Immunogenic polypeptides and vaccine compositions against diverse sarbecoviruses

Abstract

ABSTRACT IMMUNOGENIC POLYPEPTIDES AND VACCINE COMPOSITIONS AGAINST DIVERSE SARBECOVIRUSES The present disclosure relates to immunogenic polypeptides having Sarcebovirus 5 derived stabilized receptor binding domain (RBD) fragments. The immunogenic polypeptides comprise specific stabilizing mutations at various positions on the amino acid sequence. Further, the present invention also provides vaccine compositions comprising one or more of the immunogenic polypeptides, methods and uses thereof. 10

Description

FIELD OF INVENTION

[001] The present disclosure broadly relates to the field of immunobiology, andparticularly discloses immunogenic peptides, and vaccine compositions foreliciting immune response against Sarbecoviruses. The present disclosure alsorelates to a method for producing the vaccine composition as well as the methodof eliciting an immune response against diverse Sarbecoviruses, in a subject, byadministering the subject with an effective amount of said vaccine composition.BACKGROUND OF THE INVENTION

[002] Coronaviruses (CoVs) have consistently posed a significant risk ofdissemination. Zoonotic viruses such as betacoronaviruses continue to represent aglobal hazard through spillover events into human population. This has beenwitnessed multiple times in the last 30 years by severe acute respiratorysyndrome coronavirus 1 (SARS CoV-1), Middle East respiratory syndromecoronavirus (MERS CoV) outbreaks, and the latest severe acute respiratorysyndrome coronavirus 2 (SARS CoV-2) pandemic. The unprecedentedappearance and spread of coronavirus disease of 2019 (COVID-19) at the globallevel accelerated the development of various vaccines utilizing multiple platforms,out of which some were approved by the regulatory agencies and rolled out forpublic usage. However, the rapid evolution of SARS CoV-2 resulted in emergenceof variants which exhibited immune escape and rendered the vaccines lesseffective (Li et al., 2024; Liu et al., 2024; Lyke et al., 2022).

[003] Sarbecoviruses are a subgenus of betacoronaviruses that includes the SARSCoV, MERS-CoV, and the more recently identified SARS-CoV-2, which causesCOVID-19. In Sarbecoviruses such as SARS CoV-1 and SARS CoV-2, the viralspike protein (S) is the principal target of neutralising antibodies that preventinfection. Within spike, the immunodominant receptor-binding domain (RBD) isthe primary target of neutralising antibodies as observed in COVID-19convalescent sera and vaccine recipients (Premkumar et al., 2020; Yang et al.,2020).

[004] The attachment and entry of the viral particles into host cells is facilitated bythe S-proteins through their binding to ACE2 (angiotensin-converting enzyme-2).Protein S is comprised of two distinct subunits, namely S1 and S2. The S1 subunit,positioned in the N-terminal region, encompasses RBD responsible forrecognizing the host cell receptor (Huang et al., 2020; Lan et al., 2020). The RBDsof SARS-CoV-2, SARS CoV-1 as well as MERS CoV have been demonstrated tobe highly suitable targets for the development of vaccines across various platformsowing to their exceptional level of antigenicity and capability to effectivelystimulate robust immune responses (Ahmed et al., 2021; Malladi, Patel, et al.,2021; Tai et al., 2020, 2023).

[005] Human infection with viruses against which pre-existing immunity is notprevalent in the population can result in epidemics and even pandemics withsubstantial morbidity and mortality. These outbreaks can have a negative influenceon public health and the economy worldwide. Considering the number of viruseswhich have various zoonotic reservoirs and the potential to infect humans, futurepandemic preparedness is of utmost importance to mitigate these risks.

