Disulphide-linked oligomerization domain, polypeptides, and uses thereof

Abstract

The present disclosure relates to disulphide-linked oligomerization domains for oligomerization of polypeptide fragments. The disulphide-linked 5 oligomerization domain, according to embodiments herein, facilitate correct folding and assembly of proteins into their functional oligomeric forms, and enhances the stability and immunogenicity of oligomeric proteins. Also disclosed are polypeptides having the disulphide-linked oligomerization domains linked to antigenic peptides, and a pharmaceutically acceptable carrier. 10 The present disclosure further relates to compositions, nucleotides, recombinant host cells, and methods for producing the polypeptides as disclosed herein.

Description

DISULPHIDE-LINKED OLIGOMERIZATION DOMAIN, POLYPEPTIDES, AND USES THEREOFFIELD OF INVENTION

[0001] The present disclosure broadly relates to the field of immunobiology, and particularly discloses disulphide-linked oligomerization domain, polypeptide fragments or proteins comprising the disulphide-linked oligomerization domain, methods of preparation, and uses thereof.BACKGROUND OF THE INVENTION

[0002] Protein stabilization is a crucial aspect of protein engineering, particularly in the context of therapeutic proteins and protein subunit vaccines. Oligomerization domains play a crucial role in facilitating the correct folding and assembly of proteins into their functional oligomeric form, enhancing the stability and structural integrity of oligomeric proteins. Two widely used protein oligomerization domains are the isoleucine zipper (IZ), derived from the GCN4 transcriptional activator in Saccharomyces cerevisiae. and the foldon domain from the bacteriophage T4 fibritin protein (Fd) (Harbury et al, 1993, 1994; Giithe et al, 2004). These domains play a crucial role in facilitating the correct folding and assembly of proteins into their functional oligomeric form, enhancing the stability and structural integrity of oligomeric proteins.

[0003] Foldon has been used as a trimer domain in several vaccine antigens and therapeutic molecules to enhance stability, solubility, and immunogenicity of the Influenza hemagglutinin stem domain (Lu etal, 2014; Sutton etal, 2017) and hemagglutinin ectodomain (Krammer et al, 2012; Yu et al, 2015; Du et al, 2011), HIV envelope glycoprotein (Li et al, 2019; Ringe et al, 2015), F protein of Respiratory Syncytial Virus (McLellan et al, 2013; Che et al, 2023), SARS- CoV-2 Spike glycoprotein (Lu etal, 2022; Hsieh etal, 2020; Schaub etal, 2021; Xiong et al, 2020) and Receptor Binding Domain (Tai et al, 2016; Vogel et al, 2021) and ACE2 (Xiao et al, 2021). Despite the extensive use of heterologousoligomerization agents like Foldon in serval vaccine preclinical studies, their self-immunogenicity after repeated application has not been evaluated in depth.

[0004] However, in a few cases, the incorporation of glycan into heterologous oligomerization domains like Isoleucine Zipper (IZ) and Foldon subdued self-immune responses in vaccinated animals (Sliepen etal, 2015). The isoleucine zipper (IZ) comprises a-helices arranged in a coiled-coil pattern with heptad repeats. The first “a” and fourth “d” amino acid residues within each heptad repeat are crucial in determining the protein's oligomerization state (Harbury et al, 1993). The immunogenicity of oligomerization domains, including the IZ oligomerization domain, is a crucial aspect of protein engineering for therapeutic and vaccination purposes. There exists a need for development of a versatile and powerful motif in protein engineering.SUMMARY OF INVENTION

[0005] In an aspect of the present disclosure, there is provided a disulphide- linked oligomerization domain comprising a Domain A attached to a Domain B, wherein the Domain A is a disulphide forming motif having an amino acid sequence of at least 95% identity to a sequence selected from SEQ ID NO. 109 or SEQ ID NO. 180; and wherein the Domain B is an oligomerization motif having an amino acid sequence of at least 95% identity to the sequence selected from SEQ ID NO. 162, SEQID NO. 181, or SEQID NO. 182.

[0006] In an aspect of the present disclosure, there is provided a disulphide- linked oligomerization domain comprising a polypeptide having an amino acid sequence as set forth in Formula I: E-I-(S / N)-E-(E / T)-D-P-C-E-C-K-S-I-K-K- (E / N)-I-(E / T)-(N / A)-I-(T / K)-K-E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-A (Formula I) (SEQ IN NO. 99).

[0007] In another aspect of the present disclosure, there is provided a polypeptide B (a fusion peptide) comprising the disulphide-linked oligomerization domain as disclosed herein, attached to a polypeptide A at the N-terminal or C-terminal end of the polypeptide A for oligomerization of the polypeptide A.

[0008] In another aspect of the present disclosure, there is provided a polynucleotide encoding the disulphide-linked oligomerization domain or the polypeptide B as disclosed herein.

[0009] In another aspect of the present disclosure, there is provided a recombinant vector having the polynucleotides, as disclosed herein, operably linked to a promoter.

[0010] In another aspect of the present disclosure, there is provided a recombinant host cell comprising the recombinant vector as described herein.

[0011] In yet another aspect of the present disclosure, there is provided a composition comprising the polypeptide B as disclosed herein, and a pharmaceutically acceptable carrier.

[0012] In another aspect of the present disclosure, there is provided a method for producing the polypeptide B, comprising culturing the recombinant host cell as disclosed herein under suitable conditions to express the polypeptide B.

[0013] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0014] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0015] Figure 1 is a representation illustrating the design of disulphide- linked oligomerization domain (cCMP-IZm): wherein (a) depicts three- dimensional structure of cCMP (PDB ID: 1AQ5), showing the cCMP aminoacid sequence (i.e. EEDPCECKS); and Disulphide-linked cysteines, (b) depicts helical wheel representation of the coiled-coil heptad repeats in IZm, wherein the d positions contain isoleucine residue essential for oligomerization. Predicted glycosylation sites at positions c and g, featuring asparagines (N), are indicated by dotted circles, (c) depicts amino acid sequences of the heptad repeat synthetic peptide (IZm) and variant, in accordance with embodiments herein.

[0016] Figure 2 depicts protein SDS-PAGE profile of oligomerizing DS variants (V0, VI, V2, V3, and V4) when fused at the N-terminal of RBD1- DM37 (SEQ ID NO. 98) and PhH3HA10 (SEQ ID NO. 100), wherein a) depicts results for SARS CoV-2 RBD, and b) depicts results for Influenza A virus-mutated stem, in accordance with embodiments herein.

[0017] Figure 3 depicts the protein SDS-PAGE profile of fusion proteins, according to embodiments herein, upon storage. The Coomassie-stained protein gel is shown in panels (a), (b), and (c) for 0, 24, and 96-hour samples, and panel (d) provides the lane information, in accordance with embodiments herein.

[0018] Figure 4 depicts comparative oligomer profile analysis of Influenza A-virus-mutated stem protein fused with Foldon and DSV2, according to embodiments herein. Expi293 cell produced proteins absorbance at 280 nm (mAU) versus elution volume (mL) is plotted using GraphPad Prism software, wherein a) depicts the size exclusion chromatography profile of the Gel Filtration Standard (Bio-Rad, Cat #1511901), and b) depicts the log MW vs. elution volume plot. The overlapped SEC profiles of mPhH3HA10-Foldon (SEQ ID NO. 71) and DSV2-mPhH3HA10 (SEQ ID NO. 75) are shown in panel (c), in accordance with embodiments herein.

[0019] Figure 5 depicts comparative oligomer profile analysis of RBD1- DM37 domain (SEQ ID NO. 98) and its DSV2 fused oligomer (DSV2-RBD1- DM37, SEQ ID NO. 27). Expi293 cell produced proteins absorbance at 280nm (mAU) versus elution volume (ml) is plotted using GraphPad Prism software, a) The size exclusion chromatography profile of Gel Filtration Standard (BioRad, Cat #1511901) and b) Log MW Vs elution volume plot is shown. Theoverlapped SEC profile of RBD1-DM37 and DSV2-RBD1-DM37 is shown in panel c), in accordance with embodiments herein.

[0020] Figure 6 depicts pseudoviral neutralization titers of mice sera raised against RBD1 -DM37 fused with DS variants (V0, VI, V2, V3, V4) (as depicted in SEQ ID NO. 19, SEQ ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 31 and SEQ ID NO. 35), wherein (a) is a schematic representation depicting the immunization protocol in BALB / c mice. In panel (b), neutralization ID50 titers plot against B.l, (c) Delta, and (d) Omicron BA.l pseudoviruses are shown against each antigen boost sera (day 35), in accordance with embodiments herein.

[0021] Figure 7 depicts comparative immunogenicity of the Spike-6p- DM37 fused with the Foldon (SEQ ID NO. 57) and DSV2 (SEQ ID NO. 59) oligomerization domains in Mice, wherein (a) is a schematic representation depicting the immunization protocol. ELISA end point titers are depicted in (b) 2 weeks prime sera, (c) 5-week boost sera, and (d) 16 weeks extended boost sera, raised against the above-mentioned antigens and analyzed on various SARS-CoV-2 protein immobilized plates, in accordance with embodiments herein.

[0022] Figure 8 depicts the comparative immunogenicity of the mECTO- H3fused with the Foldon (SEQ ID NO. 63) and DSV2 (SEQ ID NO. 67) oligomerization domains in mice, wherein (a) is a schematic representation depicting the immunization protocol. ELISA endpoint titers are depicted in (b) 2 weeks prime sera, (c) 5 week boost sera, and (d) 16-weeks extended boost sera, raised against the above-mentioned antigens and analyzed on various SARS- CoV-2 protein immobilized plates, in accordance with embodiments herein.

[0023] Figure 9 depicts the comparative immunogenicity of the PhH3HAl 0 fused with the Foldon (SEQ ID NO. 71) and DSV2 (SEQ ID NO. 75) oligomerization domains, wherein (a) is a schematic representation depicting the immunization protocol. ELISA endpoint titers as depicted in (b) 2 weeks prime sera, (c) 5-week boost sera, and (d) 16 weeks extended boost sera, raisedagainst the above-mentioned antigens and analyzed on various SARS-CoV-2 protein immobilized plates, in accordance with embodiments herein.

[0024] Figure 10 depicts pseudoviral neutralization titers of mice sera raised against Spike-6p-DM37 fused with Foldon (SEQ ID NO. 57) and DSV2 (SEQ ID NO. 59), wherein (a) is a schematic representation depicting the immunization protocol, (b) depicts the neutralization ID50 titers plot against B.l, & Omicron BA. l pseudoviruses shown against each antigen boost sera, in accordance with embodiments herein.

[0025] Figure 11 depicts comparative immunogenicity of the Spike-6p- DM37 the Foldon (SEQ ID NO. 57) and DSV2 (SEQ ID NO. 59) oligomerization domains in Hamster, wherein (a) is a schematic representation depicting the immunization protocol, (b) depicts ELISA end point titers of 2 weeks prime sera and 5-week boost sera, raised against the above-mentioned antigens and analyzed on various SARS-CoV-2 protein immobilized plates, (c) depicts comparison of body weight loss post SARS-CoV-2 Beta virus challenge 105PFU / lOOpl in these immunized animals are shown. PBS+SWE was used as mock immunization groups, and (d) showing lung histopathology from one of the representative animals from panel (c), in accordance with embodiments herein.

[0026] Figure 12 depicts (a) Gel filtration standard curve used for molecular weight estimation, and (b) Size-exclusion chromatography (SEC) elution profiles of mECTO-H3-Foldon and mECTO-H3-DSV2, in accordance with embodiments herein.

