Protein oligomers for active immunization

Abstract

The present invention pertains to protein oligomers for active immunization. Specifically, the invention relates to protein oligomers comprising at least a first monomer and a second 5 monomer, said at least first and second monomer comprising, in N- to C-terminal order, at least one first RBD, an immunoglobulin Fc (Ig Fc), and at least one second RBD, wherein a) Ig Fc has enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, compared to wildtype Ig Fc; and b) RBD is a receptor binding domain from or derived from SARS-CoV-2 spike protein, wherein the receptor binding domain comprises the amino acid sequence of SEQ ID NO. 13, or a fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO. 13. The invention further relates to a vaccine comprising said protein oligomer, preferably for use in active immunization and / or booster vaccination in a subject.

Description

[0001] Protein oligomers for active immunization

[0002] The present invention pertains to protein oligomers for active immunization. Specifically, the invention relates to protein oligomers 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 RBD, an immunoglobulin Fc (Ig Fc), and at least one second RBD, wherein a) Ig Fc has enhanced affinity for the neonatal Fc receptor (FcRn) at mucosal pH, compared to wildtype Ig Fc; and b) RBD is a receptor binding domain from or derived from SARS-CoV-2 spike protein, wherein the receptor binding domain comprises the amino acid sequence of SEQ ID NO. 13, or a fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO. 13. The invention further relates to a vaccine comprising said protein oligomer, preferably for use in active immunization and / or booster vaccination in a subject.

[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 two four now, the COVID- 19 pandemic has been ongoing. As of February 2024, there have been more than 774 million confirmed cases of COVID-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. Due to the ongoing and fast emergence of new variants, 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.

[0004] In active immunization, vaccines stimulate the immune system to produce a protective immune response, which mimics the host’s response to a natural infection. The immunity after active immunization lasts for months to many years depending on the nature of the vaccine, the type of immune response and host factors.

[0005] In view of the ongoing SARS-CoV-2 pandemic, novel strategies have been attempted to induce long-lasting active immunization. In one approach, a monomeric lipopeptide fusion inhibitor against SARS-CoV-2 has been generated that reduces viral transmission by blocking membrane fusion between the viral and host cell membranes. These peptides correspond to the highly conserved heptad repeat (HR) domain at the C terminus of the SARS-CoV-2 transmembrane spike glycoprotein (S) and can inhibit fusion by preventing the required conformational rearrangement in S, including refolding steps that leads directly to membrane fusion and viral entry (see De Vries RD, Schmitz KS, Bovier FT et al. „Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS.VoV-2 transmission in ferrets”. Science (2021), vol. 371, issue 6536, pp. 1379-1382, DOI: 10.1126 / science.abf4896). In another study, Wang et al. showed effective neutralization of SARS-CoV-2 by engineered dimeric IgA antibodies (see Wang Z, Lorenzi JCC, Muecksch F et al. „Enhanced SARS-CoV-2 neutralization by dimeric IgA”. Science Translational Medicine (2020), vol. 13, issue 577, DOI: 10.1126 / scitranslmed.abfl555.). Cohen et al. developed mosaic nanoparticles that display the receptor-binding domains (RBDs) from SARS-CoV-2 and seven other animal sarbecoviruses. These nanoparticle constructs showed improved protection against both SARS-CoV-2 and SARS-CoV challenges in animal models (see Cohen AA, Van Doremalen N, Greaney AJ et al. “Mosaic RBD nanoparticles protect against challenge by diverse sarbecoviruses in animal models”. Science (2022), vol. 377, issue 6606, DOI. 10.1126 / science.abq0839).

[0006] Further approaches include also the search of effective adjuvants alongside vaccines. For instance, Lei et al. developed cationic nanocarriers as potent adjuvants for recombinant S-RBD vaccine of SARS-CoV-2 that showed strongly increased IgM and IgG titers in serum (Lei H, Alu A, Yang J et al. “Cationic nanocarriers as potent adjuvants for recombinant S-RBD vaccine of SARS-CoV-2”. Signal Transduction and Targeted Therapy 5, 291 (2020), https : / / doi. org / 10.1038 / s41392-020-00434-x).

[0007] In addition, different administration routes have been tested by several groups. Wong et al. for example developed a prototype two-dose vaccine (BReC-CoV-2) by combining the receptor binding domain (RBD) antigen via conjugation to Diphtheria toxoid (EcoCRM). When administered intranasally (i.n.), the vaccine was capable to effectively protect mice against lethal SARS-CoV-2 challenge (see Wong TY, Lee KS, Russ BR et al. “Intranasal administration of BReC-CoV-2 COVID-19 vaccine protects K18-hACE2 mice against lethal SARS-CoV-2 challenge”. Nature, npj Vaccines 7, 36(2022), https: / / doi.org / 10.1038 / s41541- 022-00451-7). Similarly, Lei et al. developed an adjuvanted intranasal RBD vaccine, which induced and maintained high levels of neutralizing IgG antibodies in the sera for at least 1 year after administering this vaccine in a three-dose intranasal immunization scheme (see. Lei H, Alu A, Yang J et al. “Intranasal administration of a recombinant RBD vaccine induces longterm immunity against Omicron-included SARS-CoV-2 variants. Signal Transduction and Targeted Therapy 7, 159 (2022). https: / / doi.org / 10.1038 / s41392-022-01002-l). Another group developed a chimeric triple-RBD-based mucosal vaccine harbouring one Delta RBD and two Omicron RBDs within a novel protein scaffold. Intranasal immunization with these constructs elicited coordinated mucosal IgA and higher neutralizing antibody specificity against the Omicron variant (see Yang J, Liu MQ, Liu L et al. “A triple RBD-based mucosal vaccine provided broad protection against SARS-CoV-2 variants of concern”. Cellular & Molecular Immunology 19, 1279-1289 (2022), https: / / doi.org / 10.1038 / s41423-022-00929-3).

[0008] Although these strategies to induce active immunization showed remarkable success, there is still a dire need for the development of novel vaccines that could meet the constant global demand for effective vaccines.

[0009] The technical problem underlying the present invention could be seen as the provision of means and methods, which comply with the aforementioned needs. This technical problem has been solved by the embodiments characterized in the claims and herein below.

[0010] Summary of the invention

[0011] 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 domain (RBD), an immunoglobulin Fc (Ig Fc), and at least one second receptor binding domain (RBD), wherein a) 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 is from or derived from SARS-CoV-2 spike protein, wherein the receptor binding domain comprises the amino acid sequence of SEQ ID NO. 13, or a fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO. 13. Preferably, the sequence identity is calculated over the entire length of the RBD or fragment thereof.

[0012] 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.

[0013] The protein oligomer of the invention is particularly suitable for use as a vaccine, preferably a mucosal vaccine, against the SARS-CoV-2 coronavirus, and can be advantageously used for active immunization and / or booster vaccination in a subject, as demonstrated in the Examples.

[0014] 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 angiotensin 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) (with only two N-Glycosylation sites N331 / 343). Later studies confirmed that >90% of neutralizing antibodies in SARS-CoV-2 infected humans are indeed directed against this RBD domain (Piccoli et al., Cell, 2020). The RBD sequence used in the protein oligomers and vaccines of the invention included amino acid 320-541. The corresponding amino acid sequence is shown in SEQ ID NO. 13. The rationale behind this decision to have a slightly larger fragment as originally assigned RBD region was to conserve the protein structure as much as possible e.g., by preserving structurally relevant disulfide bridges (including C391-C525) and further include potentially relevant candidate immunogenic epitopes at extended N-and C-terminal regions. Surprisingly, clinical translation of a parallel development of an mRNA-based vaccine design (BNT162bl) targeting RBD failed due to enhanced toxicity compared to the whole spike approach in early phase human trials (Walsh et al.,N Engl J Med, 2020). Interestingly, to increase immunogenicity, multivalent display of the RBD antigen expressed by BNT162bl was achieved by the addition of the bacteriophage T4 fibritin-derived foldon trimerization domain (Mulligan et al., Nature, 2020). This strategy was shown to be fruitful for SARS-CoV-1 protein-based vaccines published in (Li et al., Viral Immunol, 2013). However, later studies indicated potential immunogenicity of foldon trimerization domain (Sliepen et al., J Biol Chem, 2015). This might, among others, provide a plausible explanation for the surprising inferior toxicity profile of the foldon-RBD based BNT162bl vs. entire spike BNT162b2 approach. In contrast, BioVac works with a tetrameric RBD both at N- and C-terminus of an IgGl Fc domain compared to 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 obvious that combined N- and C-terminal fusion of RBD to the Fc molecule will result into efficient expression of a highly functional and stable protein - the BioVac. Moreover, BioVac 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. Finally, 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 BioVac in-vivo persistence and therefore improved stability, 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 substation was hijacked for mucosal adaptation. To the inventors’ knowledge, this is the first report on a successful combination of DHS Fc within a tetrameric Fc fusion protein, 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. 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 18 disulfide bounds and 10 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, high BioVac protein expression titers were achieved 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 ACE2) and high immunogenicity due to multivalent display underscore the relevance of BioVac as a novel principle for active mucosal and systemic immunization.

[0015] In a 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, more preferably IgGl Fc.

[0016] Preferably, said Ig Fc is human Ig Fc, more preferably selected from the group consisting of human IgG Fc, human IgA Fc, and human IgM Fc, even more preferably human IgGl Fc or human IgG3 Fc, and most preferably human IgGl Fc.

[0017] In another 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 ofHA-TF, ZW1, DD-KK, 7.8.60, SEED, EW-RVT, and Al 07.

[0018] In a further preferred embodiment of the protein oligomer of the invention, enhanced affinity of Ig Fc for the neonatal Fc receptor (FcRn) at mucosal pH, in comparison 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), or combinations of said mutations in (i) to (v), in the Ig Fc.

[0019] In a still further 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.

[0020] 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.

[0021] In yet another preferred embodiment of the protein oligomer of the invention, the receptor binding domain (RBD) or fragment thereof is the same or different, (i) within the at least first monomer of the protein oligomer, or (ii) within the at least second monomer of the protein oligomer, or (iii) both within the at least first monomer and within the at least second monomer of the protein oligomer.

[0022] Preferably, the RBD or fragment thereof is from one or more variants of concern (VOC), selected from the group consisting of SARS-CoV-2 variant alpha (B. l.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 and SARS-COV-2 variant omicron BA.2.87.1.

[0023] In a further 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 receptor binding domain(s) (RBD), preferably as defined herein.

[0024] 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 (GGGGSfi (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).

[0025] 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 and p53 tetramerization domain.

[0026] In still another preferred embodiment of the protein oligomer of the invention, the protein oligomer comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1 to 12, or 35 to 43 or 55, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NO. 1 to 12, or 35 to 43, or 55. Preferably, the sequence identity is calculated over the entire length of the protein oligomer of the invention.

[0027] The invention further relates to a vaccine comprising the protein oligomer of the invention.

[0028] In a preferred 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.

[0029] Preferably, the adjuvant is selected from the group consisting of 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, and combinations thereof.

[0030] Preferably, the adjuvant is associated with said at least first monomer or said at least second monomer in the protein oligomer, more preferably at least one RBD is linked enzymatically or chemically to the adjuvant.

[0031] In another preferred embodiment of the vaccine of the invention, said vaccine is administered intranasally (i.n.) and / or via the intramuscular (i.m.) route, preferably said vaccine is coadministered intranasally and via the intramuscular route.

[0032] In still another preferred embodiment of the vaccine of the invention, said vaccine is for use in active immunization in a subject, preferably for use in active immunization in a human subject.

[0033] Preferably, said vaccine is for use in

[0034] (i) systemic immunization of a human subject with pre-existing systemic immunity against SARS-CoV-2 infection;

[0035] (ii) mucosal immunization of a human subject with pre-existing systemic immunity against SARS-CoV-2 infection;

[0036] (iii) systemic immunization of a human subject with naive immunity; or

[0037] (iv) mucosal immunization of a human subject with naive immunity. In yet another embodiment of the vaccine of the invention, said vaccine is for use in booster vaccination in a subject, preferably a human subject.

[0038] Preferably, the human subject is selected from the group consisting of:

[0039] (i) a vaccination refractory human subject, preferably a non-seroconverted human subject;

[0040] (ii) a human subject under immunosuppression, such as a transplant patient, a chemotherapy patient, a patient with hematological malignancy or a hemodialysis patient; and

[0041] (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.

[0042] Detailed description of the protein oligomer of the invention

[0043] 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.

[0044] Immunoglobulin Fc (Ig Fc)

[0045] The center is formed by an immunoglobulin (Ig) Fc (crystallizable fragment) domain, in the monomer of the protein oligomer of the invention.

[0046] 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.

[0047] 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. For instance, the IgG Fc can be human IgGl Fc or human IgG3 Fc, in this case.

[0048] The Ig Fc domain can be a monomeric Ig Fc domain, or the Ig Fc domain can be capable of dimerization.

[0049] 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, in connection with the ablation of the two disulfide bridges in human IgGl Fc and the corresponding entire hinge region. 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).

[0050] The Ig Fc domain can form a homodimer or a heterodimer, in the protein oligomer of the invention.

[0051] Dimerization in the Ig Fc can be, for instance, via homodimerization.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Dimerization in the Ig Fc can also be via heterodimerization.

[0057] 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).

[0058] 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.

[0059] 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.

[0060] 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).

[0061] 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.

[0062] 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; see Figures 9 and 10. 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 5.3 to 7.0, preferably of about 5.5 to 6.6, more preferably of about 6.3 to 6.5, or most preferably 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 that 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 anti-drug 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.

[0063] 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).

[0064] 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, 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 physiological pH such as in extracellular space like lamina propria of the mucosa or serum pH (about pH 7.35-7.4), 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.

[0065] The Fc gamma receptor can be selected from the group consisting of FcgammaRI, FcgammaRIIa, FcgammaRIIc, FcgammaRIIIa, and FcgammaRIIIb.

[0066] 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).

[0067] 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; see Figures 9 and 10. 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 in patients with post vaccine or infection (post / long-Covid) syndromes.

[0068] Receptor binding domain (RBD)

[0069] A “receptor binding domain (RBD)” as used herein is a structural and functional unit in a 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 non-limiting example of a fragment of a receptor binding domain (RBD) as used herein is a receptor binding motif (RBM). 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 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.

[0070] 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.

[0071] To give a specific example, 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).

[0072] For instance, it has been found thatHeLa cells expressing ACE2 are susceptible to SARS-CoV- 2 infection whereas those without ACE2 are not. In vitro binding measurements also showed that the SARS-CoV-2 receptor binding domain binds to ACE2 with an affinity in the low nanomolar range, indicating that the receptor binding domain is a key functional component within the SI subunit that is responsible for binding of SARS-CoV-2 by ACE2.