[006] Therefore, there remains a dire need for the development of a comprehensivethermostable, safe and effective pan-sarbecovirus vaccine formulation, which canelicit a targeted immune response and is also capable of eliciting a broadlyneutralizing cross reactive response against diverse Sarbecoviruses.SUMMARY OF INVENTION

[007] In an aspect of the present disclosure, there is provided an immunogenicpolypeptide comprising a polypeptide selected from the group consisting of: apolypeptide having an amino acid sequence of at least 95% sequence identity tothe sequence selected from SEQ ID NO: 1 or SEQ ID NO: 59; a polypeptidehaving an amino acid sequence of at least 95% sequence identity to the sequenceselected from SEQ ID NO: 3 or SEQ ID NO: 61; a polypeptide having an aminoacid sequence of at least 95% sequence identity to the sequence selected from SEQID NO: 5 or SEQ ID NO: 63; a polypeptide having an amino acid sequence of atleast 95% sequence identity to the sequence selected from SEQ ID NO: 7 or SEQID NO: 65; a polypeptide having an amino acid sequence of at least 95% sequenceidentity to the sequence selected from SEQ ID NO: 9 or SEQ ID NO: 67, and apolypeptide having an amino acid sequence of at least 95% sequence identity tothe sequence selected from SEQ ID NO: 85 or SEQ ID NO: 69, wherein thepolypeptide comprises substitution mutations A17P, Y34W, and P196L.

[008] In an aspect of the present disclosure, there is provided a polynucleotideencoding the immunogenic polypeptide as described herein.

[009] In another aspect of the present disclosure, there is provided a recombinantvector containing the polynucleotide as disclosed herein operably linked to apromoter.

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

[0011] In another aspect of the present disclosure, there is provided a vaccinecomposition comprising the immunogenic polypeptide as disclosed herein, and apharmaceutically acceptable carrier.

[0012] In an aspect of the present disclosure, there is provided a method forproducing the vaccine composition comprising: (a) culturing the recombinant hostcell as described herein, under suitable conditions to obtain the immunogenicpolypeptide; (b) subjecting the immunogenic polypeptide to purification; and (c)contacting the immunogenic polypeptide of step (b) with a pharmaceuticallyacceptable carrier, to obtain the vaccine composition.

[0013] In an aspect of the present disclosure, there is provided a method ofeliciting an immune response against diverse Sarbecoviruses, in a subject,comprising administering the subject with an effective amount of the vaccinecomposition as disclosed herein.

[0014] In an aspect of the present disclosure, there is provided a kitcomprising the immunogenic polypeptide or the vaccine composition as describedherein, and an instruction leaflet.

[0015] These and other features, aspects, and advantages of the presentsubject matter will be better understood with reference to the following descriptionand appended claims. This summary is provided to introduce a selection ofconcepts in a simplified form. This summary is not intended to identify keyfeatures or essential features of the claimed subject matter, nor is it intended to beused to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0016] The following drawings form a part of the present specification andare included to further illustrate aspects of the present disclosure. The disclosuremay be better understood by reference to the drawings in combination with thedetailed description of the specific embodiments presented herein.

[0017] Figure 1 depicts the results related to expression and purification ofSarbecoviruses RBD derivatives. Coomassie stained profiles of SarbecovirusesRBDs purified from Expi293F cells are shown, in accordance with theembodiments herein.

[0018] Figure 2 depicts relative thermal stability of Sarbecoviruses RBDderivatives: Nano-DSF profiles showing thermal melting temperature forSarbecoviruses RBD derivatives: wild type (WT) and stabilized (St), inaccordance with the embodiments herein.

[0019] Figure 3 depicts relative thermal tolerance of Sarbecoviruses RBDderivatives: Purified proteins were incubated at temperatures ranging from 4-70°Cfor 2 hours followed by Nano-DSF profiles of (A) wild type and (B) stabilizedRBD derivatives, in accordance with the embodiments herein.

[0020] Figure 4 depicts immunogenicity studies in mice: Endpoint ELISAIgG titers against different Sarbecoviruses RBDs obtained after twoimmunizations with (A) individual stabilized RBD oligomers and (B) stabilizedRBD derivative cocktail formulated with adjuvant, in accordance with theembodiments herein.

[0021] Figure 5 depicts immunogenicity studies in mice: Pseudovirusneutralization titers against (A) SARS CoV-1, (B) SARS CoV-2, (C) BtKY72 and(D) heterologous pseudoviruses including LYRa3, SHC014 and Khosta-2 fromsera obtained after two immunizations, in accordance with the embodimentsherein.

[0022] Figure 6 illustrates efficacy of Stabilized RBD trimer and RBD-S2monomer cocktail formulation, wherein A) is a schematic representation of theimmunization protocol, and (B) depicts body weight change and lung viral titresin mice post SARS-CoV-1 challenge and (C) depicts body weight change andlung viral titres in mice post SARS-CoV-2 BA.5 challenge, in accordance with theembodiments herein.

