respiratory tract infections, only somewhat less frequent than human respiratory syncytial virus (hRSV), in children under 5 years of age (Williams JV, et al., Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children. N. Engl. J. Med. 2004;350:443-450. doi: 10.1056 / NEJMoa025472). Both hMPV and hRSV are also a frequent cause of morbidity and mortality in the elderly, and immunocompromised adults. Indeed, hMPV and hRSV share not only clinical symptoms but also biological traits that led to their recent classification in the metapneumovirus and orthopneumovirus genera, respectively, of the newly created Pneumoviridae family, detached from the original Paramyxoviridae family.

[0106] The hMPV genome is a single-stranded negative-sense RNA molecule that encodes nine different gene products, including three glycoproteins: G, F, and SH (van den Hoogen BG, Bestebroer TM, Osterhaus AD, Fouchier RA. Analysis of the genomic sequence of a human metapneumovirus. Virology. 2002;295: 119-132. doi: 10.1006 / viro.2001.1355). G and F are the main protein constituents of the virus envelope. It was originally thought that G mediated the initial interaction of hMPV virions with cell-surface proteoglycans, whereas F acted at a subsequent step to promote fusion of the viral and cell membranes and hence entry of the viral ribonucleoprotein into the cell. However, the precise pathway of hMPV entry into cells is still a matter of debate.

[0107] The F glycoprotein is synthesized as an inactive precursor, F0, that requires proteolytic processing to become fusion competent. Whereas the hRSV F0 precursor is cleaved twice inside the cell at two polybasic sites recognized by furin-like proteases, the hMPV F0 precursor is cleaved only once by trypsin-like proteases outside the cell, as is the case for the precursor of most paramyxovirus F proteins. Cleavage generates two subunits, F2 and Fl, that remain covalently linked by disulfide bonds. The mature hMPV F is a trimer of disulfide-linked heterodimers that is incorporated into virions in a metastable pre-fusion conformation. During membrane fusion, the F glycoprotein refolds through a series of unstable intermediates into a highly stable post-fusion conformation. hMPV F membrane fusion is enhanced in a minority of viral strains by exposure to acidic conditions. Therefore, although low pH is probably not a general mechanism for activation of hMPV F, studies of low pH dependency have identified regions of hMPV F that might be critical for the structural rearrangements that occur during membrane fusion.

[0108] Recent determination of hRSV F crystal structures, folded in either the pre-fusion or post-fusion conformation, revealed the drastic metamorphosis that F undergoes during membrane fusion (Krarup A, et al., A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nat.Commun. 2015;6:8143. doi: 10.1038 / ncomms9143; McLellan JS, et al., Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science. 2013;342:592-598. doi: 10.1126 / science.1243283; McLellan JS, et al., Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science. 2013;340: 1113-1117. doi: 10.1126 / science.1234914; McLellan JS, Yang Y, Graham BS, Kwong PD. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J. Virol. 2011;85:7788- 7796. doi: 10.1128 / JVI.00555-11; Swanson KA, et al. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc. Natl Acad. Sci. USA. 2011;108:9619-9624. doi: 10.1073 / pnas.1106536108). Among other changes, the pre-fusion-to-post-fusion transition includes refolding of heptad repeat A (HRA) sequences of the Fl subunit into one long a-helix, and insertion of the fusion peptide - located at the N-terminus of HRA — into the target-cell membrane. Refolding of this fusion intermediate promotes assembly of HRA and HRB sequences into a stable six-helix bundle that drives membrane fusion and is characteristic of post-fusion F. In addition, the structural studies with hRSV F identified antigenic sites unique to the pre-fusion conformation that are recognized by potent neutralizing antibodies that account for most of the neutralizing activity found in human serum. Antibody-mediated hRSV neutralization is thought to involve blockade of the structural changes that occur in the F protein during membrane fusion, in agreement with the high neutralizing potency of antibodies that bind unique epitopes of pre-fusion hRSV F. However, there are also neutralizing antibodies that bind epitopes shared by pre-fusion and post-fusion hRSV F, explaining the capacity of purified post-fusion hRSV F to induce neutralizing antibodies and afford protection against hRSV challenge in mice. Recently, the structure of a soluble form of post-fusion hMPV F was determined, revealing extensive similarity with post-fusion hRSV F despite having only -38% sequence identity. The purified post-fusion hMPV F protein was able to elicit high titers of neutralizing antibodies in mice, suggesting that it may be a promising vaccine candidate. Although a few monoclonal antibodies capable of neutralizing both hRSV and hMPV have been reported, no significant cross-neutralization was detected in polyclonal antibody responses elicited by soluble postfusion forms of either hRSV F or hMPV F. While the responses to a pre-fusion form of hMPV F may be different than to a post-fusion form, it is likely that a pan-pneumovirus vaccine will require two antigens, or a single chimeric antigen displaying antigenic sites from each virus.

[0109] Amino acid residues 66 to 87 contained within the F2 fragment comprise the receptor binding domain of pre-fusion F protein; see Huang et al., PLoS Pathog. 2020 Oct; 16(10): el008942. Published online 2020 Oct 9. doi: 10.1371 / joumal.ppat.1008942; Huang J et al., J Virol. 2021 Sep; 95(18): e00593-21.

[0110] The amino acid sequences of alpha 5 integrin and beta 1 integrin are depicted in UniProt P08648 and P05556, respectively.

[0111] Parvovirus

[0112] Human parvovirus B19 (B19V) belongs to the genus Erythroparvovirus in the Parvoviridae family. It packages a linear single-stranded DNA (ssDNA) genome of approximately 5,600 nucleotides (nt). B19V infection causes fifth disease in children, persistent anemia in immunocompromised patients, transient aplastic crises, hydrops fetalis in pregnant women, and arthropathy. B19V initially infects human respiratory tracts through an unknown mechanism and eventually reaches the bone marrow, where it causes infection of erythroid progenitor cells. However, infections of other tissues or cells, such as endothelial cells, have been reported. The clinical manifestations of B19V infection, as seen in transient aplastic crisis, pure red cell aplasia, chronic anemia, and hydrops fetalis, are direct outcomes of the infection and death of the human erythroid progenitor cells where B19V replicate. Up to the present, neither a vaccine nor a specific antiviral has been developed to prevent or treat B19V-caused diseases.

[0113] The B19V capsid consists of 60 structural subunits, of which ~95% are VP2 (58 kDa) and ~5% are VP1 (83 kDa). VP1 is identical to VP2 with the exception of an additional N-terminal region of 227 amino acid (aa) residues, referred to as the VP1 unique region (VPlu). Although the VP2 protein is the major capsid protein, in contrast to other parvoviruses, B19V VPlu is critical for eliciting an efficient immune response. The N-terminal 1-80 aa of VPlu is rich in neutralizing epitopes, highlighting the critical role of VPlu during the initial process of infection. The middle VPlu of 128-160 aa harbors a secretory phospholipase A2 (PLA2) motif, which executes PLA2 enzymatic activity for efficient escape of the virus from late endosomes after entry. The function of the C terminus (161-227 aa) of the VPlu is currently unknown. In matured virions, the N-terminal part of VPlu is not external to the capsid; however, a brief exposure to mild temperatures or low pH rendered this region accessible and triggered the VPlu PLA2 activity, indicating that VPlu can be exposed in the extracellular milieu before entry into cells. Later, it was found that VPlu is externalized and becomes accessible to antibodies when the virus binds to the primary P-antigen glycan receptor.

[0114] The VPlu exposure outside the virion prior to virus internalization explains how an originally inaccessible region of the capsid can harbor neutralizing epitopes. Recombinant VP2-only capsids were unable to be internalized into B19V permissive cells; however, the interaction between the capsid and the P-antigen receptor induces a conformational change of VPlu that is necessary for subsequent interactions with host cells leading to virus internalization. Moreover, a naturally derived neutralizing human monoclonal antibody, which recognizes an epitope (SO- 42 aa) of VPlu, blocks virus internalization when it is present during virus binding but not after virus binding. These lines of evidence further support that the externalized N-terminal part of VPlu participates in virus binding to cells and is required for virus internalization. The N- terminal region of the VPlu has been proven necessary and sufficient for cellular binding and internalization, and the VPlu 5-80 aa or 5-68 aa has been proposed as receptor-binding domain required for B19V internalization; see Bieri et al., PLOS Pathog. 17(4).el009434, 2021; Zou et al., J Virol. 2021 Jun 24;95(14):e00466-21. doi: 10.1128 / JVI.00466-21. Epub 2021 May 5.

[0115] The amino acid sequence of VP1 is shown in UniProt Q9JGS0. Amino acids 5-68 or 5-80 comprise the receptor binding domain of VP1.

[0116] The amino acid sequence of tyrosine protein kinase receptor UFO (AXL) is shown in UniProt P30530.

[0117] SARS-CoV (SARS-CoV-1) and SARS-CoV-2

[0118] The ongoing outbreak of coronavirus disease 2019 (COVID-19) originated in China in December 2019 and became a global pandemic by March 2020. COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Two other coronaviruses have caused worldwide outbreaks in the past two decades, namely SARS-CoV (or SARS-CoV-1 - both names are used interchangeably herein) (2002-2003) and Middle East respiratory syndrome coronavirus (MERS-CoV) (2012-present). The surface spike (S) glycoprotein, which is critical for virus entry through engaging the host receptor and mediating virus-host membrane fusion, is the major antigen of coronaviruses. The S proteins of SARS- CoV-2 and SARS-CoV, which are phylogenetically closely related, have an amino acid sequence identity of -77% (P. Zhou, X.-L. Yang, X.-G. Wang, B. Hu, L. Zhang, W. Zhang, H - R. Si, Y. Zhu, B. Li, C.-L. Huang, H.-D. Chen, J. Chen, Y. Luo, H. Guo, R.-D. Jiang, M.-Q. Liu, Y. Chen, X.-R. Shen, X. Wang, X.-S. Zheng, K. Zhao, Q.-J. Chen, F. Deng, L.-L. Liu, B. Yan, F.-X. Zhan, Y.-Y. Wang, G.-F. Xiao, Z.-L. Shi, A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273 (2020)). Such a high degree of sequence similarity raises the possibility that cross-reactive epitopes may exist.

[0119] The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global COVID-19 pandemic with a series of SARS-CoV-2 waves. This has resulted in the deployment of an effective vaccine within less than a year from the beginning of the pandemic. Following the completion of 2 years of the COVID-19 pandemic, thus far, more than 180 vaccine candidates are in clinical trials from various manufacturers. Of these, more than 20 vaccines with different platforms have been currently approved globally, and 11 of them have WHO emergency use listing and authorization. As of February 2023, 13.3 billion doses have been administered globally leading to 70.81% of the world population receiving at least one dose of a COVID-19 vaccine (https: / / www.bing. com / covid?vert=vaccineTracker accessed on 5 February 2023). Most of these vaccines are designed to elicit an immune response against the spike protein that is critical for SARS-CoV-2 binding and cell entry. Similar to SARS-CoV-2 infection, COVID-19 vaccines elicit early production of humoral immune responses and also stimulate long-lasting memory B- and T-cell responses. Subsequently, clinical trials and real- world data showed COVID- 19 vaccines are highly effective against SARS-CoV-2 symptomatic diseases. Thus, the global rollout of CO VID-19 vaccines has played a critical role in reducing pandemic spread, disease severity, hospitalizations, and deaths. More recently, the world has seen a plateau in severe infections, hospitalizations, and deaths which has been largely attributed to the rapid development and deployment of COVID-19 vaccines; see Sunagar et al., Vaccines (Basel). 2023 Apr; 11(4): 849. Published online 2023 Apr 15. doi: 10.3390 / vaccinesl 1040849.

[0120] As one could learn from the Covid- 19 pandemic, a variant of concern denotes a viral variant which seems to pose a greater thread to public health due to enhanced transmissibility or infectivity.

[0121] All viruses, including SARS-CoV-2, change over time. Most changes have little to no impact on the virus’ properties. However, some changes may affect the virus’s properties, such as how easily it spreads, the associated disease severity, or the performance of vaccines, therapeutic medicines, diagnostic tools, or other public health and social measures. WHO, in collaboration with partners, expert networks, national authorities, institutions and researchers have been monitoring and assessing the evolution of SARS-CoV-2 since January 2020. During late 2020, the emergence of variants that posed an increased risk to global public health prompted the characterisation of specific “Variants of Interest” (VOIs) and “Variants of Concern” (VOCs), in order to prioritise global monitoring and research, and ultimately to inform the ongoing response to the COVID-19 pandemic.

[0122] For instance, SARS-CoV-2 variants of concern have been described under https: / / www.ecdc.europa.eu / en / covid-19 / variants-concern, https: / / www.who.int / activities / tracking-SARS-CoV-2-variants or in the Stanford University Coronavirus Antiviral and Resistance Database. The aforementioned WHO website also includes VOC profiles of spike protein amino acid changes.

[0123] If not indicated otherwise, the established nomenclature systems for naming and tracking SARS-CoV-2 genetic lineages by GISAID, Nextstrain and Pango is used herein; see, e.g., WHO website https: / / www.who.int.

[0124] SARS-CoV-2 variants of concern include, for example, SARS-CoV-2 variant alpha (B.1.1.7), SARS-CoV-2 variant beta (B.1.351), SARS-CoV-2 variant gamma (P. l), SARS-CoV-2 variant delta (B.1.617.2), SARS-CoV-2 variant omicron, such as SARS-COV-2 variant omicron BA.l, SARS-COV-2 variant omicron BA.2, SARS-COV-2 variant omicron BA.2.3.20, SARS-COV- 2 variant omicron BA.2.75, SARS-COV-2 variant omicron BA.3, SARS-COV-2 variant omicron BA.4, SARS-COV-2 variant omicron BA.5, BJ1, BA.4.6, or XBD, or the FDA / WHO recommended (2025 / 26) relevant SARS-CoV-2 variants, LP8.1 and NB.1.8.1 (see e.g., Guo C, Yu Y, Liu J, Jian F, Yang S, Song W, Yu L, Shao F, Cao Y. Antigenic and virological characteristics of SARS-CoV-2 variants BA.3.2, XFG, and NB.1.8.1. Lancet Infect Dis. 2025 Jun 5:81473-3099(25)00308-1. doi: 10.1016 / S1473-3099(25)00308-L Epub ahead of print. PMID: 40484018; Liu J, Yu Y, Yang S, Jian F, Song W, Yu L, Shao F, Cao Y. Virological and antigenic characteristics of SARS-CoV-2 variants LF.7.2.1, NP.l, and LP.8.1. Lancet Infect Dis. 2025 Mar;25(3):el28-el30. doi: 10.1016 / 81473-3099(25)00015-5. Epub 2025 Jan 28. PMID: 39889723.

[0125] Most knowledge that was cumulated for vaccine design of SARS-CoV-2 relied on a decade of existing science of SARS-CoV-1. Fortunately, the impact of angiontensin converting enzyme 2 (ACE2) as key entry receptor was soon confirmed to be even more pronounced in SARS- CoV-2 by several investigators (Lan et al., Nature, 2020). In contrast to the entire Spike that is heavily glycosylated (Hoffmann et al.,EMBO J, 2021; Watanabe et al., Science, 2020), one strategy for the virus to hide immune response, the present inventors decided to reduce the immunogen to the most relevant receptor binding domain (RBD).

[0126] Amino acid residues 306 to 527 of GenBank accession number ABF65836.1 contain the RBD of SARS-CoV spike protein.

[0127] The amino acid sequence of the SARS-CoV-2 spike protein is shown e.g. in GenBank accession number QHD43416.1 or UniProt accession number P0DTC2.

[0128] SEQ ID NO. 13 shows the amino acid sequence of the receptor binding domain (RBD) from SARS-CoV-2 spike protein (corresponding to amino acid residues 320 to 541 of the amino acid sequence of the SARS-CoV-2 spike protein). Also amino acid residues 320 to 541 of GenBank accession number QHD43416.1 contain the RBD of SARS-CoV-2 spike protein.

[0129] SEQ ID NO. 14 depicts the amino acid sequence of the receptor binding motif (RBM) from SARS-CoV-2 spike protein (corresponding to amino acid residues 438 to 506 of the amino acid sequence of the SARS-CoV-2 spike protein).

[0130] SEQ ID NO. 78 shows the amino acid sequence of the receptor binding domain (RBD) from the spike protein of SARS-CoV-2 (LP8.1).

[0131] SEQ ID NO. 79 depicts the amino acid sequence of the receptor binding domain (RBD) from the spike protein of SARS-CoV-2 (NB.1.8.1). These latter two sequences are from strains corresponding to the FDA / WHO recommended (2025 / 26) relevant SARS-CoV-2 variants, LP8.1 and NB.1.8.1 among Sarbecoviruses; see also Figures 13 and 14.

[0132] The amino acid sequence of Angiotensin converting enzyme ACE2 is shown in UniProt Q9BYF1.

[0133] Further relevant coronaviruses

[0134] Figures 13 and 14 also show the receptor binding domain (RBD) of the spike protein from other relevant coronavirus strains, such as the receptor-binding domain (RBD) of the spike protein from "seasonal cold" viruses, including Alphacoronaviruses (e.g., hCoV-NL63) and Betacoronavirus lineage B, e.g., HCoV-OC43. Figure 13 additionally provides the sequences of predicted RBD regions from additional coronaviruses, including merbecoviruses (betacoronavirus lineage C, e.g., MERS-CoV) and bat coronaviruses (e.g., NeoCoV, PDF- 2180, HKU5-CoV-2, and HtHKU5-CoV-2 / HtHKU5-CoV-2-441); see also SEQ ID Nos. 69 to 76.

[0135] Receptor binding domain (RBD)

[0136] A “receptor binding domain (RBD)” as used herein is a structural and functional unit in a respiratory viral protein which is capable of binding to a receptor or co-receptor on a viral target cell in a subject, e.g. a mucosal cell or epithelial cell of a human subject. A receptor binding domain (RBD) as referred to herein can comprise the complete receptor binding domain, or fragments of the receptor binding domain, as defined elsewhere herein. A nonlimiting example of a fragment of a receptor binding domain (RBD) as used herein is a receptor binding motif (RBM). The receptor binding domain (RBD) can also be comprised by a respiratory viral protein, for instance, in cases where the exact boundaries of the receptor binding domain (RBD) are not yet known. To provide an example, the RBD of rhinoviral capsid protein is embedded in the amino acid sequence encoded by nucleotides 2305 to 3126 of GenBank accession number EF582385.1; see McIntyre et al., 2013.

[0137] Mucosal surfaces - such as the lining of the gut or the reproductive tract or the nasal mucosa - are the main point of entry for viruses into the body. For instance, the nose is not only the mere entry site but also the main target of SARS-CoV-2. Almost all respiratory viruses interact with epithelial cells, and make use of the normal epithelial signalling and trafficking pathways of the host cell. In addition to protein receptors or co-receptors, carbohydrate chains of proteoglycans and epithelial-membrane glycosphingolipids have emerged as a new class of receptors for viral attachment to the target cell, as explained elsewhere herein.

[0138] Coronaviruses (CoV) use the homotrimeric spike glycoprotein comprising a SI subunit and S2 subunit in each spike monomer on the envelope to bind to their cellular receptors. Such binding triggers a cascade of events that leads to the fusion between cell and viral membranes for cell entry. To give specific examples, Coronavirus SARS-CoV-2 causing Coronavirus disease 2019 (COVID-19) uses the receptor binding domain of its spike glycoprotein to interact with host cell angiotensin converting enzyme 2 (ACE2) sites to initiate a cascade of events that culminate in severe acute respiratory syndrome, in some individuals. Such interaction also mediates viral attachment of, fusion with and entry into the target cell, of SARS-CoV-2; see e.g. Lan et al., Nature, volume 581, pages 215-220 (2020).

[0139] Lan et al. (Nature (2020), volume 581, pages 215-220) have elucidated the interaction between the receptor binding domain of the spike protein of SARS-CoV-2 and ACE2 at a higher resolution, by determining the structure of the SARS-CoV-2 receptor binding domain-ACE2 complex using X-ray crystallography.

[0140] The receptor binding motif (RBM) of the SI subunit in the spike monomer of SARS-CoV-2 has been shown to interact directly with ACE2, by Wrapp et al. (Cryo-EM structure of the 2019- nCoV spike in the prefusion conformation. Science 367, 1260-1263 (2020)).

[0141] Further, it has been found that neuropilin- 1 (NRP-1), a member of a family of signaling proteins, serves as an entry factor and potentiate SARS-CoV-2 infectivity; see e.g. Mayi et al., PLoS Pathog. 2021 Jan 4;17(l):el009153. doi: 10.1371 / journal.ppat.1009153. eCollection 2021 Jan, Cantuti-Castelvetri et al., Science. 2020 Nov 13;370(6518):856-860. doi: 10.1126 / science.abd2985. Epub 2020 Oct 20. PMID: 33082293. So NRP-1 serves as a coreceptor for SARS-CoV-2 entry.

[0142] The amino acid sequence of the SARS-CoV-2 spike protein and its receptor binding domain is also described, e.g., by Lan et al. (Nature. 2020 May;581(7807):215-220. doi: 10.1038 / s41586- 020-2180-5. Epub 2020 Mar 30) or by Wen-Hsiang Chen, Peter J. Hotez & Maria Elena Bottazzi (2020) Potential for developing a SARS-CoV receptor-binding domain (RBD) recombinant protein as a heterologous human vaccine against coronavirus infectious disease (COVID)-19, Human Vaccines &Immunotherapeutics, 16:6, 1239-1242, DOI:

[0143] 10.1080 / 21645515.2020.1740560.

[0144] Specically, the receptor binding domain (RBD) from SARS-CoV-2 spike protein, comprises amino acid residues 320 to 541 of GenBank accession number QHD43416.1. It binds to angiotensin-converting enzyme 2 (ACE2) (UniProt Q9BYF1), on human target cells.

[0145] The receptor binding domain of the SARS-CoV-2 and SARS-CoV (RBD219-N1) share substantial amino acid sequence similarity (75% identity, 83% similarity). As SARS-CoV-2, SARS-CoV uses the human receptor angiotensin converting enzyme 2 (ACE2) for cell entry. Previous cryo-electron microscopy studies of the SARS-CoV spike protein and its interaction with the cell receptor ACE2 have shown that receptor binding induces the dissociation of the SI with ACE2, prompting the S2 to transit from a metastable pre-fusion to a more-stable postfusion state that is essential for membrane fusion. Therefore, binding to the ACE2 receptor is a critical initial step for SARS-CoV to enter into target cells. A SARS-CoV receptor-binding domain (RBD) recombinant protein was developed and manufactured under current good manufacturing practices (cGMP), in 2016. The bulk drug substance has been stored frozen (-70°C to -80°C) and is under stability testing since its manufacturing, so far remaining stable. The protein known as RBD219-N1 was expressed in yeast, and purified to optimize expression yield, antigenicity, and functionality, as well as immunogenicity in mice when formulated on alum. Moreover, alum-adj uvanted RBD219-N1 induced protective immunity against homologous virus challenge with SARS-CoV (MAI 5 lethal strain), with minimal immunopathology, lessening potential safety concerns; see, e.g., Chen et al., Optimization of the production process and characterization ofthe yeast-expressed SARS-CoV Recombinant Receptor-BindingDomain (RBD219-N1), a SARS vaccine candidate. J Pharm Sci.2017; 106(8): 1961-70. doi: 10.1016 / j.xphs.2017.04.037; Jiang S, et al., Roadmap to developing a recombinantcoronavirus S protein receptor-binding domain vaccine for severeacute respiratory syndrome. Expert Rev Vaccines. 2012; 11(12): 1405— 13. doi: 10.1586 / erv.12.126; Chen WH, et al., Yeast-expressed recombinant protein of the receptorbinding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate. Hum Vaccin Immunother. 2014;10(3):648-58. doi:10.4161 / hv.27464.

[0146] The receptor binding domain (RBD) from SARS-CoV (or SARS-CoV- 1) spike protein comprises or consists of amino acid residues 306 to 527 of GenBank accession number ABF65836.1, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acid residues 306 to 527 of GenBank accession number ABF65836.1. Said receptor binding domain (RBD) from SARS-CoV (or SARS-CoV-1) spike protein binds to angiotensin-converting enzyme 2 (ACE2) (UniProt Q9BYF1), on human target cells.