[0073] 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.

[0074] 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)).

[0075] 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. l009153. 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.

[0076] Figure 1 shows the structure of the SARS-CoV-2 spike protein.

[0077] The amino acid sequence of the SARS-CoV-2 spike protein and its receptor binding domain is 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:

[0078] 10.1080 / 21645515.2020.1740560.

[0079] 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.

[0080] 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).

[0081] 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).

[0082] SEQ ID NO. 15 depicts the amino acid sequence of human ACE2. If not indicated otherwise, “ACE2” as used herein means human ACE2.

[0083] SEQ ID NO. 16 depicts the amino acid sequence of human Neuropilin- 1.

[0084] 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 of the 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 recombinant coronavirus S protein receptor-binding domain vaccine for severe / acute 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. Specifically, the receptor binding domain (RBD) as used herein is a receptor binding domain from or derived from SARS-CoV-2 spike protein, or a fragment of said receptor binding domain, wherein the receptor binding domain comprises or consists of the amino acid sequence of SEQ ID NO. 13, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity to SEQ ID NO. 13. Preferably, the sequence identity is calculated over the entire length of the RBD or fragment thereof. Preferably, the fragment of the receptor binding domain or the amino acid sequence having at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity to SEQ ID NO. 13 is able to bind to ACE2 in the nanomolar range, such as with a dissociation constant Kd of between 1 nM and 990 nM, or 50 nM to 500 nM, or even in the picomolar range, more preferably with a dissociation constant of between 0.1 pM and 990 pM, 1 pM and 950 pM, 10 pM and 900 pM, 20 pM and 800 pM, 30 pM and 700 pM or 40 pM to 600 pM, or 50 pM and 500 pM, as determined by surface plasmon resonance (SRP). Preferably, the fragment of the receptor binding domain has a length of about 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, or 70 amino acid residues.

[0085] One example for a fragment of the receptor binding domain (RBD) (SEQ ID NO. 13) is the receptor binding motif (RBM). The corresponding amino acid sequence of the RBM is shown in SEQ ID NO. 14. It has a length of 68 amino acid residues. The receptor binding motif (RBM) as used herein is preferably a receptor binding motif from or derived from SARS-CoV-2 spike protein, or a fragment of said receptor binding motif, wherein the receptor binding motif comprises or consists of the amino acid sequence of SEQ ID NO. 14, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity to SEQ ID NO. 14. Preferably, the sequence identity is calculated over the entire length of the RBM or fragment thereof.

[0086] 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.

[0087] As one could learn from the Covid- 19 pandemic, a variant of concern denotes a viral variant which seems to pose a greater threat to public health due to enhanced transmissibility or infectivity.

[0088] 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 characterization of specific “Variants of Interest” (VOIs) and “Variants of Concern” (VOCs), in order to prioritize global monitoring and research, and ultimately to inform the ongoing response to the COVID-19 pandemic.

[0089] 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 amino acid changes.

[0090] 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.

[0091] 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, 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 and SARS-COV-2 variant omicron BA.2.87.1.

[0092] 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 viral protein(s) used to bind to the target cell and the target cell. Methods for identification of receptor binding domains in viral proteins such as the SARS-CoV-2 spike protein, binding to receptors such as ACE2 or neuropilin- 1 (NRP-1), in the target cell include, for instance, yeast two-hybrid system, mutational analysis, protein microarrays, X-ray crystallography, flow cytometric 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).

[0093] A fragment of the receptor binding domain (RBD) (SEQ ID NO. 13) as used herein comprises at least 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, or 70 amino acid residues of the receptor binding domain, and is still capable of binding to the viral receptor ACE2 or neuropilin- 1 (NRP-1), on the target cell of a subject, such as a mucosal cell or epithelial cell, thereby preventing the binding of the SARS-CoV-2 virus to said target cell and / or mediating the uptake of the protein oligomer of the invention into said target cell of the subject. One non-limiting example of a fragment of a receptor binding domain (RBD) is a receptor binding motif (RBM).

[0094] The expression “a receptor binding domain derived from SARS-CoV-2 spike protein” as used herein encompasses also variants of a receptor binding domain from SARS-CoV-2 spike protein. Said expression can comprise a receptor binding domain from a SARS-CoV-2 spike protein of specific “Variants of Interest” (VOIs) and “Variants of Concern” (VOCs), as defined herein. A variant of a receptor binding domain can be, e.g., as sequence variant, such as a sequence variant of a receptor binding domain from a SARS-CoV-2 spike protein of specific “Variants of Interest” (VOIs) and “Variants of Concern” (VOCs), as defined 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 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. The above explanations and embodiments apply mutatis mutandis to the expression “a receptor binding motif derived from SARS-CoV- 2 spike protein” as used herein.

[0095] 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. It is particularly preferred that the said sequence variant of the receptor binding domain or receptor binding motif 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 SEQ ID NO. 13 or 14, over the entire length of the receptor binding domain or receptor binding motif.

[0096] Methods for producing and identifying functional variants of receptor binding domains or receptor binding motifs of viral proteins such as the SARS-CoV-2 spike protein 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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 ACE2 or neuropilin on the SARS-CoV-2 viral target cell of a subject, such as a mucosal cell or epithelial cell, thereby preventing the binding of the SARS-CoV-2 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 ACE2 receptor in the SARS-CoV-2 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.

[0101] Monomer 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:

[0102] N-terminus [first RBD] (Ig Fc) [second RBD] C-terminus

[0103] The receptor binding domain (RBD) and the immunoglobulin Fc (Ig Fc) are defined elsewhere herein.

[0104] 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.

[0105] 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.

[0106] The “linker” as used herein can be, for instance, a variable linker amino acid sequence.

[0107] 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, 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)i (SEQ ID NO. 33) or (GGGGS)? (SEQ ID NO. 49) has reduced sensitivity to protease cleavage, in comparison to (GGGGS)s (SEQ ID NO. 50). SEQ ID NOs. 17, 18, 34 and 52 to 54 show further appropriate linker sequences. The linker can also be an Fc hinge region-derived linker; see, e.g., US patent 6,165,476. 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.

[0108] 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.

[0109] 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 reactogenicity 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.

[0110] 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 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. 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.

[0111] 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.

[0112] 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.

[0113] As 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.

[0114] 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.

[0115] 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 C-CAS Sting agonist) can be conjugated to an enzyme-cleavable linker, in the monomer of the protein oligomer of the invention; see e.g. Figure 7. 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.

[0116] 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. 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.

[0117] 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.

[0118] 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:

[0119] N-terminus [RBD-Z-]n(Ig Fc, preferably human Ig Fc) [-Z-RBD]nC-terminus

[0120] (Formula I)

[0121] The receptor binding domain (RBD) and the immunoglobulin Fc (Ig Fc) are defined elsewhere herein.

[0122] As 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.

[0123] [RBD-Z-] moiety (at the N-terminus)

[0124] 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.

[0125] The [RBD-Z-] moiety can be structured as follows.

[0126] 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.

[0127] The linker is defined elsewhere herein.

[0128] 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, as illustrated in Figures 4 and 5.

[0129] The oligomerization domain is defined elsewhere herein. 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 ACE2 on the SARS-CoV-2 target cell of a subject, such as a human subject.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] [-Z-RBD] moiety (at the C-terminus)

[0134] 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.

[0135] The [-Z-RBD] moiety can be as follows.

[0136] 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.

[0137] The linker is defined elsewhere herein.

[0138] 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; see, e.g., Figures 4 and 5.

[0139] The oligomerization domain is specified elsewhere herein.

[0140] In view of the above, the skilled person will appreciate that the [-Z-RBD] moiety can comprise or consist of RBD only.

[0141] 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. 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.

[0142] [RBD-Z-] moiety (at the N-terminus) and [-Z-RBD] moiety (at the C -terminus)

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] These are non-limiting examples. Encompassed by the invention are any possible combinations comprised by Formula I, as defined herein.

[0150] “n”

[0151] “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.

[0152] Structure of the monomer

[0153] In the protein oligomer of the invention, the monomer can comprise or consist of a structure selected from:

[0154] [RBD-]n(Ig Fc) [-RBD]n(Formula I-a) [RBD-linker-]n(Ig Fc) [-RBD]n(Formula I-b)

[0155] [RBD-]n(Ig Fc) [-linker-RBD]n(Formula I-c)

[0156] [RBD-linker-]n(Ig Fc) [-linker-RBD]n(Formula I-d)

[0157] [RBD-oligomerization domain-]n(Ig Fc) [-RBD]n(Formula I-e)

[0158] [RBD-]n(Ig Fc) [-oligomerization domain-RBD]n(Formula I-f)

[0159] [RBD-oligomerization domain-]n(Ig Fc) [-oligomerization domain-RBD]n(Formula I-g)

[0160] [RBD-oligomerization domain-linker-]n(Ig Fc) [-RBD]n(Formula I-h)

[0161] [RBD-]n(Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-i)

[0162] [RBD-oligomerization domain-linker-]n(Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-j)

[0163] [RBD-linker-oligomerization domain-]n(Ig Fc) [-oligomerization domain-RBD]n(Formula I-k)

[0164] [RBD-oligomerization domain-]n(Ig Fc) [-oligomerization domain-linker-RBD]n(Formula 1-1)

[0165] [RBD-linker-oligomerization domain-]n(Ig Fc) [-oligomerization domain-linker-RBD]n(Formula I-m)

[0166] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [-RBD]n(Formula I-n)

[0167] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [linker-RBD]n(Formula I-o)

[0168] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [-oligomerization domain-RBD]n(Formula I-p)

[0169] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-q)

[0170] [RBD-linker-oligomerization domain-linker]n(Ig Fc) [-oligomerization domain-linker-RBD]n

[0171] (Formula I-r)

[0172] [RBD-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0173] (Formula I-s)

[0174] [RBD-linker-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0175] (Formula I-t)

[0176] [RBD-oligomerization domain-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0177] (Formula I-u)

[0178] [RBD-linker- oligomerization domain-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0179] (Formula I-v)

[0180] [RBD-oligomerization domain-linker-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-w)

[0181] [RBD-linker-oligomerization domain-linker-]n(Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-x) The above formulas are indicated from N-terminus to C-terminus.

[0182] Ig Fc as defined herein is preferably a human Ig Fc.

[0183] Particularly preferred is the core structure with the formula, from the N-terminus to the C- terminus:

[0184] [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.

[0185] RBD within one monomer

[0186] 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, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or even more receptor binding domains (RBDs).

[0187] The receptor binding domain (RBD) can be the same or different, in the monomer of the protein oligomer of the invention.

[0188] 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 variant alpha (B.1.1.7), in the at least first monomer of the protein oligomer of the invention.

[0189] Or the at least one first receptor binding domain can be from SARS-CoV-2 variant alpha (B. l.1.7), and the at least one second receptor binding domain can be from SARS-COV-2 variant omicron BA.5, in the at least first monomer of the protein oligomer of the invention.

[0190] The receptor binding domain (RBD) can be the same or different, in the at least second monomer of the protein oligomer of the invention.

[0191] 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 variant delta (B.1.617.2), in the at least second monomer of the protein oligomer of the invention.

[0192] Or the at least one first receptor binding domain can be from SARS-CoV-2 variant delta (B.1.617.2), and the at least one second receptor binding domain can be from SARS-COV-2 variant omicron B A.2, in the at least second monomer of the protein oligomer of the invention. It is envisaged by the invention, that the receptor binding domains can freely be 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.

[0193] 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. l.1.7), 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.

[0194] 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. l.1.7), 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 SARS-CoV-2 variant delta (B.1.617.2), in the at least second monomer of the protein oligomer of the invention.

[0195] Or the at least one first receptor binding domain can be from SARS-CoV-2 variant alpha (B. l.1.7), and the at least one second receptor binding domain can be from SARS-COV-2 variant omicron BA.5, 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 SARS-CoV-2 variant delta (B.1.617.2), in the at least second monomer of the protein oligomer of the invention.

[0196] To provide further non-limiting examples, the receptor binding domain can be from SARS- CoV-2 variant alpha (B. l.1.7) 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), at the C-terminus of the at least first monomer, or vice versa.

[0197] The receptor binding domain can be from SARS-CoV-2 variant omicron BA.5 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 SARS-CoV-2 variant omicron 2.75, at the C-terminus of the at least second monomer, or vice versa.

[0198] Or the receptor binding domain is from SARS-CoV2 variant omicron BA.l, at the N-terminus of the at least first monomer, and the receptor binding domain is from SARS-CoV-2 variant omicron BA.2, at the C-terminus of the at least first monomer, or vice versa.

[0199] And the receptor binding domain is from SARS-CoV2 variant omicron BA.5, at the N-terminus of the at least second monomer, and the receptor binding domain is from SARS-CoV-2 variant omicron BA.2.75, at the C-terminus of the at least second monomer, or vice versa.

[0200] Protein oligomer 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.

[0201] For instance, dimerization can be achieved by the Ig Fc. Oligomerization can be achieved by the usage of an oligomerization domain, as defined herein.

[0202] 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.

[0203] 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.

[0204] As will be appreciated by those of skill in the art, each monomer can differ from each other in number or structure of its constituents, for instance, in the number of linkers, oligomerization domains, adjuvants, and / or receptor binding domains, in the protein oligomer of the invention.

[0205] 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 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.

[0206] 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 SARS-CoV-2 viral target cell of a subject. The SARS-CoV- 2 viral target cell of the subject expresses the receptor ACE2 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 ACE2, in the picomolar range, as set forth elsewhere herein, (ii) Furthermore, protein oligomers formed by said variants have enhanced affinity for the neonatal Fc receptor (FcRn) at a pH of about pH 6.0 to 6.7, preferably about pH 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 SARS-CoV-2 infection, i.e. Covid-19 vaccination, of a subject, such as a human.

[0207] For instance, such variants of the protein oligomer of the invention comprise an amino acid sequence of any one of SEQ ID NO. 1 to 12, or 36 to 44, or 57, or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NO. 1 to 12, or 35 to 43, or 55. Preferably, the sequence identity is calculated over the entire length. Said variants of the protein oligomer of the invention are able to bind to ACE2, preferably human ACE2, in the picomolar range, more preferably with a dissociation constant (Kd) of between 0.1 pM and 990 pM, 1 pM and 950 pM, 10 pM and 900 pM, 20 pM and 800 pM, 30 pM and 700 pM or 40 pM to 600 pM, or 50 pM and 500 pM, as determined by surface plasmon resonance (SRP). The dissociation constants (Kd) for some BioVac constructs are shown in the Examples.