[0023] Figure 6 illustrates long-term thermal stability of the RBD cocktailimmunogens wherein (A) depicts thermal denaturation profile and (B) depictsbinding affinity of the RBD cocktail immunogens from day0- day 30 for proteinsincubated at 4°C and 37°C determined by Nano-DSF and BLI experimentsrespectively. Lyophilized proteins were incubated at the indicated temperaturesand reconstituted in PBS prior to measurements, in accordance with theembodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0024] Those skilled in the art will be aware that the present disclosure issubject to variations and modifications other than those specifically described. Itis to be understood that the scope of the present disclosure includes all suchvariations and modifications that may be apparent to a person skilled in the art inlight 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 suchsteps or features.Definitions

[0025] 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 andunderstood as by a person of skill in the art. The terms used herein have themeanings recognized and known to those of skill in the art, however, forconvenience and completeness, particular terms and their meanings are set forthbelow.

[0026] The articles "a", "an" and "the" are used to refer to one or to morethan one (i.e., to at least one) of the grammatical object of the article.

[0027] The terms "comprise" and "comprising" are used in the inclusive,open sense, meaning that additional elements may be included. It is not intendedto be construed as "consists of only".

[0028] Throughout this specification, unless the context requires otherwisethe word "comprise", and variations such as "comprises" and "comprising", willbe understood to imply the inclusion of a stated element or step or group of elementor steps but not the exclusion of any other element or step or group of element orsteps.

[0029] The term "including" is used to mean "including but not limited to"."Including" and "including but not limited to" are used interchangeably.

[0030] The term "pharmaceutically acceptable carrier" refers to any knowncarriers, excipients, adjuvants known to a person skilled in the art, which can beused in therapeutic and vaccine formulations. The term "pharmaceuticallyeffective amount" or "effective amount", as used herein, refers to an amount thatis effective in eliciting an immune response, in a subject.

[0031] The term "vaccine composition" refers to a composition that elicits aprophylactic or therapeutic immune response in a subject. Typically, a vaccinecomposition elicits an antigen-specific immune response to an antigen of apathogen, for example a viral pathogen, or to a cellular constituent correlated witha pathological condition.

[0032] The term "subject" refers to any animal, including a mammal, e.g.,human and non-human mammals. Examples of non-human animals include nonhuman primates, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mice, rats,hamsters, guinea pigs and etc. Unless otherwise noted, the terms "patient" or"subject" are used herein interchangeably. Preferably, the subject is human.

[0033] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinary skillin the art to which this disclosure belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in the practice or testingof the disclosure, the preferred methods, and materials are now described. Allpublications mentioned herein are incorporated herein by reference.

[0034] Embodiments herein provide immunogenic polypeptides havingSarcebovirus derived stabilized receptor binding domain (RBD) fragments. Thewildtype RBD fragments obtained from various Sarbecoviruses, particularlySARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b), RmYNO2(Clade 2), BtKY72 (Clade 3), and SARS CoV-2 XBB1.5 are stabilized byintroducing specific stabilizing mutations at various positions on the amino acidsequence. The present inventors observed that the introduction of these mutationssubstantially improved thermal stability by about ~7°C, and enhanced purifiedyield by about 3-23-fold, relative to corresponding wildtype (WT) RBDs, withoutaffecting their binding to conformation specific ligands.

[0035] Further, in some embodiments, the immunogenic polypeptidescomprise Sarbecovirus derived stabilized receptor binding domain (RBD)fragment, and a fragment of S2 subunit of SARS-CoV-2 spike protein . Thefragment of S2 subunit may be attached N-terminally or C-terminally to thestabilized RBD, preferably via a linker. In an embodiment, the fragment of S2subunit is attached C-terminally to the stabilized RBD. The fragment of S2 subunitof SARS-CoV-2 spike protein may be engineered to include substitution mutationsat positions 817, 892, 899, 942, 969 and 973, positions in respect of the wildtypeS2 subunit of SARS-CoV-2 spike protein (Accession Id: YP_009724390.1). In anembodiment, the amino acid sequence of the fragment of S2 subunit of SARSCoV-2 spike protein is engineered to substitute amino acid residue at positions817, 892, 899, and 942 with proline (i.e. F817P, A892P, A899P, and A942Prespectively) and substitute amino acid residue at positions 969 and 973 withaspartic acid (i.e. N969D and I973D respectively). In an embodiment, thefragment of S2 subunit of SARS-CoV-2 spike protein has an amino acid sequenceas depicted in SEQ ID NO: 83 (corresponding nucleotide sequence is depicted inSEQ ID NO: 84).