[0147] The receptor binding domain (RBD) from SARS-CoV-2 spike protein comprises or consists of amino acid residues 320 to 541 of GenBank accession number QHD43416.1, or SEQ ID NO. 13, or SEQ ID NO. 14, or SEQ ID NO. 78, or SEQ ID NO. 79, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acid residues 320 to 541 of GenBank accession number QHD43416.1, or to SEQ ID NO. 13, or SEQ ID NO. 14, or SEQ ID NO. 78, or SEQ ID NO. 79. Said receptor binding domain (RBD) from SARS-CoV-2 spike protein binds to angiotensinconverting enzyme 2 (ACE2) (UniProt Q9BYF1), on human target cells.

[0148] The receptor binding domain (RBD) from the spike protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) comprises or consists of amino acid residues 367 to 606 of UniProt accession number K9N5Q8, or SEQ ID NO. 69, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acid residues 367 to 606 of UniProt accession number K9N5Q8, or to SEQ ID NO. 69. Said receptor binding domain (RBD) from the spike protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) binds to the receptor Dipeptidyl peptidase IV (DPP4), or CD26 (UniProt P27487), on human target cells.

[0149] The receptor binding domain (RBD) from hCoV-NL63 spike protein comprises or consists of SEQ ID NO. 70, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 70. Said receptor binding domain (RBD) from hCoV-NL63 spike protein binds to angiotensinconverting enzyme 2 (ACE2) (UniProt Q9BYF1), on human target cells.

[0150] The receptor binding domain (RBD) from HCoV-OC43 spike protein comprises or consists of SEQ ID NO. 75 or 76, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least

[0151] 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

[0152] 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

[0153] 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO.

[0154] 75 or 76. Said receptor binding domain (RBD) from HCoV-OC43 spike protein binds to sialoside receptor (Hulswit RIG, Lang Y, Bakkers MJG, Li W, Li Z, Schouten A, Ophorst B, van Kuppeveld FJM, Boons GJ, Bosch BJ, Huizinga EG, de Groot RJ. Human coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-binding site in spike protein domain A. Proc Natl Acad Sci U S A. 2019 Feb 12; 116(7):2681-2690; Tortorici MA, Walls AC, Lang Y, Wang C, Li Z, Koerhuis D, Boons GJ, Bosch BJ, Rey FA, de Groot RJ, Veesler D. Structural basis for human coronavirus attachment to sialic acid receptors. Nat Struct Mol Biol. 2019 Jun;26(6):481-489).

[0155] The receptor binding domain (RBD) from NeoCoV spike protein comprises or consists of SEQ ID NO. 71, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least

[0156] 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least

[0157] 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0158] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

[0159] 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 71.

[0160] The receptor binding domain (RBD) from PDF-2180 spike protein comprises or consists of SEQ ID NO. 72, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 72. The receptor binding domain (RBD) from HKU5-CoV-l spike protein comprises or consists of SEQ ID NO. 73, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 73.

[0161] The receptor binding domain (RBD) from HtHKU5-CoV-2 or HtHKU5-CoV-2-441 spike protein comprises or consists of SEQ ID NO. 74, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least

[0162] 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least

[0163] 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

[0164] 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 74.

[0165] The receptor binding domain (RBD) from NeoCoV spike protein, PDF-2180 spike protein, HKU5-CoV-l spike protein, HtHKU5-CoV-2 spike protein or HtHKU5-CoV-2-441 spike protein binds to angiotensin-converting enzyme 2 (ACE2) (UniProt Q9BYF1) or bat orthologues thereof, or to Dipeptidyl peptidase IV (DPP4), or CD26 (UniProt P27487).

[0166] The receptor binding domains (RBD) from Coronaviruses as referred to herein that can be used in the protein oligomer of the invention are also depicted, in Figures 13 and 14.

[0167] The receptor binding domain (RBD) from influenza virus hemagglutinin comprises or consists of amino acid residues 63 to 286 of GenBank accession number ACQ99608, or an amino acid sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 77, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acid residues 63 to 286 of GenBank accession number ACQ99608, or to an amino acid sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 77. Said receptor binding domain (RBD) from influenza virus hemagglutinin binds to sialic acid-containing proteins or sialylated glycoconjugates, on human target cells.

[0168] The receptor binding domains (RBD) from influenza viruses referred to herein that can be used in the protein oligomer of the invention are also depicted, in Figures 12 and 14. The receptor binding domain (RBD) from parainfluenza virus hemagglutinin-neuraminidase (HN) comprises or consists of amino acid residues 54 to 572 of GenBank accession number AY283063, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least

[0169] 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least

[0170] 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0171] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

[0172] 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acid residues 54 to 572 of GenBank accession number AY283063. Said receptor binding domain (RBD) from parainfluenza virus hemagglutinin-neuraminidase (HN) binds to sialic acid-linked receptors or sialylated glycoconjugates, on human target cells.

[0173] The receptor binding domain (RBD) from rhinovirus capsid protein VP1 is encoded by nucleotides 2305 to 3126 of GenBank accession number EF582385.1, or by a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

[0174] 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

[0175] 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0176] 98%, or at least 99% sequence identity to nucleotides 2305 to 3126 of GenBank accession number EF582385.1, encoding the receptor binding domain (RBD) from rhinovirus capsid protein VP1. Said receptor binding domain (RBD) from rhino virus capsid protein VP1 binds to intercellular adhesion molecule 1 (ICAM-1) receptor (UniProt P05362), very low density lipoprotein (VLDL)-receptor (VLDL-R) (UniProt Q9Y679) or Cadherin Related Family Member 3 (CDHR3) (UniProt Q6ZTQ4), on human target cells.

[0177] The receptor binding domain (RBD) from human adenovirus fiber protein comprises or consists of amino acid residues 386-581 of UniProt accession number Pl 1818, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

[0178] 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

[0179] 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0180] 98%, or at least 99% sequence identity to amino acid residues 386-581 of UniProt accession number Pl 1818. Said receptor binding domain (RBD) from human adenovirus fiber protein binds to coxsackie and adenovirus receptor (CAR) (UniProt P78310), membrane cofactor protein (MCP) or CD46 (UniProt Pl 5529), GDI a glycan, polysialic acid, or desmogl ein-2 (DSG-2) (UniProt Q 14126), on human target cells.

[0181] The receptor binding domain (RBD) from respiratory syncytial virus glycoprotein G comprises or consists of amino acid residues 64-298 of UniProt accession number P03423, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acid residues 64-298 of UniProt accession number P03423. Said receptor binding domain (RBD) from respiratory syncytial virus glycoprotein G binds to CX3C motif chemokine receptor 1 (CX3CR1) (UniProt P49238), heparan sulfate proteoglycans (HSPG), or Nucleolin (UniProt P19338), on human target cells.

[0182] The receptor binding domain (RBD) from human metapneumovirus fusion (F) protein comprises or consists of amino acid residues 66 to 87 contained within the F2 fragment of prefusion F protein (LIKTELDLTKSALRELRTVSAD) (SEQ ID NO. 56), or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

[0183] 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

[0184] 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0185] 98%, or at least 99% sequence identity to SEQ ID NO. 56. Said receptor binding domain (RBD) from human metapneumovirus fusion (F) protein binds to heparan sulfate, glucosaminoglycan, or integrin a5pi (UniProt P08648; P05556) (hypothesized), on human target cells.

[0186] The receptor binding domain (RBD) from parvovirus B19 VP1 unique region comprises or consists of amino acid residues 5 to 68 of UniProt accession number Q9JGS0, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

[0187] 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

[0188] 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0189] 98%, or at least 99% sequence identity to amino acid residues 5 to 68 of UniProt accession number Q9JGS0. Said receptor binding domain (RBD) from parvovirus B 19 VP1 unique region binds to glycosphingolipid (GSL) globoside (Gb4), or tyrosine protein kinase receptor UFO (AXL) (UniProt P30530), on human target cells.

[0190] Sequence identity between, e.g., two amino acid sequences can be calculated by determining the number of identical amino acids between said sequences wherein the sequences are aligned so that the highest order match is obtained. It can be calculated using published techniques or methods codified in computer programs such as, for example, BLASTP or FASTA (Altschul 1990, J Mol Biol 215, 403). The percent identity values are, in one aspect, calculated over the entire amino acid sequence or over a sequence stretch of at least 50% of the query sequence. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (Higgins 1989, CABIOS 5, 151) or the programs Gap and BestFit (Needleman 1970, J Mol Biol 48; 443; Smith 1981, Adv Appl Math 2, 482), which are part of the GCG software packet (Genetics Computer Group 1991, 575 Science Drive, Madison, Wisconsin, USA 53711), may be used. The sequence identity values recited above in percent (%) are to be determined, in another aspect of the invention, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. Preferably, the sequence identity is calculated over the entire length of the RBD as referred to herein.

[0191] Encompassed are also fragments of the receptor binding domains (RBDs) as used herein. A fragment of the receptor binding domain (RBD) as used herein comprises at least five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120 or 130 amino acid residues of the receptor binding domain, and is still capable of binding to the respiratory viral receptor, on the target cell of a subject, such as a mucosal cell or epithelial cell, thereby preventing the binding of the respiratory virus to said target cell and / or mediating the uptake of the protein oligomer of the invention into said target cell of the subject.

[0192] One example for a fragment of the receptor binding domain (RBD) is the receptor binding motif (RBM) from SARS-CoV-2 spike protein. The amino acid sequence of said RBM is depicted in SEQ ID NO. 14. As appreciated by the skilled person, the receptor binding domain (RBD) or receptor binding motif (RBM) as used herein encompasses also the use of the receptor binding domains or receptor binding motifs (RBM) of the spike protein of variants of interest (VOI) or viral variants of concern (VOCs) of SARS-CoV-2.

[0193] The receptor binding domain or receptor binding motif referred to herein can be identified by any method known in the art based on the interaction formed between the respiratory viral protein(s) used to bind to the target cell and the target cell. Methods for identification of receptor binding domains in respiratory viral proteins include, for instance, yeast two-hybrid system, mutational analysis, protein microarrays, X-ray crystallography, flow cytometic analysis, mass spectrometry (MS), or MS and limited proteolysis (LP) of the receptor binding domain-receptor complex; see Mertinkova et al., Scientific Reports volume 10, Article number: 1163 (2020); Du et al., J Virol. 2013 Sep;87(17):9939-42. doi: 10.1128 / JVI.01048-13. Epub 2013 Jul 3; Qian et al., J Virol. 2015 Sep 1; 89(17): 8816-8827. Published online 2015 Jun 17. doi: 10.1128 / JVI.03737-14; Tai et al., Cellular & Molecular Immunology volume 17, pages613- 620 (2020).

[0194] Further encompassed are sequence variants of the receptor binding domains (RBDs) as used herein. A sequence variant of a receptor binding domain as used herein differs from the specific amino acid sequence or a specific nucleic acid sequence as specified before by one, two, three, four, five, six, seven, eight, nine, ten, or even more nucleotide or amino acid substitutions, additions or deletions, or combinations thereof. To provide a non-limiting example, a sequence variant of a receptor binding domain from the spike protein of the SARS-COV-2 variant omicron BA.5 can contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or even more amino acid substitutions, in comparison to the amino acid sequence of the native or non-modified receptor binding domain from the spike protein of the SARS-COV-2 variant omicron BA.5.

[0195] A sequence variant of the receptor binding domain or receptor binding motif as used herein is preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the specific nucleic acid sequence or amino acid sequence of the native or non-modified receptor binding domain or native or non-modified receptor binding motif, preferably over the entire length.

[0196] Methods for producing and identifying functional variants of receptor binding domains or receptor binding motifs of respiratory viral proteins are well described in the literature; see, e.g. Lan et al., Nature volume 581, pages 215-220 (2020); Sambrook et al., Molecular cloning: a laboratory manual / Sambrook, Joseph; Russell, David W. — . 3rd ed. — New York: Cold Spring Harbor Laboratory, 2001. Ausubel et al., Current Protocols in Molecular Biology.

[0197] Another example for a variant of a receptor binding domain or receptor binding motif as used herein is a peptidomimetic of the receptor binding domain or receptor binding motif. As known in the art, peptidomimetics are compounds whose essential elements (pharmacophore) mimic a natural peptide or protein in 3D space and which retain the ability to interact with the biological target or target cell in a subject and produce the same biological effect; see, e.g., the review by Vagner et al. 2008, Current Opinion in Chemical Biology 12, Pages 292-296. Peptidomimetics are designed to circumvent some of the problems associated with a natural peptide, e.g., stability against proteolysis (duration of biological activity) and poor bioavailability. Certain other properties, such as selectivity and / or specificity for the biological target or target cell in a subject as referred to herein, or potency of the biological activity often can be substantially improved.

[0198] The variants of a receptor binding domain or receptor binding motif as used herein can also be synthetic long peptides; see, e.g., Melief, C., van der Burg, S. Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines. Nat Rev Cancer 8, 351-360 (2008). https: / / doi.org / 10.1038 / nrc2373.

[0199] The variants such as sequence variants or peptidomimetics of the receptor binding domain or receptor binding motif as used herein are still capable of binding to the receptor on the respiratory viral target cell of a subject, such as a mucosal cell or epithelial cell, thereby preventing the binding of the virus to said target cell of the subject and / or mediating the uptake of the protein oligomer of the invention into said target cell of the subject. Preferably, said variants have better binding affinities to the receptor in the viral target cell of the subject than the native or non-modified receptor binding domain or native or non-modified receptor binding motif, and / or mediate a better, e.g. faster, uptake of the protein oligomer of the invention into the target cell of the subject.

[0200] Monomer

[0201] The monomer, such as the at least first or second monomer included in the protein oligomer of the invention, comprises a structure of the generic Formula:

[0202] N-terminus [first RBD] (Ig Fc) [second RBD] C-terminus

[0203] The receptor binding domain (RBD) and the immunoglobulin Fc (Ig Fc) are defined elsewhere herein. The receptor binding domain (RBD) can be associated directly, with the immunoglobulin Fc (Ig Fc), i.e. without intervening moiety, in the monomer of the protein oligomer of the invention. A direct association can mean, for example, that the RBD is covalently bound to the Ig Fc. For instance, the first and second RBD can be associated directly, with the Ig Fc.

[0204] The receptor binding domain (RBD) can be fused to the immunoglobulin Fc (Ig Fc), via a linker as defined herein. For instance, the first and / or second receptor binding domain (RBD) can be fused to the immunoglobulin Fc (Ig Fc), via a linker. It is envisaged by the invention that the first RBD can be fused to the Ig Fc, via a linker, and the second RBD is associated directly with the Ig Fc. Or the second RBD can be fused to the immunoglobulin Ig, via a linker, and the first RBD is associated directly with the Ig Fc. Or the second RBD can be fused to the immunoglobulin Ig, via a linker, and the first RBD can be fused to the immunoglobulin Ig, via a linker, in the monomer of the protein oligomer of the invention.

[0205] The “linker” as used herein can be, for instance, a variable linker amino acid sequence.

[0206] The linker can be an independently selected variable linker amino acid sequence which means that the linker linking the first RBD to the Ig Fc can be different from the linker linking the second RBD to the Ig Fc. It is, however, envisaged by the invention that the linker linking the first RBD to the Ig Fc can be the same linker as the linker linking the second RBD to the Ig Fc. For example, a linker in accordance with the invention may be of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or even more amino acid residues long. Linkers are often composed of flexible amino acid residues, for example, but not limited to glycine and serine so that the adjacent protein domains are free to move relative to one another. The design of a linker that enables proper folding of the various domains of a protein is well described in the art. The linker as used herein is preferably a flexible linker and can be used to link together, e.g., the Ig Fc to RBD, or the Ig Fc to an oligomerization domain, or the RBD to an oligomerization domain, as defined elsewhere herein. The linker can advantageously have reduced sensitivity to protease cleavage, especially for in vivo applications of the protein oligomer of the invention as a vaccine as described elsewhere herein. For example, it is known that, e.g., the linker (GGGGS)l (SEQ ID NO. 33) or (GGGGS)2 (SEQ ID NO. 49) or GGGGGSGGGGS (SEQ ID NO. 54), has reduced sensitivity to protease cleavage, in comparison to (GGGGS)s (SEQ ID NO. 50). SEQ ID NOs. 17, 18, 34, 52 or 53 show further appropriate linker sequences. The linker can also be an Fc hinge region-derived linker; see, e.g., US patent 6,165,476.

[0207] Property, design and functionality of suitable linkers are well known in the art; see, e.g., Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. For instance, Table 3 and references cited therein of this publication show examples of linkers and their functionalities.

[0208] The receptor binding domain (RBD) can also be fused to the immunoglobulin Fc (Ig Fc), via an oligomerization domain as defined herein. For instance, the first RBD can be fused to the Ig Fc, via an oligomerization domain, and the second RBD is associated directly with the Ig Fc. Or the second RBD can be fused to the immunoglobulin Ig, via an oligomerization domain, and the first RBD is associated directly with the Ig Fc. Or the second RBD can be fused to the immunoglobulin Ig, via an oligomerization domain, and the first RBD can be fused to the immunoglobulin Ig, via an oligomerization domain, in the monomer of the protein oligomer of the invention.

[0209] The “oligomerization domain” as used herein can be, for instance, the non-triple helical trimerization domain of human collagen 18, the C-terminal oligomerization domain of human C4b-binding protein, coiled coils, oligomeric mini-proteins, short peptides with discrete protein-like structures, trimerization domain of the bacteriophage T4 fibritin (foldon), TNFalpha trimerization domain, zinc finger or p53 tetramerization domain; see, e.g., Ali and Imperial! 2005, Bioorganic and Medicinal Chemistry 13, 5013. The skilled person will acknowledge that the use of human oligomerization domains in the monomer of the protein oligomer of the invention does not elicit off-target immunogenicity, after administration to a human subject, in contrast to non-human oligomerization domains such as the T4 bacteriophage T4 fibritin (foldon). In fact, the use of foldon might provide one plausible explanation for the enhanced reactogenecity vs. the SARS-CoV-2 spike protein sequence and discontinuation of the top candidate vaccine of BioNTech; see, e.g., Walsh et al., N Engl J Med 2020; 383:2439- 2450; DOI: 10.1056 / NEJMoa2027906. Accordingly, a non-human oligomerization domain can preferably be immunosilenced or de-immunogenized, for human immunogenization, in the monomer of the protein oligomer of the invention, by methods known in the art. For instance, de-immunization may be achieved by unspecific shielding approaches, which include PEGylation, fusion to polypeptides (e.g., XTEN or PAS), reductive methylation, glycosylation, and polysialylation. Alternatively, the identification of epitopes for T cells or B cells and their subsequent deletion through site-directed mutagenesis represent promising deimmunization strategies and can be accomplished through either experimental or computational approaches, see, e.g., Zinsli et al., Computational and Structural Biotechnology Journal, Volume 19, 2021, Pages 315-329; Sliepen et al. J Biol Chem. 2015 Mar 20; 290(12): 7436-7442. Published online 2015 Jan 29. doi: 10.1074 / jbc.Ml 14.620534.

[0210] As appreciated by the skilled person, combinations of linker and oligomerization domains, as defined herein, can be used to fuse the receptor binding domain (RBD) to the immunoglobulin Fc (Ig Fc). Accordingly, the first and / or second receptor binding domain (RBD) can be fused to the immunoglobulin Fc (Ig Fc), via an oligomerization domain and a linker, via a linker and an oligomerization domain, or via a linker, an oligomerization domain, and a linker, as defined herein.

[0211] The receptor binding domain (RBD) can be fused to an adjuvant as specified herein, in addition to being bound to an Ig Fc, a linker, an oligomerization domain, or combinations thereof.

[0212] For instance, the first receptor binding domain (RBD) or the second receptor binding domain (RBD), or both, can be linked enzymatically or chemically to an adjuvant, in the monomer of the protein oligomer of the invention. If the first receptor binding domain (RBD) and the second receptor binding domain (RBD) are linked to an adjuvant, the adjuvant can be the same or can be different.

[0213] An adjuvant as used herein can be, for instance, oil-in-water emulsion of squalene, mRNA, ds mRNA, one or more peptides for T cell response, a sting agonist (bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP or cdGMP)), dsDNA, ssRNA, GM-CSF, CSF, TNF alpha, interferons, CpG oligonucleotides, Toll-like receptor (TLR) agonists, or combinations thereof. Said adjuvants are elucidated elsewhere herein.

[0214] As also appreciated by the skilled person, the adjuvant can also be conjugated, e.g., to the linker, the oligomerization domain or Ig Fc, in the monomer of the protein oligomer of the invention.

[0215] The linker can be a non-cleavable linker or can be a cleavable linker, i.e. the linker can comprise one or more protease cleavage sites to allow controlled linker cleavage, e.g., at a specific target site or cell compartment; see, e.g., Bargh et al., Chem. Soc. Rev., 2019,48, 4361-4374; Bargh et al., Chem. Commun, 2021, 57, 3457-3460; Bargh et al., Chem. Sci., 2020, 11, 2375-2380. Enzyme-cleavable linkers are also commercially available, e.g. from BroadPharm, WuXi Biologies, and other suppliers. To give an example for an enzyme-cleavable linker, one protease sensitivity strategy described in the art utilizes predominant proteases found in lysosomes of tumor cells to recognize and cleave specific peptide sequences in the linker. For instance, the valine-citrulline (VC) dipeptide is known as an intracellular cleavage mechanism by cathepsin B.

[0216] Cleavage of the cleavable linker comprised by the monomer of the protein oligomer of the invention by the respective protease allows for, e.g., the release of adjuvant of the protein oligomer of the invention into a cell compartment, such as the cytosol. It is also within the scope of the present invention that combinations of different adjuvants are used. For example, one adjuvant (e.g. a TLR agonist) can be conjugated to a non-cleavable linker, and another adjuvant (e.g. a sting agonist) can be conjugated to an enzyme-cleavable linker, in the monomer of the protein oligomer of the invention. In such example, the TLR agonist conjugated to the non-cleavable linker is then active in the cellular endosome of the target cell, whereas the sting agonist conjugated to an enzyme-cleavable linker is released to and active in the cytosol of the target cell.

[0217] Advancing new vaccines such as mucosal vaccines and improving existing vaccines requires innovative adjuvant approaches and delivery strategies. In addition, many adjuvants that are effective by injection are not optimal for mucosal delivery. These problems can be circumvented by joining the receptor binding domain(s), Ig Fc, linker, and / or oligomerization domain to an adjuvant, in the monomer of the protein oligomer of the invention.

[0218] It is further envisaged by the invention that the first receptor binding domain (RBD) can be fused at the N-terminus to one, two, three, four, five, six, seven, eight, nine, ten, or even more further receptor binding domains (RBDs) as defined herein.

[0219] Similarly, the second receptor binding domain (RBD) can be fused at the C-terminus to one, two, three, four, five, six, seven, eight, nine, ten, or even more further receptor binding domains (RBDs). This is explained elsewhere herein, in more detail.

[0220] More specifically, the monomer, such as the at least first or second monomer included in the protein oligomer of the invention, comprises a structure of Formula I: N-terminus [RBD-Z-]n(Ig Fc, preferably human Ig Fc) [-Z-RBD]nC-terminus

[0221] (Formula I)

[0222] The receptor binding domain (RBD) and the immunoglobulin Fc (Ig Fc) are defined elsewhere herein.

[0223] As further appreciated by the skilled person, the monomer, such as the at least first and second monomer, comprises a human Ig Fc, in the protein oligomer of the invention in order to avoid an unwanted immune response against the Ig Fc, if the protein oligomer is used for vaccination of human subjects.

[0224] [RBD-Z-] moiety (at the N-terminus)

[0225] Ig Fc can be flanked N-terminally by at least one [RBD-Z-] moiety, but it can be flanked N- terminally also by two, three, four, five, six, seven, eight, nine, or ten, or even more [RBD-Z-] moieties, in the monomer of the protein oligomer of the invention.

[0226] The [RBD-Z-] moiety can be structured as follows.

[0227] Z can comprise an optional linker, i.e. the [RBD-Z-] moiety may or may not comprise a linker, in the monomer of the protein oligomer of the invention.

[0228] The linker is defined elsewhere herein.

[0229] Z can comprise an optional oligomerization domain, i.e. the [RBD-Z-] moiety may or may not comprise an oligomerization domain, in the monomer of the protein oligomer of the invention. The oligomerization domain can be used, e.g., to form oligomers of [RBD-Z-] moieties, such as [RBD-Z-] moiety dimers, [RBD-Z-] moiety trimers, [RBD-Z-] moiety tetramers, and so forth, in the monomer of the protein oligomers of the invention.