[0027] Figure 13 depicts representative negative-stain transmission electron microscopy (NS-TEM) images of mECTO-H3-DSV2, wherein panel (i) shows particle distribution at a 100 nm scale bar, while panel (ii) displays representative 2D class averages derived for each protein, highlighting trimeric structural features (scale bar: 10 nm), in accordance with embodiments herein.

[0028] Figure 14 depicts characterization of DSV2-(RBD1-DM37)-S2 (also referred to herein as DSV2-RS2) immunogen. DSV2-(RBD1-DM37)-S2 and (RBD1-DM37)-S2 (also referred to herein as RS2) proteins were expressedand purified from the supernatant of Expi293 cells, wherein panel (a) presents the SDS-PAGE profile of the purified proteins under both reducing and nonreducing conditions, panel (b) presents the SEC profile of these proteins, while panel (c) displays the thermal unfolding profile, in accordance with embodiments herein.

[0029] Figure 15 depicts comparative immunogenicity of DSV2-(RBD1- DM37)-S2 (also referred to herein as DSV2-RS2) and (RBD1-DM37)-S2 (also referred to herein as RS2) proteins in hamsters, where immunogenicity data is depicted for DSV2-RS2 and RS2 protein antigens in a hamster model, following an immunization and viral challenge protocol, in accordance with embodiments herein.

[0030] Figure 16 depicts protection of hamsters after immunization with DSV2-(RBD1-DM37)-S2 (also referred to herein as DSV2-RS2) and -(RBD1- DM37)-S2 (also referred to herein as RS2) post-live beta virus challenge, in accordance with embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0031] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the scope of the present disclosure includes all such variations and modifications that may be apparent to a person skilled in the art in light of the present disclosure. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.Definitions

[0032] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The termsused herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0033] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0034] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0035] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0036] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably. The term “pharmaceutically acceptable carrier”, as used herein, refers to any carrier, excipients, adjuvants known to a person skilled in the art, for use in therapeutic or vaccine compositions

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0038] In general, when more than one alternatives amino acid residues are possible at a position, it may be indicated as P / S which is intended to mean that any of proline or serine may be present at that position. Similarly, N / I may indicate that any of asparagine or isoleucine may be present at that position, and so on. The representation “X” in an amino acid chain, as used herein is intended to refer to alternative amino acid residues that may be present at that position,for eg: X3 in an amino acid chain means that any amino acid residue may be present at position 3 of the amino acid chain. Such notations are generally known to a person skilled in the art and generally used in representing amino acid residues / substitutions in a given amino acid sequence. Also, amino acids are generally represented by single letter and three letter abbreviations, for example “Alanine” is represented by single letter code “A” and three letter code “ala” or “Ala”. Similarly, the single letter code include R (Arginine), N (Asparagine), D (Aspartic acid), C (Cysteine), E (Glutamic acid), Q (Glutamine), G (Glycine), H (Histidine), I (Isoleucine), L (Leucine), K (Lysine), M (Methionine), F (Phenylalanine), P (Proline), S (Serine), T (Threonine), W (Tryptophan), Y (Tyrosine), and V (Valine). Such representations are generally used and well understood by a person skilled in the art. The present disclosure in describing the present invention employs such representations or phrases which is intended to mean the generally acceptable meaning in the art.

[0039] Embodiments herein provide disulphide-linked oligomerization domains for oligomerization of polypeptide fragments. The term “oligomerization domain” or “oligomerization motif’, as used herein, refers to a domain or polypeptide fragment that is capable of forming oligomers. The term “disulphide-linked oligomerization domain”, as used herein, refers to a domain or polypeptide fragment that is capable of forming disulphide linked oligomers. The disulphide-linked oligomerization domain, according to embodiments herein, facilitate correct folding and assembly of proteins into their functional oligomeric forms, and enhances the stability and immunogenicity of oligomeric proteins. The disulphide-linked oligomerization domain, as disclosed herein, when fused with peptides such as antigenic peptides are capable of improving thermal stability, oligomerization, and affecting immunogenicity of such peptides. By enhancing the stability and homogeneity of the oligomeric protein antigens, the disulphide-linked oligomerization domain has the potential to improve the potency and durability of immune responses elicited by protein subunit vaccines.

[0040] Unlike existing oligomerization domains, the present disclosure provides an oligomerization domain which incorporate unique structural motifs that enhance its stability, ensuring the integrity of proteins under various physiological conditions, including high temperatures and varying pH levels. This stability is crucial for maintaining the efficacy of vaccines and therapeutic proteins during storage and transportation.Disulphide-linked oligomerization domain

[0041] The disulphide-linked oligomerization domain, according to embodiments herein, comprises a fusion of a disulphide forming motif and an oligomerization motif, which when introduced with N-glycosylation sites achieves disulphide linked oligomers having reduced immunogenicity.

[0042] Accordingly, in an embodiment, there is provided a disulphide- linked oligomerization domain comprising a combination of a Domain A and a Domain B.

[0043] The Domain A, according to embodiment herein, is a polypeptide fragment capable of forming a disulphide linkage, also referred to herein as “disulphide forming motif’. The Domain A comprises at least two cysteine residues. In an embodiment, the Domain A is a disulphide forming motif having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from SEQ ID NO. 109 or SEQ ID NO. 180, and comprising cysteine residues at position 5 and 7 of the Domain A.

[0044] According to embodiments herein, the Domain A may be fused to Domain B covalently, at the N-terminal or C-terminal end, with or without one or more linkers. In an embodiment, there is provided a disulphide-linked oligomerization domain, wherein the Domain A is covalently attached at the N- terminal end of the Domain B. In a preferred embodiment Domain A comprises amino acid residues “EIS” or “EIN” covalently attached at the N-terminal end of the Domain A. Accordingly, in an embodiment, there is provided a disulphide-linked oligomerization domain comprising a Domain A attached toa Domain B, wherein the Domain A comprises amino acid residues “EIS” or “EIN” covalently attached at the N-terminal end of the Domain A, and comprising cysteine residues at position 8 and 10 of the Domain A.

[0045] The Domain B, according to embodiment herein, is a polypeptide fragment capable of oligomerization, also referred to herein as an “oligomerization motif’. In an embodiment, the Domain B is a synthetic polypeptide comprising heptad repeats having Isoleucine residues. In an embodiment, the Domain B is an oligomerization motif having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence selected from SEQ ID NO. 162, SEQID NO. 181, or SEQID NO. 182. In an embodiment, the Domain B is an oligomerization motif having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence selected from SEQ ID NO. 162, SEQID NO. 181, or SEQID NO. 182, and comprising one or more N-glycosylation sites having amino acid sequence “NXT”, wherein “X” is selected from “E” or “I”.

[0046] In an embodiment, the Domain A is a disulphide forming motif having an amino acid sequence selected from the group consisting of SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120, SEQ ID NO. 121, SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 126, SEQ ID NO. 127, SEQ ID NO. 128, SEQ ID NO. 129, SEQ ID NO. 130, SEQ ID NO. 131, SEQ ID NO. 132, SEQ ID NO. 133, SEQ ID NO. 134, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 137, SEQ ID NO. 138, SEQ ID NO. 139, SEQ ID NO. 140, SEQ ID NO. 141, SEQ ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 145, SEQ ID NO. 146, and SEQ ID NO. 180.

[0047] In an embodiment, the Domain B is an oligomerization motif having an amino acid sequence selected from the group consisting of SEQ ID NO. 147,SEQ ID NO. 148, SEQ ID NO.149, SEQ ID NO.150, SEQ ID NO.151, SEQ ID NO.152, SEQ ID NO.153, SEQ ID NO.154, SEQ ID NO.155, SEQ ID NO.156, SEQ ID NO.157, SEQ ID NO.158, SEQ ID NO.159, SEQ ID NO. 160, SEQ ID NO. 161, SEQ ID NO. 162, SEQID NO. 181, or SEQID NO. 182.

[0048] In an embodiment, the disulphide-linked oligomerization domain comprises a Domain A attached to a Domain B, wherein the Domain A is a disulphide forming motif having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from SEQ ID NO. 109 or SEQ ID NO. 180, and wherein the Domain B is an oligomerization motif having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence selected from SEQ ID NO. 162, SEQID NO. 181, or SEQID NO. 182.

[0049] In an embodiment, the disulphide-linked oligomerization domain comprises a Domain A attached to a Domain B, wherein the Domain A is a disulphide forming motif having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from SEQ ID NO. 109 or SEQ ID NO. 180, and comprising cysteine residues at position 5 and 7 of the Domain A, and wherein the Domain B is an oligomerization motif having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence selected from SEQ ID NO. 162, SEQID NO. 181, or SEQID NO. 182, and comprising one or more N-glycosylation sites. In an embodiment, the Domain B comprises N-glycosylation sites having amino acid sequence “NXT” at amino acid positions selected from the group consisting of:(i) positions 7 to 9, and 25 to 27 of Domain B; and(ii) positions 4 to 6, and 25 to 27 of Domain B, wherein “X” is selected from “E” or “I”.

[0050] In an embodiment, there is provided a disulphide-linked oligomerization domain, wherein the Domain A is attached to the Domain B atthe N-terminal of Domain B, and wherein the Domain A has an amino acid sequence having cysteine residues at positions 5 and 7, and wherein the Domain B comprises N-glycosylation sites having amino acid sequence “NXT” at amino acid positions selected from the group consisting of:(iii) positions 7 to 9, and 25 to 27 of Domain B; and(iv) positions 4 to 6, and 25 to 27 of Domain B.

[0051] The disulphide-linked oligomerization domain, according to embodiments herein, may comprise various combinations of the Domain A with Domain B, fused at C-terminal or N-terminal end. For example, the disulphide- linked oligomerization domain may comprise a combination of SEQ ID NO. 102 and SEQ ID NO. 147, a combination of SEQ ID NO. 103 and SEQ ID NO. 148, a combination of SEQ ID NO. 102 and SEQ ID NO. 148, a combination of SEQ ID NO. 103 and SEQ ID NO. 147, a combination of SEQ ID NO. 109 and SEQ ID NO. 181, a combination of SEQ ID NO. 180 and SEQ ID NO. 182, and so on.

[0052] The disulphide-linked oligomerization domain may include engineered N-glycosylation sites at various positions on the amino acid sequence.

[0053] In an embodiment, the disulphide-linked oligomerization domain comprises a combination of the N-terminal region of chicken cartilage matrix protein (cCMP) (Domain A) and a 4-heptad repeat synthetic peptide sequence (IZm) (Domain B), with engineered N-glycosylation sites introduced at specific residues within the coiled-coil structure. In an embodiment, the polypeptide comprising the combination of the N-terminal region of chicken cartilage matrix protein (cCMP) and a 4-heptad repeat synthetic peptide sequence (IZm) has the amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence as set forth in SEQ ID NO. 3, also referred to herein as “DSV0”, “V0”, or “cCMP-IZm-V0”.

[0054] The term “N-linked glycosylation”, as used herein, refers to the attachment of a glycan moiety to the nitrogen atom of an asparagine (N) residue on an amino acid chain. Accordingly, the term “N-linked glycosylation site”refers to the site of glycosylation on the amino acid chain. For example, N- glycosylation sites may have an amino acid sequence “NET”, at positions “3 to 5” on an amino acid chain. Such representation is intended to mean that a site having the amino acid residues “N”, “E”, and “T”, is a N-glycosylation site present at positions 3, 4, and 5, respectively, on the amino acid chain. The disulphide-linked oligomerization domains, according to embodiments herein, have been engineered to include such N-glycosylation sites at various positions. The glycans are positioned at non-interfering residues, ensuring they do not disrupt oligomerization. Additionally, disulfide bonds may be included to further stabilize the oligomeric structure through interprotomer interaction.