[0208] Encompassed by the invention are also peptidomimetics of the monomers of the protein oligomer of the invention.

[0209] 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 variant omicron BA.l, and a second protein oligomer of the invention can comprise the receptor binding domains of the spike protein from SARS-CoV-2 variant omicron BA.2. Such combinations of different protein oligomers of the invention can be used for active immunization of subjects, as described herein.

[0210] Method for producing the protein oligomer of the invention

[0211] 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.

[0212] As shown in the following examples, 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. 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] A 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] Properties and advantages of the protein oligomer of the invention

[0222] (i) Proof of concept for BioVac constructs The present inventors have developed several 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”, such as HD-Bio VacOOl (SEQ ID NO. 1), HD- BioVac002 (SEQ ID NO. 2), HD-Bio Vac004 (SEQ ID NO. 35), HD-BioVac006 (SEQ ID NO. 36) and HD-Bio Vac008A (SEQ ID NO. 37) and BioVac008B (SEQ ID NO. 38). Further sequences of BioVac constructs are shown in SEQ ID Nos. 3 to 12, 39 to 43, and 55, disclosed elsewhere herein.

[0223] Each of these constructs contains a „core“ structure as shown in Figure 1 and 2. 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 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.

[0224] 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; see e.g., Figures 12 and 13. 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.

[0225] 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 powerful immunogen for effective vaccination. Figure 11 shows expression efficacy, size and purity in HEK (Expi) expression system for construct HD-BioVac002.

[0226] 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 harboring 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 and in the Examples.

[0227] (ii) Multiple receptor binding domains in the protein oligomers of the invention

[0228] 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 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 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 Figure 14 and 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. Thus, the protein oligomers of the invention can advantageously be used for active immunization.

[0229] 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 / Figures.

[0230] Furthermore, the protein oligomers of the invention can be quickly 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.

[0231] (iii) Optimized Fc domains in the protein oligomers of the invention

[0232] 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; see Figure 10. 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. Further benefits of using DHS mutations in the protein oligomer of the invention are maintenance 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.

[0233] 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

[0234] 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).

[0235] 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.

[0236] 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.

[0237] 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 SARS-CoV-2; see, e.g., Florian Krammer, Nature (2020), Vol 586, p. 516-527.

[0238] 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.

[0239] 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; see Figure 15.

[0240] 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.

[0241] 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.

[0242] Further, the protein oligomers of the present invention are very safe with respect to toxicity, as demonstrated in human vaccinations.

[0243] 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 reactogenicity 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.

[0244] Finally, 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, nebulizer / nebulizator / inhalator to reach deeper respiratory regions, or the like.

[0245] 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.

[0246] Preferred embodiments

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] For example, an 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.

[0256] To provide another example, an 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 nonmodified or native human IgG3 Fc domain, over the entire length.

[0257] 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.

[0258] 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.

[0259] 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. In a preferred embodiment of the protein oligomer of the invention, Ig Fc is a homodimer or a heterodimer, as explained elsewhere herein.

[0260] 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.

[0261] 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 / pentamer 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, as set forth elsewhere herein; see Figures 6 and 7.

[0262] 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 7.35 to 7.40, preferably of about pH 7.4, in comparison to wildtype, non-modified or native Ig Fc.

[0263] 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. 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-h!gGl,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.

[0264] 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.

[0265] 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).

[0266] Table 1:

[0267] 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.

[0268] The used numbering is according to EU numbering; see, e.g., https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] The equilibrium dissociation constant (KD or Kd) of interaction of wildtype IgG with the neonatal Fc receptor (FcRn) at pH 5.8, is preferably between about 500 and 800 nM.

[0274] 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 .

[0275] 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 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.

[0276] 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 150 nM.

[0277] 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.

[0278] 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.

[0279] In another preferred embodiment, the Ig Fc has enhanced affinity for the neonatal Fc receptor (FcRn) at a mucosal pH of 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; see Figures 9 and 10.

[0280] 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.

[0281] 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; see Figure 9. 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 high affinity and specific 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 similarities to RBD as antigen. One example for such erroneous immune responses is the formation of autoantibodies etc. with undesired off target effects.

[0282] LALAPG (L234A, L235A, P329G) mutations in human IgGl are described, e.g., in Bailey et al., Nature Communications volume 9, Article number: 4560 (2018).

[0283] 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.

[0284] Preferably, said LALAPG mutations as used herein comprise L234A / L235A / P329G in human IgGl Fc.

[0285] 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.

[0286] 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.

[0287] 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). 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.

[0288] Preferably, 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 and SARS-COV-2 variant omicron BA.2.87.1.

[0289] 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 current 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 subtle structural alterations over viral evolution.

[0290] 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 current 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.

[0291] 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

[0292] 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.

[0293] 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.

[0294] 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 an 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 or 52 to 53.

[0295] 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.

[0296] 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.

[0297] 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.

[0298] 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:

[0299] [RBD-]n(human Ig Fc) [-RBD]n(Formula I-a)

[0300] [RBD-linker-]n(human Ig Fc) [-RBD]n(Formula I-b)

[0301] [RBD-]n(human Ig Fc) [-linker-RBD]n(Formula I-c)

[0302] [RBD-linker-]n(human Ig Fc) [-linker-RBD]n(Formula I-d)

[0303] [RBD-oligomerization domain-]n(human Ig Fc) [-RBD]n(Formula I-e)

[0304] [RBD-]n(human Ig Fc) [-oligomerization domain-RBD]n(Formula I-f)

[0305] [RBD-oligomerization domain-]n(human Ig Fc) [-oligomerization domain-RBD]n(Formula I-g)

[0306] [RBD-oligomerization domain-linker-]n(human Ig Fc) [-RBD]n(Formula I-h)

[0307] [RBD-]n(human Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-i)

[0308] [RBD-oligomerization domain-linker-]n(human Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-j)

[0309] [RBD-linker-oligomerization domain-]n(human Ig Fc) [-oligomerization domain-RBD]n(Formula I-k) [RBD-oligomerization domain-]n(human Ig Fc) [-oligomerization domain-linker-RBD]n(Formula 1-1)

[0310] [RBD-linker-oligomerization domain-]n(human Ig Fc) [-oligomerization domain-linker-RBD]n(Formula I-m)

[0311] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [-RBD]n(Formula I-n)

[0312] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [linker-RBD]n(Formula I-o)

[0313] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [-oligomerization domain-RBD]n(Formula I-p)

[0314] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [-linker-oligomerization domain-RBD]n(Formula I-q)

[0315] [RBD-linker-oligomerization domain-linker]n(human Ig Fc) [-oligomerization domain-linker-RBD]n(Formula I-r)

[0316] [RBD-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0317] (Formula I-s)

[0318] [RBD-linker-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n

[0319] (Formula I-t)

[0320] [RBD-oligomerization domain-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-u)

[0321] [RBD-linker- oligomerization domain-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-v)

[0322] [RBD-oligomerization domain-linker-]n(human Ig Fc) [-linker-oligomerization domain-linker-RBD]n(Formula I-w)

[0323] [RBD-linker-oligomerization domain-linker-]n(human Ig Fc) [-linker-oligomerization domain-linker- RBD] „

[0324] (Formula I-x)

[0325] Evidently, the oligomerization domain can be placed either directly in proximity to Fc, or after RBD, in the mentioned monomers.

[0326] 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] !, and the second monomer can comprise or consist of [RBD-]2 (human Ig Fc) [- oligomerization domain-RBD]2. 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.

[0327] 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.

[0328] In a particularly preferred embodiment of the protein oligomer of the invention, the protein oligomer comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO. 1 to 12, or 36 to 44, or 57, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NO. 1 to 12, or 35 to 43, or 55. Preferably, the sequence identity is calculated over the entire length. Preferably, said protein oligomer comprising the aforementioned amino acid sequence binds to the SARS- CoV-2 viral receptor ACE2 on the target cell of a subject such as a mucosal cell or an epithelial cell, in the picomolar range. Said protein oligomer of the invention or amino acid sequences with the indicated minimal percentage sequence identity are able to bind to ACE2, preferably human ACE2, preferably with a dissociation constant (Kd) of between 0.1 pM and 1 nM, 0.1 pM and 990 pM, 1 pM and 950 pM, 10 pM and 900 pM, 20 pM and 800 pM, 30 pM and 700 pM or 40 pM to 600 pM, or 50 pM and 500 pM, as determined by surface plasmon resonance (SRP) or an alternative method, such as pseudoviral neutralization. Preferably, the subject is a human. The dissociation constants (Kd) of some BioVac constructs are shown in the Examples.

[0329] 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.

[0330] In addition, the invention relates to a nucleotide sequence encoding the protein oligomer of the invention, preferably to a nucleotide sequence encoding the amino acid sequence of a protein oligomer selected from the group consisting of SEQ ID NO. 1 to 12, or 35 to 43, or 55, or a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a nucleotide sequence encoding an amino acid sequences of any one of SEQ ID NO. 1 to 12, or 35 to 43, or 55. Preferably, sequence identity is calculated over the entire length.

[0331] The invention 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.

[0332] Vaccine comprising the protein oligomer of the invention

[0333] The invention further relates to a vaccine comprising the protein oligomer of the invention, as defined herein. In one embodiment, the protein oligomer of the invention can be administered as a vaccine per se see e.g. basic immunization approaches in Figures 29 and 30.

[0334] 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.

[0335] 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.

[0336] Stabilizers are employed to prevent alterations of the vaccine when exposed to e.g. heat, light, acidity or humidity. Non-limiting examples of often used stabilizers include monosodium glutamate (MSG) and 2-phenoxy ethanol.

[0337] 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.

[0338] Compositions comprising such pharmaceutically acceptable carriers, stabilizers 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.

[0339] 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 / 978-3-030-35910-2_4k 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).

[0340] 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.

[0341] Adjuvants

[0342] 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.

[0343] Preferably, 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.

[0344] 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.

[0345] 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.

[0346] Further examples of adjuvants that can be used to this end are set out below, in the Examples and the Figures 6 to 8.

[0347] 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.

[0348] 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.

[0349] RIG-I and MDA5 are two cytosolic helicases induced by IFN 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 Cardiff 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.

[0350] 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 picomaviruses 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.

[0351] 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.

[0352] Further known ds RNA sensors are TLR7 and TLR9.

[0353] Sequences for suitable adjuvants are well described in the art, as evident from the cited literature.

[0354] 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 defined herein.

[0355] 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.

[0356] 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 would pathophysiologically occur) and endosomally 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.

[0357] 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 (via the Fc component). For instance, ds mRNA, ds DNA, ss RNA, TLR agonist or cGas / sting agonist, T cell peptides or spike mRNA (entire or immunogenic fragments thereof) can act like adjuvants so that it is no longer necessary to find and test appropriate adjuvant approaches; see Figures 7 and 8.

[0358] In addition, it is important to note that there are at least four different possibilities to conjugate up to four different payloads in the HD-BioVac constructs 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) chemical conjugation sites (cysteine conjugation inter- / intrachain that can be produced by a standard antibody-drug conjugate technique). The fifth possibility for conjugating a payload could come from the oligomerization domain(s), in the monomer of the protein oligomer of the invention.

[0359] Protein oligomer of the invention for use in active immunization and / or booster vaccination in a subject

[0360] 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.

[0361] 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 such as the BioVac constructs, for different immunization scenarios and based on the immunological status of a human subject could be, for example:

[0362] (i) systemic immunization of a subject with pre-existing systemic immunity against SARS- CoV-2 infection; this could be to broaden the immunity via SARS-COV-2 variant specific BioVac constructs to combat novel / feature variants of concern (VoC) towards generating broad neutralizing antibodies and deep antiviral immunity, or to generate immunity for otherwise less immunogenic variants using bi- or multivalent BioVac constructs consisting of an immunogenic and less-immunogenic RBD in the same molecule (as demonstrated for delta- omicron hybrid BioVac), or to generate neutralizing antibodies via induction of an effective protein immunogen based humoral immune response in otherwise immunosuppressed populations (e.g. elderly, transplant and cancer patients) not responding to different types of vaccines e.g. mRNA / Vector based immunizations, or to refresh or boost immune response generating high quality memory T- and B-cell responses with BioVac (± Fc-silenced, ± adjuvant free), or to overcome original antigenic sin (e.g. induced by repeated potent Wuhan whole spike mRNA vaccinations), or to restrain the immune response to specifically focus on RBD e.g. to reduce potential off-target autoimmune responses associated with highly glycosylated spike regions exposed by infection or prior vaccinations (post / long covid, post Vac).

[0363] (ii) mucosal immunization of a subject with pre-existing systemic immunity against SARS- CoV-2 infection; mucosal immunity is limited with all systemic immunization principles used so far, therefore, mucosal vaccination is required for “sterile” immunization preventing (re)infection, moreover; it is not clear how long mucosal immunity last even after mucosal immunization e.g., by an infection (depending on parameters like recognition of novel infectious agent by existing immunity) highlighting the need for repeated mucosal immunization especially in the infection seasons in analogy to flue / influenza and common “cold” seasons induced by rhino- / corona etc. virus families. Cross protection against other viral infection could also be envisioned due to local activation of innate immune response at mucosal infection portals e.g., nose, naso- / oro-pharynx.

[0364] (iii) systemic immunization of a subject with naive immunity; here at least two potential approaches are demonstrated, a fast “pandemic” scheme with only three weeks interval between prime and boost vs. an ideal immunization scheme with ~ 3 months interval between prime and booster immunization when the antibody response declines at least sufficiently and the immune response is mature to benefit most from a boost / refreshing and long-lasting memory immunization. and / or

[0365] (iv) mucosal immunization of a subject with naive immunity; the present inventors show that the presence of a systemic immunizing facilitates mucosal immunization efficacy tremendously in preclinical model, under these conditions BioVac forms an excellent immunogen by complexing e.g., with pre-existing antibodies to form a potent antibody-multivalent immunogen complex. However, adjuvanting BioVac as demonstrated by L3A enhances mucosal immunization efficacy which could be further strengthen by more specific and potent adjuvants such as the described conjugation (preferred) or mixing of BioVac with mRNA, TLR- and / or cGAS-STING agonists etc.

[0366] A fifth approach in pandemic situation would be to induce a systemic immune response with i.m. administration of BioVac and to bridge the ideal time to second i.m. vaccination of approximately three months by repeated passive i.n. immunization with BioVac.

[0367] 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 from or derived from SARS-CoV-2 spike 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 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.

[0368] As demonstrated in the following Examples, 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; see also e.g. Figures 16, 19, 20 and 22 (human) and Figure 27 (mouse). 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.