[0230] The oligomerization domain is defined elsewhere herein.

[0231] The rationale for including oligomerization domains into the monomer of the protein oligomer of the invention is to increase the number of receptor binding domains, thereby improving binding affinity and / or avidity of the protein oligomer of the invention to the corresponding receptor on the target cell of a subject, such as a human subject.

[0232] In view of the above, the skilled person will appreciate that the [RBD-Z-] moiety can comprise or consist of RBD only. RBD corresponds to the receptor binding domain or fragment thereof or receptor binding motif, as defined herein.

[0233] Z can comprise also a linker and an oligomerization domain, or an oligomerization domain and a linker, or a linker and an oligomerization domain and a linker, in addition to RBD, in the [RBD-Z-] moiety. Z can comprise an adjuvant, as defined herein, instead or in addition to a linker and / or an oligomerization. An [RBD-Z-] moiety can, for example, comprise or consist of RBD, RBD-adjuvant, RBD- linker, RBD-oligomerization domain, RBD-linker-oligomerization domain, or RBD-linker- oligomerization domain, or RBD-linker-oligomerization domain-linker, in the monomer of the protein oligomer of the invention.

[0234] [-Z-RBD] moiety (at the C-terminus)

[0235] Ig Fc is flanked C-terminally by at least one [-Z-RBD] moiety, but it can be flanked C- terminally also by two, three, four, five, six, seven, eight, nine, or ten, or even more [-Z-RBD] moieties, in the monomer of the protein oligomer of the invention.

[0236] The [-Z-RBD] moiety can be as follows.

[0237] Z can comprise an optional linker, i.e. the [-Z-RBD] moiety in the monomer of the protein oligomer of the invention may or may not comprise a linker.

[0238] The linker is defined elsewhere herein.

[0239] Z can comprise an optional oligomerization domain, i.e. the [-Z-RBD] moiety in the monomer of the protein oligomer of the invention may or may not comprise an oligomerization domain. The oligomerization domain can be used, e.g., to form oligomers of [-Z-RBD] moieties, such as [-Z-RBD] moiety dimers, [-Z-RBD] moiety trimers, [-Z-RBD] moiety tetramers, and so forth, in the monomer of the protein oligomers of the invention.

[0240] The oligomerization domain is specified elsewhere herein.

[0241] In view of the above, the skilled person will appreciate that the [-Z-RBD] moiety can comprise or consist of RBD only.

[0242] Z can comprise also a linker and an oligomerization domain, or an oligomerization domain and a linker, or a linker and an oligomerization domain and a linker, in addition to RBD, in the [-Z- RBD] moiety. Z can comprise an adjuvant, as defined herein, instead or in addition to a linker and / or an oligomerization.

[0243] An [-Z-RBD] moiety can, for example, comprise or consist of RBD, RBD-adjuvant, RBD- linker, RBD-oligomerization domain, RBD-linker-oligomerization domain, or RBD-linker- oligomerization domain, or RBD-linker-oligomerization domain-linker, in the monomer of the protein oligomer of the invention.

[0244] [RBD-Z-] moiety (at the N-terminus) and [-Z-RBD] moiety (at the C-terminus)

[0245] Ig Fc can be flanked N-terminally by one [RBD-Z-] moiety, and C-terminally by one, two, three, four, five, six, seven, eight, nine, ten, or even more [-Z-RBD] moieties, in the monomer of the protein oligomer of the invention.

[0246] Ig Fc can be flanked C-terminally by one [-Z-RBD] moiety, and N-terminally by one, two, three, four, five, six, seven, eight, nine, ten, or even more [RBD-Z-] moieties, in the monomer of the protein oligomer of the invention. To provide a further example, Ig Fc can be flanked N-terminally by two [RBD-Z-] moieties, and C-terminally by one, two, three, four, five, six, seven, eight, nine, ten, or even more [-Z- RBD] moieties in the monomer of the protein oligomer of the invention.

[0247] Ig Fc can be flanked C-terminally by two [-Z-RBD] moieties, and N-terminally by one, two, three, four, five, six, seven, eight, nine, ten, or even more [RBD-Z-] moieties, in the monomer of the protein oligomer of the invention.

[0248] In a still further example, Ig Fc can be flanked N-terminally by three, four, five, six, or even more [RBD-Z-] moieties, and C-terminally by one, two, three, four, five, six, seven, eight, nine, ten, or even more [-Z-RBD] moieties.

[0249] Ig Fc can be flanked C-terminally by three, four, five, six, or even more [-Z-RBD] moieties, and N-terminally by one, two, three, four, five, six, seven, eight, nine, ten, or even more [RBD- Z-] moieties, in the monomer of the protein oligomer of the invention.

[0250] These are non-limiting examples. Encompassed by the invention are any possible combinations comprised by Formula I, as defined herein.

[0251] “n”

[0252] “n” is a value of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and indicates the number of [RBD-Z-] or [-Z- RBD] moieties, contained in the monomer of the protein oligomer of the invention. is a covalent bond, in the monomer of the protein oligomer of the invention.

[0253] Structure of the monomer

[0254] In the protein oligomer of the invention, the monomer can comprise or consist of a structure selected from:

[0255] [RBD-]n(Ig Fc) [-RBD]n(Formula I-a)

[0256] [RBD-linker-]n(Ig Fc) [-RBD]n(Formula I-b)

[0257] [RBD-]n(Ig Fc) [-linker-RBD]n(Formula I-c)

[0258] [RBD-linker-]n(Ig Fc) [-linker-RBD]n(Formula I-d)

[0259] [RBD-oligomerization domain-]n(Ig Fc) [-RBD]n(Formula I-e)

[0260] [RBD-]n(Ig Fc) [-oligomerization domain-RBD]n(Formula I-f)

[0261] [RBD-oligomerization domain-]n(Ig Fc) [-oligomerization domain-RBD]n(Formula I-g)

[0262] [RBD-oligomerization domain-linker-]n(Ig Fc) [-RBD]n(Formula I-h)

[0263] [RBD-]n(Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-i)

[0264] [RBD-oligomerization domain-linker-]n(Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-j) [RBD-linker-oligomerization domain-]n(Ig Fc) [-oligomerization domain-RBD]n(Formula I-k)

[0265] [RBD-oligomerization domain-]n(Ig Fc) [-oligomerization domain-linker-RBD]n(Formula 1-1)

[0266] [RBD-linker-oligomerization domain-]n(Ig Fc) [-oligomerization domain-linker-RBD]n(Formula I-m)

[0267] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [-RBD]n(Formula I-n)

[0268] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [linker-RBD]n(Formula I-o)

[0269] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [-oligomerization domain-RBD]n(Formula I-p)

[0270] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-q)

[0271] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [-oligomerization domain-linker-RBD]n(Formula I-r)

[0272] [RBD-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0273] (Formula I-s)

[0274] [RBD-linker-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0275] (Formula I-t)

[0276] [RBD-oligomerization domain-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0277] (Formula I-u)

[0278] [RBD-linker- oligomerization domain-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0279] (Formula I-v)

[0280] [RBD-oligomerization domain-linker-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-w)

[0281] [RBD-linker-oligomerization domain-linker-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-x).

[0282] The above formulas are indicated from N-terminus to C-terminus.

[0283] Ig Fc as defined herein is preferably a human Ig Fc.

[0284] Particularly preferred is the core structure with the formula, from the N-terminus to the C- terminus:

[0285] [RBD-linker]n(human Ig Fc) [linker-RBD]n(Formula I-d) wherein n is 1, as exemplified by the monomers contained, e.g., in BioVacOOl and BioVac002. Even more preferably, the protein oligomer of the invention comprises or consists of the amino acid sequence shown in any one of SEQ ID Nos. 1-12, 35-43, 55, or 80-111, or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

[0286] 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

[0287] 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0288] 98%, or at least 99% sequence identity to any one of SEQ ID Nos. 1-12, 35-43, 55 or 80-111, or combinations thereof.

[0289] RBD within one monomer

[0290] The monomer, such as the at least first or second monomer included in the protein oligomer of the invention comprises at least two receptor binding domains (RBDs), as defined herein. Since the protein oligomer of the invention comprises at least a first and a second monomer, said protein oligomer comprises at least four receptor binding domains (RBDs), in total, but can comprise more than four receptor binding domains (RBDs), such as five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fiveteen, sixteen, seventeen, eighteen, nineteen, twenty, or even more receptor binding domains (RBDs).

[0291] The receptor binding domain (RBD) can be the same or different, in the monomer of the protein oligomer of the invention.

[0292] For instance, the receptor binding domain (RBD) can be the same or different, in the at least first monomer of the protein oligomer of the invention. To give a non-limiting example, the at least one first receptor binding domain and the at least one second receptor binding domain can be from SARS-CoV-2 spike protein, such as from SARS-CoV-2 variant alpha (B.l.1.7), in the at least first monomer of the protein oligomer of the invention. Or the at least one first receptor binding domain and the at least one second receptor binding domain can be from the spike protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), or from influenza virus hemagglutinin, or from human adenovirus fiber protein.

[0293] To provide a further example, the at least one first receptor binding domain can be from SARS- CoV-2 spike protein, such as from SARS-CoV-2 variant alpha (B. l.1.7), and the at least one second receptor binding domain can be from influenza virus hemagglutinin, in the at least first monomer of the protein oligomer of the invention.

[0294] The receptor binding domain (RBD) can be the same or different, in the at least second monomer of the protein oligomer of the invention.

[0295] For instance, the at least one first receptor binding domain and the at least one second receptor binding domain can be from SARS-CoV-2 spike protein, such as from SARS-CoV-2 variant delta (B.1.617.2), in the at least second monomer of the protein oligomer of the invention.

[0296] Or the at least one first receptor binding domain can be from SARS-CoV-2 variant delta (B.1.617.2) spike protein, and the at least one second receptor binding domain can be from SARS-COV-2 variant omicron BA.2 spike protein, in the at least second monomer of the protein oligomer of the invention. Or the at least one first receptor binding domain can be from parainfluenza virus hemagglutinin-neuraminidase (HN), and the at least one second receptor binding domain can be from respiratory syncytial virus glycoprotein G.

[0297] It is envisaged by the invention, that the receptor binding domains can be freely combined, in the at least first and at least second monomer of the protein oligomer of the invention. So the receptor binding domain (RBD) can be the same or different, in the at least first monomer and the at least second monomer of the protein oligomer of the invention, as will be exemplified, without limitation, in the following. It is, thus, encompassed by the invention that two, three or four different receptor binding domains as defined herein can be used in the at least first monomer and second monomer of the protein oligomer of the invention.

[0298] The at least one first receptor binding domain and at least one second receptor binding domain can be from SARS-CoV-2 spike protein, such as SARS-CoV-2 variant alpha (B.1.1.7) spike protein, or the at least one first receptor binding domain and at least one second receptor binding domain can be from human metapneumovirus fusion (F) protein, or the at least one first receptor binding domain and at least one second receptor binding domain can be from parainfluenza virus hemagglutinin-neuraminidase (HN), in the at least first monomer and second monomer of the protein oligomer of the invention. So both monomers contain the same receptor binding domain, in this example.

[0299] Or the at least one first receptor binding domain and at least one second receptor binding domain can be from SARS-CoV-2 variant alpha (B.1.1.7) spike protein, in the at least first monomer of the protein oligomer of the invention, and the at least one first receptor binding domain and at least one second receptor binding domain can be from from SARS-CoV-2 variant delta (B.1.617.2) spike protein, in the at least second monomer of the protein oligomer of the invention.

[0300] Or the at least one first receptor binding domain can be from the spike protein of SARS-CoV- 2 variant omicron BA.2.86, and the at least one second receptor binding domain can be from SARS-COV-2 variant omicron BA.5 spike protein, in the at least first monomer of the protein oligomer of the invention, and the at least one first receptor binding domain and at least one second receptor binding domain can be from from influenza virus hemagglutinin, in the at least second monomer of the protein oligomer of the invention.

[0301] To provide further non-limiting examples, the receptor binding domain can be from influenza virus hemagglutinin at the N-terminus of the at least first monomer of the protein oligomer of the invention, and the receptor binding domain can be from SARS-CoV-2 variant delta (B.1.617.2) spike protein, at the C-terminus of the at least first monomer, or vice versa.

[0302] The receptor binding domain can be from SARS-CoV-2 variant omicron BA.5 spike protein at the N-terminus of the at least second monomer of the protein oligomer of the invention, and the receptor binding domain can be from spike protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), at the C-terminus of the at least second monomer, or vice versa. Or the receptor binding domain is from SARS-CoV2 variant omicron BA.5 spike protein, at the N-terminus of the at least first monomer, and the receptor binding domain is from respiratory syncytial virus glycoprotein G, at the C-terminus of the at least first monomer, or vice versa.

[0303] And the receptor binding domain is from parvovirus B19 VP1 unique region, at the N-terminus of the at least second monomer, and the receptor binding domain is from human metapneumovirus fusion (F) protein, at the C-terminus of the at least second monomer, or vice versa.

[0304] So the receptor binding domains or receptor binding motif of the respiratory viral proteins as referred to herein can be freely combined, within the at least first monomer of the protein oligomer of the invention, within the at least second monomer of the protein oligomer of the invention, or both within the at least first monomer and the at least second monomer of the protein oligomer of the invention.

[0305] Specific non-limiting examples for combinations of receptor binding domains are shown, e.g., in BioVac constructs HD-BioVac 125, 126, 127, 128, and 121-123; see also Figure 15.

[0306] Protein oligomer

[0307] The protein oligomer of the invention comprises at least a first and a second monomer, as defined herein. The term “at least a first and a second monomer” means that the protein oligomer of the invention can comprise more than these two monomers, for example, three, four, five, six, seven, eight, nine, ten, or even more monomers, as specified herein.

[0308] For instance, dimerization can be achieved by the Ig Fc. Oligomerization can be achieved by the usage of an oligomerization domain, as defined herein.

[0309] Accordingly, the protein oligomer can be, e.g., a dimer (comprising the at least first and second monomers), trimer (comprising three monomers, e.g, the at least first and second monomers, and a third monomer), tetramer (comprising four monomers, e.g. the at least first and second monomers, and a third and a fourth monomer), pentamer (comprising five monomers), hexamer (comprising six monomers), heptamer (comprising seven monomers), octamer (comprising eight monomers), nonamer (comprising nine monomers), decamer (comprising ten monomers), and so forth. The protein oligomer can be, e.g., a homo-dimer, homo-trimer, homo-tetramer, homo-pentamer, homo-hexamer, homo-heptamer, or homo-octamer etc., which means that the monomers forming the oligomer are the same, or a hetero-dimer, hetero-trimer, hetero-tetramer, hetero-pentamer, hetero-hexamer, hetero-heptamer, or hetero-octamer etc., which means that the monomers forming the protein oligomer are different from each other. For instance, at least two monomers, or three, or four, or five, or six, or seven, or eight, or nine, or even ten monomers can be different from each other, in a hetero-oligomer.

[0310] So the monomers can be identical, and the monomers preferably comprise the structure of the monomers listed herein, in the protein oligomer of the invention. Alternatively, the monomers can be different from each other, and comprise preferably combinations of the structures of the monomers listed herein, in the protein oligomer of the invention. As will be appreciated by those of skill in the art, each monomer can differ from each other in number or structure of its consituents, for instance, in the number of linkers, oligomerization domains, adjuvants, and / or receptor binding domains, in the protein oligomer of the invention.

[0311] To give a further example, the Ig Fc, linkers, oligomerization domains, adjuvants and / or receptor binding domains can differ in structure, within each monomer, or can differ in structure in one of the monomers of the protein oligomer of the invention, compared to the other monomer(s). For instance, the receptor binding domains can be or be derived from different viruses or different viral variants of concern, in the protein oligomer of the invention, or the human Ig Fc can differ, in the monomers. Or the different linkers, oligomerization domains and / or adjuvants can differ, in the monomers.

[0312] It will be understood by the skilled person that the present invention also encompasses variants of the amino acid sequences of the protein oligomer of the invention, or nucleic acid sequences encoding them, as long as these variant sequences also allow for the formation of a protein oligomer. Protein oligomers formed by said variants have preferably at least one, preferably at least two, more preferably at least three, particularly preferred all of the biological activities, properties and advantages of the protein oligomer of the invention, as defined elsewhere herein. It is preferred that (i) the variants are able to mediate attachment, fusion and entry, of the protein oligomer of the invention into the viral target cell of a subject. The viral target cell of the subject expresses the receptor bound by the receptor binding domain in the protein oligomer of the invention, such as a mucosal cell or an epithelial cell of a subject. Preferably, said variant of the protein oligomer of the invention binds to the receptor, in the picomolar range, as demonstrated for the BioVac constructs, (ii) Furthermore, protein oligomers formed by said variants have enhanced affinity for the neonatal Fc receptor (FcRn) at a pH of about 6.0 to 6.7, more preferably of about 6.17 to 6.65, and even more preferably of about 6.5, and preferably show no or essentially no detectable binding to FcRn at serum pH (about pH 7.35 to 7.40; preferably about pH 7.4). (iii) In addition, the variants of the protein oligomers of the invention are able to elicit a systemic immune response, in addition to local immune responses, in the mucosa of the subject. In light of these properties, said variants can advantageously be used for active immunization against respiratory viral infection, such as Covid- 19 vaccination or vaccination against any other respiratory virus as referred to herein, of a subject, such as a human.

[0313] Encompassed by the invention are also peptidomimetics of the monomers of the protein oligomer of the invention.

[0314] As appreciated by the skilled person, encompassed by the invention are also combinations of different protein oligomers of the invention. For instance, a first protein oligomer of the invention can comprise the receptor binding domains from the spike protein of SARS-CoV-2 such as from variant omicron XBB.1 ,5-like, and a second protein oligomer of the invention can comprise the receptor binding domains of the spike protein from influenza virus hemagglutinin or from parainfluenza virus hemagglutinin-neuraminidase (HN). To give a further example, a first protein oligomer of the invention can comprise the receptor binding domains from rhinovirus capsid protein VP1, and a second protein oligomer of the invention can comprise the receptor binding domains of the spike protein from human metapneumovirus fusion (F) protein or from parvovirus B 19 VP1 unique region.

[0315] Such combinations of different protein oligomers of the invention can be used for immunization of subjects, as described herein.

[0316] Method for producing the protein oligomer of the invention

[0317] The invention also relates to a method for producing the protein oligomer of the invention, comprising (a) culturing a host cell comprising a nucleic acid sequence encoding the protein oligomer of the invention, (b) obtaining from the host cell of step (a) the protein oligomer, and, optionally, (c) purifying and / or storing the protein oligomer.

[0318] It has been found by the present inventors, that the protein oligomers of the invention are stable at 4° C for a long period of time, thereby simplifying logistics, in contrast to, e.g., mRNA-based vaccines of the art which have to be stored at -80° C.

[0319] The invention further relates to recombinant DNA constructs comprising the polynucleotides or nucleic acid sequences encoding the protein oligomer of the invention, or variants, homologues or derivatives thereof, as defined herein. The recombinant DNA constructs of the invention may further comprise additional elements such as promoters, regulatory and control elements, translation, expression and other signals, operably linked to the nucleic acid sequence encoding the protein oligomer of the invention. As used herein, the term “recombinant DNA” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding the protein oligomer of the invention.

[0320] Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system. This typically includes a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of RNA expression, a sequence that encodes a suitable ribosome binding site, RNA splice junctions, sequences that terminate transcription and translation and so forth. Expression vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell.

[0321] Accordingly, the term control and regulatory elements includes promoters, terminators and other expression control elements. For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding any desired protein using the method of this invention.

[0322] A vector may additionally include appropriate restriction sites, antibiotic resistance or other markers for selection of vector-containing cells. Plasmids are the most commonly used form of vector but other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. A „host cell“ as used herein as encompasses preferably eukaryotic host cells known in the art; see, e.g., Sambrook et al., Molecular cloning : a laboratory manual / Sambrook, Joseph; Russell, David W. — . 3rd ed. — New York: Cold Spring Harbor Laboratory, 2001. Ausubel et al., Current Protocols in Molecular Biology.

[0323] An eukaryotic host cell, in an aspect, is a cell which comprises the polynucleotide encoding the monomer(s) of the protein oligomer of the invention wherein said polynucleotide is expressed in the host cell in order to generate the monomer(s) of the protein oligomer of the invention. The polynucleotide may be introduced into a host cell either transiently or stably. In an aspect, the eukaryotic host cell may be a cell of a eukaryotic host cell line which stably expresses the polynucleotide encoding the monomer(s) of the protein oligomer of the invention. In another aspect, the host cell is a eukaryotic host cell which has been transiently transfected with the polynucleotide encoding the monomer(s) of the protein oligomer of the invention and which expresses said polynucleotide. In another aspect, the said cell is a cell which has been genetically engineered to produce the monomer(s) of the protein oligomer of the invention. How such cells can be genetically engineered by molecular biology techniques is well known to the skilled person; see, e.g., Sambrook et al., Molecular cloning: a laboratory manual / Sambrook, Joseph; Russell, David W. — . 3rd ed. — New York: Cold Spring Harbor Laboratory, 2001. Ausubel et al., Current Protocols in Molecular Biology.

[0324] The method for producing the protein oligomer of the invention can be assisted by automation. Specifically, in an aspect, step a) and / or b) and / or c) of this method may be assisted by robotic devices and automated reader systems for mixing compounds and measuring the protein oligomer formation. Suitable systems are known in the art and depend on the type of response to be determined. Moreover, the method may comprise additional steps pertaining to the generation of the protein oligomer of the invention.

[0325] In a preferred embodiment, the protein oligomer of the invention is produced in HEK cells (human) or in Chinese hamster ovary (CHO) cells. Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics. CHO cells are widely used for the manufacture of biologies, with a CHO- based system for vaccine manufacture avoiding some issues associated with primary chick embryo fibroblasts, such as risk of contamination, inherent batch-to-batch variation, lack of cell banking options, and restricted scale-up capacities. In addition, CHO cells allow for manufacture using suspension cultures, which permits rapid scale-up of production in bioreactors; see, e.g., Eldi et al., Molecular Therapy, Vol. 25, Issue 10, P2332-2344, OCTOBER 04, 2017; Fischer et al., The art of CHO cell engineering: a comprehensive retrospect and future perspectives. Biotechnol. Adv. 2015; 33: 1878-1896; Jayapal K.P., Wlashchin K.F., Hu W.S., Yap M.G.S. Recombinant protein therapeutics from CHO cells — 20 years and counting. Chem. Eng. Prog. 2007; 103: 40-47; Genzel Y. Designing cell lines for viral vaccine production: where do we stand? Biotechnol. J. 2015; 10: 728-740; Wurm FM (2004). "Production of recombinant protein therapeutics in cultivated mammalian cells". Nature Biotechnology. 22 (11): 1393— 1398. doi: 10.1038 / nbtl026. PMID 15529164. S2CID 20428452. CHO are the most effective industrial eukaryotic expression systems that are used in protein production. They reach the highest titers (from a commercial perspective) and are long-term genetically stable and their glycosylation pattern especially for antibodies (most commercial mAbs are CHO expressed) are well known in context of human compatibility.

[0326] In another preferred embodiment, the protein oligomer of the invention is produced in HEK293 cells; see, e.g., Thomas and Smart, Journal of Pharmacological and Toxicological Methods, Volume 51, Issue 3, May- June 2005, Pages 187-200.

[0327] It is important to note that there are at least four different possibilities to conjugate up to four different payloads in the protein oligomers of the invention, due to (i) protein engineering (e.g. a Sortase recognition peptide at C-terminus RBD of the protein oligomers of the invention, (ii) a natural glutaminase site (in Ig Fc), (iii) a natural glycosylation site, and (iv) a chemical conjugation site (cysteine conjugation inter / intrachain that can be produced by a standard antibody-drug conjugate technique). The fifth possibility for conjugating a fifth payload could come from the oligomerization domain(s), in the monomer of the protein oligomer of the invention.

[0328] Payloads as used herein means molecules that can be conjugated to the protein oligomers of the inventions. Such molecules can be - for example, and without limitation - potent immune stimulants such as mRNA, proteins, small molecules or peptides. More specifically, such payload can be, for instance, adjuvants, interleukins, TNF, cGAS-STING agonists, mRNA, interferons (IFN), TLR-agonists, GM-CSF, or complimentary MHCI / II class peptides enhancing the immune repertoire.