[0055] The sequence of the disulphide-linked oligomerization domains, according to embodiments herein, is engineered to contain at least two N- glycosylation sites at exposed residue locations in the coiled-coil that should not interfere with coiled-coil formation. For example, in the heptad repeat (SEQ ID NO. 162), isoleucines at positions (a) and (d) play a crucial role in helix oligomerization, contributing to the stability and formation of the helical structure (Figure 1). Conversely, positions (c), (e) and (g) in the heptad repeat are considered potential sites for glycosylation, as these positions are less likely to interfere with the isoleucines involved in oligomerization. To introduce glycosylation, potential sites are identified based on their spatial arrangement and structural context. Specifically, the glycosylation sites may be introduced at SEE NET (a region disordered in the cCMP structure), EIE NIT (which corresponds to the gab region in the heptad repeat), and AIK —> NIT (representing the cde region in the heptad repeats) (Figure 1), of the cCMP-IZm- V0.

[0056] Accordingly, in an embodiment, the disulphide-linked oligomerization domain comprises a polypeptide having a sequence as set forth in SEQ ID NO. 3 (cCMP-IZm-V0) and having engineered N-glycosylation sites of amino acid sequence “NXT”, wherein “X” is selected from “E” or “I”, at amino acid positions selected from the group consisting of:(i) positions 19 to 21, and 37 to 39;(ii) positions 16 to 18, and 37 to 39;(iii) positions 3 to 5, 19 to 21, and 37 to 39; and(iv) positions 3 to 5, 16 to 18, and 37 to 39.

[0057] In an embodiment, the disulphide-linked oligomerization domain also referred to herein as “cCMP-IZm-Vn” (wherein n =1, 2, 3, or 4) achieves disulphide linked oligomerization of peptide monomers. The two cysteine residues at amino acid positions 8 and 10 on the disulphide linked oligomerization domain (cCMP-IZm-Vn) polypeptide facilitate the interchain disulphide linked oligomerization.

[0058] The disulphide linked oligomerization domain, according to embodiments herein, may further be linked to a peptide or protein antigen at the N-terminal or C-terminal end of the peptide or protein antigen (also referred to herein as Polypeptide A). The peptide may be an antigenic peptide which may be fused with the disulphide linked oligomerization domain to obtain an oligomeric immunogen (also referred to herein as Polypeptide B). The glycan- shielded oligomeric immunogen, according to embodiments herein, demonstrates reduced immunogenicity of the oligomerization domain compared to non-glycosylated oligomerization domains. The glycan-shielded oligomeric immunogen also shows increased stability and homogeneity relative to immunogens using traditional oligomerization domains such as foldon and provides enhanced protection and immune response in animal models. The combination of cCMP and IZm, along with glycosylation and disulfide bonds, achieves self-scaffold titres and enhanced stability in the disulphide linked oligomerization domain for protein antigens.

[0059] In an embodiment of the present disclosure, the disulphide linked oligomerization domain has an amino acid sequence as set forth in Formula 1 :E-I-(S / N)-E-(E / T)-D-P-C-E-C-K-S-I-K-K-(E / N)-I-(E / T)-(N / A)-I- (T / K)-K-E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-A (Formula 1).

[0060] In general, when more than one alternatives amino acid residues are possible at a position, it may be indicated as P / S which is intended to mean that any of proline or serine may be present at that position Similarly, N / I may indicate that any of asparagine or isoleucine may be present at that position, and so on. Such notations are generally known to a person skilled in the art and generally used in representing amino acid residues / substitutions in a given amino acid sequence. Also, amino acids are generally represented by single letter and three letter abbreviations, for example “Alanine” is represented by single letter code “A” and three letter code “ala” or “Ala”. Similarly, the single letter code include R (Arginine), N (Asparagine), D (Aspartic acid), C (Cysteine), E (Glutamic acid), Q (Glutamine), G (Glycine), H (Histidine), I (Isoleucine), L (Leucine), K (Lysine), M (Methionine), F (Phenylalanine), P (Proline), S (Serine), T (Threonine), W (Tryptophan), Y (Tyrosine), and V (Valine). Such representations are generally used and well understood by a person skilled in the art. The present disclosure in describing the present invention employs such representations or phrases which is intended to mean the generally acceptable meaning in the art.

[0061] The amino acid sequence of Formula 1 is also set forth as SEQ ID NO. 99. The Formula 1 as set forth in SEQ IN NO. 99 is as follows:E-I-X3-E-X5-D-P-C-E-C-K-S-I-K-K-XI6-I-XI8-XI9-I-X2I-K-E-Q-E- A-I-K-K-K-I-E-A-I-E-K-N-I-T-A, whereinX3 is selected from S or N;X5 is selected from E or T;Xi6 is selected from E or N;Xis is selected from E or T;X19 is selected from N or A; andX21 is selected from T or K.

[0062] In an embodiment of the present disclosure, the disulphide linked oligomerization domain has an amino acid sequence as set forth in Formula I:E-I-(S / N)-E-(E / T)-D-P-C-E-C-K-S-I-K-K-(E / N)-I-(E / T)-(N / A)-I- (T / K)-K-E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-A (Formula I) (SEQ IN NO. 99), wherein indicates a peptide bond.

[0063] In an embodiment of the present disclosure, the disulphide linked oligomerization domain comprises engineered N-glycosylation sites of amino acid sequence “NXT”, wherein “X” is selected from “E” or “I”. In an embodiment, the engineered N-glycosylation sites are at amino acid positions 19 to 21, and 37 to 39 of Formula 1. In another embodiment, the N- glycosylation sites are at amino acid positions amino acid positions 16 to 18, and 37 to 39 of Formula 1. In an embodiment, the engineered N-glycosylation sites are at amino acid positions 3 to 5, 19 to 21, and 37 to 39 of Formula 1. In an embodiment, the engineered N-glycosylation sites are at amino acid positions 3 to 5, 16 to 18, and 37 to 39 of Formula 1. In another embodiment, the disulphide linked oligomerization domain comprises cysteine residues at amino acid positions 8 and 10 of Formula 1.

[0064] Accordingly, in another embodiment, the disulphide-linked oligomerization domain has an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence as set forth in Formula 1 :E-I-(S / N)-E-(E / T)-D-P-C-E-C-K-S-I-K-K-(E / N)-I-(E / T)-(N / A)-I- (T / K)-K-E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-AFormula 1 (SEQ IN NO. 99), wherein the disulphide-linked oligomerization domain comprises cysteine residues at amino acid positions 8 and 10, and wherein the disulphide-linked oligomerization domain comprises N- glycosylation sites having amino acid sequence “NXT” at amino acid positions selected from the group consisting of:(i) positions 19 to 21, and 37 to 39;(ii) positions 16 to 18, and 37 to 39;(iii) positions 3 to 5, 19 to 21, and 37 to 39; and(iv) positions 3 to 5, 16 to 18, and 37 to 39, wherein “X” is selected from “E” or “I”.

[0065] The present disclosure provides various variants of the disulphide linked oligomerization domain as disclosed herein. In an embodiment, the disulphide linked oligomerization domain comprises an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity identity to a sequence: E-I-N-E-T-D-P-C-E-C-K-S-I-K-K-E-I-E-N-I- T-K-E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 5), also referred to herein as “DSV1”, “VI”, or “cCMP-IZm-Vl”.

[0066] In another embodiment, the disulphide linked oligomerization domain has an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence: E-I-S-E-E-D-P-C-E- C-K-S-I-K-K-E-I-E-N-I-T- K-E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 7), also referred to herein as “DSV2”, “V2”, or “cCMP-IZm-V2”.

[0067] In another embodiment, the disulphide linked oligomerization domain has an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence: E-I-S-E-E-D-P-C-E- C-K-S-I-K-K-N-I-T-A-I-K- K-E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 9), also referred to herein as “DSV3”, “V3”, or “cCMP-IZm-V3”.

[0068] In another embodiment, the disulphide linked oligomerization domain has an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence: E-I-S-E-E-D-P-C-E- C-K-S-I-K-K-N-I-T-A-I-K- K-E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 11), also referred to herein as “DSV4”, “V4”, or “cCMP-IZm-V4”.

[0069] In another embodiment, there is provided a disulphide linked oligomerization domain having an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of:(a) E-I-N-E-T-D-P-C-E-C-K-S-I-K-K-E-I-E-N-I-T-K-E-Q-E-A-I-K-K-K- I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 5);(b) E-I-S-E-E-D-P-C-E-C-K-S-I-K-K-E-I-E-N-I-T-K-E-Q-E-A-I-K-K-K- I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 7);(c) E-I-S-E-E-D-P-C-E-C-K-S-I-K-K-N-I-T-A-I-K-K-E-Q-E-A-I-K-K-K- I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 9); and(d) E-I-S-E-E-D-P-C-E-C-K-S-I-K-K-N-I-T-A-I-K-K-E-Q-E-A-I-K-K-K- I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 11).Polypeptide B

[0070] Embodiments herein provide a polypeptide B comprising the disulphide linked oligomerization domain. In an embodiment, the polypeptide B is a fusion protein. In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain as described herein, attached to a polypeptide A at the N-terminal or C-terminal end of the polypeptide A for oligomerization of the polypeptide A. The term “polypeptide A”, as used herein, refers to a polypeptide fragment intended to form an oligomer. In an embodiment, the polypeptide A is an antigenic peptide or therapeutic peptide. In an embodiment, the polypeptide B is an oligomeric protein. Accordingly, embodiments herein provide an oligomeric protein comprising the disulphide linked oligomerization domain as described herein. In an embodiment, the oligomeric protein comprises disulphide linked oligomerization domain attached to the N-terminal or C-terminal of polypeptide A. Various antigenic or therapeutic peptides are known and may be used as polypeptide A in various embodiments herein. Examples of such antigenic peptide include, but are not limited to, fragments of RBD of Sarbecovirus, fragments of Spike protein of Sarbecovirus, fragments of Hemaglutinin (HA) ectodomain of Influenza, fragment of the stem region of HA of Influenza, and / or fragments of S2 ectodomain fragment of spike protein of Coronavirus. In an embodiment, the oligomerization is disulfide-linked oligomerization. Accordingly, in an embodiment, the polypeptide A is selected from a fragment of RBD ofSarbecovirus, fragment of Spike protein of Sarbecovirus, a fragment of Hemaglutinin (HA) ectodomain of Influenza, a fragment of the stem region of HA of Influenza, a fragment of S2 ectodomain fragment of spike protein of Coronavirus, or combination thereof.

[0071] The disulphide linked oligomerization domain, according to embodiments herein, may be attached to the polypeptide A with or without a linker. In an embodiment, the disulphide linked oligomerization domain is attached to the polypeptide A by a linker at the N-terminal or C-terminal end of the polypeptide A.

[0072] In an embodiment, there is provided polypeptide B comprising a disulphide-linked oligomerization domain attached to a polypeptide A at the N- terminal or C-terminal end of the polypeptide A for oligomerization of the polypeptide A.

[0073] In an embodiment, there is provided polypeptide B comprising a disulphide-linked oligomerization domain attached to a polypeptide A at the N- terminal or C-terminal end of the polypeptide A for oligomerization of the polypeptide A, wherein the disulphide-linked oligomerization domain has an amino acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence as set forth in Formula 1.