[0369] Accordingly, the protein oligomer or vaccine of the invention can be used, e.g., for active immunization to protect a subject against infection by SARS-CoV-2 viruses or any strains or descendent lineages or variants thereof, referred to herein. Thereby, said protein oligomer or vaccine protects subjects against the SARS-CoV-2-associated disease Covid- 19.

[0370] 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 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about

[0371] 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about

[0372] 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about

[0373] 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about

[0374] 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; see e.g. Figures 16, 19, and 20.

[0375] 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. in Fig. 16, 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.

[0376] 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. 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.

[0377] 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.

[0378] 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.

[0379] Preferably, the IgG is IgGl .

[0380] 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).

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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.

[0385] 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.

[0386] 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, as defined herein.

[0387] 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 strains, descendent lineages or variants thereof, as defined herein. The neutralizing antibodies can provide protection against infection by said virus, thereby preventing Covid- 19.

[0388] 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.

[0389] 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.

[0390] Definition of subject

[0391] 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).

[0392] As appreciated by the skilled person, there is no need for an Ig species switch if the protein oligomers of the invention are used in non- human subjects because binding and immunogenicity is via the receptor binding domain. So mice could be vaccinated with human Fc BioVac constructs, as shown in the Examples.

[0393] More preferably, the subject is a human subject. Even more preferably, the human subject is selected from the group consisting of: (i) a vaccination refractory human subject, preferably a non-seroconverted human subject;

[0394] (ii) a human subject under immunosuppression, such as a transplant patient, a chemotherapy patient, a patient with hematological malignancy or a hemodialysis patient; and

[0395] (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.

[0396] Routes of administration

[0397] 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.

[0398] 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.

[0399] 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.

[0400] Mucosal tissues (e.g. nasal, oral, ocular, rectal, vaginal tissues) cover a large surface of the body. Since many viral infections such as SARS-CoV-2 infection are initiated at mucosal sites, the present invention provides strategies for neutralizing 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.

[0401] 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.

[0402] Most intranasal applications are at a pH between 6.17-6.65, in the medical field.

[0403] 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.

[0404] 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.

[0405] 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.

[0406] 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.

[0407] 1ml nasal spray, solution contains 50 mg dexpanthenol. One spay (corresponding to 0.14ml nasal spray, solution) contains 7 mg dexpanthenol.

[0408] 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.

[0409] 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

[0410] 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.

[0411] 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.

[0412] 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.

[0413] 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 subcutaneous / 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 SARS-CoV-2 or strains or variants thereof, in the subject.

[0414] 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

[0415] (i) systemic immunization of a subject with pre-existing systemic immunity against SARS- CoV-2 infection; adjuvanted booster could be utilized if the level of immune response e.g. antibody titer is limited or below detection in immunocompromised individuals by repeated i.m. application, whereas the immune response to the first injection should be awaited up to 4 weeks e.g. by antibody titer and then subsequently boosted up to ideal neutralization titer is achieved. Of note, in elderly individuals without antibody response to SARS-COV-2, a single i.m. injection was sufficient if a supportive T-Cell immunity was present e.g., due to mRNA or vector vaccines. Adjuvanted or adjuvant free booster to broaden the immune repertoire e.g., against variant, i.m. injection could be administered on top of preexisting B / T-Cell response induced by previous immunization (vaccination or infection). Ideal time point is, however, to await the humoral response to decline back from the peak, allow immune maturation, usually between 3-6 months post last exposure.

[0416] (ii) mucosal immunization of a subject with pre-existing systemic immunity against SARS- CoV-2 infection; mucosal immunization e.g. by i.n. or oral (pharyngeal) administration could take place at any time, preferably if a humoral response is present e.g. at least 3 weeks after prime or 1 week after booster. At presence of a humoral immunity i.n. administration could take place ± adjuvant with higher doses (> 50pg per nose whole - Img, preferable 200pg) in one-week to one-month intervals for four repetitions. Or at lower doses <50pg per nose hole, once per day or bid (ca. 12h apart) for 5 consecutive days schedules.

[0417] (iii) systemic immunization of a subject with naive immunity; here at least two potential approaches are demonstrated, a fast “pandemic” scheme with only three weeks interval between prime and boost vs. an ideal immunization scheme with ~ 3 months interval between prime and booster immunization when the antibody response decline at least sufficiently and the immune response is mature to benefit most from a boost / refreshing and long-lasting memory immunization.

[0418] (iv) mucosal immunization of a subject with naive immunity; the same schedule as described in ii). In completely immune naive individual, novel approaches could be utilized to potentiate immunogenicity by e.g. utilizing conjugation (preferred) or mixing of BioVac with mRNA, TLR- and / or cGAS-STING agonists. In this case, the prime i.n. immunization could take place with a single maximum tolerated dose (MTD) and further booster if required based on assessment of the IgA response in blood and / or saliva. The MTD will primarily depend on adjuvant reactogenicity, as adjuvant free mucosal application shows no MTD in mouse or human subjects.

[0419] (v) a fifth approach in pandemic situation would be to induce a systemic immune response with i.m. administration of BioVac and to bridge the ideal time to second i.m. vaccination of approximately three months by repeated passive i.n. immunization with BioVac.

[0420] Dosages and Schemes of administration

[0421] 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.

[0422] 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.

[0423] 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0424] Tests of effect of vaccination

[0425] 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 SARS-CoV-2 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 viral nucleic acid such as SARS-CoV-2 nucleic acid, or antibody assays to detect the presence of 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-SARS-CoV-2 antibodies in the subject. Further assays include, e.g. the pseudoviral neutralization assay (see e.g. Nie, J., Li, Q., Wu, 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 ACE2-receptor binding domain binding.

[0426] 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 a protein oligomer which comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1 to 12, or 35 to 43, or 55, or a vaccine comprising a protein oligomer which comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity to SEQ ID NO. 1 to 12, or 35 to 43, or 55. Preferably, the sequence identity is calculated over the entire length. Preferably, said protein oligomer comprised by said vaccine binds to ACE2 on the target cell of a subject as defined herein, such as a mucosal cell or an epithelial cell of a subject, preferably a human subject, in the picomolar range. Said protein oligomer or vaccine of the invention is able to bind to ACE2, preferably human ACE2, in the picomolar range, more preferably with a dissociation constant (Kd) of between 0.1 pM and 1 nM, 0.1 pM and 990 pM, 1 pM and 950 pM, 10 pM and 900 pM, 20 pM and 800 pM, 30 pM and 700 pM or 40 pM to 600 pM, or 50 pM and 500 pM, as determined by surface plasmon resonance (SRP) or an alternative method, such as a pseudoviral neutralization assay. Preferably, said vaccine is for use in active vaccination in a subject, preferably a human as defined herein.

[0427] Definitions and still further embodiments

[0428] 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).

[0429] 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.

[0430] 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.

[0431] 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.

[0432] 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.

[0433] 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.

[0434] 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. 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, Glu, 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, Glu); 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, Glu, Asp), and xiii) Hydrophobic amino acids (Leu, He, Vai).

[0435] 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 polypeptides made according to the present invention.

[0436] 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.

[0437] The invention also encompasses any homologues of the polypeptides specifically defined by their amino acid sequence according to the invention. 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, and any derivatives, enantiomers and fusion proteins thereof.

[0438] 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.

[0439] 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.

[0440] 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.

[0441] 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.

[0442] 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 useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0443] Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents reverses the charge of the lysinyl residues. Other suitable reagents for derivatizing a-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chlorob orohydri de; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxyl ate.

[0444] Arginyl residues are modified by reaction with one or several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Such derivatization requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine c-amino group.

[0445] Modification of tyrosyl residues has permits introduction of spectral labels into a peptide. This is accomplished by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to create 0-acetyl tyrosyl species and 3-nitro derivatives, respectively.

[0446] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R' — N — C — N — R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Conversely, glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Deamidation can be performed under mildly acidic conditions. Either form of these residues falls within the scope of this invention. Derivatization with bifunctional agents is useful for cross-linking the peptide to a waterinsoluble support matrix or other macromolecular carrier. Commonly used cross-linking agents include 1,1 -bi s(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy succinimide esters, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N- maleimido-l,8-octane. Derivatizing agents such as methyl-3-[(p- azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.

[0447] Other chemical modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (Creighton, supra), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl.

[0448] Such chemically modified and derivatized moieties may improve the (poly)peptide's solubility, absorption, biological half-life, and the like. These changes may eliminate or attenuate undesirable side effects of the proteins in vivo.

[0449] It should be appreciated that the invention further encompass any of the (poly)peptides of the invention or disclosed herein, any serogates thereof, any salt, base, ester or amide thereof, any enantiomer, stereoisomer or disterioisomer thereof, or any combination or mixture thereof. Pharmaceutically acceptable salts include salts of acidic or basic groups present in (poly) peptides or compounds of the invention or as disclosed herein. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-(2- hydroxy-3 -naphthoate)) salts. Certain compounds of the invention can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.

[0450] The present invention encompasses any fragment, derivative or analogue of the protein oligomer of the invention. In certain embodiments, any of the protein oligomer of the invention and derivatives thereof can bind the viral receptor or co-receptor of the target cell as well as neutralizing antibodies (nAb / s), and more importantly, possess the ability to elicit the production of neutralizing antibodies in a subject vaccinated by the protein oligomer of the invention. Thus, in some embodiments the protein oligomer of the invention is a (poly)peptide possessing the ability to elicit the production of neutralizing antibodies, and / or inducing immunity against SARS-CoV-2. In certain embodiments, the protein oligomer of the invention may be fused to additional peptide sequences, such as adjuvants or tags for purification or labeling, as explained elsewhere herein. The invention further encompasses any fusion protein comprising the protein oligomer of the invention as described herein. More specifically, additional peptide sequences can be added to the protein oligomer of the invention thereby forming fusion proteins, which act to promote stability, purification, and / or detection. For example, a reporter peptide portion (e.g., green fluorescent protein (GFP), P-galactosidase, or a detectable domain thereof) can be used. Purification-facilitating peptide sequences include those derived or obtained from maltose binding protein (MBP), glutathione-S-transferase (GST), or thioredoxin (TRX). Other examples fur such purification-facilitating peptide sequences have been mentioned elsewhere herein.

[0451] It should be noted that the protein oligomer of the invention can be produced either synthetically, or by recombinant DNA technology. Methods for producing polypeptides peptides are well known in the art and also described elsewhere herein.

[0452] 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.

[0453] An “isolated polypeptide” is 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.

[0454] A “fragment” of a protein or peptide as used herein means a fragment which 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 protein or peptide, exhibiting the same biological properties or activities as the protein or peptide. A non-limiting example of a fragment of a receptor binding domain (RBD) as defined herein is a receptor binding motif which is still capable of binding to the ACE2 receptor or co-receptor on the viral target cell of a subject, such as a mucosal cell or epithelial cell.

[0455] A “domain” of a protein or polypeptide as used herein means a distinct functional and / or structural unit in a protein. Usually, a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions. For example, Src homology 3 (SH3) domains are small domains of around 50 amino acid residues that are involved in protein-protein interactions. SH3 domains have a characteristic 3D structure. They occur in a diverse range of proteins with different functions, including adaptor proteins, phosphatidylinositol 3 -kinases, phospholipases and myosins. Another example for a protein domain is an Fc domain, as defined elsewhere herein.

[0456] A still further example for a protein domain is a receptor binding domain (RBD). Viral infection of a subject is initiated upon binding of a viral protein with the receptor or co-receptor on the target cell surface of the subject, which occurs via a receptor binding domain (RBD). For instance, coronaviruses 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. For instance, cryo-electron microscopy studies of the SARS-CoV spike protein and its interaction with the cell receptor angiotensin converting enzyme 2 (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 post-fusion 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 of a subject.

[0457] Accordingly, a “receptor binding domain” (RBD) as used herein means a structural and functional unit in a viral protein which mediates viral attachment of, fusion with and entry into the target cell of a subject, by binding to its receptor or co-receptor on the target cell. Due to its important role in viral attachment, fusion and entry, the receptor binding domain (RBD) serves as an attractive target for designing antiviral vaccines. For instance, it has been found that vaccines based on the receptor binding domain (RBD) in the spike protein of SARS-CoV induced the most potent neutralizing antibody responses and protective immunity in vaccinated animals; see, e.g., Emerging Microbes and Infections (2012) 1, el3; doi: 10.1038 / emi.2012.1; published online 8 August 2012.

[0458] A “receptor binding motif (RBM)” as used herein means a distinct amino acid sequence stretch in the receptor binding domain which contains most of the contacting residues to the receptor or co-receptor. For example, the receptor binding motif (RBM) of the SARS-CoV-2 spike monomer is a distinct region within the receptor binding domain (RBD) corresponding to amino acid residues 438-506, located in the SI subunit of the SARS-CoV-2 spike monomer which contains most of the residues contacting ACE2.

[0459] The term “oligomerization domain” as used herein refers generally to a protein domain which mediates the sub-unit assembly of the receptor binding domain, a fragment of the receptor domain, or a receptor binding motif in the monomer(s) of the protein oligomer of the invention, as defined herein. The oligomerization domain mediates dimerization, trimerization, or tetramerization and so on, of the receptor binding domain, a fragment of the receptor domain, or a receptor binding domain, in said monomer(s). Such oligomerization leads, e.g., to functional advantages of multivalency and high binding strength, increased structure stabilization and combined functions of different domains, resulting in enhanced biological activity, such as improved or increased affinity and avidity, of the protein oligomer of the invention.

[0460] The oligomerization domain can comprise, e.g., 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; see, e.g., Ali and Imperial! 2005, Bioorganic and Medicinal Chemistry 13, 5013.

[0461] In biochemistry, an oligomer is a molecule that consists of a few similar or identical repeating units which could be derived, actually or conceptually, from copies of a smaller molecule or repeating unit, its monomer. The name is composed of Greek elements oligo-, "a few" and - mer, "parts". In the context of the present invention, an “oligomer” is to be understood as a “protein oligomer” or “(poly)peptide oligomer” that comprises a few monomer units, e.g., two, three, four, five, six, seven, eight, nine, ten or even more monomer units.