[0329] Conjugation principles for immune armed protein oligomers of the invention are well known in the art. In contrast to mRNA-based vaccines that may become decapsulated and elicit harm over circulation by e.g. unintended uptake in liver, heart and other organs, the protein oligomers of the invention enable to direct a large repertoire of candidate payloads to cells expressing the putative receptor e.g., ACE2-positive cells for SARS-Cov-2-RBD, immune cells with either RBD specific receptors (BCRs) or uptake / activation over FcR (dendritic and other antigen presenting cells, APCs), the latter can be specifically modulated e.g., by silencing the FcR, if bystander immune activation is not required. Current advanced vaccine adjuvant strategies often elicit a non-specific immune activation e.g., via formation of an immunogenic surface by oil-water emulsions, or mimicking bacterial DNA (CpG). They are mixed with the antigen and injected i.m. / s.c. In contrast, a targeted payload-based immune stimulation may elicit several advantages. For example, more potent immune stimulators may be used, or used in sufficiently high localized doses, to specifically modulate the immune response towards desired polarization state (Ml / Thl vs. M2 / Th2 etc.) that might be crucial for the protective effect of a vaccine, or to induce a sufficiently robust immune response in otherwise immune- compromised / impaired populations e.g., senescent / elderly populations, cancer- or transplant patients etc. Potent immune stimulants like interleukins, interferons or TNF are currently not employed, or could not be used at sufficiently high doses, due to their high reactogenicity and risk for uncontrolled systemic distribution leading to undesired systemic toxicities. In addition, utilization of multiple payloads representing different relevant immune activation pathways may become crucial for effective immunization. The protein oligomers of the invention allow conjugations not only via linkage of the payload to its RBD moieties but also over multiple linkage sites like the intramolecular disulfide bridges (standard chemical conjugation), glycosylation and glutaminase sites naturally existing in its IgG Fc moiety or via engineering of recognition sites for enzymatic conjugation e.g., for sortase or glutaminase. Together with a broad repertoire for conjugation of payloads like cGAS-STING agonists, mRNA, interferons (IFN), TLR-agonists, complimentary MHCI / II class peptides enhancing the immune repertoire that are representatively shown, armed protein oligomers of the invention emerge as a novel and highly versatile vaccination principle. Moreover, these versatile modification options could also be utilized to specifically suppress immune cells targeting RBD or mimetic self-epitopes such as spike protein mimetic self-epitopes (for coronaviruses), to fine tune or ablate potential undesired immune responses, as reported, for instance, in patients with post vaccine or infection (e.g. post / long-covid) syndromes.

[0330] Properties and advantages of the protein oligomer of the invention

[0331] (i) Proof of concept for Bio Vac constructs

[0332] The present inventors have developed a series of constructs of a novel protein oligomer which exhibit superior properties, in comparison to known vaccines. The protein oligomer of the invention has been exemplified by constructs named ”HD-BioVac” or briefly “BioVac” or “BioVac constructs”, such as HD-BioVacOOl (or HD-BioVacl) (SEQ ID NO. 1), HD- Bio Vac002 (SEQ ID NO. 2), HD-BioVac004 (SEQ ID NO. 35), HD-BioVac006 (SEQ ID NO. 36) and HD-BioVac008A (SEQ ID NO. 37) and BioVac008B (SEQ ID NO. 38). Further sequences of BioVac constructs are shown in SEQ ID Nos. 3 to 12 and 39 to 43, and 55. These constructs comprise receptor binding domains from the SARS-CoV-2 spike protein.

[0333] Production and characterization of BioVac constructs 001-012, 035-043, and 055, including their use for active immunization, passive immunization and therapy and their superior properties is described in great detail, in co-pending PCTZEP2025 / 066857 and PCTZEP2025 / 066860, the disclosure content of which is incorporated herein by reference.

[0334] Each of these BioVac constructs contains a „core“ structure. The core structure contains two protein monomers. Each monomer is formed by a centrical human IgGl Fc domain which is flanked both N-terminally and C-terminally by a receptor binding domain (RBD) and / or receptor binding motif (RBM) of the SI subunit of the spike protein of SARS-CoV-2. The receptor binding domain (RBD) of the SI subunit of the spike protein of SARS-CoV-2 is depicted in SEQ ID NO. 13. The receptor binding domain can also comprise amino acid residues 320 to 541 of GenBank accession number QHD43416.1, which is indentical to SEQ ID NO. 13. The receptor binding motif (RBM) of the SI subunit of the spike protein of SARS- CoV-2 is depicted in SEQ ID NO. 14. This receptor binding domain or motif binds specifically to the cell receptor angiotensin converting enzyme 2 (ACE2). The amino acid sequence of the SARS-CoV-2 spike protein is also shown e.g. in GenBank accession number QHD43416.1 or UniProt accession number P0DTC2. The amino acid sequence of ACE2 is shown in SEQ ID NO. 15; see also e.g. Lan et al., Nature volume 581, pages 215-220 (2020). The Fc domain and the receptor binding domain (RBD) are connected via a flexible glycine-serine linker, as disclosed elsewhere herein.

[0335] Specifically, HD-BioVacOOl (SEQ ID NO. 1) contains the receptor binding domain (RBD) of the SI subunit of the spike protein of SARS-CoV-2 (Wuhan). The Fc domain is from human IgGl which includes DHS mutations, mediating enhanced affinity of said Fc domain for the neonatal Fc receptor (FcRn) at mucosal pH of about 6.5, compared to wildtype human IgGl Fc. Further, HD-BioVacOOl (SEQ ID NO. 1) shows preserved binding to ACE2 but no detectable binding to FcRn at physiologic human serum and extracellular pH ~7.4. The DHS mutations correspond to L309D / Q311H / N434S in the amino acid sequence of human IgGl Fc. The amino acid sequence of the human IgGl Fc domain carrying the DHS mutations included in HD-BioVacOOl is shown in SEQ ID NO. 45. The linker connecting the Fc domain and the receptor binding domain (RBD) is a linker with the amino acid sequence GGGGSGGGGS (SEQ ID NO. 49) known to be less protease sensitive than, e.g., the (GGGGS)s linker (SEQ ID NO. 50). HD-BioVacOOl binds via the receptor binding domain (RBD) of the SI subunit of the spike protein of SARS-CoV-2 (SEQ ID NO. 13) to human ACE2 (SEQ ID NO. 15), in the picomolar range. The corresponding amino acid sequence of the HD-BioVacOOl construct is depicted in SEQ ID NO. 1. This tetrameric RBD, IgG FcyR intact (immune stimulating) and FcRn (mucosal uptake) enhanced construct is particularly suitable for active immunization against SARS-CoV-2, especially for prime immunization.

[0336] HD-BioVac002 (SEQ ID NO. 2) corresponds to HD-BioVacOOl, with the exception that it contains additional immune silencing LALAPG mutations, in human IgGl Fc. Said LALAPG mutations are for ablating Fc-Fc gamma receptor-mediated immunological effector functions. The amino acid sequence of the human IgGl Fc domain carrying the DHS mutations and LALAPG mutations included in HD-BioVac002 is shown in SEQ ID NO. 46. The linkers are the same as in HD-BioVacOOl . The corresponding amino acid sequence of HD-BioVac002 is depicted in SEQ ID NO. 2. This construct is particularly suitable for active immunization against SARS-CoV-2, especially for boost immunization or to correct aberrant immune responses to narrow high affinity RBD binding T / B Cell-receptors. Silencing of the FcyR immune effector function in BioVac002 prohibits non-specific uptake of the immunogen by immune cells (T / B and antigen presenting cells, APC) and therefore prevents paracrine stimulation of low affinity non-specific adaptive immune response (anergy) while B- and T- cell receptors with high affinity will bind and expand to produce effective and enduring (memory) immune responses. A particular advantage of BioVac concept in booster setting is further that the tetrameric immunogen could form high order complexes with existing humoral response (antibodies induced after prime immunization), large multivalent protein display constitute the most powerfull immunogen for effective vaccination.

[0337] HD-BioVac004 (SEQ ID NO. 35) contains the receptor binding domain (RBD) of the SI subunit of the spike protein of SARS-CoV-2, including a N501Y substitution (as included, e.g., in the UK or alpha variant). The Fc domain includes the DHS mutations, as described for HD- BioVacOOl. The linkers are the same as in HD-BioVacOOl. HD-BioVac004 binds to ACE2 (SEQ ID NO. 15), via the receptor binding domain (RBD) of the SI subunit of the spike protein of SARS-CoV-2 carrying the N501Y substitution, in the picomolar range. The corresponding amino acid sequence of HD-BioVac004 is depicted in SEQ ID NO. 35. This construct is particularly suitable for all SARS-COV-2 subvariants haboring this pivotal N501Y RBD mutation. The present inventors have developed novel protein oligomers that can advantageously be used for active immunization of subjects, including prime and / or boost immunization of subjects. The novel protein oligomers of the invention are superior in comparison to known vaccines, in different aspects, as set forth elsewhere herein.

[0338] It is also and advantage that the BioVac constructs can be used for prophylactic administration for avoiding SARS-CoV-2 infection, i.e. for passive immunization, in (a) subject(s).

[0339] Furthermore, the BioVac constructs can advantageously be used for treating of symptoms associated with Covid- 19 caused by SARS-CoV-2 infection, in a subject, preferably a human subject.

[0340] The proof of concept has been shown for the BioVac constructs described herein. In replacing the receptor binding domains (RBD) of the SI subunit of SARS-CoV-2 spike protein by receptor binding domains (RBD) of other respiratory viral proteins as referred to herein in the BioVac constructs, the use of these protein oligomers for immunization can be expanded to many other respiratory viruses as well as the treatment of symptoms and diseases caused by said viruses, as described herein.

[0341] To provide a non-limiting example, in replacing the receptor binding domain (RBD) of the SI subunit of SARS-CoV-2 spike protein by the receptor binding domain (RBD) of influenza virus hemagglutinin as defined herein in the BioVac constructs, such protein oligomers of the invention can be used not only for immunization against influzenza viruses, but also for treating symptoms and diseases caused by said influzenza viruses, in influenza virus-infected subjects.

[0342] The universal principle behind BioVac construct design is shown for key respiratory viruses and successful application of BioVac for passive and active immunization against SARS-CoV- 2 is demonstrated. Influenza virsus is here depecited to further demonstrate the universality of BioVac strategy applied to other viruses that are of high relevance for human and veterinary disease burden and frequently contribute to pandemics. Due to high mutational frequency (genetic drift) the sequences for influenza viruses to be used for vaccination are annually updated with current recommendation of the World Health Organization (WHO) Global Influenza Programme being a trivalent vaccine for the northern hemisphere 2024 / 25 influenza season containing an A / Wisconsin / 67 / 2022 (HlNl)pdmO9-like virus; (EPI ISL 15928563), an A / Massachusetts / 18 / 2022 (H3N2)-like virus; (EPI_ISL_13897304) and a B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus. (EPI_ISL_983345). The EPI_ numbers refer to publicly available GISAID accession numbers (https: / / gisaid.org). The receptor-binding domain (RBD) is part of influenza virus hemagglutinin (HA) surface glycoprotein. Like the RBD in SARS-CoV-2 spike protein, HA-RBD is a trimeric envelope molecule and the immunodominant key region for vaccination against influenza viruses. HA- RBD is confined to amino acid residues 63 to 286 (residues 55 to 271 in H3 numbering) (DuBois R. et al. J Virol 2011 Jan;85(2):865-72. https: / / doi.org / 10.1128 / jvi.01412-10), as representatively shown in SEQ ID NO. 57 (sequence shown below) for Influenza A virus hemagglutinin (A / Mexico / 4603 / 2009(H1N1), GenBank accession number ACQ99608). Recommended numbering scheme for Influenza A HA Subtypes is reported by Burke DF, Smith DJ (2014) PLoS ONE 9(11): el 12302. https: / / doi.org / 10.1371 / joumal.pone.0112302). There are multiple approaches to cover HA from different subtypes utilizing different scaffolds to induce broad neutralizing immunity that may overcome annual adaption and protect against pandemics. The use of computationally optimized broadly reactive antigens (COBRAs) HA might be one of these promising strategies (Dziminaski J et al. Commun Biol. 2023 Apr 25;6(1):454, https: / / doi.org / 10.1038 / s42003-023-04793-3 ). Accordingly, BioVac could at least present the HA-RBD of four different influenza viruses (tetravalent RBD display) or homotetrameric display of broadly reactive HA-RBDs. SEQ ID NO. 58 (shown below) represents HA-RBD amino acid sequence derived from COBRA Pl. COBRA Pl was constructed using a combination of human H1N1 sequences spanning the years 1933-1957 and 2009-2011, along with swine sequences from 1931 to 1998. Restriction of the immunogen from whole HA to only the RBD region combined with Fc fusion may boost expression efficacy and presentation of this key area to the immune system. Moreover, in analogy to SARS-CoV-2, tetramerization in BioVac may provide an advantage over natural trimeric HA presentation by influenza viruses when passive immunization is aimed to block the receptor and viral entry.

[0343] SEQ ID NO. 57:

[0344] GVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQ LSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLS KSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKV RDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISD

[0345] SEQ ID NO. 58:

[0346] GIAPLQLGKCNIAGWLLGNPECESLLSARSWSYIVETPNSENGTCYPGDFIDYEELREQ LSSVSSFERFEIFPKESSWPNHNTTKGVTAACSHAGKSSFYRNLLWLTKKGGSYPKLS KSYVNNKGKEVLVLWGVHHPSTSTDQQSLYQNENAYVSVVSSNYNRRFTPEIAERP KVRGQAGRMNYYWTLLEPGDTIIFEATGNLIAPWYAFALSRGSGSGIITSN

[0347] Potential cross-species transmission of highly pathogenic avian influenza H5 subtype to humans is considered one of the hottest candidates for the next pandemic threat and therefore calls for development universal influenza vaccines using broad reactive HA. Interestingly, mutations of key amino acid residue in the HA-RBD region seem to be of high relevance for H5N1 infection of humans (Huang, P., Sun, L., Li, J. et al. Cell Discov 9, 58 (2023). https: / / doi.org / 10.1038 / s41421-023-00571-x ). In line, most recently Guan L, Eisfeld A, Pattinson D, et al. reported a new highly pathogenic avian influenza virus (HP Al) of the H5N1 subtype in Cow’s milk (Guan N Engl J Med. 2024, DOI: 10.1056 / NEJMc2405495). The consensus sequences of these viruses have been deposited in GISAID (accession numbers EPI ISL 19091701, EPI ISL 19091702, EPI ISL 19091703, EPI ISL 19091704, EPI ISL 19091705, EPI ISL 19091706, EPI ISL 19091707, and EPI ISL 19091708) followed by a detailed report of the American Center for Disease Control and Prevention (CDC, https: / / www.cdc.gov / bird-flu / spotlights / h5nl-analysis-texas.html). First attempts to develop broad cross-neutralizing antibodies against H1N1 as well as H5N1 revealed important insights to the key relevance of distinct regions within HA-RBD that might be instructive in design of next generation broad reactive antigens (Li, T., Chen, J., Zheng, Q. et al. Nat Commun 13, 5182 (2022). https: / / doi.org / 10.1038 / s41467-022-32926-5). Therefore, heterotetrameric or broad- reactive HA-RBD based BioVac may exhibit great potential to induce cross neutralizing antibodies and immune response against a spectrum of influenza viruses once used as an active vaccination principle. Intranasal application of such influenza BioVac (iBioVac) may further enhance the mucosal immunity and systemic protection. Moreover, in analogy to data presented for SARS-CoV-2, mucosal application of a high affinity HA-RBD based iBioVac may outcompete and block viral entry, irrespective of subtypes, hence providing an effective passive immunization strategy. Finally, heterotetramerization of Influenza HA-RBD with SARS-CoV-2 or other viruses against which a potent immunization, e.g. due to infection or vaccination, is present, may further facilitate immune recognition and assist to form of a broad and deep immunity.

[0348] The sequences of constructs HD-BioVaclOO to BioVacl28 are depicted in SEQ ID Nos. 80 to 111, disclosed elsewhere herein. These constructs comprise receptor binding domains from the spike protein of further coronaviruses (see Figures 13 and 14) or from hemagglutinin (HA) of influenza viruses (see Figures 12 and 14). Figure 15 depicts homotetrameric and heterotetrameric constructs HD-BioVaclOO to BioVacl28. The knob-in-hole Fc(KiH) mutation was used to express up to four different receptor binding domains (RBDs) of Corona viruses or Influenza viruses in a single BioVac molecule. The STR mutation, analogous to LALAPG (see BioVac002), was used for FcRy silencing to confine immunogenicity solely to the RBD regions. These constructs were well-expressed in the CHO system and demonstrated high- affinity binding to their cognate receptors and high immunogenicity in the classical prime / boost immunization scheme; see Examples.

[0349] Particularly preferred HD-BioVac constructs are shown in SEQ ID Nos. 1-12, 35-43, 55 and 80-111.

[0350] As explained elsewhere herein, production and characterization of BioVac constructs 001-012, 035-043, and 055, including their use for active immunization, passive immunization and therapy and their superior properties is described in great detail, in co-pending PCT / EP2025 / 066857 and PCT / EP2025 / 066860, the disclosure content of which is incorporated herein by reference.

[0351] BioVac constructs 100-128 depicted in SEQ ID Nos. 80-111 are also shown, e.g., in Figure 15.

[0352] (ii) Multiple receptor binding domains in the protein oligomers of the invention

[0353] The presence of four (or even more) receptor binding domains in the protein oligomer of the invention leads to functional advantages of multivalency and high binding strength, increased structure stabilization and combined functions of the receptor binding domains and Fc domains, resulting in improved or increased affinity, and broader coverage of respiratory viral RBD subvariants, and improved or increased avidity, of the protein oligomer of the invention, in comparison to a vaccine comprising only one receptor binding domain. This is reflected, inter alia, by the fact that the protein oligomer of the invention comprising the RBD of SARS-CoV- 2 binds to the human ACE2 receptor with exceptionally high binding affinities. This has been demonstrated for the constructs HD-BioVacOOl and HD-BioVac004 which bind to ACE2 via the receptor binding domain (RBD) of the SI subunit of the spike protein of SARS-CoV-2, in the picomolar range, as tested by ELISA and surface plasmon resonance (SPR) analysis. Such binding affinity outperforms those of other SARS-CoV-2 receptor binding domain constructs described in the art, e.g. spike (trimeric RBD), dimeric RBD-Fc or Fc-RBD constructs; see Extended Data Table 2 of the publication by Shang et al., Nature. 2020 May; 581(7807): 221- 224. doi: 10.1038 / s41586-020-2179-y.

[0354] Thus, the protein oligomers of the invention can advantageously be used for active immunization.

[0355] Furthermore, the protein oligomers of the invention can be fastly adapted to display multiple viral variants in the same molecule, hence the presence of immunity against one variant may enhance immunogenicity of a novel variant. This was demonstrated e.g. for enhanced immunogenicity of omicron by co-presentation together with delta in the same BioVac molecule.

[0356] Due to specific focus on multivalent RBD amino acid sequence, potential to display different variants in several or the same molecule (successful expression of tetrameric BioVac with four different subvariants was demonstrated - see Fc-KiH constructs) a potent, deep and broad immunization is induced with BioVac principle, as demonstrated in the corresponding human and preclinical data.

[0357] Another aspect which is important to mention is the fact that the protein oligomers of the invention can contain receptor binding domains (RBD) from different respiratory viral proteins, as explained elsewhere herein. Thus, the protein oligomers of the invention can be used e.g. for immunization against different respiratory viruses, at the same time.

[0358] It is within the scope of the invention, that a combination of different protein oligomers of the invention is used for immunization and / or therapy. For instance, one protein oligomer of the invention can carry the receptor binding domain (RBD) of the SI subunit of SARS-CoV-2 spike protein as defined herein, and another protein oligomer of the invention can contain the receptor binding domain (RBD) of influenza virus hemagglutinin as specified herein. Said different protein oligomers of the invention can then be co-administered to a subject for immunization against the SARS-CoV-2 and influenza viruses and / or for therapy of symptoms and diseases causes by said respiratory viruses.

[0359] (iii) Optimized Fc domains in the protein oligomers of the invention

[0360] Furthermore, the protein oligomers of the present invention contain a monomeric or dimeric Fc domain which has an increased binding affinity to FcRn. In addition, the protein oligomers of the present invention are optimized for mucosal uptake and penetration in that they comprise an engineered Ig Fc domain having enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, and no or essentially no detectable binding to FcRn at serum pH (about pH 7.35 to 7.40; preferably about pH 7.4), compared to wildtype human Ig Fc. Such enhanced affinity for FcRn is mediated, e.g., by DHS mutations (L309D / Q311H / N434S in human IgGl Fc), YTE (M252Y / S254T / T256E in human IgGl Fc), LS mutations (N428L / N434S in human IgGl Fc), KF mutations (H433K / N434F in human IgGl Fc), or DE mutations (S239D / I332E in human IgGl Fc), in the Ig Fc. Due to this genetic modification, the protein oligomers of the present invention are able to bind to FcRn at the apical site of the mucosa at acidic pH (pH of about 6.0 to 6.7, preferably about 6.5). Instead of being directed to the endolysosomal compartment for degradation, the protein oligomer :FcRn complexes are sorted into tubules originating from sorting endosomes and directed to the plasma membrane at the lamina propria. Upon fusion with the plasma membrane, the intracellular fluid within the tubules is released and rapidly equilibrates with the extracellular pH 7.4. At extracellular pH, the affinity of FcRn for the Fc domain of the protein oligomers of the invention is so low that said protein oligomers are released into the lamina propria. In the lamina propria, the Ig Fc domain of the protein oligomers of the invention is bound by antigen presenting cells (APC) via Fc gamma receptors. Said antigen presenting cells process and present antigens of the receptor binding domains (RBD) included in the protein oligomers of the invention, for recognition by lymphocytes such as CD4 T cells. When an antigen-presenting cell displays a peptide antigen of said receptor binding domains on MHC class II proteins, CD4 T cells aid immune cells such as macrophages, B cells or CD8 T cells through a combination of cell-to-cell interactions and through cytokines. In view of this, the protein oligomer of the invention advantageously induces protective immune responses at the relevant mucosal sites of pathogen entry by mucosal delivery of said protein oligomer, in active immunization approaches.

[0361] Further benefits of using DHS mutations in the protein oligomer of the invention are maintainance of the immune effector functions in the Ig Fc and easy adaptation to different Ig platforms such as human IgG Fc platforms. In addition, the aggregation potential is lower compared to other FcRn enhancement strategies described in the art.

[0362] The Ig Fc such as human Ig Fc can further comprise e.g. LALAPG mutations (L234A, L235A, and P329G, in human IgGl), LALA mutations (L234A / L235A in human IgGl Fc), and / or STR mutations (L234S / L235T / G236R in human IgGl Fc), for ablating Fc-Fc gamma receptor- mediated effector functions without essentially affecting affinity for Fc gamma receptor. These mutations abolish the interaction of the Fc domain with Fcgamma receptors and complement proteins. Such an approach can be used for shutting off known Fc functions or complement; see, e.g., Hezareh, M., Hessell, A. J., Jensen, R. C., van de Winkel, J. G. & Parren, P. W., J. Virol. 75, 12161-12168 (2001); Vafa, O. et al., Methods San Diego Calif. 65, 114-126 (2014); Schlothauer, T. et al., Protein Eng. Des. Sei.29, 457-466 (2016). iv) Further modes of action and properties of the protein oligomers of the invention

[0363] Briefly, mucosal and systemic immune responses to natural infection with respiratory viruses and to vaccination can be summarized, as set out below. The lower human respiratory tract is thought to be mostly protected by IgG (IgGl is most prevalent), the main type of antibody in serum, which is transported into the lung. The upper respiratory tract is thought to be mostly protected by secretory IgAl (slgAl).

[0364] Natural infection with respiratory viruses induces both a systemic immune response, dominated by IgGl, as well as a mucosal immune response in the upper respiratory tract that is dominated by slgAl. This process can lead to sterilizing immunity for many respiratory viruses, including SARS-CoV-2.