[0074] The linker, according to embodiments herein, may be a linker of amino acid length in the range of 1 to 20 amino acids. Examples of linkers that may be used include “RVQPTESITRPN” (SEQ ID NO. 83), “GS”, “ASGS”, “G”, “GSAGAG” (SEQ ID NO. 163), “RS”, “AS”, or combination thereof. The sequence and length of linker may vary. Various linkers are known and may be suitably used in various embodiments herein.

[0075] In an embodiment, the disulphide linked oligomerization domain is attached at N-terminal of the polypeptide A, using a linker sequence derived from SARS-CoV-2 B.l RBD (receptor binding domain) from position 319 to 331 (position in respect of the wildtype RBD) having V327T substitution and F329 deletion. In an embodiment, the linker has an amino acid sequence as set forth in SEQ ID NO. 83 (also referred to herein as SARS-CoV-2 linker).

[0076] In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached at N-terminal end of the polypeptide A by a linker, preferably RVQPTESITRPN (SEQ ID NO. 83). In an embodiment, the polypeptide B has an amino acid sequence as set forth in SEQ ID NO. 79 or SEQ ID NO. 179.

[0077] In another embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached at C-terminal end of the polypeptide A by a linker, preferably “GSAGAG”. In an embodiment, the polypeptide B has an amino acid sequence as set forth in SEQ ID NO. 78.

[0078] In yet another embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached at C-terminal end of the polypeptide A by a linker, preferably “GS”. In an embodiment, the polypeptide B has an amino acid sequence as set forth in SEQ ID NO. 77.

[0079] In an embodiment, the polypeptide B has an amino acid sequence selected from a group consisting of SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 179, and SEQ ID NO. 79.

[0080] In another embodiment, the polypeptide B has an amino acid sequence selected from SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 171, SEQ ID NO. 172, SEQ ID NO. 173, SEQ ID NO. 174, SEQ ID NO. 175, SEQ ID NO. 176, SEQ ID NO. 177, SEQ ID NO. 178, and SEQ ID NO. 179.

[0081] In an embodiment, there is provided a polypeptide B, wherein the oligomerization is disulfide-linked oligomerization, or wherein cysteine (C) residues in the disulphide-linked oligomerization domain are linked by a disulphide linkage.

[0082] In an embodiment, there is provided a polypeptide B, wherein one or more asparagine (N) residues in the disulphide-linked oligomerization domain are N glycosylated.

[0083] The polypeptide B, according to embodiments herein, may further comprise a peptide selected from TPA signal peptide, HRV3C proteasecleavage site or a portion thereof, one or more linkers, His-tag, or combinations thereof.

[0084] The TPA signal peptide, according to embodiments herein, may be at the N-terminal or C-terminal end of the polypeptide B. In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached at N-terminal end of the polypeptide A; and a TPA signal peptide at the N-terminal end of the disulphide linked oligomerization domain, wherein the disulphide linked oligomerization domain is attached to the N-terminal end of the polypeptide A by a linker, preferably RVQPTESITRPN (SEQ ID NO. 83).

[0085] In another embodiment, the polypeptide B comprises a disulphide linked oligomerization domain attached at C-terminal end of the polypeptide A; and a TPA signal peptide at the N-terminal end of the polypeptide A, wherein the disulphide linked oligomerization domain is attached at C-terminal end of the polypeptide A by a linker, preferably “GSAGAG” or “GS”. In an embodiment, the TPA signal peptide has an amino acid sequence as set forth in SEQ ID NO. 80.

[0086] The HRV3C protease cleavage site or portion thereof, according to embodiments herein, is preferably attached to the polypeptide B at the C- terminal end of the polypeptide B. In an embodiment, the HRV3C protease cleavage site has an amino acid sequence as set forth in SEQ ID NO. 85. The term “portion thereof’, as used herein, refers to a portion of the HRV3C protease cleavage site, for eg: a portion having the amino acid sequence “LEVLFQ” which is a vector derived sequence. In an embodiment, the polypeptide B comprises a disulphide linked oligomerization domain attached at N-terminal end of the polypeptide A; HRV3C protease cleavage site; and optionally a TPA signal peptide, wherein the HRV3C protease cleavage site is attached to the polypeptide A, wherein the disulphide linked oligomerization domain is attached at the N-terminal end of the polypeptide A by a linker, preferably RVQPTESITRPN (SEQ ID NO. 83), wherein the HRV3C proteasecleavage site is attached to the polypeptide A by a linker, preferably “GS”, “AS” and / or “ASGS”.

[0087] In an embodiment, the polypeptide B comprises a disulphide linked oligomerization domain attached at C-terminal end of the polypeptide A; HRV3C protease cleavage site; and optionally, a TPA signal peptide, wherein the HRV3C protease cleavage site is attached to the polypeptide A, wherein the disulphide linked oligomerization domain is attached at the C-terminal end of the polypeptide A by a linker, preferably “GSAGAG” or “GS”, wherein the HRV3C protease cleavage site is attached to the disulphide linked oligomerization domain by a linker, preferably “GS”, “AS” or “ASGS”.

[0088] The polypeptide B, according to embodiments herein, may further comprise a His-tag at the N-terminal or C-terminal end of the polypeptide B. The His-tag may be attached to the polypeptide B by a linker such as “GS” or “G”, at the C-terminal end of the polypeptide B. The His-tag, in an embodiment, may be a lOxHis-tag or 8xHis-tag. In an embodiment, the His-tag has an amino acid sequence selected from SEQ ID NO. 91 or SEQ ID NO. 92.

[0089] The polypeptide B, according to embodiments herein, comprises the disulphide linked oligomerization domain attached to the polypeptide A at the C-terminal or N-terminal end of the polypeptide A. In an embodiment, the polypeptide A is an antigenic peptide selected from the group consisting of a fragment of RBD of Sarbecovirus (also referred to herein as RBD1-DM37), fragment of Spike protein of Sarbecovirus (also referred to herein as Spike-6P- DM37), a fragment of Hemaglutinin (HA) ectodomain of Influenza (also referred to herein as mECT0-H3), a fragment of the stem region of HA of Influenza (also referred to herein as PhH3HA10), a fragment of S2 ectodomain fragment of spike protein of Coronavirus (also referred to herein as S2), or combination thereof. The polypeptide A having a combination of RBD1-DM37 and S2 is referred to herein as RBD1-DM37-S2. In an embodiment, the polypeptide A has an amino acid sequence selected from the group consisting of SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 169, or SEQ ID NO. 170.

[0090] In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached to a polypeptide A at the C-terminal or N-terminal end of the polypeptide A; and optionally, a peptide selected from a TPA signal peptide, HRV3C protease cleavage site or a portion thereof, one or more linkers, His-tag, or combinations thereof, wherein the polypeptide A is as set forth in SEQ ID NO. 101, wherein the disulphide linked oligomerization domain has an amino acid sequence selected from a group consisting of SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 11. In an embodiment, the polypeptide B has an amino acid sequence selected from the group consisting of: SEQ ID NO. 65, SEQ ID NO. 67, and SEQ ID NO. 78.

[0091] In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached to a polypeptide A at the C-terminal or N-terminal end, preferably N-terminal, of the polypeptide A; and optionally a peptide selected from a TPA signal peptide, HRV3C protease cleavage site or a portion thereof, one or more linkers, His-tag, or combinations thereof, wherein the polypeptide A is as set forth in SEQ ID NO. 98, wherein the disulphide linked oligomerization domain has an amino acid sequence selected from a group consisting of SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, and SEQ ID NO. 11. In an embodiment, the polypeptide B has an amino acid sequence selected from the group consisting of SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and SEQ ID NO. 35. In an embodiment, the polypeptide B has an amino acid sequence selected from the group consisting of SEQ ID NO. 25, and SEQ ID NO. 27.

[0092] In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached to a polypeptide A at the C-terminal or N-terminal end, preferably N-terminal, of the polypeptide A; and optionally a TPA signal peptide, HRV3C protease cleavage site or a portion thereof, one or more linkers, His-tag, or combinations thereof, wherein the polypeptide A is as set forth in SEQ ID NO. 100, wherein the disulphide linked oligomerizationdomain has an amino acid sequence selected from a group consisting of SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, and SEQ ID NO. 11.

[0093] In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached to a polypeptide A at the C-terminal or N-terminal end, preferably C-terminal, of the polypeptide A; and optionally a TPA signal peptide, HRV3C protease cleavage site or a portion thereof, one or more linkers, His-tag, or combinations thereof, wherein the polypeptide A is as set forth in SEQ ID NO. 97, wherein the disulphide linked oligomerization has an amino acid sequence selected from a group consisting of SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, and SEQ ID NO. 11. In an embodiment, the polypeptide B has an amino acid sequence selected from the group consisting of SEQ ID NO. 59, and SEQ ID NO. 77.

[0094] In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached to a polypeptide A at the C-terminal or N-terminal end, preferably N-terminal, of the polypeptide A; and optionally a TPA signal peptide, HRV3C protease cleavage site or a portion thereof, one or more linkers, His-tag, or combinations thereof, wherein the polypeptide A is as set forth in SEQ ID NO. 100, wherein the disulphide linked oligomerization domain has an amino acid sequence selected from a group consisting of SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, and SEQ ID NO. 11. In an embodiment, the polypeptide B has an amino acid sequence selected from the group consisting of SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 73, and SEQ ID NO. 75. In an embodiment, the polypeptide B has an amino acid sequence selected from the group consisting of SEQ ID NO. 45, and SEQ ID NO. 47.

[0095] In an embodiment, the polypeptide B comprises the disulphide linked oligomerization domain attached to a polypeptide A at the C-terminal or N-terminal end of the polypeptide A; and optionally a TPA signal peptide, HRV3C protease cleavage site or a portion thereof, one or more linkers, His- tag, or combinations thereof, wherein the polypeptide A is as set forth in SEQID NO. 169 or SEQ ID NO. 170, wherein the disulphide linked oligomerization has an amino acid sequence selected from a group consisting of SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, and SEQ ID NO. 11. In an embodiment, the polypeptide B has an amino acid sequence selected from the group consisting of SEQ ID NO. 179, and SEQ ID NO. 166.

[0096] In an embodiment, the polypeptide B has an amino acid sequence selected from the group consisting of SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 59, SEQ ID NO. 77, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 78, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 73, SEQ ID NO. 75, and SEQ ID NO. 166.

[0097] In an embodiment, the polypeptide B has an amino acid sequence selected from SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 59, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 78, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 166, SEQ ID NO. 171, SEQ ID NO. 172, SEQ ID NO. 173, SEQ ID NO. 174, SEQ ID NO. 175, SEQ ID NO. 176, SEQ ID NO. 177, SEQ ID NO. 178, or SEQ ID NO. 179.Polynucleotide

[0098] Further, the embodiments herein provide polynucleotides encoding the disulphide linked oligomerization domain as disclosed herein, or the polypeptide B as disclosed herein. In an embodiment, the polynucleotides encode the disulphide linked oligomerization domain comprising various combinations of Domain A and Domain B. In another embodiment, the polynucleotides encode the polypeptide B comprising the disulphide linked oligomerization domain, wherein the disulphide linked oligomerization domainhas a combination of Domain A and Domain B. The polynucleotides, according to embodiments herein, may be deoxyribose nucleic acid (DNA), ribose nucleic acid (RNA), or mRNA (messenger RNA) fragment.

[0099] In an embodiment of the present disclosure, the polynucleotide encoding the disulphide linked oligomerization domain has a nucleotide sequence of at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, and SEQ ID NO. 12.