[0462] A “monomer” (mono-, "one" and -mer, "part") as used herein is a molecule that can react together with other monomer molecules to form a larger oligomer chain or three-dimensional network in a process called “oligomerization”. Oligomerization can be mediated by specialized domains which ensure dimerization, trimerization or oligomerization, so called dimerization, trimerization or oligomerization domains, as further defined herein below, and well known in the art. As an indispensable component of recombinant fusion proteins, “linkers” have shown increasing importance in the construction of stable, bioactive fusion proteins. The general properties of “linkers” as used herein derived from naturally-occurring multi-domain proteins can be considered as the foundation in linker design. Empirical linkers designed by researchers are generally classified into three categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles; see, e.g., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357— 1369; Protein Sci. 2013 Feb; 22(2): 153-167; published online 2012 Dec 6. doi: 10.1002 / pro.2206. Linkers or spacers are short amino acid sequences created in nature to separate multiple domains in a single protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. However, Glycine (Gly)-rich linkers are flexible, connecting various domains in a single protein without interfering with the function of each domain. The advent of recombinant DNA technology made it possible to fuse two interacting partners with the introduction of artificial linkers. Often, independent proteins may not exist as stable or structured proteins until they interact with their binding partner, following which they gain stability and the essential structural elements. Gly-rich linkers have been proven useful for these types of unstable interactions, particularly where the interaction is weak and transient, by creating a covalent link between the proteins to form a stable protein-protein complex. Gly-rich linkers are also employed to form stable covalently linked dimers, and to connect two independent domains that create a ligand-binding site or recognition sequence. The lengths of linkers vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners. Various structures of covalently linked protein complexes have been described using X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy techniques.

[0463] The term “payload” 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; see, e.g. Figure 6.

[0464] An “immunoglobulin” as used herein means an antibody (Ab) which is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. Immunoglobulin G (IgG) is most abundant in human serum, of the five immunoglobulin isotypes, IgG, IgD, IgA, IgE and IgM. The four IgG subclasses, IgGl, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcyR) and complement component Clq. As a result, the different subclasses have different effector functions, both in terms of triggering FcyR-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces. However, FcRn is also expressed in myeloid cells, where it participates in both phagocytosis and antigen presentation together with classical FcyR and complement. These properties, i.e. IgG- polymorphisms and post-translational modification of the antibodies in the form of glycosylation, affect IgG-function; see, e.g., Vidarsson, Front Immunol. 2014; 5: 520; Remesh et al., Structure, Volume 26, Issue 7, 3 July 2018, Pages 1007-1014. e2.

[0465] An “IgG immunoglobulin” or an “IgG immunoglobulin molecule” as used herein consists of four polypeptide chains, composed of two identical 50 kDa y heavy (H) chains and two identical 25 kDa K or light (L) chains, linked together by inter-chain disulfide bonds. Each heavy chain consists of an N-terminal variable domain (VH) and three constant domains (CHI, CH2, CH3), with an additional “hinge region” between CHI and CH2. Similarly, the light chains consist of an N-terminal variable domain (VL) and a constant domain (CL). The light chain associates with the VH and CHI domains to form a Fab arm (“Fab” = fragment antigen binding), and functionally, the V regions interact to form the in antigen-binding region - acquired through differential assembly of Variable, Diversity (VH only), and Joining gene segments and inclusion of somatic mutations, although their relative contribution to antigen binding varies greatly. Two heavy chain-light chain heterodimers (HL) combine into a single antibody molecule (H2L2) via disulfide bonds in the hinge region and non-covalent interactions between the CH3 domains. The part of the antibody formed by the lower hinge region and the CH2 / CH3 domains is called “Fc” (“fragment crystalline”) or “Fc domain” or “Fc region”.

[0466] The term “Fc” or “Fc domain” or “Fc region” or “Fc part” (all terms are used interchangeably, if not indicated otherwise) as used herein means the fragment crystallizable region which is the tail region of an antibody or immunoblobulin that interacts with cell surface receptors, i.e. Fc receptors, and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc domains contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc domains of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor- mediated activity. The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisecting GlcNAc and a-2,6 linked sialic acid residues. Fusion of the Fc domain of immunoglobulins to proteins has been found to enhance the production and secretion of the fusion proteins in mammalian cells (see, e.g., Lo et al., 1998, Protein Eng. 11, 495, Capon et al., 1989, Nature 337, 525). In addition, linking of angiogenesis inhibitors to an immunoglobulin Fc domain have shown to increase the half life of said inhibitors (see, e.g., Capon et al. 1989, Nature 337, 525; Gordon et al., 2001, J. Clin. Oncol. 19, 843; Holash et al., 2002, Proc. Natl. Acad. Sci. USA 99, 11393). However, the Fc domain can not only be used for purification, solubilization and / or detection purposes but alters advantageously the biological and pharmacokinetic properties of a fusion protein or protein oligomer such as the protein oligomer of the invention, as set forth herein and in the following Examples. As evident to those skilled in the art, in principle, any Ig isotype or IgG isoform can be used to generate the protein oligomer of the invention. Even sub-fragments or single chains of the Fc domain of IgG can be used in the protein oligomer of the invention.

[0467] The human Ig Fc domain as referred to herein can be from human IgG or be derived from human IgG, such as human IgGl or IgG4, more preferably from human IgGl or derived from human IgGl (Bergers and Javaherian Science 1999; Lee et al Clin Cane Res 2008). For example, the human Fc domain from IgGl can comprise or consist of an amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 24 of WO 2019 / 201892.

[0468] The human Ig Fc domain can be modified, as explained in detail elsewhere herein. For instance, enhanced affinity of human Ig Fc for the neonatal Fc receptor (FcRn) at mucosal pH, compared to human wildtype Ig Fc, can be 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).

[0469] To give a further example for a Fc domain modification, the Ig Fc domain can comprise 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.

[0470] Immunoglobulin “Fc receptors (FcRs)” as used herein are membrane molecules expressed by several hematopoietic cells that recognize the Fc domain (or Fc or Fc region or Fc part) of several immunoglobulin (Ig) classes and subclasses. The prior art distinguishes FcR for IgG (FcyRI / CD64, FCYRII / CD32, and FcyRIII / CDl 6), IgE (FceRI), IgA (FcaRI / CD89), IgM (FcpR), and IgA / IgM (Fca / pR). Several other receptors expressed on different cell types also bind Ig molecules: neonatal FcR for IgG (FcRn) on intestinal epithelium, placenta, and endothelium, low affinity FccR (FccRII / CD23) on B cells and macrophages, and polymeric Ig receptor (plgR) on mucosal epithelium.

[0471] The neonatal Fc receptor (FcRn) is distinctively a beta (P)-2-microglobulin (P2m) associated protein that is structurally related to the major histocompatibility class I (MHC-I) family, yet it is unable to present antigenic peptides to T cells. Further, FcRn has a quasi-ubiquitous expression pattern, possesses a predominantly intracellular localization, is monomorphic, and binds another, structurally and functionally unrelated protein to IgG, namely albumin. While the subtypes of IgG are fundamental in immune responses, albumin functions as a carrier protein in addition to being an important regulator of oncotic blood pressure. Despite these differences, IgG and albumin are the two most abundant serum proteins that possess a long serum half-life owing to their interaction with FcRn, which rescues them from intracellular degradation through a cellular recycling mechanism. Another of FcRn's functions is to transport IgG from mother to offspring thereby providing to the naive and immature immune system of the newborn the experience and protection developed in the adult progenitor. This process is developmentally regulated in that it occurs antenatally in rodents and humans through the inverted yolk sac or placenta, respectively, but uniquely continues at significant levels in the early post-natal life of rodents due to the high levels of FcRn expression in the intestinal epithelium. This functional expression of FcRn and its ability to transcytose IgG is not limited to the newborn but persists throughout life and permits the targeted delivery of IgG to sites where the presence of this type of antibody reinforces immunity, a process widely exploited by IgG-based therapeutics. Finally, the functions of FcRn are differentially determined by whether IgG is a single molecule, and thus monomeric, or present as an immune complex (IC). In the latter case, FcRn has been shown to critically regulate the innate immune responses as well as processing and presentation of antigens contained within IgG IC (see e.g. Pyzik et al. Front. Immunol., 10 July 2019 |https: / / doi.org / 10.3389 / fimmu.2019.01540).

[0472] The protein family of Fc receptors for IgG (FcyRs) are broadly expressed by cells of hematopoietic origin. They can be divided into inhibitory (FcyRIIB) and activating receptors (FcyRI, FcyRIIA, FcyRIIC, FcyRIIIA, and FcyRIIIB). Through binding of IgG via the Fc portion, FcyRs are essential for regulating responses to infections and controlling inflammation (Hargreaves CE, Rose-Zerilli MJ, Machado LR, Iriyama C, Hollox EJ, Cragg MS, et al. Fcgamma receptors: genetic variation, function, and disease. Immunol Rev. (2015) 268:6-24. doi: 10.1111 / imr.12341; Koenderman L, Inside-out control of Fc-receptors. Front Immunol. (2019) 10:544. doi: 10.3389 / fimmu.2019.00544).

[0473] “Fc-based therapeutics” are widely used in therapy and prevention of various diseases. Fc plays multiple roles in dimerization for formation of Y-shaped structure of Ig and maintenance of the structure, and Fc-mediated effector functions and extension of serum half-life. There are two domains: second constant domain (CH2) and third constant domain (CH3) in monomeric Fc of IgG. CH2 domain has a single N-linked glycan at N297 (antibody residues are numbered according to EU numbering). Two CH3 domains interact strongly with each other to form homodimer resulting in dimerization of Fc. These elements contribute to the physicochemical characteristics of Fc. Through the binding of Fc in IgG to its receptor Fc-gamma receptors (FcyRs), immune leukocytes presenting FcyRs on their surface membrane are recruited and activated, which subsequently triggers antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis to kill and clear target cells (e.g., tumor cells). In addition, Fc can bind to the serum complement molecule (Clq) to initiate the assembly of membrane attack complex formed by complement cascade proteins to destroy target cells, which is termed complement-dependent cytotoxicity (CDC). These effector functions are important for the pharmaceutical efficacies of Fc-based therapeutics. Besides mediation of effector functions, Fc can also bind to neonatal Fc receptor (FcRn) in a pH-dependent manner, which leads to the extension of the serum half-life of IgG. In addition, binding of Fc to immune- related molecules such as Fc receptors can regulate immune response in vivo. Taking together, Fc part in an Fc-based therapeutic protein plays important roles in biological and pharmacological properties including (i) increased stability and aggregation resistance; (ii) acquired multivalent binding to the target; (iii) enhanced Fc-mediated effector functions; (iv) extended serum half-life; and (v) modulated immunogenicity.

[0474] As known in the art, “affinity” is the strength of a single bond or interaction. For instance, when it comes to the antibody-antigen relationship, the binding affinity is the strength of the interaction between the antigen’s epitope and the antibody’s paratope at a singular binding site. Thermodynamically, affinity is the total of all forces that result in increased binding strength (Kon) minus all of the forces that result in decreased binding strength (Koff).

[0475] When an antigen is encountered for the first time, the affinity of the antibodies produced is low. Once the body is familiar with the antigen, the immune response adapts and the binding affinity increases.

[0476] Another example of affinity is the interaction between a biomolecule and its ligand, such as a receptor or co-receptor bound by a virus, and a drug designed to bind to it, such as the protein oligomer of the invention.

[0477] The non-covalent interactions that participate in the affinity of a binding site include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic forces. These interactions at the binding site between the two molecules and the presence of other molecules influences the affinity.

[0478] Accordingly, the affinity between antigens and antibodies is caused by numerous chemical interactions between the two molecules.

[0479] Antibodies and antigens are multivalent, meaning they possess more than one binding site. The measure of the total binding strength of an antibody at every binding site is termed “avidity”. Avidity is also known as the functional affinity.

[0480] Avidity is determined by three factors: (i) The binding affinity: The strength of the relationship at a singular binding site, (ii) The valency: The total number of binding sites involved. And (iii) the structural arrangement: For instance, the structure of the antigen and antibody involved.

[0481] To give an example, two of the antibodies found within the human body are IgE and IgM. IgE antibodies have just two binding sites, while IgM antibodies have ten. The affinity of each of these antibodies is the strength at just one binding site, while the avidity is the total strength of the binding interactions at the two IgE binding sites or the ten IgM binding sites. The valency of IgM is five times greater than that of IgE, so the difference between the affinity and the avidity will be greater for IgM antibodies than IgE antibodies.

[0482] Another factor that can change the avidity is the structural arrangement of either the antibody or antigen. For instance, if a non-competitive inhibitor binds to the IgM antibody, its avidity towards its antigen would decrease.

[0483] Affinity and avidity are both measures of binding strength. While affinity is the measure of the binding strength at a single binding site, avidity is a measure of the total binding strength. Antibodies have between two and ten binding sites. Antibodies with fewer binding sites tend to have high affinity and low avidity, while those with greater binding sites tend to have low affinity and high avidity.

[0484] Affinity of the protein oligomer of the invention can be defined as strength of the binding interaction between the receptor binding domain comprised by said protein oligomer of the invention and the receptor to which the receptor binding domain binds to. It depends on the closeness of the stereochemical fit between receptor binding domain sites and receptor determinants, the size of the area of contact between them, and the distribution of charged and hydrophobic groups. As well known in the art, binding affinity is a measure of dynamic equilibrium of the ratio of on-rate (kon) and off-rate (koff) under specific concentrations of reactants. The affinity constant, Ka, is the inverse of the dissociation constant, Kd. The strength of complex formation in solution is related to the stability constants of complexes, however in case of large biomolecules, such as receptor binding domain-receptor pairs, their interaction is also dependent on other structural and thermodynamic properties of reactants plus their orientation and immobilization. KD is the equilibrium dissociation constant, a ratio of koff / kon, e.g., between the antibody and its antigen, or between the receptor binding domain comprised by said protein oligomer of the invention and the receptor. KD and affinity are inversely related. For instance, the KD value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the antibody.

[0485] Quantitative and qualitative measurements of biomolecule associations are well known in the art. Qualitative methods such as ELISAs, pull-down assays, and gel shift assays work by immobilizing one protein to a substrate and applying another protein, typically containing a label or reporter tag, to it. If the two proteins bind, they release a detectable signal. These methods merely provide a yes / no answer as to whether binding occurred. In contrast, quantitative methods provide a scalar readout of binding affinity, releasing a signal that indicates the strength of the interaction. Biosensor-based methodologies work by immobilizing a binding partner to a surface and presenting the test partner to interact with it. The change in signal is observed and recorded by the instrument. Other quantitative methods use capillaries or tubes instead of immobilizing proteins to a surface. Such quantitative methods include, for example, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), BioLayer Interferometry (BLI), or MicroScale Thermophoresis (MST).

[0486] Avidity of the protein oligomer of the invention refers to the accumulated strength of multiple affinities of individual non-covalent binding interactions, such as between the receptor binding domains comprised by said protein oligomer of the invention and their receptors, and is commonly referred to as functional affinity. Avidity differs from affinity, which describes the strength of a single interaction.