[0365] Intramuscular or intradermal vaccination leads in many cases to a strong induction of serum IgG but not to an induction of mucosal IgA. Although some IgG can also be found on the mucosal surfaces of the upper respiratory tract, the lack of slgA often leaves an individual vulnerable to infection of the upper respiratory tract.

[0366] Intranasal vaccination can efficiently induce mucosal antibody responses, thereby potentially providing sterilizing immunity in the upper respiratory tract. However, systemic immune responses are often lower after this type of vaccination. Although mucosal immunity might not be required to protect from severe or even symptomatic disease, it could be required to achieve optimal protection from infection and onward transmission of, e.g., SARS-CoV-2; see, e.g., Florian Krammer, Nature (2020), Vol 586, p. 516-527.

[0367] While many vaccines including the currently approved SARS-CoV-2 vaccines induce systemic immune responses, they probably do not evoke “mucosal immunity” in form of mucosal, secretory immunoglobulin A (IgA) or tissue-resident memory T cells (TRM). However, this is important because secretory, polymeric IgA can neutralize incoming viral particles at the mucosal surface before infection of epithelial cells takes place, which is important for an optimal protection against respiratory virus infections. Furthermore, IgA enables specific effector functions by cross-linking the Fca-receptor, and polymeric forms of IgA might increase antibody avidity.

[0368] In contrast, the protein oligomers of the invention have advantageously been shown to elicit humoral immunity after intramuscular application, and mucosal immunity after nasal application, in human subjects. In particular, they evoke a response in form of mucosal, secretory immunoglobulin A (IgA) and / or tissue-resident memory T cells (TRM). In addition, the protein oligomers of the invention emulate natural viral infections.

[0369] The protein oligomers of the invention can be produced cheaply and efficiently, so that it is possible to produce and use them in low-income countries. They are are stable at 4° C for a long period of time, thereby simplifying logistics, in contrast to, e.g., mRNA-based vaccines which have to be stored at -80° C.

[0370] The protein oligomers of the invention can be produced in very high yields as indicated by preliminary 500L BioVacOOl upscaling with up to ~ 10 g / L protein yields.

[0371] Further, the protein oligomers of the present invention are very safe with respect to toxicity, as demonstrated in human vaccinations.

[0372] As a still further advantage, adjuvants can be coupled to constituents of the monomer of the protein oligomer of the invention, such as the Ig Fc, oligomerization domain or linker, so that the adjuvant does not have to be co-administered with said protein oligomer. Hence, more potent adjuvants that are otherwise not used for their uncontrolled systemic distribution and consequent undesired reactogenecity could be directed via BioVac to the ACE2 positive cells e.g. in mucosa or high affinity RBD targeting T / B-Cell receptors for a specific immune activation.

[0373] Furthermore, the protein oligomers of the present invention have high potential as over-the- counter (OTC) medicines, for instance in the form of a nasal spray, inhalator / nebulizer to reach deeper respiratory regions, or the like.

[0374] The protein oligomers of the present invention can advantageously be used as immunogens for active immunization of subjects, i.e. used as immunogen for SARS-CoV-2 vaccination or for vaccination against other respiratory viruses as referred to herein.

[0375] Accordingly, the protein oligomers of the invention advantageously induce protective immune responses at the relevant mucosal sites of pathogen entry by mucosal delivery of said protein oligomers, in active immunization approaches.

[0376] The protein oligomers of the invention are able to induce both a systemic immune response, dominated by IgGl, as well as a mucosal immune response in the upper respiratory tract that is dominated by slgAl . In light of this, the administration of the protein oligomers of the invention can lead to immunity for many respiratory viruses, thereby providing optimal protection from respiratory viral infection.

[0377] The protein oligomers of the invention can also be used for blocking the interaction between the respiratory virus and the viral receptor on the target cell of a subject, due to the high avidity and affinity of the protein oligomers of the invention to the viral receptor on the viral target cell of a subject. Thus, it is a further advantage that the protein oligomers of the invention can be used for prophylactic administration for avoiding or preventing respiratory viral infection, i.e. for passive immunization, in (a) subject(s) because they prevents efficiently the respiratory virus from binding to its receptor on viral target cells of a subject, thereby blocking viral entry into said target cells in the subject. Specifically, the protein oligomer of the invention advantageously induces protective immune responses at the relevant mucosal sites of pathogen entry by mucosal delivery of said protein oligomer.

[0378] Key relevant design features of protein oligomers of the invention or BioVac constructs for passive immunization include 1) identification of the viral receptor binding domain (RBD), i.e., minimal amino acid sequence that contains the receptor binding motif (RBM) but provides sufficient structural stability, functional integrity and expression efficacy for large scale production, and at the same time avoids non-specific immune responses e.g., human homologue and / or highly glycosylated regions. 2) In a second step, the RBD sequence is further optimized to fit into a tetrameric IgG Fc-fusion protein. These include, minimal linker strategies for bland C-Terminal fusion to Fc preventing protease degradation and still sufficiently flexible for a proper presentation / binding of the BioVac RBD to putative receptors. 3) Fc engineering for enhanced uptake at mucosal pH (FcRn) preferably via DHS mutation. 4) Engineering of FcyR immune effector functions via silencing (e.g., STR or LALAPG substitutions) for an RBD dominant function vs. enhanced immune effector functions to generate an inflammatory mucosal niche e.g., to establish an immune barrier against a broad spectrum of infective agents. What is more, the protein oligomer of the invention can advantageously be used for treating subjects suffering from infections by respiratory viruses and to ameloriate or even cure symptoms and diseases causes by said respiratory viruses in said subjects.

[0379] The novel protein oligomers of the invention are superior in comparison to known vaccines, in different aspects, as set forth elsewhere herein.

[0380] Preferred embodiments

[0381] Ig Fc as used herein is preferably selected from the group consisting of IgG Fc, IgA Fc, and IgM Fc, preferably IgGl Fc or IgG3 Fc, more preferably IgGl Fc.

[0382] In a preferred embodiment of the protein oligomer of the invention, Ig Fc is human Ig Fc. Preferably, human Ig Fc is selected from the group consisting of human IgG Fc, human IgA Fc, and human IgM Fc, more preferably human IgGl Fc or human IgG3 Fc, most preferably human IgGl Fc.

[0383] As appreciated by those of skill in the art, it is desirable to use human or human-derived sequences for producing the monomers of the protein oligomer of the invention, except for the receptor binding domain. Such an approach increases the chances for obtaining a specific immune response against the virus-derived receptor binding domain sequence only, and to avoid an unwanted immune response against the other constituents of the monomers, such as the oligomerization domain or Ig Fc.

[0384] The amino acid sequences of human IgA Fc are shown in SEQ ID NO. 30 and 31. The amino acid sequence of human IgGl Fc is shown in SEQ ID NO. 26. The amino acid sequence of human IgG2 Fc is shown in SEQ ID NO. 27. The amino acid sequence of human IgG3 Fc is shown in SEQ ID NO. 28. The amino acid sequence of human IgG4 Fc is shown in SEQ ID NO. 29. The amino acid sequence of human IgM is shown in SEQ ID NO. 32.

[0385] A human Ig Fc as used herein encompasses also sequences derived from human Ig Fc sequences, such as human IgG Fc, human IgA Fc, or human IgM Fc sequences, or preferably human IgGl Fc, or human IgG3 Fc, more preferably human IgGl Fc.

[0386] An Fc domain “derived from” human Ig Fc as used herein means that such a Fc domain is identical to or can differ from the corresponding amino acid sequence of the native or nonmodified (poly)peptide in the human Ig Fc domain, in one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, or even more amino acid residues, while maintaining, or altering, or even exceeding the biological activity of the corresponding native human Ig Fc domain. This includes, for instance, the dimerization property, Fc receptor binding of the Fc domain (e.g., binding to FcRn and / or Fc gamma receptor), and modifying the pharmacokinetic properties of the Fc domain or induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Such mutations and methods for producing them are well known in the art and include, e.g., one or more substitutions, additions and / or deletions in the nucleic acid or amino acid sequence of the native (or wildtype) human Ig Fc domain; see, e.g., Sambrook et al., Molecular cloning : a laboratory manual / Sambrook, Joseph; Russell, David W. — . 3rd ed. — New York: Cold Spring Harbor Laboratory, 2001. Ausubel et al., Current Protocols in Molecular Biology. Encompassed are also Fc domains carrying combinations of such mutations, e.g., a human Ig Fc domain having three substitutions and one deletion, in its nucleotide or amino acid sequence.

[0387] For instance, an Fc domain “derived from” human IgGl or human IgG3 as used herein means that such a Fc domain is identical to or can differ from the corresponding amino acid sequence of the native or non-modified (poly)peptide in the human IgGl Fc domain or human IgG3 Fc domain, in one, two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 35, 40, 50, or even more amino acid residues, while maintaining, or altering, or even exceeding the biological activity of the corresponding native human IgGl Fc domain or human IgG3 Fc domain. This includes, for instance, the dimerization property, Fc receptor binding of the Fc domain (e.g., binding to FcRn and / or Fc gamma receptor), modifying the pharmacokinetic properties of the Fc domain and / or induction of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and / or inflammation via the induction of cytokine secretion. Such mutations are well known in the art and include, e.g., one or more substitutions, additions and / or deletions in the nucleic acid or amino acid sequence of the native (or wildtype or non-modified or non-mutated) human IgGl Fc or human IgG3 Fc domain. As mentioned above, encompassed are also human IgGl Fc or human IgG3 Fc domains carrying combinations of such mutations.

[0388] The mentioned term “Fc domain derived from the human Ig Fc domain” comprises variants or variant sequences of the human Ig Fc domain. Preferably, said variant sequence is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the specific amino acid sequence of the non-modified or native human Ig domain, over the entire length.

[0389] For example, a Fc domain “derived from” the human IgGl Fc domain comprises variants or variant sequences of the human IgGl Fc domain. Preferably, the said variant sequence is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the specific amino acid sequence of the non-modified or native human IgGl Fc domain, over the entire length.

[0390] To provide another example, a Fc domain “derived from” the human IgG3 Fc domain comprises variants or variant sequences of the human IgG3 Fc domain. Preferably, the variant sequence is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the specific amino acid sequence of the non-modified or native human IgG3 Fc domain, over the entire length. As indicated above, said variant sequence maintains, or alters, or even exceeds at least one of the aforementioned biological activities of the corresponding native or non-modified human Ig Fc domain.

[0391] Methods for engineering of Fc domains with optimized physicochemical properties are well described in the art; see, e.g. Yang et al., Front. Immunol., 08 January 2018, https: / / doi.org / 10.3389 / fimmu.2017.01860. For instance, engineering the human Ig Fc domain to improve pharmacokinetic (PK) properties (manifest as an increased area under the plasma drug concentration-time curve (AUC), a lower clearance rate and a longer P-phase Tl / 2) is of great interest for therapeutic purposes. Better PK enable less frequent administration and lower dosing, which in turn translate into improved patient compliance and lower costs. Mutations of the Fc domain that enhance the affinity for FcRn at both the endosomal and physiological pH have been shown to result in greater antibody clearance. In contrast, Fc mutations that preferentially enhance FcRn affinity at pH 5.8 confer increased antibody half-life in circulation.

[0392] Specific mutations in Fc domains that can be advantageously be used in the Ig Fc such as the human Ig Fc of the protein oligomer of the invention are explained in more detail elsewhere herein.

[0393] In a preferred embodiment of the protein oligomer of the invention, Ig Fc is a homodimer or a heterodimer, as explained elsewhere herein.

[0394] Preferably, the Ig Fc heterodimer comprises Fc domains from knobs-into-holes (KiH)- engineered IgG, more preferably from knobs-into-holes (KiH)-engineered IgGl or IgG3.

[0395] For instance, heteromeric implementation of an N-terminal oligomerization domain on one of the two KiH-engineered Fc, such as KiH-engineered IgGl or IgG3 Fc, could further enhance the backbone RBD tetramerization towards formation of additional valences, e.g. N-RBD- oligmerization domain-Fc(hole)-RBD-C together with RBD-Fc(knob)-RBD together with RBD-oligomerization, may form a trimer / tetramer / pentarmer on hole N-terminus of RBD and the tetrameric RBD backbone. One could also imagine the same with N-oligomerization domain-RBD-Fc-RBD-C, then the backbone remains and the oligomerization domain can be added to one arm, two, three, or all four arms. The same could take place by simple RBD multimerization, RBD-linker-RBD-linker-RBD etc., like string on the beads on one to each of the four arms. Furthermore, T-cell epitopes or molecules functioning as adjuvant could be conjugated at the N- or C-termini via the same principles, either simply via a linker or via oligomerization domain to enhance their valency (potency). Moreover, N- / C- terminal recognition sites or within the linker modifications for enzymatic (glutaminase) attachment of immune enhancing molecules, e.g. mRNA, cGAS-STING agonist, Interferons etc. could be a strategy to enhance immunogenicity. Finally, in addition to, e.g., sortase sites at N / C terminus, an intra Fc glutaminase motif or glycosylation sites could be used for linker-payload attachments without amino acid engineering at the N / C terminus.

[0396] In a still further preferred embodiment of the protein oligomer of the invention, Ig Fc exhibits enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH. Further, said Ig Fc exhibits preferably no or essentially no detectable binding to FcRn at serum pH of about pH 7.35 to 7.40, preferably about pH 7.4, in comparison to wildtype, non-modified or native Ig Fc.

[0397] IgG isotype antibodies avoid clearance by endolysosomal degradation by virtue of their pH- dependent binding to the neonatal Fc receptor (FcRn), which is expressed by cells in nearly every organ in mammals. The FcRn receptor consists of the glycosylated heavy a-chain polypeptide, an MHC I class family member which associates with p2-microglobulin (P2m). IgG internalized by pinocytosis binds to FcRn at endosomal pH (5.5-6.0) and as a result, instead of being directed to the endolysosomal compartment for degradation, IgG:FcRn complexes are sorted into tubules originating from sorting endosomes and directed to return to the plasma membrane. Upon fusion with the plasma membrane, the intracellular fluid within the tubules is released and rapidly equilibrates with the extracellular pH 7.4. At extracellular pH, the affinity of FcRn for the Fc domain is so low that antibodies are released back into circulation. This process occurs readily, despite the strong avidity effects of both the high local concentration of FcRn at the site of vesicle fusion, and the 2: 1 stoichiometric binding of FcRn to IgG. Biophysical studies have elucidated the molecular details of the IgG:FcRn interaction including the role of residues in the CH2-CH3 interface of the Fc domain in contact with FcRn, the pivotal role of His310 and His435 on pH-dependent binding, and the significance of protein dynamics.

[0398] Lee et al. (Nat Commun. 2019; 10: 5031) have reported the engineering of a human IgG Fc domain which, by virtue of having moderately higher affinity for FcRn at pH 5.8 but no detectable binding at pH 7.4 under high avidity conditions, confers improved antibody PK properties compared to the clinical stage YTE and LS variants, both in the commonly used hFcRnTgmouse model (hemizygotic 276) as well as in their new knock-in model (hFcRn-hp2m- hFcyRs-hIgGl,K mice). Importantly, the authors show that antibodies utilizing the engineered ultra-long half-life Fc domain display the full range of effector functions as the wildtype (wt) Fc domain while exhibiting far favorable biophysical properties for clinical development. Specifically, Lee and co-workers (Nat Commun. 2019; 10: 5031) found that YTE- and LS-IgGl showed moderate and significant binding, at pH 7.4, respectively when SPR analysis was performed with medium or high hFcRn:hp2m densities (see Fig. lb, c, Table 1, in the mentioned publication). In contrast, no binding of DHS- or wt-IgGl to hFcRn:hp2m at physiological conditions at even the highest density tested could be detected.

[0399] The authors of Lee et al. (Nat Commun. 2019; 10: 5031) further report that Fc domains containing the amino acid substitutions M428L / N434S (LS mutant), M252Y / S254T / T256E (YTE mutant), or H433K / N434F (KF mutant) confer 10- to 12-fold higher affinity for FcRn at pH 5.8, result in the greatest reported increase in antibody half-life (2- to 4-fold in circulation) in mice and in non-human primates, and are being evaluated in multiple clinical trials; see, e.g., Lee et al. (Nat Commun. 2019; 10: 5031); Zalevsky J, et al., Nat. Biotechnol. 2010;28: 157- 159; Vaccaro C. et al., Proc. Natl. Acad. Sci. USA. 2006;103: 18709-18714; Ko S-Y, et al., Nature. 2014;514:642-645; Zhu Qing, et al., Science Translational Medicine. 2017;9(388):eaaj 1928. Table 1 shows the KD values for binding of DHS, YTE, LS variants or wildtype (WT) IgGl to hFcRn:hp2m dimer at pH 5.8 and 7.4; adopted from Lee et al. (Nat Commun. 2019; 10: 5031).

[0400] Table 1:

[0401] In a preferred embodiment of the protein oligomer of the invention, enhanced affinity of Ig Fc for the neonatal Fc receptor (FcRn) at mucosal pH, and no or essentially no detectable binding to FcRn at serum pH (about pH 7.35 to 7.40; preferably about pH 7.4), compared to wildtype Ig Fc, is mediated by (i) DHS mutations (L309D / Q311H / N434S in human IgGl Fc), (ii) YTE (M252Y / S254T / T256E in human IgGl Fc), (iii) LS mutations (N428L / N434S in human IgGl Fc), (iv) KF mutations (H433K / N434F in human IgGl Fc), and / or (v) DE mutations (S239D / I332E in human IgGl Fc), in the Ig Fc.

[0402] The used numbering is according to EU numbering; see, e.g., https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0403] The Ig Fc domain in the protein oligomer of the invention binds to FcRn on target cells such as mucosal or epithelial cells. The Ig Fc domain in the protein oligomer of the invention is engineered to bind to FcRn with increased affinity, which is mediated by DHS, YTE, KF and / or LS mutations in the Ig Fc domain.

[0404] The use of DHS mutations is particularly advantageous in the Fc domain of the protein oligomer of the invention because it has enhanced affinity for FcRn at acidic endosomal or mucosal pH and no detectable binding to FcRn at serum pH 7.4; see Table 1. Further, said Fc domain with DHS mutations maintains immune effector functions of IgG, and it can be adapted to different IgG platforms. In addition, the aggregation potential of such Fc domain with DHS mutations is lower, in comparison to other FcRn enhancement strategies.

[0405] Human FcRn binds human IgGl with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, using Surface Plasmon Resonance / Biacore; see Abdiche et al., MAbs. 2015;7(2):331-43. doi: 10.1080 / 19420862.2015.1008353.

[0406] In another study it has been found that wt-IgGl has a KD of 550 ± 50 nM to hFcRn:hp2m dimer at pH 5.8, also using Surface Plasmon Resonance; see Table 1 of Lee et al., Nat Commun. 2019; 10: 5031.

[0407] The equilibrium dissociation constant (KD or Kd) of interaction of wildype IgG with the neonatal Fc receptor (FcRn) at pH 5.8, is preferably between about 500 and 800 nM.

[0408] Table 1 of the publication by Lee et al. (Nat Commun. 2019; 10: 5031) shows KD values for binding of DHS, YTE, LS variants to hFcRn:hp2m dimer at pH 5.8 and 7.4. DHS-IgGl has a KD of 110 ± 20 nM to hFcRn:hp2m dimer at pH 5.8. YTE-IgGl has a KD of 23 ± 1 nM to hFcRn:hp2m dimer at pH 5.8. LS-IgGl has a KD of 55 ± 3 nM to hFcRn:hp2m dimer at pH 5.8. As set forth above, wt-IgGl has a KD of 550 ± 50 nM to hFcRn:hp2m dimer at pH 5.8. Accordingly, the DHS variant had a ~5-fold better affinity for hFcRn at pH 5.8 compared to wt IgGl . The YTE variant had a ~24-fold better affinity for hFcRn at pH 5.8 compared to wt IgGl . And the LS variant had a ~10-fold better affinity for hFcRn at pH 5.8 compared to wt IgGl .

[0409] In a preferred embodiment of the protein oligomer of the invention, the affinity of the Ig Fc for the neonatal Fc receptor (FcRn) at mucosal pH such as a pH of about 6.5, is preferably enhanced or increased by at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, by at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 15-fold, at least about 20-fold, at least about 24-fold, or at least about 25-fold, compared to wildtype Ig Fc.

[0410] An Ig Fc with enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, preferably a pH of about 6.5 compared to wildtype Ig Fc, as used herein, means preferably a KD of between about 20 nM and about 150 nM.

[0411] Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD). Means and methods for determining the KD are known in the art and include, for instance, Surface Plasmon Resonance / Biacore.

[0412] Modifications of the Ig Fc resulting in enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, compared to wildtype Ig Fc, are described in the literature and set forth elsewhere herein.

[0413] In another preferred embodiment, the Ig Fc has enhanced affinity for the neonatal Fc receptor (FcRn) at a mucosal pH of about 6.5, and no or essentially no detectable binding to FcRn at serum pH (about pH 7.35 to 7.40; preferably about pH 7.4), compared to wildtype human Ig Fc.

[0414] In a still further preferred embodiment of the protein oligomer of the invention, the human Ig Fc further comprises LALAPG mutations (L234A / L235A / P329G in human IgGl Fc), LALA mutations (L234A / L235A in human IgGl Fc), or STR mutations (L234S / L235T / G236R in human IgGl Fc), for ablating Fc-Fc gamma receptor-mediated effector functions, without essentially affecting affinity for Fc gamma receptor. Preferably, the Fc gamma receptor is selected from the group consisting of FcgammaRI, FcgammaRIIa, FcgammaRIIc, FcgammaRIIIa, and FcgammaRIIIb.

[0415] Advantageously, the invention also provides protein oligomers in which the Fc-Fc gamma receptor-mediated effector functions have been shut off. Accordingly, these protein oligomers no longer have the capability of mediating Fc-Fc gamma receptor-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or inflammation via the induction of cytokine secretion, for instance in the lamina propria.

[0416] The rationale for using LALA, LALAPG, or STR mutations is to reduce any Fc-effector functions to restrict recognition of the immunogen by adaptive immune cells (B and T cells) only via BCR / TCR binding to the RBD immunogenic epitopes, in the protein oligomer of the invention. This might restrain immunogenicity in boost situations confined to immunogen instead of, e.g., uptake over FcRs and other type of activation. In boost situations, where there is already a basal immunization one would like to select for only those BCRs and TCRs that recognize and potently signal, and not amplification of moderate to low affinity immune cell binder. The aim is to avoid non-specific and potentially erroneous immune responses that could, e.g., lead to binding on other proteins with structural similiarities to RBD as antigen. One example for such erroneous immune responses is the formation of autoantibodies etc. with undesired off target effects.

[0417] LALAPG (L234A, L235A, P329G) mutations in human IgGl are described, e.g., in Bailey et al., Nature Communications volume 9, Article number: 4560 (2018).

[0418] Also Wilkinson et al. (PLoS ONE, 16(12): e0260954 (2021). https: / / doi.org / 10.1371 / journal. pone.0260954) describe a set of variants having specific amino acid substitutions in the Fc region at L234 and L235 combined with the substitution G236R. They show no detectable binding to Fey receptors or to Clq, are inactive in functional cell-based assays and do not elicit inflammatory cytokine responses.

[0419] Preferably, said LALAPG mutations as used herein comprise L234A / L235A / P329G in human IgGl Fc.

[0420] Such LALAPG mutations (L234A / L235A / P329G in human IgGl Fc), LALA mutations (L234A / L235A in human IgGl Fc), or STR mutations (L234S / L235T / G236R in human IgGl Fc) can be introduced into human Ig Fc, for example, by site-directed mutagenesis, PCR, directed evolution, alanine scanning, or structure-guided design have been used to identify these mutations or other methods known in the art; Sambrook et al., Molecular cloning : a laboratory manual / Sambrook, Joseph; Russell, David W. — . 3rd ed. — New York: Cold Spring Harbor Laboratory, 2001. Ausubel et al., Current Protocols in Molecular Biology; Saunders, Front. Immunol. 10: 1296, 07 June 2019 | https: / / doi.org / 10.3389 / fimmu.2019.01296.

[0421] Preferably, the Fc-Fc gamma receptor-mediated effector function ablated by the aforementioned mutations in the human Ig Fc is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and / or inflammation via the induction of cytokine secretion.