[0100] In another embodiment of the present disclosure, the polynucleotide encoding the polypeptide B has a nucleotide sequence of at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 60, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 74, SEQ ID NO. 76, and SEQ ID NO. 167.

[0101] In another embodiment of the present disclosure, the polynucleotide encoding the polypeptide B has a nucleotide sequence of at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 60, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 74, SEQ ID NO. 76, and SEQ ID NO. 167.

[0102] In an embodiment, the polynucleotide encoding the polypeptide B has a nucleotide sequence selected from the group consisting of SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 60, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ IDNO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 74, SEQ ID NO. 76, and SEQ ID NO. 167.

[0103] Embodiments herein provide recombinant vectors comprising the polynucleotide encoding polypeptide B fragments as disclosed herein, wherein the polynucleotide is operably linked to a promoter. Examples of promoters include, but are not limited to, CMV (Cytomegalovirus) promoter, EFla (Eukaryotic translation Elongation Factor 1 Alpha) promoter, CAG (CMV early enhancer / chicken P actin) promoter, PGK (phosphoglycerate kinase) promoter, SV40 (Simian Virus 40), and UBC (human ubiquitin C) promoter.

[0104] In an embodiment, the recombinant vector comprises the polynucleotide operably linked to a promoter, wherein the polynucleotide has a sequence of at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 60, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 74, SEQ ID NO. 76, and SEQ ID NO. 167. Various vectors are generally known and may be used in achieving the recombinant vector according to embodiments herein. Examples of vectors include, but are not limited to, pcDNA series such as pcDNA3.1, pCDNA3.2, and pcDNA3.4; pcDNA5 / FRT / TO; pHDM; and pTwistCMV series.

[0105] Further, the embodiments herein provide recombinant host cells transfected with the recombinant vector comprising the polynucleotides as disclosed herein. In an embodiment, the recombinant host cell is a prokaryotic or eukaryotic cell. In an embodiment, the recombinant host cell is a bacterial cell, yeast cell, insect cell, and mammalian cell.

[0106] In an embodiment, the recombinant host cell comprises the polynucleotide having a sequence of at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 60, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 74, SEQ ID NO. 76, and SEQ ID NO. 167.

[0107] In an embodiment, the host cell is bacterial cell, yeast cell, insect cell, or mammalian cell, wherein the bacterial cell is Escherichia coli, and wherein the yeast cell is selected from the group consisting of Pichia X33, Pichia GlycoSwitch® , DSMZ 70382, GS115, KM71, KM71H, BG09, GS190, GS200, JC220, JC254, JC227, JC300-JC308, YJN165, and CBS7435, and wherein the insect cell is selected from the group consisting of Expi-Sf9®, Sf9, High Five® , Sf21, and S2, and wherein the mammalian cell is selected from the group consisting of Expi293F®, Expi-CHO-S ® , CHO-K1, CHO-S, HEK293F® , CHOBC™, SLIM™ , SPOT™, SP2 / 0 , Sp2 / 0-Agl4, CHODG44, HEK 293S, HEK 293 Gntl- / - ,HEK293-EBNA1, CHOL-NSO, A NSO.

[0108] Further, the embodiments herein provide compositions comprising the polypeptide B as disclosed herein. The composition may be a therapeutic composition. In an embodiment, the composition is a vaccine composition. In an embodiment, the composition comprises the polypeptide B and a pharmaceutically acceptable carrier. In an embodiment, the composition is a vaccine composition comprising the polypeptide B and a pharmaceutically acceptable carrier, wherein the polypeptide B has a sequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of group consisting of SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 43,SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 59, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 73, SEQ ID NO. 75, and SEQ ID NO. 166.

[0109] The pharmaceutically acceptable carrier, according to embodiments herein, may be any carrier generally known for use in pharmaceuticalcompositions. In an embodiment, the pharmaceutically acceptable carrier is selected from adjuvants, excipients, or combination thereof. In an embodiment, the adjuvant is selected from the group consisting of an oil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an aluminium hydroxide adjuvant, and combinations thereof. In an embodiment, the excipient is selected from buffer, sugar, alcohol, sugar alcohol, salt, aldehyde, amino acid and their derivatives, or combination thereof. In an embodiment the buffer is selected from PBS buffer, Tween80, Tris, or Sodium phosphate; sugar is selected from sucrose, trehalose, glucose, fructose, saccharose, or combinations thereof; sugar alcohol is selected from mannitol, ethanol, 2-phenoxyethanol, glycerol, erythritol, arabitol, adonitol, sorbitol, xylitol, or glucosyl glycerol; salt is selected from sodium chloride (NaCl), potassium chloride (KC1), or magnesium chloride (MgCE); aldehyde is formaldehyde; and amino acid sugars and their derivatives are selected from arginine hydrochloride (ArgHCl), Histidine, Arginine, Glycine, alanine, proline, glutamate, sarcosine, or trimethylamine N- oxide (TMAO).

[0110] In an exemplary embodiment of the present disclosure, the pharmaceutically acceptable carrier is selected from the group consisting of alhydrogel (aluminium hydroxide adjuvant), Alhydrogel CpG, Addavax (oil-in- water adjuvant), SWE (squalene-in-water emulsion adjuvant), CMS, and MF59.

[0111] Other pharmaceutically acceptable carriers which are known may also be used in various embodiments herein.

[0112] Embodiments herein provide a method for producing the polypeptide B as disclosed herein. In an embodiment, the method for producing the polypeptide B comprises (a) preparing the polynucleotides encoding the polypeptide B as described herein; (b) introducing the polynucleotides into a vector; transfecting a host cell with the vector; and culturing the host cell to express the polypeptide B. Various techniques for preparing polynucleotides, transfection, culturing are known and may be used in various embodiments.

[0113] The recombinant vector, according to embodiments herein, comprising the nucleotide encoding the polypeptide B may be introduced into the host cell by transfection, to obtain the recombinant host cell which may then be cultured to express the polypeptide B as described herein. Various transfection methods are known for eg: lipofection, electroporation, etc. which may be used to achieve the recombinant host cell as described herein. The expressed immunogenic polypeptide may then be purified by using protein purification methods generally known in field (for eg: Ni-NTA affinity chromatography) to obtain the purified polypeptide B. The immunogenic polypeptide may then be mixed with a pharmaceutically acceptable carrier to obtain the vaccine composition.

[0114] In an embodiment, the method of preparing the polypeptide B comprises culturing the recombinant host cell under suitable conditions to express the polypeptide B. The suitable conditions for culturing would vary depending on the host cells, and would be apparent to a person skilled in the art.

[0115] Embodiments herein provide a method for eliciting an immune response in a subject, comprising administering the polypeptide B as disclosed herein. In an embodiment, the method for eliciting an immune response in a subject comprises administering the polypeptide B as disclosed herein to a subject.

[0116] The term “subject” refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include non-human primates, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mice, rats, hamsters, guinea pigs, etc. Unless otherwise noted, the terms “patient” and “subject” are used herein interchangeably. Preferably, the subject is human.

[0117] Figure 1 depicts design of disulphide-linked oligomerization domain (cCMP-IZm): wherein (a) depicts three-dimensional structure of cCMP (PDB ID: 1 AQ5), showing the cCMP amino acid sequence (i.e. EEDPCECKS); and Disulphide-linked cysteines, (b) depicts helical wheel representation of the coiled-coil heptad repeats in IZm, wherein the d positions contain isoleucine residue essential for oligomerization. Predicted glycosylation sites at positionsc and g, featuring asparagines (N), are indicated by dotted circles, (c) depicts amino acid sequences of the heptad repeat synthetic peptide (IZm) and variant, according to embodiments herein. The original cCMP sequence (i.e. EEDPCECKS) is fused with IZmin the cCMP-IZm_vO construct, along with engineered variants (cCMP-IZm_Vl to V4) featuring N-glycosylation sites for improved stability and reduced immunogenicity. Glycosylation sites are highlighted. Sequences are aligned according to the heptad repeat (abcdefg) pattern, essential for preserving the coiled-coil structure and ensuring that modifications do not disrupt oligomerization.

[0118] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0119] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.Example 1:Nucleotide and amino acid sequences as disclosed in the present disclosure

[0120] Table 1 illustrates the amino acid sequence and corresponding nucleotide sequences of disulphide linked oligomerization domains, according to embodiments herein. Also provided are the amino acid sequence and corresponding nucleotide sequences of exemplary polypeptide B comprising the disulphide linked oligomerization domain and polypeptide A attached at the N-terminal or C-terminal end of the polypeptide A, according to embodiments herein. The polypeptide B designs having the TPA signal peptide, HRV3C cleavage sites, His-tag, attached using various linkers have been illustrated in Table 1. Exemplary polypeptide B having foldon (SEQ ID NO. 13) attached to the polypeptide A were also created and tested as comparison to the polypeptide B having the oligomerization domain of the present disclosure. The polynucleotide for foldon has a sequence as depicted in SEQ ID NO. 14, SEQ ID NO. 15, or SEQ ID NO. 16

[0121] The polypeptide A in the various exemplary polypeptide B were obtained from the viral surface proteins: RBD1-DM37 (SARS CoV-2 RBD (332-532), mutated stem of Hemagglutinin from the Influenza A virus- A / Philippines / 2 / 1982 / (H3N2)), Spike 6P-DM37 (stabilized and soluble SARS- CoV-2 Spike-B.l (1-1208)), mECTO-H3 (Soluble ectodomain for HA protein of Influenza A virus A / Hong Kong / 45 / 2019 (H3N2)-like virus, 2020-21), PhH3HA10 (Mutated stem of Hemagglutinin from the Influenza A virus- A / Philippines / 2 / 1982 / (H3N2)), and RBD1-DM37-S2 (a combination of SARS CoV-2 RBD (from positions 332-532) linked to S2 ectodomain fragment of spike protein of (from positions 698-1163) SARS CoV-2, positions are in respect of wildtype sequences).

[0122] Exemplary fusion protein designs of the viral surface proteins fused to oligomerizations domains (DSV) or foldon were as follows:1. All DS variant (DSV0 (SEQ ID NO. 3), DSV1 (SEQ ID NO. 5), DSV2 (SEQ ID NO. 7), DSV3 (SEQ ID NO. 9) and DSV4 (SEQ ID NO. 11), disulphide-linked oligomerization domain) were fused at N terminal of the peptides (polypeptides A) RBD1-DM37 (SARS CoV-2 RBD (332-532) and mutated stem of Hemagglutinin from the Influenza A virus-A / Philippines / 2 / 1982 / (H3N2). The exemplary polypeptides B are as depicted in SEQ ID NO. 19, SEQ ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 31, and SEQ ID NO. 35.2. The peptide (polypeptide A) Spike 6P-DM37 (stabilized and soluble SARS-CoV-2 Spike-B. l (1-1208)) was fused C terminally with Foldon to obtain SEQ ID NO. 57 or DSV2 to obtain SEQ ID NO. 59.3. The peptide (polypeptide A) mECTO-H3 (Soluble ectodomain for HA protein of Influenza A virus A / Hong Kong / 45 / 2019 (H3N2)-like virus, 2020-21) was fused C terminally with Foldon to obtain SEQ ID NO. 63) or DSV2 to obtain SEQ ID NO. 67.4. The peptide (polypeptide A) PhH3HA10 (Mutated stem of Hemagglutinin from the Influenza A virus- A / Philippines / 2 / 1982 / (H3N2)) was fused with Foldon to obtain SEQ ID NO. 71) and DSV2 to obtain SEQ ID NO. 75.5. The peptide (polypeptide A) RBD1-DM37-S2 (SARS CoV-2 RBD (332- 532) fused to S2 ectodomain fragment of spike protein (698-1163) of SARS CoV-2 (as depicted in SEQ ID NO. 169) was fused with DSV2 of SEQ ID NO. 7 to obtain polypeptide B of SEQ ID NO. 166. SEQ ID NO. 164 comprises polypeptide A RBD1-DM37-S2 without DSV2.