[0487] Half maximal effective concentration (EC50) as used herein refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after a specified exposure time. EC50 is a measure of concentration, expressed in molar units (M), where 1 M is equivalent to 1 mol / L. The IC50 is the concentration of drug or vaccine required for 50% inhibition. IC50 is an operational term dependent on the assay conditions, whereas kon, koff, and KI are intrinsic to a bimolecular interaction. For example, the IC50 for a competitive inhibitor will increase with increasing substrate concentrations even though the KI, kon, and koff are unchanged. The change in IC50 due to competition is mathematically described by the Cheng-Prusoff relationship: IC50 / KI = 1 + [S] / Km, where [S] is the substrate concentration and Km is the Michaelis constant. The IC50 is equal to KI for noncompetitive inhibition.

[0488] One example for an “immunogenic composition” is a vaccine such as the protein oligomer of the invention. Thus, in some embodiments, the immunogenic composition or vaccine may induce an immune response in a subject. “Immune response” as used herein means the activation of a host's immune system, e.g., that of a mammal such as a human, in response to the introduction of antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0489] As used herein, the terms “administering” and “administration” refer to any method of providing a drug or a vaccine to a subject. Such methods are well known to those skilled in the art and include, but are not limited to: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intramural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition. In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, or an efficacious route of administration so as to treat a subject. In some aspects, administering comprises exposing. For instance, exposing a subject to alternating electrical fields means administering alternating electrical fields to the subject.

[0490] The human “respiratory tract” as used herein is a complex organ system divided into the upper respiratory tract, that includes the nasal cavity, pharynx and larynx, and the lower respiratory tract comprising the conducting airways (trachea, bronchi and bronchioles) and the respiratory zone (respiratory bronchioles and alveoli).

[0491] “Mucosa” as used herein means the soft tissue that lines the body's canals and organs in the digestive, respiratory and reproductive systems. Mucosa comprises nasal mucosa, eye surface and canalis lacrimalis as well as paranasal (maxillary) and frontal sinuses, oral mucosa, naso / oro / hypo pharynx, esophageal mucosa, bronchial mucosa, gastric mucosa, intestinal mucosa, , and mucosa of the reproductive tract.

[0492] Mucosal tissues contain components of both the innate and acquired immune system are strategically located in areas where external pathogens enter the body. Immune cells that reside in mucosal tissues protect against the entry of infectious agents. The immune responses arising in these regions must be very selective so as to avoid mucosal damage that would impair functions such as gas exchange in the lungs.

[0493] The mucosa of the human respiratory tract is also referred to herein as human airway mucosa which comprises the oral mucosa, nasal mucosa, nasopharyngeal or pharyngeal mucosa, laryngeal mucosa, tracheal mucosa, and bronchial mucosa.

[0494] The mucosa as used herein is preferably the nasal, naso- / oro- / hypo-pharynx and oral mucosa and the mucosa of the eye, if not indicated otherwise.

[0495] The pH for healthy nasal or nasopharyngeal mucosa in human is a moderately acidic pH of about 5.3 to 7.0. Preferably, mucosal pH as referred to herein is nasal or nasopharyngeal pH, with 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.

[0496] The pH of the eye mucosa is almost neutral between about 7 and 7.3.

[0497] The pH for oral mucosa is about 6.8.

[0498] In comparison, the pH of the plasma or serum is about 7.35-7.40.

[0499] The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency or other generally recognized pharmacopoeia for use in subjects like vertebrate animals, mammals or human, preferably in human.

[0500] The term "pharmaceutically acceptable carrier" relates to non-toxic excipients, including e.g. solid, semisolid or liquid fillers, diluents, binding agents, lubricants, various types of wetting agents, encapsulating material or formulation auxiliaries of any type. Examples of suitable pharmaceutically acceptable carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc.

[0501] A “vaccine” as used herein is a composition preferably an immunogenic composition that is used to stimulate the human or animal body’s immune response against diseases, such as infectious diseases caused by viruses. Vaccines are usually administered through different routes such as needle injections, but some can be administered by mouth or sprayed into the nose.

[0502] A “mucosal vaccine” as used herein means a vaccine which can be administered via one or more mucosal sites of a human or animal, i.e. a mucosal vaccine is able to enter the human or animal body via mucosal membranes or surfaces, and is capable of induce mucosal immunity. Mucosal vaccines offer the potential to trigger robust protective immune responses at the predominant sites of pathogen infection by mucosal immunization through oral, nasal, rectal, vaginal, sublingual, or transcutaneous routes. An example for a mucosal vaccine inducing mucosal immunity is a vaccine comprising the protein oligomer of the invention. “Vaccination” as used herein means the act of introducing a vaccine such as the protein oligomer of the invention into the human or animal body to produce protection from a specific disease which may be caused, e.g., by a virus.

[0503] The term “active vaccination” or “active immunization” as known in the art means administering an immunogen that might be live / attenuated, killed / inactivated, toxoid or subunit in origin.

[0504] In simple terms, when e.g. the human body is exposed to a novel disease agent such as a virus, B cells create antibodies that assist in destroying or neutralizing the disease agent. When B cells encounter a pathogen, they create memory cells in addition to antibodies. Memory cells are a type of B cell produced following the primary infection that can recognize the pathogen. Memory cells can survive for decades, waiting within the body until the pathogen invades again. When the human body is exposed to the pathogen for a second time, the immune response is more robust, quickly addressing the disease agent.

[0505] The protein oligomers of the invention can be used for active vaccination or active immunization, i.e. they act as an immunogen to elicit an immune response, when administered to a subject. The protein oligomer of the invention can be used for active vaccination or active immunization in that it elicits a systemic and mucosal immune response against the receptor binding domain or receptor binding motif comprised by the protein oligomers of the invention in the vaccinated subject, as explained elsewhere herein and shown in the following Examples.

[0506] An “adjuvant” as used herein means a substance which enhances the animal or human body's immune response to an antigen or a vaccine. Accordingly, adjuvants are included in vaccines or vaccine formulations to enhance the immunogenicity and efficacy of vaccines. Preferably, the adjuvant is for activating or enhancing in vitro and / or in vivo the antigen presenting function of antigen presenting cells for a therapeutic or prophylactic intervention. That means, the adjuvant can stimulate macrophages, can stimulate or enhance the humoral immune response, e.g. enhancing or stimulating the production of antibodies. In addition, the adjuvant can also enhance or stimulate the cellular immune response, e.g. increasing the proliferation of T-cells.

[0507] “Over-the-counter (OTC) drugs” as used herein are drugs sold directly to a consumer without a requirement for a prescription from a healthcare professional, as opposed to prescription drugs, which may be supplied only to consumers possessing a valid prescription. In many countries, OTC drugs are selected by a regulatory agency to ensure that they contain ingredients that are safe and effective when used without a physician's care. OTC drugs are usually regulated according to their active pharmaceutical ingredient (API) rather than final products. By regulating APIs instead of specific drug formulations, governments allow manufacturers the freedom to formulate ingredients, or combinations of ingredients, into proprietary mixtures.

[0508] “Over-the-counter (OTC) vaccines” as used herein are vaccines sold directly to a consumer without a requirement for a prescription from a healthcare professional. The protein oligomers of the invention are particularly suitable as OTC vaccines. The BioVac protein is very stable at RT and 4 °C for prolonged period, it could be administered repeatedly as a nasal spray, especially in adjuvant-free setting and has shown no MTD after short or long-term repeated use, i.e. demonstrates low reactogenicity and very low toxicity profile e.g. nose dryness after repeated administration that was compensated by co-administration of dexpanthenol. Therefore, it is conceivable that BioVac could be one day administered in the same manner as other nasal sprays with alpha sympathomimetic e.g. are utilized currently in the clinical practice as OTC.

[0509] “Immunization” as used herein is a process by which a human or animal becomes protected against a disease such as a disease caused by a virus through vaccination. This term is often used interchangeably with vaccination or inoculation.

[0510] “Immunity” as used herein means protection from an infectious disease, in particular an infectious disease caused by a virus. If a human or animal is immune to a disease, the human or animal can be exposed to it without becoming infected.

[0511] Innate immunity means that everyone is bom with innate (or natural) immunity, a type of general protection. For example, the skin acts as a barrier to block pathogens from entering the body.

[0512] Adaptive (or active) immunity develops throughout human lives. Humans develop adaptive immunity when they are exposed to diseases or when they are immunized against them with vaccines such as the protein oligomer of the invention.

[0513] Passive immunity is "borrowed" from another source and it lasts for a short time. For example, antibodies in a mother's breast milk give a baby temporary immunity to diseases the mother has been exposed to.

[0514] “Humoral immunity” as used herein means that virus and / or virus-infected cells or a vaccine such as the protein oligomer of the invention can stimulate B lymphocytes to produce antibodies specific for e.g. viral antigens such as the receptor binding domains used in the protein oligomer of the invention. Antibody neutralization is most effective when virus is present in large fluid spaces, e.g., serum, or on moist surfaces, e.g., the gastrointestinal and respiratory tracts.

[0515] “Mucosal immunity” as used herein means that a vaccine evokes a response in form of mucosal, secretory immunoglobulin A (IgA) such as slgAl and / or tissue-resident memory T cells (TRM).

[0516] A ”pathogen“ as used herein is any organism or substance, especially a microorganism, capable of causing disease, such as bacteria, viruses, protozoa or fungi.

[0517] A “virus” as used herein is a virus which enters the human or animal body via mucosal surfaces, such as respiratory viruses. As explained elsewhere herein, 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 human or animal body.

[0518] Viruses have evolved several pathways to initiate entry through epithelial barriers. Viruses can enter and infect epithelial cells by accessing the cell cytosol using one of two mechanisms — direct entry at the epithelial plasma membrane, or entry through the epithelial endocytic pathway. By contrast, some viruses do not need to infect epithelial cells to spread — they are internalized by epithelial cells and cross the epithelial barrier using transcytosis, as has been described recently for SARS-CoV-2; see, e.g., Knyazev et al., Front. Immunol., 07 September 2021 | https: / / doi.org / 10.3389 / fimmu.2021.636966.

[0519] Most viruses contain two or three elements: the genome, in the form of single-stranded (ss) or double-stranded (ds) DNA or RNA; the capsid, which consists of viral proteins; and, possibly, an envelope, which originates from the host cell and consists of host-cell lipids that are organized as a bilayer. Viral-envelope glycoproteins as well as, in some cases, selected hostcell proteins can be recruited to the envelope. “Naked” viruses contain only the genetic material surrounded by the capsid. By contrast, in “enveloped” viruses, the genome is surrounded by a capsid and is protected further by the viral envelope.

[0520] A “coronavirus” (CoV) as used herein means a coronavirus infecting mammalian subject(s) including human, avian subject(s) or any other vertebrates. Thus, the term coronaviruses (designated herein CoVs) for the purposes of the present invention encompasses four main subgroupings of coronaviruses, known as Alpha, Beta, Gamma, and Delta. More specifically, under this term is meant the enveloped viruses with a positive-sense RNA genome (ssRNA+) and with a nucleocapsid of helical symmetry; and also large RNA viruses with the genomic size of ranges from approximately 26 to 32 kilobases; and further viruses with the characteristic morphology of large, bulbous surface projections under electron microscopy, which is created by the viral spike (S) proteins, i.e. viral surface proteins determining host tropism and immunogenicity. Encompassed are the novel coronaviruses that have emerged in humans in the twenty-first century: severe acute respiratory syndrome coronavirus (SARS-CoV and SARS- CoV-2) which cause acute respiratory distress syndrome (ARDS) and are associated with high mortality rates. Thus, the development of effective and improved therapeutic and preventive strategies that can be readily applied to new emergent strains is a research priority.

[0521] In the specific case of the SARS CoV or SARS-CoV-2, a defined receptor binding domain on S mediates the attachment of the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2) (see below). Some CoVs, specifically the members of Beta CoV subgroup A, also have a shorter spike-like protein called hemagglutinin esterase (HE).

[0522] The following CoVs can infect humans, specifically the Alpha CoVs 229E and NL63, and Beta CoVs OC43, HKU1, SARS-CoV, and SARS-CoV-2. Specifically for the Beta-CoVs, which are of the greatest clinical importance concerning humans, these are OC43, and HKU1 of the A lineage, SARS-CoV of the B lineage, and SARS-CoV-2 of A and B lineage. Rambaut et al. have suggested a dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology; see, e.g., Nature Microbiology volume 5, pages 1403-1407 (2020). The Alpha and Beta CoVs genera descend from the bat gene pool. It should be further noted that human CoVs are difficult to grow in the laboratory. Coronaviruses infecting animals are also contemplated, in particular the bat coronaviruses HKU4 and HKU5.

[0523] Within the above group of human CoVs, of particular relevance to the present invention are the SARS-CoV-2 associated with Severe Acute Respiratory Syndrome, as for being the primary causes of life-threatening infectious diseases and epidemics in humans. The other human CoVs are believed to cause a significant percentage of all common colds in human adults (primarily in the winter and early spring seasons). In certain individuals CoVs may further be a direct or indirect cause of pneumonia, i.e. direct viral pneumonia or a secondary bacterial pneumonia.

[0524] A “variant of concern” as used herein means a viral variant which seems to pose a greater threat to public health due to enhanced transmissibility or infectivity. SARS-CoV-2 variants of concern as of 15 March 2024 are described, e.g. under https: / / www.ecdc.europa.eu / en / covid- 19 / variants-concern, or other references cited herein.

[0525] A “viral disease” as used herein means an infectious disease caused by a virus such as a respiratory virus. A viral disease (or viral infection) occurs when a human or animal's body is invaded by pathogenic viruses, and infectious virus particles (virions) attach to and enter susceptible cells, via their receptors or co-receptors. For instance, SARS CoV and SARS-CoV- 2 are associated with Severe Acute Respiratory Syndrome. This syndrome is associated with high mortality rates. As set forth above, the other human CoVs are believed to cause a significant percentage of all common colds in human adults. In certain individuals, CoVs may further be a direct or indirect cause of pneumonia, i.e. direct viral pneumonia or a secondary bacterial pneumonia.

[0526] A “target cell” or “viral target cell” as used herein is a cell of a subject which is infected by a virus. A target cell can be a mucosal cell or epithelial cell. The subject can be a human or a mammal or an animal. The target cell contains a receptor or co-receptor which is bound by a viral protein and which facilitates viral entry into the target cell. For instance, an epithelial cell of a human can carry ACE2 receptors on its membrane which are bound by the spike protein of SARS-CoV-2 and then infected by said virus. Other co-receptors or low affinity receptors of SARS-Cov-2 like neuropilins expressing cells could be further targeted by BioVac RBD.