[0422] The effect of such mutations in human IgG such as IgGl can be tested, for example, by Fc- FcyR engagement assays or Fc effector cell bioasssays. Binding to Clq can be tested by using, e.g., ELISA. Cytokine release can be tested by a Luminex or O-Link or ELISA assays or other commercially available assays; see also Bailey et al., Nature Communications volume 9, Article number: 4560 (2018) and Wilkinson et al. (PLoS ONE, 16(12): e0260954 (2021). https: / / doi.org / 10.1371 / journal. pone.0260954; Lee, CH., Kang, T.H., Godon, O. et al. An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence. Nat Commun 10, 5031 (2019). https: / / doi.org / 10.1038 / s41467-019-13108-2).

[0423] In another preferred embodiment of the protein oligomer of the invention, the RBD or fragment thereof is the same or different, (i) in the at least first monomer of the protein oligomer, or (ii) in the at least second monomer of the protein oligomer, or (iii) both in the at least first monomer and in the at least second monomer of the protein oligomer.

[0424] Preferred RBD are shown, e.g., in Figures 12 to 15 and defined and specified by sequence elsewhere herein and in the claims.

[0425] Preferably, if the RBD is from SARS-CoV-2 spike protein, the RBD or fragment thereof in the protein oligomer of the invention is from one or more variants of concern (VOC), selected from the group consisting of: SARS-CoV-2 variant alpha (B.1.1.7), SARS-CoV-2 variant beta (B.1.351), SARS-CoV-2 variant gamma (P. l), SARS-CoV-2 variant delta (B.1.617.2), SARS- CoV-2 variant omicron, such as SARS-COV-2 variant omicron BA. l, SARS-COV-2 variant omicron BA.2, SARS-COV-2 variant omicron BA.2.3.20, SARS-COV-2 variant omicron BA.2.75, SARS-COV-2 variant omicron BA.3, SARS-COV-2 variant omicron BA.4, SARS- COV-2 variant omicron BA.5, SARS-COV-2 variant omicron BJ1, SARS-COV-2 variant omicron BA.4.6, SARS-COV-2 variant omicron XBD, SARS-COV-2 variant omicron XBB.1.5-like, SARS-COV-2 variant omicron XBB.1.5-like + F456L, SARS-COV-2 variant omicron XBB.1.5-like + L455F + F456L, SARS-COV-2 variant omicron BA.2.86, SARS- COV-2 variant omicron BA.2.87.1, SARS-CoV-2 (LP8.1) and SARS-CoV-2 (NB.1.8.1).

[0426] Presentation of heterotypic molecules with, e.g., four different RBD variants can increase the formation of broad neutralizing antibodies and avoid the development of original antigenic sin. Original antigenic sin (also known as antigenic imprinting, or immunological imprinting) is the propensity of the immune system to preferentially use immunological memory based on a previous infection when a second slightly different version of that foreign pathogen (e.g. a virus) is encountered. This leaves the immune system "trapped" by the first response it has made to each antigen, and unable to mount potentially more effective responses during subsequent infections. Antibodies or T-cells induced during infections with the first variant of the pathogen are subject to repertoire freeze, a form of original antigenic sin. This phenomenon has also been described in relation to the SARS-CoV-2 pandemic: The imprinting of the immune system to the original Wuhan SARS-CoV-2 spike protein may restrict the recognition of subtle modifications of the spike protein structure introduced by subsequent mutations. The original highly immunogenic sites and relative high immune activation by mRNA may reduce the potential of immune response to adapt to these subtile structural alterations over viral evolution.

[0427] Heterotypic presentation of immunogen in the same construct can guide the immune cells to generate a stronger immune response by otherwise less immunogenic variants. Again, to put this back in the context of the SARS-CoV-2 pandemic, omicron variants used to be less immunogenic (evolutionary advantage to escape immune response led to their spread in infected / vaccinated populations), a heterotypic molecule delta-Fc-omicron, however, could potently induce a variant specific immune response whereby the delta component is well recognized by the infected / vaccinated immune system functioning as an intramolecular adjuvant for the new immune escape omicron variant. This also provides a perspective towards development of adjuvant free booster approaches. Large scale production of non-specific immune activating adjuvants was and is a limiting factor for mass immunization in such situations. The same applies if an immunogen (payload, adjuvants like e.g. GMCSF / GCSF / Interferon) is expressed within the protein oligomer of the invention.

[0428] It has been shown by the present inventors that using different RBD from Coronavirus families may also improve original antigenic sin towards preferential selection for BCR clones that recognized common epitopes across Coronaviruses to generated broad-neutralizing antibodies

[0429] In boost situations, existing antibodies will bind to the protein oligomer of the invention or the HD-BioVac constructs defined herein at least tetrameric RBD backbone forming an antigenantibody like large immune complex, this is known as one of the best immunogens for generation of enduring immunogenic response via activation of memory cells, which can lead to an improved while prolonged interval between immunization where vaccine are sparse such as, e.g., in pandemic situation. Moreover, the risk for additional vaccine-related side effects could be reduced.

[0430] In another preferred embodiment of the protein oligomer of the invention, (i) said at least first monomer, or (ii) said at least second monomer, or (iii) both said at least first and said at least second monomer further comprise(s) a) at least one linker and / or b) at least one oligomerization domain and / or c) at least one, or two, three, four, five, six, seven, eight, nine, or ten additional RBD, preferably an RBD as defined herein. In still another preferred embodiment of the protein oligomer of the invention, the linker is an independently selected variable linker amino acid sequence. Preferably, the linker has reduced sensitivity to protease cleavage, more preferably the linker comprises or is the linker (GGGGS)i (SEQ ID NO. 33) or (GGGGS)2(SEQ ID NO. 49) or GGGGGSGGGGS (SEQ ID NO. 54), and the reduced sensitivity to protease cleavage is in comparison to (GGGGS)s (SEQ ID NO. 50). The linker can also comprise or consist of a Fc hinge region-derived linker, or another glycineserine linker known in the art. Further linkers that can be used are depicted in SEQ ID NO. 17, 18, 34, 52 or 53.

[0431] In another preferred embodiment of the protein oligomer of the invention, the oligomerization domain is selected from the group consisting of: The non-triple helical trimerization domain of human collagen 15 (SEQ ID NO. 25) or human collagen 18 (SEQ ID NO. 24), the C-terminal oligomerization domain of human C4b-binding protein, the GCN4-pII isoleucine zipper (SEQ ID NO. 21), the IZN4 trimerization domain (SEQ ID NO. 22), coiled coils, oligomeric miniproteins, short peptides with discrete protein-like structures, trimerization domain of the bacteriophage T4 fibritin (foldon) (SEQ ID NO. 19, 20), TNF alpha trimerization domain, zinc finger, or p53 tetramerization domain (SEQ ID NO. 23); see Ali and Imperial! 2005, Bioorganic and Medicinal Chemistry 13, 5013.

[0432] Also sequence variants of the aforementioned oligomerization domains can be used, in the monomer of the protein oligomer of the invention. A sequence variant of an oligomerization domain as used herein differs from the wild-type, non-modified or native amino acid sequence of an oligomerization domain or a nucleic acid sequence encoding said oligomerization domain as specified before, by one, two, three, four, five, six, seven, eight, nine, ten, or even more nucleotide or amino acid substitutions, additions or deletions, or combinations thereof. For example, a sequence variant of an oligomerization domain can contain an amino acid substitution and a deletion of an amino acid residue, in comparison to the amino acid sequence of the wild-type, native or non-modified oligomerization domain. Methods for producing such sequence variants are known in the art; see, e.g., Sambrook et al., Molecular cloning: a laboratory manual / Sambrook, Joseph; Russell, David W. — . 3rd ed. — New York: Cold Spring Harbor Laboratory, 2001. Ausubel et al., Current Protocols in Molecular Biology.

[0433] Said sequence variant of the oligomerization domain is preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the specific nucleic acid sequence or amino acid sequence of the native or non-modified oligomerization domain, preferably over the entire length. It is particularly preferred that the said variant sequence is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequences depicted in any one of SEQ ID NO: 19 to 25. Preferably, the sequence identity is calculated over the entire length of the oligomerization domain. Evidently, said sequence variants of oligomerization domains are still capable of forming oligomers, such as dimers, trimer, tetramers, and so on, or have even better oligomerization properties than the wildtype, native or non-modified oligomerization domain.

[0434] In another preferred embodiment of the protein oligomer of the invention, the at least first and / or second monomer comprises or consists of a structure selected from the group consisting of

[0435] [RBD-]n(human Ig Fc) [-RBD]n(Formula I-a)

[0436] [RBD-linker-]n(human Ig Fc) [-RBD]n(Formula I-b)

[0437] [RBD-]n(human Ig Fc) [-linker-RBD]n(Formula I-c)

[0438] [RBD-linker-]n(human Ig Fc) [-linker-RBD]n(Formula I-d)

[0439] [RBD-oligomerization domain-]n(human Ig Fc) [-RBD]n(Formula I-e)

[0440] [RBD-]n(human Ig Fc) [-oligomerization domain-RBD]n(Formula I-f)

[0441] [RBD-oligomerization domain-]n(human Ig Fc) [-oligomerization domain-RBD]n(Formula I-g)

[0442] [RBD-oligomerization domain-linker-]n(human Ig Fc) [-RBD]n(Formula I-h)

[0443] [RBD-]n(human Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-i)

[0444] [RBD-oligomerization domain-linker-]n(human Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-j)

[0445] [RBD-linker-oligomerization domain-]n(human Ig Fc) [-oligomerization domain-RBD]n(Formula I- k)

[0446] [RBD-oligomerization domain-]n(human Ig Fc) [-oligomerization domain-linker-RBD]n(Formula 1-1)

[0447] [RBD-linker-oligomerization domain-]n(human Ig Fc) [-oligomerization domain-linker-RBD]n(Formula I-m)

[0448] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [-RBD]n(Formula I-n)

[0449] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [linker-RBD]n(Formula I-o)

[0450] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [-oligomerization domain-RBD]n(Formula I-p)

[0451] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-q)

[0452] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [-oligomerization domain-linker-RBD]n(Formula I-r)

[0453] [RBD-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0454] (Formula I-s)

[0455] [RBD-linker-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0456] (Formula I-t) [RBD-oligomerization domain-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-u)

[0457] [RBD-linker- oligomerization domain-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-v)

[0458] [RBD-oligomerization domain-linker-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-w), and

[0459] [RBD-linker-oligomerization domain-linker-]n(human Ig Fc) [-linker-oligomerization domain-linker- RBD] „

[0460] (Formula I-x).

[0461] Evidently, the oligomerization domain could be placed either directly in proximity to Fc, or after RBD, in the mentioned monomers.

[0462] The above formulas are indicated from N-terminus to C-terminus, and their constituents and meaning have been explained elsewhere herein. The at least first and second monomer can comprise or consists of the same structure. For example, both the at least first and second monomer can comprise or consists of [RBD-]i (human Ig Fc) [-RBD]i. Or the at least first monomer can differ from the second monomer, in the protein oligomer of the invention. For instance, the at least first can comprise or consists of [RBD-linker-] i (human Ig Fc) [-linker- RBD] i, and the second monomer can comprise or consist of [RBD-]2 (human Ig Fc) [- oligomerization domain-RBD]2.

[0463] As appreciated by those of skill in the art, also combinations of different receptor binding domains, or fragment thereof, or receptor binding motifs, as defined herein can be used, within one monomer, or within the at least two monomers of the protein oligomer of the invention.

[0464] As set forth elsewhere herein, the protein oligomers of the invention have been exemplified by the BioVac constructs. Production and characterization of BioVac constructs 001-012, 035- 043, and 055, including their use for active immunization, passive immunization and therapy and their superior properties is described in great detail, in co-pending PCT / EP2025 / 066857 and PCT / EP2025 / 066860, the disclosure content of which is incorporated herein by reference.

[0465] The BioVac constructs comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO. 1 to 12, 35 to 43, 55, and 80 to 111. For instance, SEQ ID NO. 1 to 12, 35 to 43, 55 bind to the SARS-CoV-2 viral receptor ACE2 on the target cell of a subject such as a human mucosal cell or a human epithelial cell, in the picomolar range, as determined by surface plasmon resonance (SRP) or pseudoviral neutralization. The dissociation constants (Kd) of some BioVac constructs are shown in co-pending PCT / EP2025 / 066857 and PCT / EP2025 / 066860. In addition, Figure 15 depicts homotetrameric and heterotetrameric HD-BioVac constructs named HD-BioVaclOO to 128 (SEQ ID Nos. 80 to 111). The knob-in-hole Fc(KiH) mutation was used to express up to four different receptor binding domains (RBDs) of Corona viruses or Influenza viruses in a single BioVac molecule. The STR mutation, analogous to LALAPG (see BioVac002), was used for FcRy silencing to confine immunogenicity solely to the RBD regions. These constructs are well-expressed in the CHO system and demonstrate high-affinity binding to their cognate receptors and high immunogenicity in the classical prime / boost immunization scheme; see Examples.

[0466] Means and methods and conditions for determination Kon / Koff / KD are well known in the art; see, e.g., https: / / www.bio-rad.com / webroot / web / pdf / lsr / literature / Bulletin_6044A.pdf or O’Shannessy et al., Anal Biochem. 1993 Aug l;212(2):457-68. doi: 10.1006 / abio.1993.1355.

[0467] In addition, the invention relates to a nucleotide sequence encoding the protein oligomer of the invention.

[0468] The invention further relates to any nucleic acid or nucleotide sequence encoding the protein oligomer of the invention, as well as any expression vector comprising said encoding nucleic acid sequence, or any host cell expressing the same.

[0469] Vaccine comprising the protein oligomer of the invention

[0470] The invention further relates to a vaccine comprising the protein oligomer of the invention, as defined herein.

[0471] In one embodiment, the protein oligomer of the invention can be administered as a vaccine per se.

[0472] In another embodiment of the vaccine of the invention, the vaccine further comprises one or more of the following: a pharmaceutically acceptable buffer, a pharmaceutically acceptable carrier, a surfactant, a preservative, a stabilizer, a mucosal drug delivery system, an adjuvant, or combinations thereof.

[0473] Accordingly, the protein oligomer of the invention can be present in a vaccine composition together with one or more buffer(s), carrier(s), surfactant(s), preservative(s), stabilizer(s), mucosal drug delivery system(s), diluent(s), excipient(s), additive(s) and / or adj uvant(s) that are pharmaceutically acceptable. The protein oligomer or vaccine of the invention is of a grade and purity suitable for administration to a subject as defined herein, preferably a human subject.

[0474] Stabilisers are employed to prevent alterations of the vaccine when exposed to e.g. heat, light, acidity or humidity. Non-limiting examples of often used stabilisers include monosodium glutamate (MSG) and 2-phenoxy ethanol.

[0475] Preservatives are typically added to prevent serious adverse side effects such as infection with bacteria or viruses grown in the vaccine during production or storage. Non-limiting examples of preservatives include antibiotics, formaldehyde, phenoxyethanol or thiomersal, which are usually added to vials of vaccine that contain more than one dose to prevent contamination and growth of potentially harmful bacteria / viruses.

[0476] Compositions comprising such pharmaceutically acceptable carriers, stabilisers and / or preservatives can be formulated by well known conventional methods. Pharmaceutically acceptable buffer(s), carrier(s), surfactant(s), preservative(s), stabilizer(s), diluent(s), excipient(s), additive(s) and / or adjuvant(s) are well described in the literature; see, e.g., "Remington’s Pharmaceutical Sciences" by E.W. Martin (18th ed., Mack Publishing Co., Easton, PA (1990), Pharmaceutical Manufacturing Handbook: Production and Processes; Editor(s): Shayne Cox Gad PH.D., D.A.B.T.; First published: 28 August 2007.

[0477] A mucosal drug delivery system as used herein can be nanoparticle-mediated drug delivery systems (Parvathaneni, V., Kulkami, N.S., Gupta, V. (2020). Current Status and Perspectives in Mucosal Drug Delivery of Nanotherapeutic Systems. In: Muttil, P., Kunda, N. (eds) Mucosal Delivery of Drugs and Biologies in Nanoparticles. AAPS Advances in the Pharmaceutical Sciences Series, vol 41. Springer, Cham. https: / / doi.org / 10.1007 / 978-3-030-3591Q-2_4), mucoadhesive drug delivery systems (Laffleur and Bernkop-Schnurch, NANOMEDICINE 2013, VOL. 8, NO. 12), nasal route for drug delivery (Mato, Int J Pharm. 2019 Dec 15;572: 118813. doi: 10.1016 / j.ijpharm.2019.118813).

[0478] Mucoadhesive drug delivery gives rapid absorption and good bioavailability due to its considerable surface area and high blood flow. Drug delivery across the mucosa bypasses the first-pass hepatic metabolism and avoiding the degradation of gastrointestinal enzymes. Mucoadhesive drug delivery systems are delivery systems which utilize the property of bioadhesion of certain polymers which become adhesive on hydration and hence can be used for targeting a drug to a particular region of the body for extended periods of time. Bioadhesion is an interfacial phenomenon in which two materials, at least one of which is biological, are held together by means of interfacial forces. The attachment could be between an artificial material and biological substrate, such as adhesion between a polymer and a biological membrane. In the case of polymer attached to the mucin layer of a mucosal tissue, the term “mucoadhesion” is used. Mucoadhesive drug delivery systems can be delivered by various routes such as buccal, nasal, ocular, gastro, vaginal, and rectal. Preferably, said delivery system is a nasal or oral or buccal delivery system, more preferably a nasal delivery system.

[0479] Adjuvants

[0480] As appreciated by those of skill in the art, adjuvants are included in vaccine formulations to enhance the immunogenicity and efficacy of vaccines such as the vaccine of the invention.

[0481] Preferaby, the adjuvant is selected from the group consisting of: MF59® (oil-in-water emulsion of squalene), mRNA, ds mRNA, one or more peptides for T cell response, a sting agonist (bis- (3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP or cdGMP)), dsDNA, ssRNA, GM-CSF, CSF, TNF alpha, interferons, CpG, TLR agonists, or combinations thereof, in the vaccine or protein oligomer of the invention. Said adjuvants are known in the art; see, e.g., Liang et al., Front Immunol. 2020 Nov 6;11 :589833. doi: 10.3389 / fimmu.2020.589833. eCollection 2020; Mekonnen et al., Expert Rev Vaccines. 2022 Jan;21(l):69-81. doi: 10.1080 / 14760584.2021.1991794. Epub 2021 Nov 1; Kayesh et al., Viruses. 2021 Nov 18;13(11):2302. doi: 10.3390 / vl3112302; Shi et al., Vaccine. 2019 May 27;37(24):3167-3178. doi: 10.1016 / j. vaccine.2019.04.055. Epub 2019 Apr 29; Batty et al., Adv Drug Deliv Rev. 2021 Feb; 169: 168-189. Published online 2020 Dec 13. doi: 10.1016 / j. addr.2020.12.006. Moreover, the mRNA adjuvant could be vaccination target specific e.g., entire SARS-COV-2 spike protein, short immunogenic fragments of the spike or other viral components like the nucleocapsid, and / or consist of other peptide epitopes to broaden the immune response towards induction of an effective T-cell immunity. For instance, Heitmann et al. (Nature. 2022 Jan;601(7894):617-622. doi: 10.1038 / s41586-021-04232-5) have reported a Covid-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins, combined with the Toll-like receptor 1 / 2 agonist XS 15 emulsified in Montanide ISA51 VG (adjuvant), aiming to induce profound SARS-CoV-2 T cell immunity to combat Covid-19. CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concernindependent T cell responses, supporting the ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.

[0482] Another example for an adjuvant is MF59®. MF59® is a squalene oil-in-water nanoemulsion adjuvant which has been licensed for use in pandemic and seasonal influenza vaccines in many countries. MF59® is safe and well tolerated in humans. MF59® -adjuvanted vaccination spares vaccine dose and enhances hemagglutination inhibiting antibodies against homologous and heterologous influenza virus strains. The mechanisms of MF59® involve rapid induction of chemokines, inflammatory cytokines, recruiting multiple immune cells, uric acid and benign apoptosis of certain innate immune cells. The adjuvant effects of MF59® on generating vaccinespecific isotype-switched IgG antibodies, effector CD8 T cells, and protective immunity were retained even in a CD4-deficient condition by inducing effective immune-competent microenvironment with various innate and antigen presenting cells in a mouse model. CD4- independent adjuvant effects of MF59® might contribute to improving the vaccine efficacy in children, the elderly, and immunocompromised patients as well as in healthy adults; see, e.g., Ko and Kang, Hum Vaccin Immunother. 2018; 14(12): 3041-3045. Published online 2018 Aug 29. doi: 10.1080 / 21645515.2018.1495301.

[0483] So the adjuvant MF59® (oil-in-water emulsion of squalene) can be administered with the protein oligomer or vaccine of the invention to enhance the immunogenicity and efficacy of the protein oligomer or vaccine of the invention, in one embodiment.

[0484] Further examples of adjuvants that can be used to this end are set out below.

[0485] For instance, ds mRNA can act like an adjuvant in the protein oligomer or vaccine of the invention. Cellular ds RNA sensors, such as TLR3 and the Rig-I-like receptors (RLRs) (i.e. RIG-I, MDA5, and LGP2), are important triggers for antiviral responses, such as antiviral IFN- alpha and IFN-beta. This can be exploited to enhance immune responses to the protein oligomer or vaccine of the invention; see, e.g., Swiecki et al., J Leukoc Biol. 2011 Oct; 90(4): 691-701. TLR3 is mainly expressed in hematopoietic cells, particularly dendritic cells and macrophages, but also in some stromal cells. TLR3 detects ds RNA, which gains access to the endosomal compartment by phagocytosis of virus-infected cells or apoptotic cell debris, internalization of antibodies bound to viruses, or autophagy. TLR3 transmits signals through the TRIF pathway, which results in the phosphorylation and nuclear translocation of IRF3 and the transcription and secretion of IFN-p. TLR3-TRIF signaling also activates NF-KB and the transcription of inflammatory cytokine genes. The specificity of TLR3 for ds RNA allows recognition of RNA viruses such as EMCV, influenza A virus, CVB, WNV, DV, reovirus, and rhinovirus. Moreover, TLR3 detects the DNA viruses MCMV and HSV-1, most likely through recognition of RNA intermediates, which may be generated during viral replication.

[0486] RIG-I and MDA5 are two cytosolic helicases induced by IFN-I in most cell types. RIG-I and MDA5 detect ds RNA intermediates that accumulate in the cytosol during viral replication and interact with the adaptor molecule IPS-1 (also known as mitochondrial antiviral-signaling protein, virus-induced signaling adapter, or Cardif). IPS-1 is localized on the mitochondria and recruits TRAF3, which activates TRAF family member-associated NF-KB-binding kinase 1 and IKK 8, leading to the phosphorylation and nuclear translocation of IRF3 and IRF7 and production of IFN-P and IFN-a. Additionally, IPS-1 associates with FADD protein and receptor-interacting protein- 1, which activate caspase-8 and caspase- 10, resulting in NF-KB activation and production of inflammatory cytokines. IPS-1 is also located on peroxisomes and facilitates rapid antiviral responses through IRF1.

[0487] RIG-I and MDA5 detect distinct ds RNA forms that differ in structure, length, and 5' cap structures. The distinct ligand preferences of the MDA5 and RIG-I receptors confer specific recognition of disparate viruses. RIG-I has been shown to detect paramyxoviruses (SeV, NDV, respiratory syncytial virus, and Measles virus); orthomyxovirus (influenza A and B viruses); rhabdovirus (VSV and Rabies virus); flavivirus (Japanese encephalitis virus, HCV, WNV, and DV); filovirus (Ebola virus); reovirus; and metapneumovirus. Recent studies have shown that RIG-I also recognizes DNA viruses by detecting RNA intermediates generated through the RNA polymerase Ill-mediated transcription of ds DNA. MDA5 detects picornaviruses such as EMCV, CVB, Mengo virus, and Theiler virus, as well as murine norovirus 1. MDA5 is also involved in the recognition of WNV, DV, reovirus, SeV, MHV, Measles virus, and LCMV.

[0488] LGP2 is another RLR that detects ds RNA. LGP2 does not contain any signaling domains and was initially thought to negatively regulate MDA5 and RIG-I. Accordingly, LGP2-deficient mice have more robust IFN-I responses following poly(EC) stimulation and VSV infection compared with WT mice. However, recent data have demonstrated that LGP2 may positively influence antiviral responses, as RLR-mediated IFN-I responses were impaired in mice lacking LGP2 or the LGP2 ATP -binding site.