[0123] Exemplary polypeptide B fragments having the disulphide linked oligomerization domain (DSV0, DSV1, DSV2, DSV3, and DSV4) have the amino acid sequence as set forth in: SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.59, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO. 73, SEQ ID NO.75, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.79, ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.53, SEQ ID NO. 55, SEQ ID NO. 166, SEQ ID NO. 171, SEQ ID NO. 172, SEQ ID NO. 173, SEQ ID NO. 174, SEQ ID NO. 175, SEQ ID NO. 176, SEQ ID NO. 177, SEQ ID NO. 178, and SEQ ID NO. 179.

[0124] The corresponding exemplary nucleotide sequences of the exemplary polypeptide B having the oligomerization domain (DS VO, DS VI, DSV2, DSV3, and DSV4), are as set forth in: SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.46, SEQID NO.48, SEQ ID NO.60, SEQ ID NO.66, SEQ ID NO.68, SEQ ID NO.74, SEQ ID NO.76, SEQ ID NO.30, SEQ ID NO. 32, SEQ ID NO.50, SEQ ID NO.52, SEQ ID NO. 34, SEQ ID NO.36, SEQ ID NO.54, SEQ ID NO.56, and SEQ ID NO. 167.

[0125] The exemplary polypeptide B fragments having the foldon are of amino acid sequence as are as set forth in: SEQ ID NO.57, SEQ ID NO.61, SEQ ID NO.63, SEQ ID NO.69, and SEQ ID NO.71.

[0126] The corresponding nucleotide sequences of the exemplary polypeptide B fragments having the foldon, are as set forth in: SEQ ID NO. 58, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 70, and SEQ ID NO. 72.

[0127] Table 1 depicts the sequences used in the present disclosure.Table 1:

[0128] Table 2 provides a list of exemplary amino acid sequences of Domain A and Domain B, wherein SEQ ID NOs 102 to 146 and SEQID NO. 180 depict exemplary sequences of Domain A and wherein SEQ ID NOs 147 to 162, and SEQ ID NOs 181 to 182 depict exemplary sequences of Domain B.Table 2:Example 2: Protein expression

[0129] The plasmids encoded the fusion proteins enlisted in Example 1 and were transfected in Expi293 suspension cells. From the media supernatant, the 10X histidine-tagged protein was purified using Ni-NTA column chromatography techniques. These proteins were purified for homogeneity. The protein concentration was determined using a BCA assay. Purified proteins were aliquoted and snap-frozen using Liquid N2 and stored at -80 °C for longterm storage. Information pertaining to each protein used in the study is mentioned in Table 3.

[0130] Table 3: Information of protein used in study.Example 3: Protein profile and stability assay.

[0131] Frozen purified fusion protein was slowly thawed in ice. 10 micrograms of each protein were analysed on the SDS Page and termed as a zero-hour sample. The protein SDS PAGE profile of DS variant fused at N terminal of SARS CoV-2 RBD (332-532) and Influenza stem are provided in Figure 2.

[0132] Figure 2 depicts protein profile of oligomerizing DS variants when fused at the N-terminal of RBD1-DM37 (SEQ ID NO. 98) and PhH3HA10 (SEQ ID NO. 100). RBD1-DM37 and PhH3HA10 fused with oligomerizing DS variants such as V0 (SEQ ID NO. 3), VI (SEQ ID NO. 5), V2 (SEQ ID NO. 7), V3 (SEQ ID NO. 9), and V4 (SEQ ID NO. 11). These fusion proteins were expressed transiently in the suspension Expi293 cell (recombinant host cell) supernatant and purified using Ni-NTA chromatography techniques. Proteinswere analyzed on SDS-PAGE immediately after thawing from minus 80°C freezer. Each protein sample (10 pg) was mixed with 6X reducing and nonreducing PAGE dye and analyzed on 10 % Tris-Glycine SDS PAGE provided in the following panels: a) for RBD1-DM37 (SARS CoV-2 RBD), and b) for PhH3HA10 (Influenza A virus-mutated stem).

[0133] Foldon and DSV2 fused Spike-6P-DM37, mECTO-H3, and mPhH3HA10 samples (i.e. SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 63, SEQ ID NO. 67, SEQ ID NO. 71 and SEQ ID NO. 75) were incubated further for 24 and 96 hours and analysed on the SDS page, respectively for protein stability analysis. The SDS PAGE data provided in Figure 3 clearly demonstrates that DSV2 fused oligomer was found to be stable at 4°C up to 96 hours; however, Foldon fused oligomers were partially stable.

[0134] Figure 3 depicts protein SDS-PAGE profile upon storage. Spike-6P- DM37, mECTO-H3, and mPhH3HA10 fused with the Foldon and DSV2 oligomerization domains. These fusion proteins were expressed transiently in the suspension Expi293 cell supernatant and purified using Ni-NTA chromatography techniques. These proteins were analyzed on SDS-PAGE immediately after thawing, 24 hours later, and 96 hours after 2-8 °C storage. Each protein sample (10 pg) was mixed with 6X reducing and non-reducing PAGE dye and analyzed on an 8% Tris-Glycine SDS PAGE. The Coomassie- stained protein gel is shown in panels (a), (b), and (c) for 0, 24, and 96 -hour samples. The lane information is provided in panel (d).Example 4: Protein Oligomerization analysis (Size exclusion chromatography profile)

[0135] Purified protein was subjected to Size-exclusion chromatography (SEC) analysis. IX PBS and buffered proteins 200 pl volume were injected into Superose 6 10 / 300 GL prepacked column (Cytiva). The SEC profile and its detailed analysis are provided in Figures 4 and 5, which clearly demonstrate that (1) Both foldon and DSV2 tend to form protein oligomerization, and (2) oligomers are homogenous in nature.

[0136] Figure 4 depicts comparative oligomer profile analysis of mPhH3HA10 protein fused with Foldon (SEQ ID NO. 71) and DSV2 (SEQ ID NO. 75). Expi293 cell produced proteins absorbance at 280 nm (mAU) versus elution volume (mL) is plotted using GraphPad Prism software, wherein a) shows the size exclusion chromatography profile of the Gel Filtration Standard (Bio-Rad, Cat #1511901) and b) shows the log MW vs. elution volume plot. The overlapped SEC profiles of mPhH3HA10-Foldon and DSV2-mPhH3HA10 are shown in panel (c). The calculated protein molecular weight of all proteins is shown in Table 4. Table 4:

[0137] Figure 5 depicts comparative oligomer profile analysis of RBD1- DM37 and its DSV2 fused oligomer. Expi293 cell produced proteins absorbance at 280nm (mAU) versus elution volume (ml) is plotted using GraphPad Prism software, a) The size exclusion chromatography profile of Gel Filtration Standard (Bio-Rad, Cat #1511901) and b) Log MW Vs elution volume plot is shown. The overlapped SEC profile of RBD1-DM37 (i.e. SEQ ID NO. 98) and DSV2-RBD1-DM37 (i.e. SEQ ID NO. 27) is shown in panel (c). The calculated protein molecular weight of all proteins is shown in Table 5.Table 5:Example 5: Protein immunization study in BALB / mice:

[0138] Female BALB / c mice (7- to 8-week-old) were used for immunization of all tested proteins used in this study. A pre-bleed serum was collected one day prior to immunization and termed. Prime immunization was given on day zero. 20 pg of each PBS buffered protein mixed with Sepivac SWE™ adjuvant (squalene-in-water emulsion adjuvant) in 1 : 1 ratio in 100 pl volume formulation. The intramuscular injection was administrated into both hindlimbs of animals (50pl each). After 14 days prime bleed sera were collected. After 7 days, the boost immunization was administrated with a similar dose of prime. Boost sera and extended boost sera were collected on day 35 and day 112 for ELISA and Neutralization assays.

[0139] The ELISA endpoint titers of these sera were tested on self and non- self-antigen as well as on scaffold-immobilized plates. Additional boost sera were also tested for their ability to neutralize various pseudo-typed viruses such as B.l and Beta, etc. These results were analyzed and summarized in Figures 6 to 10.

[0140] Figure 6 depicts pseudoviral neutralization titers of mice sera raised against RBD1-DM37 fused with DS variants. Antigens such as RBD1-DM37 and their N-terminal fused DS variants (VO, VI, V2, V3, V4) (as depicted in SEQ ID NO. 19, SEQ ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 31 and SEQ ID NO. 35) were purified from Expi293 cells. The Sepivac SWE adjuvanted (1 : 1) 10 pg of these antigen formulations were used to immunize 7-8-week-old female BALB / c mice intramuscularly twice on days 0 and 21, and blood was drawn on days 14 and 35 to collect sera as depicted in (a). In panel (b), neutralization ID50 titers plot against B. l, (c) Delta, and (d) Omicron BA.l pseudoviruses are shown against each antigen boost sera (day 35).

[0141] Figure 7 depicts comparative immunogenicity of the Spike 6P- DM37 fused with the Foldon and DSV2 oligomerization domains in Mice. Antigens such as Spike 6P-DM37 fused with Foldon (to obtain SEQ ID NO. 57) and with DSV2 (to obtain SEQ ID NO. 59), were purified from Expi293 cells. The Sepivac SWE adjuvanted (1 : 1) 10 pg of these antigen formulations were used to immunize 7-8-week-old female BALB / c mice intramuscularly twice on days 0 and 21, and blood was drawn on days 14 and 35 and 112 to collect sera, as depicted in (a). ELISA end point titers are depicted in (b) for 2 weeks prime sera, (c) for 5 week boost sera, and (d) for 16 weeks extended boost sera were raised against the above-mentioned antigens and analyzed on various SARS-CoV-2 protein immobilized plates. Immobilized antigens used in ELISA are provided on the X axis (top) and shown with a red arrow. The X axis represents the sera raised against mentioned antigens.

[0142] Figure 8 depicts comparative immunogenicity of the mECTO-H3 fused with the Foldon and DSV2 oligomerization domains in mice. Antigens such as mECTO-H3 -fused Foldon (to obtain SEQ ID NO. 63) and with DSV2 (to obtain SEQ ID NO. 67), were purified from Expi293 cells. The Sepivac SWE adjuvanted (1 : 1) 10 pg of these antigen formulations were used to immunize 7-8-week-old female BALB / c mice intramuscularly twice on days 0 and 21, and blood was drawn on days 14 and 35 and 112 to collect sera as depicted in (a). ELISA end point titers are as depicted in (b) 2 weeks prime sera,(c) 5-week boost sera, and (d) 16 weeks extended boost sera, raised against the above-mentioned antigens and analyzed on various SARS-CoV-2 protein immobilized plates. Immobilized antigens used in ELISA are provided on the X axis (top) and shown with a red arrow. The X axis represents the sera raised against mentioned antigens.