[0527] “Endocytosis” is a process in which a cell takes in materials from the outside by engulfing and fusing them with its plasma membrane. For instance, viruses can penetrate and infect cells of barrier tissues directly through the cytoplasmic membrane and indirectly by using cell endocytosis pathways.

[0528] “Endosome” as used herein is a membranous transport vesicle that is involved in endocytosis.

[0529] “Transcytosis” as used herein means a rapid and selective vesicular transcellular pathway that is characteristic of polarized epithelia. Cargo is transported from one pole of the cell to the opposite pole. The cargo remains enclosed in transcytotic vesicles, which precludes access to the cytosol and therefore viral infection of epithelial cells. Specifically, transcytosis is the transport of macromolecules, supramolecular complexes, and even microorganisms through the cell using membrane-bounded carriers. As a rule, the process is characteristic of polarized cells that form tissue barriers between two environments. Transcytosis can be carried out using various mechanisms; for example, one of the variants can be considered as a branch of endocytosis when the transported molecules are internalized using receptor-dependent mechanisms. This occurs most often through clathrin-coated vesicles; they enter the sorting membrane compartments of cells and then are released from cells by exocytosis, which is more typical for intestinal cells. Another variant, more typical for endothelial cells, is dependent on caveolae and a more direct route of transcytosis without entering intermediate compartments. It seems that viruses can use both types of transcytosis.

[0530] A “lamina propria” (chorion) as used herein is formed of conjunctive tissue that is traversed by blood and lymphoid vessels. It supports epithelial cells through the basal membrane.

[0531] A “subject” as used herein is an animal, such as a vertebrate or mammal, preferably a human.

[0532] “Antigen presenting cells (APCs)” as referred to herein are a heterogeneous group of immune cells that mediate the cellular immune response by processing and presenting antigens for recognition by certain lymphocytes such as T cells. Classical APCs include dendritic cells, macrophages, Langerhans cells and B cells.

[0533] “Sequence identity” as used herein is characterized by determining the number of identical nucleotides or amino acids between 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).

[0534] The term “derived from” as used herein means “is from” or “is obtained from”. For instance, the phrase “receptor binding domain (RBD) is derived from SARS-CoV-2 spike protein SARS- CoV-2” means that the receptor binding domain (RBD) is from or is obtained from the spike protein from SARS-CoV-2. So the nucleotide sequence coding for the receptor binding domain of the spike protein of SARS-CoV-2 or the amino acid sequence of this receptor binding domain of the spike protein of SARS-CoV-2 can be identical to the native or non-modified nucleotide sequence or amino acid sequence. In a broader sense, yet, the mentioned term “derived from” also encompasses modified or genetically engineered or recombinant nucleotide sequences or amino acid sequences of the receptor binding domain of the spike protein of SARS-CoV-2, or variants thereof, as defined herein. To provide an example, a receptor binding domain (RBD) derived from SARS-CoV-2 spike protein can differ from the corresponding amino acid sequence of the native receptor binding domain of SARS-CoV-2 spike protein, in one, two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, or even more amino acid residues, while at least maintaining or even exceeding the biological activity, in this case binding to the corresponding receptor ACE2 in the viral target cell of the subject, such as a mucosal cell or an epithelial cell, and / or to mediate uptake of the protein oligomer into said target cell. Encompassed by the term “derived from” are sequence variants of the receptor binding domain or receptor binding motif as used herein which are 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. It is particularly preferred that the said sequence variant of the receptor binding domain or receptor binding motif 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 SEQ ID NO: 13 or 14, over the entire length of the receptor binding domain or receptor binding motif.

[0535] The term “native” as used herein means as found in nature or wildtype. For instance, a native amino acid sequence of a human IgGl means a wildtype, non-modified, or non-genetically engineered amino acid sequence of a human IgGl .

[0536] As used herein, the term “about” when qualifying a value of a stated item, number, percentage, or term refers to a range of plus or minus 10 percent (%), 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent or 1 percent of the value of the stated item, number, percentage, or term. As regards amino acid sequences, the term “about” means plus or minus 5 amino acid residues, 4 amino acid residues, 3 amino acid residues, 2 amino acid residues or 1 amino acid residue. Preferred is a range of plus or minus 10 percent; or plus or minus 3 or 1 amino acid residue(s).

[0537] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonyms with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Evidently, the term “comprising” encompasses the term “consisting of’. More specifically, the term “comprise” as used herein means that the claim encompasses all the listed elements or method steps, but may also include additional, unnamed elements or method steps. For example, a method comprising steps a), b) and c) encompasses, in its narrowest sense, a method which consists of steps a), b) and c). The phrase "consisting of' means that the composition (or device, or method) has the recited elements (or steps) and no more. In contrast, the term “comprises” can encompass also a method including further steps, e.g., steps d) and e), in addition to steps a), b) and c).

[0538] The term “at least two” means two, three, four, five, six, seven, eight, nine, ten, or even more.

[0539] SEQUENCES

[0540] The sequences show:

[0541] SEQ ID NO: 1: HD-Bio VacOOl

[0542] SEQ ID NO: 2: HD-Bio VacOOl

[0543] SEQ ID NO: 3: HD-Bio VacOlO

[0544] SEQ ID NO: 4: HD-Bio VacOll

[0545] SEQ ID NO: 5: HD-Bio VacOll

[0546] SEQ ID NO: 6: HD-Bio Vac023

[0547] SEQ ID NO: 7: HD-Bio Vac024

[0548] SEQ ID NO: 8: HD-Bio VacO25 H

[0549] SEQ ID NO: 9: HD-Bio VacO25 K

[0550] SEQ ID NO: 10: HD-Bio Vac026

[0551] SEQ ID NO: 11: HD-Bio VacO27 H

[0552] SEQ ID NO: 12: HD-Bio VacO27 K

[0553] SEQ ID NO: 13: RBD 320-541 SEQ ID NO: 14: RBM 438-506

[0554] SEQ ID NO: 15: Human ACE2 (UniProt Q9BYF1)

[0555] SEQ ID NO: 16: Human Neuropilin (UniProt 014786)

[0556] SEQ ID NO: 17: Linker 1

[0557] SEQ ID NO: 18: Linker 2

[0558] SEQ ID NO: 19: Foldon from T4 Fibritin

[0559] SEQ ID NO: 20: T4 Bacteriophage Foldon

[0560] SEQ ID NO: 21: GCN4-pII isoleucine zipper (IZ)

[0561] SEQ ID NO: 22: IZN4

[0562] SEQ ID NO: 23: Human p53

[0563] SEQ ID NO: 24: Human COIA1

[0564] SEQ ID NO: 25: XV NCI domain (amino acids 1,135-1,198 from accession number

[0565] P39059)

[0566] SEQ ID NO: 26: IGHG1

[0567] SEQ ID NO: 27: IGHG2

[0568] SEQ ID NO: 28: IGHG3

[0569] SEQ ID NO: 29: IGHG4

[0570] SEQ ID NO: 30: IGHA1

[0571] SEQ ID NO: 31: IGHA2

[0572] SEQ ID NO: 32: IGHM

[0573] SEQ ID NO: 33: G4S linker

[0574] SEQ ID NO: 34: G4T linker

[0575] SEQ ID NO: 35: HD-Bio Vac004 Alpha-Alpha

[0576] SEQ ID NO: 36: HD-Bio Vac006 Delta-Delta

[0577] SEQ ID NO: 37: HD-BioVac008A Delta-Omicron

[0578] SEQ ID NO: 38: HD-BioVac008B Omicron-Delta

[0579] SEQ ID NO: 39: HD-Bio Vac019 Delta-BA.2

[0580] SEQ ID NO: 40: HD-Bio Vac007 Delta RBM-Delta RBM

[0581] SEQ ID NO: 41: HD-BioVac009 Omicron RBM-Omicron RBM

[0582] SEQ ID NO: 42: HD-BioVac012A Omicron RBM-Omicron RBD

[0583] SEQ ID NO: 43: HD-BioVac012B Omicron RBD-Omicron RBM

[0584] SEQ ID NO: 44: WT YP 009724390.1 surface glycoprotein [Severe acute respiratory syndrome coronavirus 2]

[0585] SEQ ID NO: 45: HD-Bio VacOOl FcDHS

[0586] SEQ ID NO: 46: HD-Bio Vac002 FcDHS-LALAPG

[0587] SEQ ID NO: 47: HD-Bio Vac017 FcLALAPG

[0588] SEQ ID NO: 48: HD-Bio Vac018 Fc wt

[0589] SEQ ID NO: 49: (G4S)2linker

[0590] SEQ ID NO: 50: (G4S)3linker

[0591] SEQ ID NO: 51: SARS-CoV-2 spike protein

[0592] SEQ ID NO: 52: Linker GGGGSGGGGSDKTHT

[0593] SEQ ID NO: 53: Linker SPGGGGGSGGGGS

[0594] SEQ ID NO: 54: Linker GGGGGSGGGGS

[0595] SEQ ID NO: 55: Bio Vac 010 Figures

[0596] The Figures show:

[0597] Figure 1: Structural elements of BioVac and rationale design of a tetrameric RBD-Fc-RBD based vaccine.

[0598] Figure 2: BioVac Core Structure and Multivalency Engineering Possibilities. The core structure of BioVac allows to work with several multivalent constructs, examples for four identical RBD (homotetrameric) or two different RBD, each dimerizing at N- vs. C-terminal (heterodimeric) using a single plasmid and Fc design is possible. Moreover, four different RBD could be expressed by defining the position of each single RBD using the N- or C- Fc fusion coordinates and directed Fc-dimerization principles, here exemplified by Fc-knob in hole (KiH) design (heterotetrameric). Evidence for realization of all these “core” structural variants and their compatibility with further Fc design elements (DHS, LALAPG etc.) was successfully demonstrated.

[0599] Figure 3: Heterotetramerization may improve protein expression, structural stability and enhance immunogenicity. Mutations leading to different Spike-RBD variants during the viral evolution may compromise expression and / or ACE2 binding efficacy in favor of immunoescape mutations as the driving force behind their evolution in immunized populations. The relatively high tetrameric RBD to IgG Fc ratio in BioVac may further amplify the impact of these alterations and contribute to a compromised expression efficacy and protein stability. We found that heterotetramerization may constitute an elegant strategy to overcome this problem. Direct expression comparison of heterotetrameric RBD consisting of N-terminal delta-RBD and C-terminal omicron-RBD is a good example for this observed phenomenon. The expression efficacy of hetero-tetrameric RBD (Delta / Omicron BioVac008A) was at ~ 113 mg / L and decreased to ~50mg / L for homotetrameric delta-RBD (BioVac006) and even stronger decrease to ~ 2mg / L for homotetrameric Omicron-RBD (BioVacOlO) in CHO transient expression model. Another trend found was that positioning more efficiently expressed RBD variants in the N-terminus of the BioVac infer an overall improved protein expression and stability while in terms of expression and structurally less advantageous variants could be positioned in the C-terminus. Moreover, the heterotetrameric molecules may elicit a much more potent immune response compared to homo-tetrameric molecules. In context of abovementioned homo- vs. heterotetramerization, the immune response elicited by homotetrameric omicron RBD (BioVacOlO) was ~ 1 / 5 of the efficacy found after i.m. injection of heterotetrameric delta-omicron RBD (BioVac008A) in mice (see Fig. 31). Moreover, heterotetramerization enables multivariant display, which may contribute to generation of “broadly” neutralizing immune responses. Indeed, broadly neutralizing sarbecovirus immunity was recently shown to be induced in mice using mosaic and cocktail nanoparticle immunogens consisting of sarbecovirus RBDs (Walls et al., Cell, 2021). Figure 4: BioVac provides a tetrameric RBD core structure. To further expand avidity or multivalency, several strategies are foreseen. First, use of oligomerization domain (OD, orange) either N- or C-terminal of the RBD. Expression of OD-RBD or RBD-OD, respectively, will result into oligomerization and alignment of these RBDs with the BioVac core structure as demonstrated for the use of a trimerization domain in this schematic. This strategy will require expression of two constructs, i.e. BioVac core structure plus the oligomerization domain consisting RBDs. In contrast, multimerization could be also achieved in a single construct by use of flexible linker (red) between an additional RBD and the core structural RBD. The same strategies could be also utilized to generate heteromultimerizations using the OD or Linker strategy. Addition of heteromeric Fc, e.g., by knob-in-hole mutations, enables expression of four distinct RBD in the core BioVac structure, and by combining these heterotetrameric core structures with OD / Linker strategies the spectrum of multivalency could be further expanded.

[0600] Figure 5: Illustrations of multimerization options based on the oligomerization domain vs. linker-based strategies as described in Fig.4 with the (oligo)multimerization executed on N- and / or C-terminal RBD of the BioVac core structure. Examples for oligomerization i.e., tri- / tetramerization domains are provided.

[0601] Figure 6: Conjugation Principles for Immune Armed BioVacs. 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, BioVac enables 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. BioVac allows 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 BioVac emerges 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 Spike mimetic self-epitopes, to fine tune or ablate potential undesired immune responses as reported in patients with post vaccine or infection (post / long-covid) syndromes.

[0602] Figure 7: Armed BioVac with Dual Immune Activation Principle. An example for a dual armed BioVac is demonstrated where a TLR-agonist is conjugated via a non-cleavable linker to BioVac for activation of the endosomal TLR pathway and a second cleavable linker is used to release a cGAS-STING agonist for activation of cytosolic DNA-sensing pathway and IFN release. This schematic shows the versatility of BioVac vaccination principle to synergistically stimulate two or more immune response compartments, hence, emulating different “infection” patterns for optimal vaccination.

[0603] Figure 8: Advantages of BioVac formulation into mRNA vaccines. BioVac could be favorably combined with mRNA-based vaccines to improve the immunization effects. As exemplified here, BioVac encapsulation into LNPs or direct conjugation of mRNA to BioVac as shown in Fig. 7 could direct the mRNA and immunization into the specific target ACE2 expressing compartment. This could be particularly crucial for e.g., mucosal like intranasal / pharyngeal or even specific pulmonary uptake of mRNA by ACE2 positive epithelial cells. Moreover, incorporation of a multivalent display protein into an mRNA vaccination could further broaden the T-Cell activation induced by the mRNA vaccine to a well-presented 3D protein structure that is needed for proper B-Cell activation and antibody response. Finally, distinct immune activation principles could synergize where mRNA induced T-cell response and reactogenicity could be utilized as a target specific immunogenic adjuvant for the protein based BioVac immunization. Not-modified mRNA such as CVnCoV could not be administrated in higher amounts due to their reactogenicity (-1 / 3-1 / 10 vs. modified mRNA utilized in Comimaty / bntl62b2 or Spikevax / mRNA-1273, respectively). Moreover, mRNA-based expression of small fragments (T-cell epitopes) is much more efficient than expression and 3D folded presentation of an entire large protein in-vivo for a proper B-Cell response. Therefore, combination of few micrograms of BioVac may overcome these limitations and synergize with mRNA vaccine to induce potent and well-tolerated T- and B-cell responses.