[0489] Further known ds RNA sensors are TLR7 and TLR9.

[0490] Sequences for suitable adjuvants are well described in the art, as evident from the cited literature. The adjuvant as defined herein can be administered separately from the protein oligomer or vaccine of the invention, in one embodiment. In this embodiment, the adjuvant as defined herein can be, for instance, co-administered with the protein oligomer or vaccine of the invention by an appropriate administration route as specified herein.

[0491] In another embodiment, the adjuvant is associated with the at least first monomer or second monomer in the protein oligomer of the invention. Preferably, at least one RBD is linked enzymatically or chemically to the adjuvant, as defined herein, in the protein oligomer of the invention, when formulated as vaccine such as a mucosal vaccine.

[0492] The adjuvant can also be conjugated, e.g., to the disulfide bridges and glycosylation sites of the Fc, the linker, the oligomerization domain, in the monomer of the protein oligomer of the invention. The linker-adjuvant payload can be released up on binding to RBD-target cells (e.g. ACE2 expressing cells in the mucosa where the viral entry of SARS-CoV-2 would pathophysiologically occur) and endosomaly released via a cleavable linker, so that the adjuvant can be released from the protein oligomer or vaccine of the invention, e.g., in specific cell compartments, as explained elsewhere herein.

[0493] Many adjuvants that are effective by injection are not optimal for mucosal delivery, as set forth elsewhere herein. Advantageously, the protein oligomer or vaccine of the invention can be used in a combined approach in that it carries and targets one or more of the adjuvants defined herein, to cells expressing the receptor or co-receptor of the virus, such as ACE2 expressing cells in the case of SARS-CoV-2, and / or immune cells. For instance, ds mRNA, ds DNA, ss RNA, TLR agonist or sting agonist, T cell peptides or spike mRNA (Curevac) can act like adjuvants so that it is no longer necessary to find and test appropriate adjuvant approaches.

[0494] In addition, it is important to note that there are at least four different possibilities to conjugate up to four different payloads in the protein oligomers of the invention, due to (i) protein engineering (e.g. a Sortase recognition peptide at C-terminus RBD of HD-BioVac, (ii) a natural glutaminase site (in Ig Fc), (iii) a natural glycosylation site, and (iv) a chemical conjugation site (cysteine conjugation inter / intrachain that can be produced by a standard antibody-drug conjugate technique). The fifth possibility for conjugating a fifth payload could come from the oligomerization domain(s), in the monomer of the protein oligomer of the invention.

[0495] Protein oligomer or vaccine of the invention for use in active immunization and / or booster vaccination in a subject

[0496] In a preferred embodiment, the protein oligomer or vaccine of the invention is for use in active immunization and / or booster vaccination in a subject, as defined herein. Preferably, the subject is a human subject.

[0497] Preferably, said protein oligomer or vaccine of the invention is for use in inducing a mucosal immunity by intranasal, oral or deep inhalation. Applications of the protein oligomer or vaccine of the invention, for different immunization scenarios and based on the immunological status of a human subject could be, for example: (i) systemic immunization of a subject with pre-existing systemic immunity against respiratory virus infection;

[0498] (ii) mucosal immunization of a subject with pre-existing systemic immunity against respiratory virus infection;

[0499] (iii) systemic immunization of a subject with naive immunity; and / or

[0500] (iv) mucosal immunization of a subject with naive immunity.

[0501] A fifth approach in pandemic situation would be to induce a systemic immune response with i.m. administration of the protein oligomer or vaccine of the invention and to bridge the ideal time to second i.m. vaccination of approximately three months by repeated passive i.n. immunization with the protein oligomer or vaccine of the invention.

[0502] Advantageously, the protein oligomer or vaccine of the invention can be used for active vaccination or immunization (both terms are used interchangeably herein) in a subject, such as a human subject. This means that the protein oligomer of the invention comprising a receptor binding domain of a respiratory viral protein as defined herein, functions as an immunogen for eliciting an immune response against said receptor binding domain, in the subject. Preferably, both a mucosal and a systemic immune response is elicited, by the protein oligomer or vaccine of the invention. The mucosal immune response comprises the production of potent humoral IgG and IgA (slgAl) and cell mediated (T-cells, innate immune cells) in the upper respiratory tract and systemic memory T / B cells.

[0503] As demonstrated for the BioVac constructs comprising RBD from SARS-CoV-2 spike protein, both a mucosal and a systemic immune response could be elicited in mice and human after a combined intranasal and intramuscular administration of the HD-BioVac constructs. Advantageously, it has been found by the present inventors that the administration of the protein oligomer of the invention as exemplified by the HD-BioVac constructs emulates natural infection with respiratory viruses and induces both a systemic immune response, dominated by IgG, as well as a mucosal immune response in the upper respiratory tract that is dominated by IgA.

[0504] Accordingly, the protein oligomer or vaccine of the invention can be used, e.g., for active immunization to protect a subject against infection by respiratory viruses or any strains or descendent lineages or variants thereof, referred to herein. Thereby, said protein oligomer or vaccine protects subjects against the respiratory virus-associated diseases or symptoms.

[0505] The mucosal immune response induced by the protein oligomer or vaccine of the invention is induced preferably, by about 1.5-fold to about 100-fold, about 2-fold to about 50-fold, or about 3 -fold to about 30-fold, in the subject, compared to a control subject or to an assay index (threshold for positivity / reactivity) or to a baseline. The mucosal immune response is induced by the protein oligomer or vaccine of the invention, by at least about 1.5-fold, at least about

[0506] 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about

[0507] 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about

[0508] 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50- fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more, in the subject, in comparison to a control subject or to an assay index threshold for positivity / reactivity or to a baseline.

[0509] Preferably, the mucosal immune response is the production of IgA in blood or secretory IgAl (slgAl) in the upper respiratory tract, respectively; see e.g. Sterlin et al., SCIENCE TRANSLATIONAL MEDICINE, 7 Dec 2020, Vol 13, Issue 577. DOI: 10.1126 / scitranslmed.abd2223. As shown e.g. for BioVacOOl, IgA levels are increasing in blood as an early sign of mucosal response preceding the IgG response. Moreover, slgA could be detected in oral saliva (collection tubes) and nasal mucosa (swap) from 3 weeks post prime and elevated levels from 1 week post boost immunization.

[0510] The level of IgA such as secretory IgAl (slgAl) antibody is preferably increased by about 1.5- fold to about 100-fold, about 2-fold to about 50-fold, or about 3-fold to about 30-fold, by the protein oligomer or vaccine of the invention in the subject, compared to a control subject or to an assay index (threshold for positivity / reactivity) or to a baseline. The level of IgA (such as secretory IgAl (slgAl)) antibody is preferably increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more, in the subject, in comparison to a control subject or to an assay index (threshold for positivity / reactivity) or to a baseline, by the protein oligomer of the invention.

[0511] Preferably, the IgA is secretory IgAl (slgAl). Secretory IgAl (slgAl) is well known in the art; see e.g. Pietrzak et al., Int J Mol Sci. 2020 Dec; 21(23): 9254. Published online 2020 Dec 4. doi: 10.3390 / ijms21239254.

[0512] More preferably, the induction of the mucosal immune response by said protein oligomer or vaccine of the invention includes an increased level of IgA, preferably secretory IgAl (slgAl), and an increased level of IgG such as IgGl antibodies, in the subject.

[0513] The level of IgG such as IgGl antibody is preferably increased by about 1.5-fold to about 100- fold, about 2-fold to about 50-fold, or about 3-fold to about 30-fold, by the protein oligomer or vaccine of the invention in the subject, compared to a control subject or to an assay index (threshold for positivity / reactivity) or to a baseline. The level of IgG such as IgGl antibody is preferably increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more, in the subject, in comparison to a control subject or to an assay index (threshold for positivity / reactivity) or to a baseline, by the protein oligomer of the invention.

[0514] Preferably, the IgG is IgGl .

[0515] The lower human respiratory tract is thought to be mostly protected by IgG (IgGl is most prevalent), the main type of antibody in serum, which is transported into the lung. The upper respiratory tract is thought to be mostly protected by secretory IgAl (slgAl).

[0516] Natural infection with respiratory viruses induces both a systemic immune response, dominated by IgGl, as well as a mucosal immune response in the upper respiratory tract that is dominated by slgAl. This process can lead to sterilizing immunity for many respiratory viruses.

[0517] Intramuscular or intradermal vaccination leads in many cases to a strong induction of serum IgG but not to an induction of mucosal IgA. Although some IgG can also be found on the mucosal surfaces of the upper respiratory tract, the lack of slgA often leaves an individual vulnerable to infection of the upper respiratory tract.

[0518] For instance, many SARS-CoV-2 vaccine candidates in clinical development have been administered intramuscularly, and only a few of the vaccine candidates in development or available vaccines are designed to induce mucosal immunity.

[0519] Intranasal vaccination can efficiently induce mucosal antibody responses, thereby potentially providing sterilizing immunity in the upper respiratory tract. However, systemic immune responses are often lower after this type of vaccination.

[0520] Advantageously, the protein oligomer or vaccine of the invention induces both a systemic immune response, dominated by IgGl, as well as a mucosal immune response in the upper respiratory tract that is dominated by slgAl. Accordingly, the administration of the protein oligomer or vaccine of the invention can lead to immunity for many respiratory viruses, thereby providing optimal protection from viral infection.

[0521] The mucosal immune response induced by the protein oligomer or vaccine of the invention in the subject is preferably cross-reactive against two or more strains of a particular virus, such as coronavirus, specifically, two or more viral variants of concern of SARS-CoV-2, or SARS-CoV and SARS-CoV-2, or SARS-CoV and MERS, or SARS-CoV-2 and MERS, as defined herein.

[0522] The mucosal immune response induced by the protein oligomer or vaccine of the invention preferably includes an increased level of neutralizing antibodies in the subject, as compared to a subject not administered the protein oligomer or vaccine of the invention. The neutralizing antibodies induced by said protein oligomer or vaccine of the invention are preferably specific for SARS-CoV-2, or any of the other respiratory viruses referred to herein, or strains, descendent lineages or variants thereof, as defined herein. The neutralizing antibodies can provide protection against infection by said viruses, thereby preventing diseases and symptoms caused by said viruses. In yet a further embodiment, the protein oligomer or vaccine of the invention induces a cellular immune response in the vaccinated subject, in addition to the humoral or mucosal response. The induced cellular immune response can include eliciting a CD8+ T cell response that includes the production of cytokines, such as interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), or any combinations thereof.

[0523] In still a further embodiment, the cellular immune response induced by said protein oligomer or vaccine of the invention includes eliciting a CD4+ T cell response. In some embodiments, the CD4+ T cells can produce IFN-y, TNF-a, IL-2, or a combination of IFN-y and TNF-a. The cellular response could be measured functionally by ELISPOT against RBD or subunit peptides, T-Cell repertoire measured by T-Cell receptor sequencing (TCR-seq) or combined transcriptome and TCR analysis via single cell RNA sequencing (scRNAseq), among others.

[0524] Protein oligomer or vaccine of the invention for use in passive immunization in a subject

[0525] In another preferred embodiment, the protein oligomer or vaccine of the invention is for use in passive immunization in a subject, as defined herein.

[0526] The protein oligomers or vaccines of the invention can be used for prophylactic administration for avoiding or preventing respiratory viral infection, i.e. for passive immunization, in (a) subject(s) because they prevents efficiently the respiratory virus from binding to its receptor on viral target cells of a subject, thereby blocking viral entry into said target cells in the subject. Specifically, the protein oligomer or vaccine of the invention advantageously induces protective immune responses at the relevant mucosal sites of pathogen entry by mucosal delivery of said protein oligomer or vaccine. Accordingly, the protein oligomers or vaccines of the invention can be used to prevent or at least impede immunescape by natural variants or mutants of respiratory viruses as referred to herein, such as the SARS-CoV-2 virus, e.g. in pandemic situations. In a preferred embodiment, the use of the protein oligomer or vaccine of the invention for passive vaccination comprises:

[0527] (i) interfering with infection in subjects exposed to a respiratory virus as referred to herein. So the protein oligomer or vaccine of the invention can be used for post-exposure prophylaxis, for instance, for treating human subjects that have been in contact with respiratory virus infected human subjects;

[0528] (ii) preventing of respiratory virus infection of subjects prior to high risk exposure situations. Accordingly, the protein oligomer or vaccine of the invention can be used for pre-exposure prophylaxis, for instance, for treating human subjects that have to use transportation by train, bus or airplane, or that visit mass events like concerts, fairs, etc.;

[0529] (iii) avoiding or ameliorating of respiratory virus infection of a subject;

[0530] (iv) avoiding or reducing of respiratory virus transmission between human subjects, such as in pandemic situations; or

[0531] (v) establishing mucosal immunity in a subject. SARS-CoV-2 infection could be successfully blocked by the protein oligomers of the invention in mice and human, as expemplified by the BioVac constructs. Said constructs carry receptor binding domains (RBD) of the SI subunit of the spike protein of SARS-CoV-2. These receptor binding domains bind specifically to the cell receptor angiotensin converting enzyme 2 (ACE2) on mucosal and epithelial cells in mice and in human, thereby preventing infection of mice or human with SARS-CoV-2 virus.

[0532] The protein oligomers or vaccines of the invention provide a broad spectrum respiratory protection for the subject by preventing respiratory viral infection, thereby preventing symptoms associated with said respiratory viruses in the subject.

[0533] Preferably, the subject is a human subject if not indicated otherwise herein.

[0534] Pharmaceutical composition comprising the protein oligomer of the invention

[0535] The invention also relates to a pharmaceutical composition comprising the protein oligomer of the invention. Said pharmaceutical composition of the invention is preferably for use in treating or ameliorating a disease caused by the respiratory viruses referred to herein, preferably a respiratory disease. The symptoms and diseases caused by the respiratory viruses referred to herein have been described in connection with the description of said respiratory viruses as elsewhere in this text.

[0536] In a preferred embodiment, the pharmaceutical composition further comprises one or more of the following: a pharmaceutically acceptable buffer, a pharmaceutically acceptable carrier, a surfactant, a preservative, a stabilizer, a mucosal drug delivery system, an adjuvant, or combinations thereof.

[0537] The pharmaceutical composition may be suitable for any mode of administration whether oral or parenteral, by injection or by topical administration, by inhalation, intranasal spray or intraocular drops. More specifically, pulmonary, oral, transmucosal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as rectal, intrathecal, direct intraventricular, intravenous, intraocular injections or any other medically acceptable methods of administration may be considered as appropriate administration mode for the compositions.

[0538] Pharmaceutical compositions, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. The pharmaceutical compositions also include, but are not limited to, emulsions and liposome-containing formulations. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.

[0539] As noted above, any of the compositions may comprise pharmaceutically acceptable carriers, vehicles, adjuvants, excipients, or diluents. As used herein pharmaceutically acceptable carriers, vehicles, adjuvants, excipients, or diluents, are well known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and one which has no detrimental side effects or toxicity under the conditions of use.

[0540] The choice of a carrier will be determined in part by the particular active agent, as well as by the particular method used to administer the composition. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0541] Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Formulations include those suitable for immersion, oral, parenteral (including subcutaneous, intramuscular, intravenous, intraperitoneal, implantation for slow release and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The nature, availability and sources, and the administration of all such compounds including the effective amounts necessary to produce desirable effects in a subject are well known in the art.

[0542] Definition of subject

[0543] As used herein, “subject” refers to the target of administration of the protein oligomer or vaccine of the invention, e.g. an animal or human. The subject can be a vertebrate, such as a mammal, preferably a human. An animal as referred to herein can be a domestic animal such as s cat, dog, rabbit, fish, hamster or guinea pig, or a farm animal such as donkey, cow, sheep, horses, goat, Arabian camel, Bactrian camel, llama and alpaca, donkey, reindeer, water buffalo, yak, Bali cattle, and Mithan, and a pig. The term does not denote a particular age or sex of the subject. Subject can be used interchangeably with “individual” or “patient” (if human). More preferably, the subject is a human subject. Even more preferably, the human subject is selected from the group consisting of:

[0544] (i) a vaccination refractory human subject, preferably a non-seroconverted human subject;

[0545] (ii) a human subject under immunosuppression, such as a transplant patient, a chemotherapy patient, a patient with hematological malignancy or a hemodialysis patient; and

[0546] (iii) elderly persons of an age of 60 years or more, preferably of an age of 70 years or more, more preferably of an age of 80 years or more, with senescent immune system.

[0547] Routes of administration

[0548] In another further preferred embodiment of the protein oligomer or vaccine of the invention, said protein oligomer or vaccine is administered mucosally, preferably intranasally, and / or via the intramuscular route. Preferably, said protein oligomer or vaccine of the invention is coadministered mucosally, preferably intranasally, and via the intramuscular route.

[0549] As indicated elsewhere herein, the protein oligomer or vaccine of the invention can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions comprising the protein oligomer or vaccine of the invention can be administered to a subject as defined herein in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.

[0550] In this embodiment, the protein oligomer or vaccine of the invention is formulated for mucosal administration to a subject as defined herein. A mucosal administration as used herein means administration to mucosal epithelium, such as - without limitation - mucosal epithelium from lungs, intestines, trachea, colon, nasal tissue, vaginal tissue, uterine tissue, and ocular mucosa. In some embodiments, administering to a mucosal epithelium is a direct or indirect administration of the protein oligomer or vaccine of the invention to one or more of the mucosal epithelium described herein. Preferably, the mucosal administration as used herein is intranasal administration, i.e. administration to nasal epithelium.

[0551] Mucosal tissues (e.g. nasal, oral, ocular, rectal, vaginal tissues) cover a large surface of the body. Since many respiratory viral infections such as SARS-CoV-2 infection are initiated at mucosal sites, the present invention provides strategies for neutralising the infectious agent at these surfaces. Mucosal vaccination involves the administration of the protein oligomer or vaccine of the invention at one or more mucosal sites leading to induction of immune responses at the mucosal site of administration, other mucosal sites, and / or systemically. Delivery systems for vaccination by mucosal routes are known in the art and defined elsewhere herein; see, e.g., Ryan et al., TRENDS in Biotechnology Vol.19 No.8 August 2001, p. 293-304.

[0552] Intranasal vaccination of the protein oligomer or vaccine of the invention can have several advantages over conventional intramuscular vaccines, not least because it can generate strong immune responses at key sites of viral exposure such as the upper respiratory tract. Intranasal and oral administration of the protein oligomer or vaccine of the invention can be carried out using preferably spray devices or alternatively for drops single or multiple use syringes without needle or incorporation into a cream for topical application. Moreover, deeper inhalation could be achieved using nebulizers including the protein oligomer or vaccine of the invention.

[0553] Most intranasal applications are at a pH between 6.17-6.65, in the medical field.

[0554] Determination of baseline human nasal pH and the effect of intranasally administered buffers are described, e.g., in the study by Washington et al., International Journal of Pharmaceutics, Volume 198, Issue 2, 5 April 2000, Pages 139-146; see also https: / / doi.org / 10.1016 / S0378- 5173(99)00442-1. For instance, the average pH in the anterior of the nose was 6.40 (+0.11, -0.15 S.D.) The pH in the posterior of the nasal cavity was 6.27 (+0.13, -0.18 S.D.). So the average baseline human nasal pH is about 6.3.

[0555] In a preferred embodiment of intranasal administration of the protein oligomer or vaccine of the invention, a buffer with pH of 6.25-6.5 is used.

[0556] To this end, for example, isotonic sea salt solutions with dexpanthenol can be used, as nasal sprays. Such sprays can contain potassium dihydrogen phosphate and dipotassium hydrogen phosphate as buffers. The pH of such sprays is 6.2.

[0557] Instead of isotonic sea salt solutions, potassium monohydrogen phosphate (Ph.Eur.) and potassium dihydrogen phosphate can be used as buffer, in such nasal sprays, pH 6.5. Water is added for injection.

[0558] 1ml nasal spray, solution contains 50 mg dexpanthenol. One spray (corresponding to 0.14ml nasal spray, solution) contains 7 mg dexpanthenol.

[0559] The protein oligomer or vaccine of the invention is preferably also suitable for further modes of administration. Possible modes of administration include, for example, oral or ophthalmic (lacrimal channel) administration, by injection or by topical administration e.g. cervix / uterus, by inhalation, or intraocular drops. More specifically, pulmonary, oral, transmucosal, intestinal or parenteral delivery, including intramuscular, subcutaneous, intradermal and intramedullary injections as well as rectal, intrathecal, direct intraventricular, intravenous, intraocular injections or any other medically acceptable methods of administration may be considered as appropriate administration mode for said protein oligomer or vaccine of the invention, depending on the subject.

[0560] As set forth above, it is preferred that the protein oligomer or vaccine of the invention is coadministered mucosally, preferably intranasally, and via the intramuscular route

[0561] Accordingly, the protein oligomer or vaccine of the invention is formulated for intramuscular administration to a subject as defined herein. Preferably, the intramuscular administration is via injection.

[0562] The protein oligomer or vaccine of the invention suitable for injectable use includes sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. Typically, the final injectable form should be sterile and should be effectively fluid for easy syringeability. The compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0563] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations.

[0564] The administration mode for the protein oligomer or vaccine of the invention is advantageously a combination of intramuscular and intranasal administration, or a combination of subcute / intradermal administration and intranasal / oral administration or intramuscular, intranasal and deep-inhalation, or topical uptake and i.m. eliciting both a mucosal and a systemic immune response against the respiratory viruses as referred to herein, such as SARS- CoV-2, or strains or variants thereof, in the subject.

[0565] The protein oligomer or vaccine of the invention can be co-administered, or administered at different times, using the mentioned combinations of administration routes. The time interval is preferably for

[0566] (i) systemic immunization of a subject with pre-existing systemic immunity against respiratory virus infection;

[0567] (ii) mucosal immunization of a subject with pre-existing systemic immunity against respiratory virus infection;

[0568] (iii) systemic immunization of a subject with naive immunity;

[0569] (iv) mucosal immunization of a subject with naive immunity;

[0570] (v) a fifth approach in pandemic situation would be to induce a systemic immune response with i.m. administration of the protein oligomer or vaccine of the invention and to bridge the ideal time to second i.m. vaccination of approximately three months by repeated passive i.n. immunization with the protein oligomer or vaccine of the invention.

[0571] Dosages and Schemes of administration

[0572] The protein oligomer or vaccine of the invention is administered in an amount sufficient to induce an immune response, preferably a mucosal and systemic immune response, in the subject as defined herein. An amount adequate to accomplish this, is defined as “effective dose”. Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of a disease of the subject, the general state of health of the subject, and the judgment of the physician.

[0573] The dose of the said protein oligomer or vaccine of the invention may range between <lpg to Img for i.m. administration based on the adjuvant / adjuvant free usage, and / or <10pg to 1g for i.n. application based on adjuvant / adjuvant free active ingredient, specifically, the said protein oligomer of the invention / kg body weight / time.

[0574] The protein oligomer or vaccine of the invention can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of doses of protein oligomer or vaccine of the invention for immunization may be

[0575] I, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0576] Tests of effect of vaccination

[0577] The efficacy of the protein oligomer or vaccine of the invention can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that the protein oligomer or vaccine of the invention is efficacious in inhibiting a respiratory virus infection in a subject by observing that the neutralizing antibodies induced thereby reduce viral load or delays or prevents a further increase in viral load. Viral loads can be measured by methods that are known in the art, for example, using PCR assays to detect the presence of respiratory viral nucleic acid such as SARS-CoV-2 nucleic acid, or antibody assays to detect the presence of respiratory viral protein such as SARS-CoV-2 protein in a sample (e.g., blood, serum, or another body fluid) from a subject, or by measuring the level of circulating anti-respiratory virus antibodies such as anti-SARS-CoV-2 antibodies in the subject. Further assays include, e.g. the pseudoviral neutralization assay (see e.g. Nie, J., Li, Q., Wu,

[0578] J. et al. Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay. Nat Protoc 15, 3699-3715 (2020)), or biochemical neutralization serum competition with respiratory virus receptor binding domain binding.

[0579] All embodiments, definitions and explanations with respect to the protein oligomer of the invention apply mutatis mutandis to a vaccine comprising said protein oligomer of the invention, or a vaccine comprising said protein oligomer of the invention.