[0143] Figure 9 depicts comparative immunogenicity of the PhH3HA10 fused with the Foldon and DSV2 oligomerization domains. Antigens such as PhH3HA10 fused Foldon (to obtain SEQ ID NO. 71) and DSV2 (to obtain SEQ ID NO. 75) were purified from Expi293 cells. The Sepivac SWE adjuvanted (1 : 1) 10 pg of these antigen formulations (vaccine composition) were used to immunize 7-8-week-old female BALB / c mice intramuscularly twice on days 0 and 21, and blood was drawn on days 14 and 35 and 112 to collect sera as depicted in (a). ELISA end point titers are as depicted in (b) 2 weeks prime sera, (c) 5-week boost sera, and (d) 16 weeks extended boost sera were raised against the above-mentioned antigens and analyzed on various SARS-CoV-2 protein immobilized plates. Immobilized antigens used in ELISA are provided on the X axis (top) and shown with a red arrow. The X axis represents the sera raised against mentioned antigens.

[0144] Figure 10 depicts pseudoviral neutralization titers of mice sera raised against Spike 6P-DM37 fused with Foldon and DSV2. Antigens such as stabilized Spike 6P-DM37 fused Foldon (to obtain SEQ ID NO. 57) and DSV2 (to obtain SEQ ID NO. 59) were purified from Expi293 cells. The Sepivac SWE adjuvanted (1 : 1) 10 pg of these antigen formulations were used to immunize 7- 8-week-old female BALB / c mice intramuscularly twice on days 0 and 21, and blood was drawn on days 14 and 35 to collect sera as depicted in (a). In panel (b) the neutralization ID50 titers plot against B.l, & Omicron BA. l pseudoviruses shown against each antigen boost sera. Boost Sera raised against antigen are provided on the X axis (top) and shown with a red arrow. SARS- CoV pseudovirus strains used in neutralization assay are represented on the X axis.

[0145] Together these experiments and their data clearly suggest the following inferences:- None of the tested proteins were found to be toxic to tested animals- Both the tested domains represent the antigenic domain at a similar extent leading to similar immunogenicity in BALB / c mice- DSV2 scaffold titer was found to be less compared to foldon scaffold titer after two immunizations 12 weeks after Boost immunization, scaffold titers for DSV2 reduced significantly as compared to foldon.Example 6: Protein immunization study in Hamster

[0146] Female Golden Syrian Hamster were used to immunize with Spike- 6P-DM37-Foldon (SEQ ID NO. 57) and Spike-6P-DM37-DSV2 (SEQ ID NO. 59) Protein. A pre-bleed serum was collected one day prior to immunization and termed. The prime immunization was given on day zero. 5 pg of each PBS buffered protein mixed with Sepivac SWE™ adjuvant (squalene-in-water emulsion adjuvant) in 1 : 1 ratio in 100 pl volume formulation (to obtain vaccine composition). The intramuscular injection was administrated into both hindlimbs of animals (50pl each). After 14 days prime bleed sera were collected. After 7 days, the boost immunization was administrated with a similar dose of prime. Boost sera collected on day 35.

[0147] These animals were challenged with 105PFU / lOOpl of Beta strain of SARS-CoV-2 (Figure 11).

[0148] Figure 11 depicts comparative immunogenicity of the Spike 6P- DM37 with the Foldon (SEQ ID NO. 57) and DSV2 (SEQ ID NO. 59) oligomerization domains in Hamster. Spike 6P-DM37 fused Foldon (to obtain SEQ ID NO. 57) and DSV2 ( to obtain SEQ ID NO. 59) were purified from Expi293 cells. The Sepivac SWE adjuvanted (1 : 1) 10 pg of these antigen formulations were used to immunize 7-8-week-old female Hamster (n=5) intramuscularly twice on days 0 and 21, and blood was drawn on days 14 and 35 collect sera and used for SARS-CoV-2 Beta virus challenge 105PFU / lOOpl depicted in (a). ELISA end point titers are depicted in (b) 2 weeks prime seraand 5-week boost sera, raised against the above-mentioned antigens and analyzed on various SARS-CoV-2 protein immobilized plates. In panel (c) comparison of body weight loss post SARS-CoV-2 Beta virus challenge 105PFU / lOOpl in these immunized animals are shown. PBS+SWE was used as mock immunization groups, and (d) showing lung histopathology from one of the representative animals from panel (c). Immobilized antigens used in ELISA are provided on the X axis (top) and shown with a red arrow. The X axis represents the sera raised against mentioned antigens.

[0149] The immunogenicity and protection against live viruses were compared and summarized in Figure 11. These data together suggested the following points:- Spike-6P-DM37-DSV2 (SEQ ID NO. 59) and Spike-6P-DM37-Foldon (SEQ ID NO. 57) immunized animals showed similar ELISA titers on Spike and RBD coated plates.- The scaffold titers from prime and boost sera of Spike-6P-DM37-DSV2 (SEQ ID NO. 59) immunized animals were near the baseline and significantly lower than Spike-6P-DM37-Foldon (SEQ ID NO. 57) immunized animals’ sera.Spike-6P-DM37-DSV2 (SEQ ID NO. 59) immunized animals were better protected from the Beta virus challenge as compared with Spike-6P-DM37- Foldon (SEQ ID NO. 57).Spike-6P-DM37-DSV2 (SEQ ID NO. 59) immunized animals show better lung histopathology than Spike-6P-DM37-Foldon (SEQ ID NO. 57) immunized animals, unlike challenged, and like to unvaccinated control animals.Results and Conclusion

[0150] The coiled-coil oligomerization domain, according to the present disclosure, is a technology that has shown promising results in enhancing the stability and immunogenicity of viral protein antigens in preclinical studies. The key findings of the present inventors are summarized below:-The DSV2 oligomerization domain was designed by fusing the N-terminal region of chicken cartilage matrix protein (cCMP) with a synthetic peptide sequence (IZm) containing engineered N-glycosylation sites.-Both the DSV2 and foldon oligomerization domains were able to oligomerize various viral antigenic peptides (polypeptide A) derived from SARS-CoV-2 Spike, Influenza A virus soluble HA ectodomain, and Influenza A- virus-mutated stem.-However, the DSV2 oligomerization domain fused antigenic peptides were found to be more stable, homogenous, and able to form oligomers more consistently across the tested antigenic peptides compared to the widely used foldon domain.-In animal studies, the DSV2-fused SARS-CoV-2 Spike showed improved protection against the Beta variant challenge compared to the foldon-fused SARS-CoV-2 Spike in hamsters.-The glycan shielding of the DSV2 domain (N-glycosylated) was designed to reduce its own immunogenicity after repeated administration in tested animals, an important consideration for protein subunit vaccines.

[0151] The improved properties of DSV2, according to the present disclosure, compared to foldon indicate its potential to enhance the efficacy of protein subunit vaccines and other oligomeric protein therapeutics in the future.Example 7: DSV2 domain and Foldon trimerized mECTO-H3 protein profile comparison.

[0152] A standard curve was generated using a Gel Filtration Standard (Bio-Rad, Cat #1511901) on an S6 column. The absorbance at 280 nm was plotted against elution volume to show the relationship between molecular weight and elution volume, which was used to estimate the molecular weights of the test proteins. Size-exclusion chromatography (SEC) of the purified proteins revealed a significant difference in molecular weight. The elution volumes were used in conjunction with the standard curve from the panel gelfiltration curve to estimate the molecular weights of the purified proteins. The mECTO-H3-Foldon protein (SEQ ID NO. 61) showed a calculated molecular weight of approximately 304 kDa, consistent with a trimeric state. In contrast, the mECTO-H3-DSV2 protein (SEQ ID NO. 67) exhibited a much larger molecular weight of approximately 2569 kDa, indicating the formation of higher-order oligomers (Figure 12).

[0153] Figure 12 depicts (a) Gel filtration standard curve used for molecular weight estimation, and (b) Size-exclusion chromatography (SEC) elution profiles of mECTO-H3-Foldon and mECTO-H3-DSV2, in accordance with embodiments herein.Negative-stain transmission electron microscopy

[0154] Negative-stain transmission electron microscopy (NS-TEM) was employed to analyze the particle distribution and structural homogeneity of the hemagglutinin (HA) protein (i.e. mECT0-H3) when fused with DSV2 trimerization domain.

[0155] Sample Preparation and Imaging: Size-exclusion chromatography (SEC) purified H3N2 hemagglutinin (HA) ectodomain protein mECT0-H3- DSV2 (SEQ ID NO. 67) was analyzed for particle distribution and overall homogeneity using negative-stain transmission electron microscopy (NS- TEM). Protein samples, diluted in lx phosphate-buffered saline (PBS) at pH 7.4, were applied to glow-discharged, carbon-coated copper TEM grids and incubated for one minute at room temperature. Excess sample was removed by blotting with Whatman filter paper. The grids were then negatively stained with a freshly prepared 1% uranyl acetate solution. Data was collected on a 120 kV Talos L120C transmission electron microscope equipped with a 4k x 4k Ceta camera.

[0156] Image Processing and Analysis: Raw micrographs of both protein samples were imported into EMAN2.2 for further processing. A total of 2,303 mECTO-H3-DSV2 particles (SEQ ID NO. 67) were manually selected and extracted using the e2boxer.py tool within the EMAN2.2 software suite.Subsequent reference-free 2D classification of these particle projections was performed using the same software to assess structural homogeneity and confirm the presence of correctly folded protein assemblies.

[0157] The mECTO-H3-DSV2 protein (SEQ ID NO. 67) formed a complex, higher-order oligomer. The structures appeared as chromosome or starfish-like complexes, where four individual HA trimers radiated from a central core. This assembly constitutes a dodecamer, a complex composed of four trimers. These constituent HA trimer bundles maintained a size of approximately 20 nm. (Figure 13).

[0158] Figure 13 depicts representative negative-stain transmission electron microscopy (NS-TEM) images of mECTO-H3-DSV2, wherein panel (i) shows particle distribution at a 100 nm scale bar, while panel (ii) displays representative 2D class averages derived for each protein, highlighting trimeric structural features (scale bar: 10 nm).Example 8: Effect of DSV2 oligomerization with high-yielding RBD-S2 fusion antigenic peptide.

[0159] The S2 ectodomain fragment (SEQ ID NO. 170) of the spike protein of SARS-CoV-2 was linked to the RBD1-DM37 (SEQ ID NO. 98) of the spike protein of SARS-CoV-2, at the N-terminal or C-terminal end of the S2 ectodomain fragment (represented herein as DSV2-(RBD1-DM37)-S2 or RS2- DSV2, respectively).

[0160] The DSV2 fusions at both the N and C-termini of the RBD-S2 fusion protein were examined. The DSV2-(RBD1-DM37)-S2 (SEQ ID NO. 166) and RS2-DSV2 proteins were expressed at approximately 500 mg / L and 250 mg / L, respectively, from the supernatant of transient suspension Expi293 cells, while RS2 was expressed at 850 mg / L. The DSV2-RS2 exhibited a homogeneous trimeric structure in contrast to the heterogeneous RS2 protein (Figure 14, panels (a)- (b)). RS2-DSV2 did not facilitate the formation of a homogeneous trimer in RS2 (data not shown). It is important to highlight that the DSV2 fusiondid not significantly alter, resulting in any considerable alteration in the melting temperature (Tm) of the RS2 protein (Figure 14, panel (c)).Comparative immunogenicity of DSV2-RS2 and RS2 proteins in hamsters.