[0604] Figure 9: Impact of Fc-Engineering on BioVac Functionality. Based on the envisioned immunization, e.g., mucosal vs. systemic, or augmented immune stimulation in prime vs. selective stimulation of high affinity T / B receptor population, BioVac provides a unique repertoire of functional adaption via protein engineering of its IgG Fc moiety. For example, DHS modification in BioVac Fc structure augments FcRn binding at optimal mucosal pH and enhance epithelial uptake, transepithelial transport to lamina propria, endosomal affinity and recycling efficacy. Whereas, FcR engineering could be used to silence or further enhance RBD independent immunogenicity and immune cell uptake / activation. The lower panel showing FcR repertoire and their respective effector function was adapted from (Musolino et al., J Immunother Cancer, 2022). Figure 10: Mucosal Optimization of BioVac by Fc-Engineering. Interestingly, the pH at the intranasal mucosa is with ~6.5 moderately acidic and in the same range found in the endosomal compartment. To our knowledge, this is the first demonstration that a tetrameric Fc-Fusion protein, harboring the 309D / Q311H / N434S (DHS) IgG Fc substitutions, behaves like a pH- toggle for optimal mucosal immunization. Not only higher affinity and uptake of BioVac to epithelial FcRn at low mucosal pH but also stabilization of endosomal BioVac-FcRn complex facilitate transepithelial transport and release of BioVac at the neutral pH at the lamina propria where the relevant immune cells reside. Here, FcR-mediated uptake of BioVac by APC / CD4 cells facilitates MHC -based antigen presentation of antigen (RBD) peptide fragments as well as generates potent bystander immunostimulatory signals for activation of B-Cells. In parallel, B-Cells with antigen (RBD) specific BCRs are selectively activated by direct binding to the tetravalent RBDs of BioVac. 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.

[0605] Figure 11: Representative BioVac expression efficacy, size and purity in HEK (Expi) expression system. SDS-Page for reduced (RBD-Fc-RBD monomer) and non-reduced BioVac002 tetrameric SarsCov2 RBD (green) with combined DHS and LALAPG substitutions in IgG Fc (blue) as well as BioVac004, where the RBD subunit was replaced by the Sars-Cov- 2 alpha variant incl. N501Y mutation. BioVac was purified by Fc-binding to protein A and subsequent size exclusion chromatography (SEC). In line with the single clean band found in SDS-Page, SEC-HPLC shows a single peak with high purity (>98%). Considering the challenges in efficient recombinant protein expression and proper folding of SARS-CoV-2 Spike, it is intriguing that the tetrameric Fc-fusion protein BioVac was efficiently expressed, folded and purified using industrial standard antibody purification methods.

[0606] Figure 12: Biochemical Binding of BioVac to ACE2 at Picomolar Range. BioVacOOl binding to human ACE2 was studied by ELISA. Coating: 5 pg / mL hACE2-His (SAE0064, Sigma) in PBS at 4°C. Blocking: 2% BSA / PBS, 2h at RT. Ab‘ : goat anti-human Fc specific (Sigma, 12136) 1 : 10000 in blocking buffer and Ab“: donkey anti-goat HRP (sc-2020, Santa Cruz) 1 :2000 in blocking buffer. The equilibrium of BioVac binding to hACE2 dissociation constant (Kd) was estimated at 120.4 pM by a sigmoidal fit of background subtracted / normalized mean ± SD intensities of BioVac 001 dilution series from n:6 experiments.

[0607] Figure 13: Effect of Tetramerization in BioVac on ACE2 Binding. Binding of tetrameric BioVacOOl vs. dimeric RBD-Fc fusion Proteins to hACE2 was studied by surface plasmon resonance (SPR) using the Biacore X-100 system. Biotinylated ACE2 was immobilized with a Biotin CAPture kit (Cytiva) and analytes were flown over with increasing concentrations of 0, 1.1, 3.3, 10, 30 and 90 nM at 30 pL / min for 120 sec and dissociation for 600 sec. 1 : 1 binding model was employed to fit the data. The Table in Figure 13 shows corresponding association rate constant (ka), dissociation rate constant (Kd) and dissociation constant (Kd) for hACE2 binding. RBD tetramerization in BioVac was associated with > 10-Fold increased hACE2 binding compared to dimeric Fc-RBD fusion protein, i.e., the Kd for RBD-Fc binding to hACE was 4205 pM (4.021 nM) vs. 307 pM for BioVacOOl in this back-to-back comparison. Figure 14: This Figure shows BioVac ACE2 binding ELISA (see Figure 12) and SPR results in context of previously reported dissociation constant (Kd) values as reported by (Shang et al., Nature, 2020).

[0608] Figure 15: Vaccination Principle - Active Immunization with BioVac. Most current vaccination schemes are based on providing the immunogen intramuscular (i.m.) for induction of a systemic immunity. This is in contrast to the natural pattern of infection that for SARS- CoV-2 a primary mucosal infection followed by systemic immune response. To emulate this natural infection pattern, first in human (FIM) for combined intranasal (i.n.) and i.m. systemic vaccination with BioVac adjuvanted with MF59 analogon (oil-in-water emulsion) is shown. The immune response in FIM study after dual i.n. and i.m. prime immunization was in line with data from patients undergoing SARSCOV2 infection response (lower right panel, adapted from (Sterlin et al.,Sci Transl Med, 2021), with a first mucosal IgA response dominance followed by the systemic IgG response. The impact of different adjuvanted BioVacs was further studied in preclinical setting using CpG and AS03.

[0609] Figure 16: First in human Mucosal and Systemic Immunization with BioVac. Combined intranasal (100 pg, i.e., 50pg BioVac per nose hole) and intramuscular (25pg BioVac adjuvanted with 0.25mL MF59) vaccination. MF59 is based on nano-emulsification of two components, i.e., Sorbitan trioleate (0.5% w / v) in squalene oil (5% v / v) and Tween 80 (0.5% w / v) in sodium citrate buffer (10 mM, pH 6.5). Immunization efficacy was assessed by IgA and IgG antibody titers against SARS-CoV-2 Spike and Nucleocapsid proteins using different sources (plasma and serum) as well as platform technologies (Euroimmun QuantiVac, Siemens ADVIA and Roche Elecsys). Given the comparability of plasma vs. serum data, post boost measurements were conducted in standard serum only. Longitudinal antibody titers post prime vaccination with BioVacOOl (Wuhan) and booster with BioVac004 (alpha variant) are shown. Potent mucosal IgA response was found reaching the saturation level of the assay (+ symbol) 18 days post prime and from 10 days post boost immunization. The peak level of IgG was reached 25 days post prime immunization with BioVac. Both IgA and IgG levels slowly declined over time after prime immunization. Accordingly, the 2nd immunization (boost) was decided to be given at 98 days post prime when the IgG and IgA antibody titer were at IgG -157 WHO BAU and IgA still - 3.44-fold above the reactive index level in Euorimmune assay. A high and persistent induction of the IgG and IgA was observed after the 2nd immunization (boost). The anti-nucleocapsid antibody level was negative over the entire 210 days indicating no natural infection occurring during this observation period. These data indicate that an optimal BioVac booster strategy that allows immune response to evolve and maturate (see Fig. 16) would require an interval of - 3 months between prime and booster immunizations. In contrast, in Covid- 19 Pandemic a much shorter interval of only few days (-21) was utilized for most vaccination regimens to induce as early as possible the highest possible antibody titer for viral neutralization. The rationale behind this strategy was that very high antibody titers may produce sterile vaccination and prevent infection. Overall, Covid- 19 pandemic has taught us that an early stage of viral evolution towards adaption to its new human “host” combined with misguided aberrant immune reaction against complex viral epitopes in infection naive immune system could result in severe sequela and death of infected patients. In this situation, fast provision of the naive immune system with minimal immunogenic antigen would be key to navigate the immune response to the right direction, but a specific and potent systemic / mucosal immunity will require establishment of memory immune cells and mucosal training which will need time and repeated exposure to properly evolve. From this FIM trial, an alternative strategy in pandemic situation is conceivable where prime immunization is given systemic (i.m.) and mucosal (i.n.) for the first exposure of human immune system to the most relevant viral epitopes, and in parallel, the immune system receives sufficient time of at least 3-4 month to evolve properly. During this interval, when the IgA start to drop ~ 3 weeks after prime until the second systemic immunization is provided, infection could be prevented and mucosal immunity trained by intranasal application of BioVac. This strategy would expose the adaptive systemic and mucosal immune systems to the relevant antigen and stimulate the evolution of a proper immune response for viral neutralization. At the same time the maturation of the immune system is bridged by repeated mucosal BioVac administrations to avoid infection until the optimal immune response strength is built. Moreover, as in this case where alpha variant was used for the booster, adaption of the antigen is further possible to broaden the immune response and avoid the risk of “antigenic sin”. Overall, this strategy may lead to maturation of a more physiologic anti-viral immunity with decreased risk for imprinting and dysregulation immune responses.

[0610] Figure 17: Biochemical Serum Neutralization Assay. Competition of serum antibodies with ACE2-RBD binding was assessed using Euroimmun assay illustrated by the left schematic. Each line illustrates the dilution series for serum collected at different days post FIM Biovac immunization. A left shift of the dilution series is found post prime immunization between day 18 and 25 indicating maturation of the immune response against the RBD. Moreover, potent induction of neutralizing antibodies and maturation after second “booster” immunization is shown at the two time points (day 107 and 126 post prime, day 10 and 19 post booster).

[0611] Figure 18: Pseudoviral Neutralization Assay. To quantify neutralizing antibodies in serum after FIM BioVac vaccination (107 days post Wuhan RBD prime and 10 days post alpha variant RBD booster), tissue culture infection dose (TCID, reporter signal of infected ACE2 expressing HEK cells) of pseudotyped lentiviral particles with the Spike protein of different SARS-CoV- 2variants (alpha, beta, delta) was determined against a serum dilution series as described in the left schematic (Adapted from (Nie et al., Nat Protoc, 2020)). These data indicate that vaccination with alternative tetrameric RBD and sufficient interval between prime and boost result in evolution of a potent and deep humoral immune response broadly neutralizing viral variants.

[0612] Figure 19: Humoral Immune Response to BioVac Post Prime and Boost Vaccinations. Violine plots represent serum antibody titers (on the natural logarithm scale) against SARS-CoV-2 Spike protein are shown for IgA (Euroimmun, ratios, >1.1 = reactive), IgG (Siemens, Index > 1 = reactive) and Euroimmun (WHO BAU > 35.2 = reactive). In Analogy to Fig.17, biochemical serum neutralization assay (Euroimmun) one week post boost was performed.

[0613] Figure 20: Humoral Immune Response to BioVac Post Prime and Boost Vaccinations. Individual serum antibody titers against SARS-CoV-2 Spike protein are shown for IgA (Euroimmun, ratios, >1.1 = reactive), IgG (Siemens, Index > 1 = reactive) and Euroimmun (WHO BAU > 35.2 = reactive). Lines connect antibody titers for each individual three weeks post prime vaccination with BioVac with one week post BioVac booster. Geometric mean values of the entire cohort for each time point are provided as red dots(n: 10). To put these responses into natural infection context, Siemens IgG Index of > 4.8 was determined by FDA as the cutoff necessary to qualify high-titer convalescent donor plasma for use in donations, i.e. to treat hospitalized patients with COVID-19 (FDA 2023; Siemens°Healthineers). Therefore, with a geometric mean Siemens index value of 9.37 ~ 21 days post prime, BioVac potently induced systemic SarscCov2 IgG titers that was 8-Fold further enhanced one week after boost immunization. Similar trends were found for Euroimmun WHO BAU valuse for IgG as well as potent IgA responses after prime and boost immunizations in this short-term pandemic scheme with only ~21 days (3 weeks) interval between ...

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 domain (RBD), an immunoglobulin Fc (Ig Fc), and at least one second receptor binding domain (RBD), wherein a) 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 is from or derived from SARS-CoV-2 spike protein, wherein the receptor binding domain comprises the amino acid sequence of SEQ ID NO. 13, or a fragment thereof, or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO. 13, preferably over the entire length.

2. The protein oligomer of claim 1, 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.

3. The protein oligomer of any one of claims 1 to 2, 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.

4. The protein oligomer of any one of claims 1 to 3, 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.

5. The protein oligomer of any one of claims 1 to 4, wherein the RBD or fragment thereof is the same or different, (i) within the at least first monomer of the protein oligomer, or (ii) within the at least second monomer of the protein oligomer, or (iii) both within the at least first monomer and within the at least second monomer of the protein oligomer.

6. The protein oligomer of claim 5, wherein the RBD or fragment thereof is from one or more variants of concern (VOC), selected from the group consisting of SARS-CoV-2 variant alpha (B. l.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 variantomicron 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 BAA, SARS-COV-2 variant omicron BA.5, SARS-COV-2 variant omicron BJ1, SARS-COV-2 variant omicron BAA.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 and SARS-COV-2 variant omicron BA.2.87.1.

7. The protein oligomer of any one of claims 1 to 6, 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 receptor binding domain(s) (RBD), preferably as defined in claim 6.

8. The protein oligomer of claim 7, 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 miniproteins, short peptides with discrete protein-like structures, trimerization domain of the bacteriophage T4 fibritin (foldon), TNFalpha trimerization domain, zinc finger or p53 tetramerization domain.

9. The protein oligomer of any one of claims 1 to 8, wherein the protein oligomer comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1 to 12, or 35 to 43, or 55, or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NO. 1 to 12, or 35 to 43, or 55.

10. A vaccine comprising the protein oligomer of any one of claims 1 to 9.

11. The vaccine of claim 10, 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.

12. The vaccine of claim 11, wherein the adjuvant is selected from the group consisting of 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.

13. The vaccine of claim 12, wherein the adjuvant is associated with said at least first monomer or said at least second monomer in the protein oligomer, preferably wherein at least one RBD is linked enzymatically or chemically to the adjuvant of claim 11 or 12.

14. The vaccine of any one of claims 10 to 13, wherein said vaccine is administered intranasally and / or via the intramuscular route, preferably wherein said vaccine is co-administered intranasally and via the intramuscular route.

15. The vaccine of any one of claims 10 to 14 for use in active immunization in a subject, preferably a human subject.

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