[0580] Definitions and further embodiments of the invention

[0581] The term “polynucleotide” or “nucleic acid” as used herein refers to single- or double-stranded DNA molecules as well as to RNA molecules. Encompassed by the said term is genomic DNA, cDNA, hnRNA, mRNA as well as all naturally occurring or artificially modified derivatives of such molecular species. The polynucleotide may be in an aspect a linear or circular molecule. Moreover, in addition to the nucleic acid or nucleotide sequence encoding the protein oligomer of the invention, a polynucleotide may comprise additional sequences required for proper transcription and / or translation such as 5'- or 3 -UTR sequences. In light of the degeneracy of the genetic code, optimized codons may be used in the nucleic acid sequences encoding the monomers of the protein oligomer of the invention. Thereby, optimal expression in, e.g., a eukaryotic host cell such as Chinese hamster ovary (CHO) cells can be achieved. Numerous codon-optimization programs and commercial services are available; see, e.g. Richardson SM, et al. GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome research. 2006;16:550-556. Villalobos A, et al. Gene Designer: a synthetic biology tool for constructing artificial DNA segments. BMC bioinformatics. 2006;7:285. Gao W, et al. UpGene: Application of a web-based DNA codon optimization algorithm. Biotechnology progress. 2004;20:443-448. Jayaraj S, et al. GeMS: an advanced software package for designing synthetic genes. Nucleic acids research. 2005;33:3011-3016. Wu G, et al. The Synthetic Gene Designer: a flexible web platform to explore sequence manipulation for heterologous expression. Protein Expr Purif. 2006;47:441-445. Bode M, et al. TmPrime: fast, flexible oligonucleotide design software for gene synthesis. Nucleic acids research. 2009;37:W214-221. Raab D, et al. The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Systems and synthetic biology. 2010;4:215-225. Gaspar P, et al. EuGene: maximizing synthetic gene design for heterologous expression. Bioinformatics. 2012;28:2683- 2684. Angov E, et al. Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host. PloS one. 2008;3:e2189. Fuglsang A. Codon optimizer: a freeware tool for codon optimization. Protein Expr Purif. 2003;31 :247-249. Qian W, et al. Balanced codon usage optimizes eukaryotic translational efficiency. PLoS genetics. 2012;8:el002603. Hatfield GW, Roth DA. Optimizing scaleup yield for protein production: Computationally Optimized DNA Assembly (CODA) and Translation Engineering. Biotechnology annual review. 2007;13:27-42. Gustafsson C, et al. Engineering genes for predictable protein expression. Protein expression and purification. 2012;83:37-46.

[0582] The invention further encompasses any nucleic acid or nucleotide sequence encoding the protein oligomer of the invention, as well as any expression vector comprising said encoding nucleic acid sequence, or any host cell expressing the same. As used herein, the term “polynucleotide” or a “nucleic acid sequence” refers to a polymer of nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As used herein, “nucleic acid” (also or nucleic acid molecule or polynucleotide) refers to any DNA or RNA polynucleotides, oligonucleotides, fragments generated by the polymerase chain reaction (PCR) and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action, either single- or double-stranded. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., alpha-enantiomeric forms of naturally-occurring nucleotides), or modified nucleotides or any combination thereof. Herein this term also encompasses a cDNA, i.e. complementary or copy DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).

[0583] The term “protein” or “polypeptide” or “(poly)peptide” or “peptide” (all terms are used interchangeably, if not indicated otherwise) as used herein encompasses isolated and / or purified (poly)peptides being essentially free of other host cell polypeptides. The term “protein” or “polypeptide” or “(poly)peptide” or “peptide” as referred to herein comprises at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or even more amino acid residues where the alpha carboxyl group of one is bound to the alpha amino group of another. A post-translational modification of the protein or peptide as used and envisaged herein is the modification of a newly formed protein or peptide and may involve one or more deletion(s), substitution(s), inversion(s) or addition(s) / insertion(s) of amino acids, or combinations thereof, chemical modification of certain amino acids, for example, amidation, acetylation, phosphorylation, glycosylation, formation of pyroglutamate, oxidation / reduction of sulfa group on a methionine, or addition of similar small molecules to certain amino acids, or addition of tags, such as labeling tags (e.g. fluorescent proteins or sitespecific labeling with chemical probes etc.) or purification / affinity tags (e.g., his (polyhistidine), FLAG, GST, and Myc tags) to certain amino acids.

[0584] The term “protein” or “polypeptide” or “(poly)peptide” or “peptide” as used herein encompasses peptidomimetics. Protein or peptide modifications as used herein include synthetic embodiments of (poly)peptides described herein. In addition, analogs (non-peptide organic molecules), derivatives (chemically functionalized (poly)peptide molecules obtained starting with the disclosed (poly)peptide sequences) and variants (homologs) of these proteins can be utilized in the means and methods and medical uses described herein. Each (poly)peptide of this disclosure is comprised of a sequence of amino acids, which may be either L- and / or D- amino acids, naturally occurring and otherwise. (Poly)peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same biological activity as the unmodified (poly)peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, can be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a Cl -Cl 6 ester, or converted to an amide of formula NR1R2 wherein R1 and R2 are each independently H or Cl -Cl 6 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups of the polypeptide, whether amino-terminal or side chain, can be in the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or can be modified to Cl -Cl 6 alkyl or dialkyl amino or further converted to an amide. Hydroxyl groups of the polypeptide side chains may be converted to Cl -Cl 6 alkoxy or to a Cl -Cl 6 ester using well-recognized techniques. Phenyl and phenolic rings of the polypeptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with Cl -Cl 6 alkyl, Cl -Cl 6 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the polypeptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the (poly)peptides of this invention to select and provide conformational constraints to the structure that result in enhanced stability.

[0585] In this connection, an “isolated polynucleotide” is a nucleic acid molecule that is separated from the genome of an organism. For example, a DNA molecule that encodes the protein oligomer of the invention or any derivative, variant, fragment or fusion protein thereof that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.

[0586] An “isolated polypeptide” is thus a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. By definition, isolated peptides are also non- naturally occurring, synthetic peptides. Methods for isolating or synthesizing peptides of interest with known amino acid sequences are well known in the art; see Sambrook et al., Molecular cloning : a laboratory manual / Sambrook, Joseph; Russell, David W. — . 3rd ed. — New York: Cold Spring Harbor Laboratory, 2001. Ausubel et al., Current Protocols in Molecular Biology. For instance, the protein oligomer of the invention is an isolated polypeptide.

[0587] An amino acid residue comprises an amino terminal part (NH2; N-) and a carboxy terminal part (COOH; C-) separated by a central part (R group) comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group. NH2 refers to the amino group present at the amino terminal end of an amino acid or (poly)peptide, and COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or (poly)peptide. The generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids of standard nomenclature are listed in 37 C.F.R. 1.822(b)(2). Examples of non-natural amino acids are also listed in 37 C.F.R. 1.822(b)(4), other non-natural amino acid residues include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues. Naturally occurring amino acids may be further modified, e.g. hydroxyproline, y-carb oxy glutamate, and O-phosphoserine. Thus, the protein oligomer of the invention may comprise natural or non-natural amino acid residues, or any combination thereof.

[0588] Further, amino acids may be amino acid analogs or amino acid mimetics. Amino acid analogs refer to compounds that have the same fundamental chemical structure as naturally occurring amino acids, but modified R groups or modified peptide backbones, e.g. homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0589] Further, the protein oligomer of the invention may comprise “equivalent amino acid residues”. This term refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and / or functionality of the polypeptide. Equivalent amino acids thus have similar properties such as bulkiness of the side-chain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic / aliphatic). As such, equivalent amino acid residues can be regarded as conservative amino acid substitutions.

[0590] In the context of the present invention, within the meaning of the term “equivalent amino acid substitution” as applied herein, is meant that in certain embodiments one amino acid may be substituted for another within the groups of amino acids indicated herein below: i) Amino acids having polar side chains (Asp, GIu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys); ii) Amino acids having non-polar side chains (Gly, Ala, Vai, Leu, He, Phe, Trp, Pro, and Met); iii) Amino acids having aliphatic side chains (Gly, Ala Vai, Leu, He); iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro); v) Amino acids having aromatic side chains (Phe, Tyr, Trp); vi) Amino acids having acidic side chains (Asp, GIu); vii) Amino acids having basic side chains (Lys, Arg, His); viii) Amino acids having amide side chains (Asn, Gin); ix) Amino acids having hydroxy side chains (Ser, Thr); x) Amino acids having sulphur-containing side chains (Cys, Met); xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr); xii) Hydrophilic, acidic amino acids (Gin, Asn, GIu, Asp), and xiii) Hydrophobic amino acids (Leu, He, Vai).

[0591] The invention further encompasses any derivatives, enantiomers, analogues, variants or homologues of any of the protein oligomer of the invention or other (poly)peptides disclosed herein. The term “derivative” is used to define amino acid sequences (polypeptide), with any insertions, deletions, substitutions and modifications to the amino acid sequences (polypeptide) that do not alter the activity of the original polypeptides. By the term “derivative” it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of a polypeptides made according to the present invention. The protein oligomer of the invention or the other polypeptides disclosed herein such as the receptor binding domain (RBD) or receptor binding motif (RBM) can be coupled (conjugated) through any of their residues to another peptide or agent. For example, the protein oligomer of the invention or the other polypeptides disclosed herein can be coupled through their N- terminus to a lauryl-cysteine (LC) residue and / or through their C-terminus to a cysteine (C) residue. Further, the protein oligomer of the invention or the other polypeptides disclosed herein may be extended at the N-terminus and / or C-terminus thereof with various identical or different amino acid residues. As an example for such extension, the polypeptide may be extended at the N-terminus and / or C-terminus thereof with identical or different amino acid residue / s, which may be naturally occurring or synthetic amino acid residue / s. An additional example for such an extension may be provided by polypeptides extended both at the N-terminus and / or C- terminus thereof with a cysteine residue. Naturally, such an extension may lead to a constrained conformation due to Cys-Cys cyclization resulting from the formation of a disulfide bond. Another example may be the incorporation of an N-terminal lysyl-palmitoyl tail, the lysine serving as linker and the palmitic acid as a hydrophobic anchor. In addition, the polypeptides may be extended by aromatic amino acid residue / s, which may be naturally occurring or synthetic amino acid residue / s, for example, a specific aromatic amino acid residue may be tryptophan. The polypeptides may be extended at the N-terminus and / or C-terminus thereof with various identical or different organic moieties, which are not naturally occurring or synthetic amino acids. As an example for such extension, the protein oligomer of the invention or the other polypeptides disclosed herein may be extended at the N-terminus and / or C-terminus thereof with an N-acetyl group.

[0592] The invention also encompasses any homologues of the polypeptides specifically defined by their amino acid sequence according to the invention.

[0593] “Homology” as used herein refers to biological features including genes and their products that are descended from a feature present in a common ancestor. Homologous features such as genes are referred to as homologs (or homologues if one follows British spelling).

[0594] Homologous genes become separated in evolution in two different ways: separation of two populations with the ancestral gene into two species or gene duplication of the ancestral gene within a lineage. Genes separated by speciation are called orthologs (orthologues). Genes separated by gene duplication events are called paralogs.

[0595] The term “homologues” is used to define amino acid sequences (polypeptide) which maintain a minimal homology to the amino acid sequences defined by the invention, e.g. preferably have at least about 50%, 60%, 65%, more preferably at least about 70%, at least about 75%, even more preferably at least about 80%, at least about 85%, most preferably at least about 90%, at least about 95% overall sequence homology with the amino acid sequence of any of the polypeptide as structurally defined herein, e.g. of a specified sequence, more specifically, an amino acid sequence of the polypeptides as denoted by any one of SEQ ID Nos. 1 to 12, or 35 to 43, or 55, or 80 to 111 and any derivatives, enantiomers and fusion proteins thereof.

[0596] More specifically, “homology” with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N-nor C- terminal extensions nor insertions or deletions shall be construed as reducing sequence identity or homology. Methods and computer programs for the alignment are well known in the art.

[0597] Amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0598] Certain commonly encountered amino acids which also provide useful substitutions include, but are not limited to, P-alanine (P-Ala) and other omega-amino acids such as 3 -aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; a-aminoisobutyric acid (Aib); s-aminohexanoic acid (Aha); 5-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGIy); ornithine (Om); citrulline (Cit); t-butyl alanine (t-BuA); t-butylglycine (t- BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (NIe); naphthylalanine (Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenyl alanine (Phe(2-F)); 3 -fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); P-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2,4- diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH.sub.2)); N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids (e.g., N-substituted glycine). Covalent Modifications of Amino Acids and the Peptide.

[0599] Covalent modifications of the (poly)peptide are included and may be introduced by reacting targeted amino acid residues of the (poly)peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

[0600] Cysteinyl residues most commonly are reacted with a-haloacetates (and corresponding amines) to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, a-bromo-P-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2- oxa-l,3-diazole. Histidyl residues are derivatized by reaction with diethylprocarbonate (pH 5.5- 7.0) which agent is relatively specific for the histidyl side chain. Bromophenacyl bromide also is usefu...

Claims

Claims1. A protein oligomer comprising at least a first monomer and a second monomer, said at least first and second monomer comprising, in N- to C-terminal order, at least one first receptor binding protein (RBD), an immunoglobulin Fc (Ig Fc), and at least one second receptor binding protein (RBD), wherein a) the Ig Fc has enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, compared to wildtype Ig Fc; and b) the receptor binding domain (RBD) is selected from the group consisting of i) the receptor binding domain (RBD) from the spike protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), wherein the receptor binding domain comprises amino acid residues 367 to 606 of UniProt accession number K9N5Q8, or SEQ ID NO. 69, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 367 to 606 of UniProt accession number K9N5Q8, or to SEQ ID NO. 69; ii) the receptor binding domain (RBD) from influenza virus hemagglutinin (HA), wherein the receptor binding domain comprises amino acid residues 63 to 286 of GenBank accession number ACQ99608, or an amino acid sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 77, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 63 to 286 of GenBank accession number ACQ99608, or to an amino acid sequence selected from the group consisting of SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, and SEQ ID NO. 77; iii) the receptor binding domain (RBD) from parainfluenza virus hemagglutinin-neuraminidase (HN), wherein the receptor binding domain comprises amino acid residues 54 to 572 of GenBank accession number AY283063, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 54 to 572 of GenBank accession number AY283063; iv) the receptor binding domain (RBD) from rhinovirus capsid protein VP1, wherein the receptor binding domain is encoded by nucleotides 2305 to 3126 of GenBank accession number EF582385.1, or an nucleotide sequence at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to nucleotides 2305 to 3126 of GenBank accession number EF582385.1 encoding a receptor binding domain (RBD) from rhinovirus capsid protein VP1; v) the receptor binding domain (RBD) from human adenovirus fiber protein, wherein the receptor binding domain comprises amino acid residues 386-581 of UniProt accession numberPl 1818, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 386-581 of UniProt accession number Pl 1818; vi) the receptor binding domain (RBD) from respiratory syncytial virus glycoprotein G, wherein the receptor binding domain comprises amino acid residues 64-298 of UniProt accession number P03423, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 64-298 of UniProt accession number P03423; vii) the receptor binding domain (RBD) from human metapneumovirus fusion (F) protein, wherein the receptor binding domain comprises amino acid residues 66 to 87 contained within the F2 fragment of pre-fusion F protein (LIKTELDLTKSALRELRTVSAD) (SEQ ID NO. 56), or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 56; viii) the receptor binding domain (RBD) from parvovirus B19 VP1 unique region, wherein the receptor binding domain comprises amino acid residues 5 to 68 of UniProt accession number Q9JGS0, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 5 to 68 of UniProt accession number Q9JGS0; ix) the receptor binding domain (RBD) from SARS-CoV (or SARS-CoV-1) spike protein, wherein the receptor binding domain comprises amino acid residues 306 to 527 of GenBank accession number ABF65836.1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 306 to 527 of GenBank accession number ABF65836.1; x) the receptor binding domain (RBD) from SARS-CoV-2 spike protein, wherein the receptor binding domain comprises amino acid residues 320 to 541 of GenBank accession number QHD43416.1, or SEQ ID NO. 13, or SEQ ID NO. 14, or SEQ ID NO. 78, or SEQ ID NO. 79, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to amino acid residues 320 to 541 of GenBank accession number QHD43416.1, or to SEQ ID NO. 13, or SEQ ID NO. 14, or SEQ ID NO. 78, or SEQ ID NO. 79; xi) the receptor binding domain (RBD) from hCoV-NL63 spike protein, wherein the receptor binding domain comprises SEQ ID NO. 70, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 70; xii) the receptor binding domain (RBD) from HCoV-OC43 spike protein, wherein the receptor binding domain comprises SEQ ID NO. 75 or 76, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 75 or 76; xiii) the receptor binding domain (RBD) from NeoCoV spike protein, wherein the receptor binding domain comprises SEQ ID NO. 71, the receptor binding domain (RBD) from PDF- 2180 spike protein, wherein the receptor binding domain comprises SEQ ID NO. 72, thereceptor binding domain (RBD) from HKU5-CoV-l spike protein, wherein the receptor binding domain comprises SEQ ID NO. 73, or the receptor binding domain (RBD) fromHtHKU5-CoV- 2 or HtHKU5-CoV-2-441 spike protein, wherein the receptor binding domain comprises SEQ ID NO. 74, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO. 71, 72, 73, or 74.

2. The protein oligomer of claim 1, wherein i) the receptor binding domain (RBD) from the spike protein of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) binds to Dipeptidyl peptidase IV (DPP4), or CD26 (UniProt P27487); ii) the receptor binding domain (RBD) from influenza virus hemagglutinin binds to sialic acidcontaining proteins or sialylated glycoconjugates; iii) the receptor binding domain (RBD) from parainfluenza virus hemagglutininneuraminidase (HN) binds to sialic acid-linked receptors or sialylated glycoconjugates; iv) the receptor binding domain (RBD) from rhinovirus capsid protein VP1 binds to intercellular adhesion molecule 1 (ICAM-1) receptor (UniProt P05362), very low density lipoprotein (VLDL)-receptor (VLDL-R) (UniProt Q9Y679) or Cadherin Related Family Member 3 (CDHR3) (UniProt Q6ZTQ4); v) the receptor binding domain (RBD) from human adenovirus fiber protein binds to coxsackie and adenovirus receptor (CAR) (UniProt P78310), membrane cofactor protein (MCP) or CD46 (UniProt P15529), GDla glycan, polysialic acid, or desmoglein-2 (DSG-2) (UniProt Q 14126); vi) the receptor binding domain (RBD) from respiratory syncytial virus glycoprotein G binds to CX3C motif chemokine receptor 1 (CX3CR1) (UniProt P49238), heparan sulfate proteoglycans (HSPG), or Nucleolin (UniProt Pl 9338); vii) the receptor binding domain (RBD) from human metapneumovirus fusion (F) protein binds to heparan sulfate, glucosaminoglycan, or integrin a5pi (UniProt P08648; P05556); viii) the receptor binding domain (RBD) from parvovirus B19 VP1 unique region binds to glycosphingolipid (GSL) globoside (Gb4), or tyrosine protein kinase receptor UFO (AXL) (UniProt P30530); ix) the receptor binding domain (RBD) from SARS-CoV spike protein binds to angiotensinconverting enzyme 2 (ACE2) (UniProt Q9BYF1); x) the receptor binding domain (RBD) from SARS-CoV-2 spike protein binds to angiotensinconverting enzyme 2 (ACE2) (UniProt Q9BYF1);xi) the receptor binding domain (RBD) from from hCoV-NL63 spike protein binds to angiotensin-converting enzyme 2 (ACE2) (UniProt Q9BYF1); xii) the receptor binding domain (RBD) from HCoV-OC43 spike protein binds to sialoside receptor; xiii) the receptor binding domain (RBD) from NeoCoV spike protein, PDF-2180 spike protein, HKU5-CoV-l spike protein, HtHKU5-CoV-2 spike protein or HtHKU5-CoV-2-441 spike protein binds to angiotensin-converting enzyme 2 (ACE2) (UniProt Q9BYF1) or bat orthologues thereof, or to Dipeptidyl peptidase IV (DPP4), or CD26 (UniProt P27487).

3. The protein oligomer of claim 1 or 2, wherein Ig Fc is selected from the group consisting of IgG Fc, IgA Fc, and IgM Fc, preferably IgGl Fc or IgG3 Fc or IgG4 Fc, more preferably IgGl Fc.

4. The protein oligomer of any one of claims 1 to 3, wherein Ig Fc is a homodimer or a heterodimer, preferably wherein the heterodimer comprises Fc domains from knobs-into-holes (KiH)-engineered IgG, more preferably from knobs-into-holes (KiH)-engineered IgGl, most preferably from knobs-into-holes (KiH)-engineered human IgGl, or heterodimeric Fc variants selected from the group consisting of HA-TF, ZW1, DD-KK, 7.8.60, SEED, EW-RVT, and A107.

5. The protein oligomer of any one of claims 1 to 4, wherein enhanced affinity of Ig Fc for the neonatal Fc receptor (FcRn) at mucosal pH, compared to wildtype Ig Fc, is mediated by (i) DHS mutations (L309D / Q311H / N434S in human IgGl Fc), (ii) YTE (M252Y / S254T / T256E in human IgGl Fc), (iii) LS mutations (N428L / N434S in human IgGl Fc), (iv) KF mutations (H433K / N434F in human IgGl Fc), or (v) DE mutations (S239D / I332E in human IgGl Fc), in the Ig Fc.

6. The protein oligomer of any one of claims 1 to 5, wherein the Ig Fc further comprises LALAPG mutations (L234A / L235A / P329G in human IgGl Fc), LALA mutations (L234A / L235A in human IgGl Fc), or STR mutations (L234S / L235T / G236R in human IgGl Fc), for ablating Fc-Fc gamma receptor-mediated effector functions, without essentially affecting affinity for Fc gamma receptor, preferably wherein the Fc gamma receptor is selected from the group consisting of FcgammaRI, FcgammaRIIa, FcgammaRIIc, FcgammaRIIIa, and FcgammaRIIIb.

7. The protein oligomer of claim 6, wherein the Fc-Fc gamma receptor-mediated effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and / or inflammation via the induction of cytokine secretion.

8. The protein oligomer of any one of claims 1 to 7, wherein the RBD or fragment thereof is the same or different, (i) in the at least first monomer of the protein oligomer, or (ii) in the atleast second monomer of the protein oligomer, or (iii) both in the at least first monomer and in the at least second monomer of the protein oligomer.

9. The protein oligomer of any one of claims 1 to 8, wherein (i) said at least first monomer or (ii) said at least second monomer or (iii) both said at least first and said at least second monomer further comprise(s) a) at least one linker and / or b) at least one oligomerization domain and / or c) at least one, or two, three, four, five, six, seven, eight, nine, or ten additional RBD.

10. The protein oligomer of claim 9, wherein the linker is an independently selected variable linker amino acid sequence, preferably wherein the linker has reduced sensitivity to protease cleavage, more preferably wherein the linker comprises or is the linker (GGGGS)i (SEQ ID NO. 33) or (GGGGS)2(SEQ ID NO. 49) or GGGGGSGGGGS (SEQ ID NO. 54), and the reduced sensitivity to protease cleavage is in comparison to (GGGGS)s (SEQ ID NO. 50), or the linker comprises a Fc hinge region-derived linker.

11. The protein oligomer of claim 9 or 10, wherein the oligomerization domain is selected from the group consisting of: The non-triple helical trimerization domain of human collagen 18, the C-terminal oligomerization domain of human C4b-binding protein, coiled coils, oligomeric mini-proteins, short peptides with discrete protein-like structures, trimerization domain of the bacteriophage T4 fibritin (foldon), TNFalpha trimerization domain, zinc finger or p53 tetramerization domain.

12. A vaccine comprising the protein oligomer of any one of claims 1 to 11.

13. The vaccine of claim 12, for passive vaccination and / or active vaccination in a subject, or for use in passive vaccination and / or active vaccination in a subject, preferably in a human subject.

14. A pharmaceutical composition comprising the protein oligomer of any one of claims 1 to 11, preferably for use in treating or ameliorating a disease caused by the respiratory viruses referred to in claim 1, preferably a respiratory disease.

15. The vaccine of claim 12 or 13, or the pharmaceutical composition of claim 14, further comprising one or more of the following: a pharmaceutically acceptable buffer, a pharmaceutically acceptable carrier, a surfactant, a preservative, a stabilizer, a mucosal drug delivery system, an adjuvant, or combinations thereof.

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