[0161] Female hamsters (n=5) received two immunizations (21 -day intervals) of DSV2-(RBD1-DM37)-S2, also referred to as DSV2-RS2, (SEQ ID NO. 166) and (RBD1-DM37)-S2, also referred to as RS2, (SEQ ID NO. 169) at a dosage of 5 pg, supplemented with SWE adjuvant (pharmaceutically acceptable carrier). The antigen-specific IgG titers were measured using RBD and S2 immobilized ELISA wells. In line with the results observed for Spike 6P-DM37-Foldon (SEQ ID NO. 57) and Spike 6P-DM37 -DSV2 (SEQ ID NO. 59), DSV2-(RBD1-DM37)-S2 (SEQ ID NO. 166) and (RBD1-DM37)-S2 (SEQ ID NO. 169) exhibited similar antigen-specific titers as well as Pseudovirus neutralization. The DSV2 self-titers remained at baseline levels during the initial immunization phase and did not exhibit notable enhancement in the booster sera (Figure 15).Protection of hamsters after immunization with DSV2-RS2 and RS2 postlive beta virus challenge.

[0162] Like the SARS-CoV-2 spike spike group, the animals immunized with DSV2-(RBD1-DM37)-S2 (SEQ ID NO. 166) and (RBD1-DM37)-S2 (SEQ ID NO. 169) were also subjected to a challenge involving the live SARS- CoV-2 Beta virus (Figure 16, (a)). The control group that underwent the viral challenge experienced a body weight loss of 9-10% five days after the challenge, while the unchallenged control group maintained stable body weight. Notably, the animals immunized with (RBD1-DM37)-S2 (SEQ ID NO. 169) or DSV2-(RBD1-DM37)-S2 (SEQ ID NO. 166) exhibited only a 2% decrease in body weight, which returned to baseline by the fifth day post-challenge (Figure 16, (b)). Histological analysis of lung tissue sections from both unchallenged and DSV2-(RBD1-DM37)-S2 (SEQ ID NO. 166) or (RBD1-DM37)-S2 (SEQ ID NO. 169) immunized animals showed distinct interstitial spaces within thelung epithelium and a reduction in immune cell infiltration, in contrast to the unimmunized group that was challenged with the Beta variant (Figure 16, (c)).

[0163] Figure 14 depicts characterization of DSV2-(RBD1-DM37)-S2 (also referred to herein as DSV2-RS2) immunogen. DSV2-(RBD1-DM37)-S2 and (RBD1-DM37)-S2 (also referred to herein as RS2) proteins were expressed and purified from the supernatant of Expi293 cells, wherein panel (a) presents the SDS-PAGE profile of the purified proteins under both reducing and nonreducing conditions, panel (b) presents the SEC profile of these proteins, while panel (c) displays the thermal unfolding profile, in accordance with embodiments herein.

[0164] Figure 15 depicts comparative immunogenicity of DSV2-(RBD1- DM37)-S2 (also referred to herein as DSV2-RS2) and (RBD1-DM37)-S2 (also referred to herein as RS2) proteins in hamsters, where immunogenicity data is depicted for DSV2-RS2 and RS2 protein antigens in a hamster model, following an immunization and viral challenge protocol. Immunization and Challenge: Female hamsters (n=5) were immunized intramuscularly on days 0 and 21 with 5 pg of either DSV2-RS2 orRS2 protein, adj uv anted with Sepivac SWE. Blood samples were collected on days 14 and 35. Following the final serum collection, the animals were challenged with 105 PFU / lOOpL of SARS-CoV-2 Beta virus, (a) ELISA end-point titers: End-point titers of sera collected on day 14 (postprime) and day 35 (post-boost) are shown. The sera were analyzed by ELISA against various SARS-CoV-2 proteins, as indicated on the x-axis. The specific antigen used for immunization is noted at the top of the x-axis (indicated by a red arrow), (b) Pseudovirus neutralization ID50 titers: Neutralization ID50 titers were measured using day 35 boost sera against Beta and Omicron BA.5 pseudoviruses. The pseudovirus strains are indicated on the x-axis. The sera were raised against the antigens specified at the top of the x-axis (indicated by a red arrow), in accordance with embodiments herein.

[0165] Figure 16 depicts protection of hamsters after immunization with DSV2-(RBD1-DM37)-S2 (also referred to herein as DSV2-RS2) and -(RBD1- DM37)-S2 (also referred to herein as RS2) post-live beta virus challenge,wherein (a) is a schematic representation depicting the immunization protocol. The female hamster that received two immunizations with the RS2 and DSV2- RS2 formulations was challenged with a heterologous SARS-CoV-2 Beta virus at a dose of 105 PFU / lOOpL, 14 days following the boost bleed, and (b) depicts comparison of body weight loss in these immunized animals after the SARS- CoV-2 Beta virus challenge, wherein panel (c) shows lung histopathology from one of the representative animals from panel (b), in accordance with embodiments herein.

Claims

I / We claim:

1. A disulphide-linked oligomerization domain comprising a Domain A attached to a Domain B, wherein the Domain A is a disulphide forming motif having an amino acid sequence of at least 95% identity to a sequence selected from SEQ ID NO. 109 or SEQ ID NO. 180; and wherein the Domain B is an oligomerization motif having an amino acid sequence of at least 95% identity to the sequence selected from SEQ ID NO. 162, SEQID NO. 181, or SEQID NO. 182.

2. The disulphide-linked oligomerization domain as claimed in claim 1, wherein the disulphide-linked oligomerization domain has an amino acid sequence of at least 95% identity to a sequence as set forth in Formula 1 : E-I-(S / N)-E-(E / T)-D-P-C-E-C-K-S-I-K-K-(E / N)-I-(E / T)-(N / A)-I-(T / K)-K- E-Q-E-A-I-K-K-K-I-E-A-I-E-K-N-I-T-AFormula 1 (SEQ IN NO. 99), wherein the disulphide-linked oligomerization domain comprises cysteine residues at amino acid positions 8 and 10, and wherein the disulphide-linked oligomerization domain comprises N- glycosylation sites having amino acid sequence “NXT” at amino acid positions selected from the group consisting of:(i) positions 19 to 21, and 37 to 39;(ii) positions 16 to 18, and 37 to 39;(iii) positions 3 to 5, 19 to 21, and 37 to 39; and(iv) positions 3 to 5, 16 to 18, and 37 to 39, wherein “X” is selected from “E” or “I”.

3. The disulphide-linked oligomerization domain as claimed in claim 2, wherein the disulphide-linked oligomerization domain has an amino acid sequence of at least 95% identity to a sequence selected from the group consisting of:(a) E-I-N-E-T-D-P-C-E-C-K-S-I-K-K-E-I-E-N-I-T-K-E-Q-E-A-I-K-K-K- I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 5);(b) E-I-S-E-E-D-P-C-E-C-K-S-I-K-K-E-I-E-N-I-T-K-E-Q-E-A-I-K-K-K- I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 7);(c) E-I-S-E-E-D-P-C-E-C-K-S-I-K-K-N-I-T-A-I-K-K-E-Q-E-A-I-K-K-K- I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 9); and(d) E-I-S-E-E-D-P-C-E-C-K-S-I-K-K-N-I-T-A-I-K-K-E-Q-E-A-I-K-K-K- I-E-A-I-E-K-N-I-T-A (SEQ ID NO. 11).

4. A polypeptide B comprising the disulphide-linked oligomerization domain as claimed in any one of claims 1 to 3, attached to a polypeptide A at the N- terminal or C-terminal end of the polypeptide A for oligomerization of the polypeptide A.

5. The polypeptide B as claimed in claim 4, wherein the oligomerization is disulfide-linked oligomerization, or wherein the cysteine (C) residues in the disulphide-linked oligomerization domain are linked by a disulphide linkage.

6. The polypeptide B as claimed in claim 4, wherein one or more asparagine (N) residues in the disulphide-linked oligomerization domain are N glycosylated.

7. The polypeptide B as claimed in claim 4, wherein the polypeptide A is selected from a fragment of RBD of Sarbecovirus, fragment of Spike protein of Sarbecovirus, a fragment of Hemaglutinin (HA) ectodomain of Influenza, a fragment of the stem region of HA of Influenza, a fragment of S2 ectodomain fragment of spike protein of Coronavirus, or combination thereof.

8. The polypeptide B as claimed in claim 4, wherein the polypeptide A is selected from a fragment having an amino acid sequence selected from a group consisting of SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 169, or SEQ ID NO. 170.

9. The polypeptide B as claimed in claim 4, wherein the polypeptide B has an amino acid sequence selected from a group consisting of SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 171, SEQ ID NO. 172, SEQID NO. 173, SEQ ID NO. 174, SEQ ID NO. 175, SEQ ID NO. 176, SEQ ID NO. 177, SEQ ID NO. 178, and SEQ ID NO. 179.

10. The polypeptide B as claimed in claim 4, further comprising a peptide selected from TPA signal peptide, HRV3C protease cleavage site or a portion thereof, one or more linkers, His-tag, or combinations thereof.

11. The polypeptide B as claimed in claim 4, wherein the linker has an amino acid sequence selected from “RVQPTESITRPN” (SEQ ID NO. 83), “GS”, “ASGS”, “G”, “GSAGAG” (SEQ ID NO. 163), “RS”, “AS”, or combination thereof, or wherein the HRV3C protease cleavage site has an amino acid sequence as set forth in SEQ ID NO. 85, or wherein the His-tag has an amino acid sequence selected from SEQ ID NO. 91 or SEQ ID NO. 92, or wherein the TPA signal peptide has an amino acid sequence as set forth in SEQ ID NO. 80.

12. The polypeptide B as claimed in claim 10, wherein the polypeptide B has an amino acid sequence selected from the group consisting of SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 59, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 73, SEQ ID NO. 75, and SEQ ID NO. 166.

13. A polynucleotide encoding the disulphide-linked oligomerization domain as claimed in any one of claims 1-3, or the polypeptide B as claimed in any one of claims 4-12.

14. The polynucleotide as claimed in claim 13, wherein the polynucleotide is a DNA, RNA, or mRNA.

15. The polynucleotide as claimed in claim 13, wherein the polynucleotide encoding the disulphide-linked oligomerization domain has a nucleotide sequence selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, and SEQ ID NO. 12.

16. The polynucleotide as claimed in claim 13, wherein the polynucleotide encoding the polypeptide B has a nucleotide sequence selected from thegroup consisting of SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 60, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 74, SEQ ID NO. 76, and SEQ ID NO. 167.

17. A recombinant vector containing the polynucleotide as claimed in any one of claims 13 to 16 operably linked to a promoter.

18. A recombinant host cell comprising the recombinant vector as claimed in claim 17.

19. The recombinant host cell as claimed in claim 17, wherein the host cell is a bacterial cell, yeast cell, insect cell, or mammalian cell.

20. A composition comprising the polypeptide B as claimed in any one of claims 4-12, and a pharmaceutically acceptable carrier.

21. The composition as claimed in claim 20, wherein the pharmaceutically acceptable carrier is selected from adjuvants, excipients, or combination thereof.

22. The composition as claimed in claim 20, wherein the adjuvant is selected from the group consisting of an oil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an aluminum hydroxide adjuvant, and combinations thereof23. A method of producing the polypeptide B as claimed in claims 4-12, said method comprising culturing the recombinant host cell as claimed in anyone of claims 18 to 19 to express the polypeptide B.

24. The disulphide-linked oligomerization domain as claimed in claim 1, wherein the Domain A is attached to the Domain B at the N-terminal of Domain B, and wherein the Domain A has an amino acid sequence having cysteine residues at positions 5 and 7, and wherein the Domain B comprises N-glycosylation sites having amino acid sequence “NXT” at amino acid positions selected from the group consisting of:(i) positions 7 to 9, and 25 to 27 of Domain B; and(ii) positions 4 to 6, and 25 to 27 of Domain B, wherein “X” is selected from “E” or “I”.

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