[0036] In an embodiment, the stabilized RBD fragments obtained fromSarbecoviruses: SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b),RmYNO2 (Clade 2), BtKY72 (Clade 3), and SARS CoV-2 XBB1.5 are depictedin SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,and SEQ ID NO: 85, respectively.

[0037] In an embodiment, the polypeptides having a combination ofsarbecovirus derived stabilized receptor binding domain (RBD) fragment, and afragment of S2 subunit of SARS-CoV-2 spike protein: Stabilized SARS CoV-1RBD-S2, Stabilized WIV-1 RBD-S2, Stabilized RaTG13 RBD-S2, StabilizedRmYNO2 RBD-S2, Stabilized BtKY72 RBD-S2, and Stabilized SARS CoV-2XBB1.5 RBD-S2 are depicted in SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69, respectively.

[0038] Substitution mutations and stabilizing mutations, according toembodiments herein, are also described herein by use of the following notation:amino acid residue substituted: amino acid position in the sequence: amino acidresidue substitute. Accordingly, an amino acid substitution with, for instance, ifthe amino acid substitution is of phenylalanine (F) with proline (P), at position 111in a sequence, it may be indicated as F111P. Further, alternative amino acidresidues substitutes are represented by the notation: amino acid substitute / aminoacid substitute / amino acid substitute. For instance, if amino acid substitution ofphenylalanine (F) is with proline (P) or serine (S), at position 111 in a sequence, itmay be indicated as F111P / S. Similarly, when more than one alternatives arepossible, it may be indicated as F111P / S / A, and so on. In general, when more thanone alternatives amino acid residues are possible at a position, it may be indicatedas P / S which is intended to mean that any of proline or serine may be present atthat position Similarly, N / I may indicate that any of asparagine or isoleucine maybe present at that position, and so on. Such notations are generally known to aperson skilled in the art and generally used in representing amino acidresidues / substitutions in a given amino acid sequence. Also, amino acids aregenerally 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 wellunderstood by a person skilled in the art. The present disclosure in describing thepresent invention employs such representations or phrases which is intended tomean the generally acceptable meaning in the art.

[0039] Embodiments herein also provide vaccine compositions comprisingone or more of the immunogenic polypeptides as described herein. It has beenobserved by the present inventors that a cocktail formulation comprising two ormore of the immunogenic polypeptides as described herein significantly augmentsthe yield, thermostability, and immunogenicity of the RBD fragments. The presentdisclosure provides stabilization of RBD fragments obtained from different cladesof Sarbecoviruses, which is combined to achieve a pan-Sarbecoviruses vaccineformulation.

[0040] The immunogenic polypeptides, according to embodiments herein,are resistant to 2-hour incubation at temperatures of up to 60°C in PBS, in contrastto corresponding WT RBDs. Further, lyophilized cocktail formulations, accordingto embodiments herein, retained antigenicity even after storage at 37°C for over15 days. In an embodiment, the vaccine composition is a lyophilized composition.Immunogenic polypeptide

[0041] Embodiments herein provide immunogenic polypeptides. In anembodiment, the immunogenic polypeptide comprises a polypeptide having anamino acid sequence of at least 95% sequence identity to the sequence selectedfrom SEQ ID NO: 1 or SEQ ID NO: 59, wherein the polypeptide comprisessubstitution mutations Y34W and P195L. In an embodiment of the presentdisclosure, the identity is at least 95%, 96%, 97%, 98%, 99%, or 99.5% to theamino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 59. In anotherembodiment of the present disclosure, the immunogenic polypeptide comprising apolypeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or SEQID NO: 59.

[0042] In an embodiment of the present disclosure, there is provided animmunogenic polypeptide comprising a polypeptide having an amino acidsequence of at least 95% sequence identity to the sequence selected from SEQ IDNO: 3 or SEQ ID NO: 61, wherein the polypeptide comprises substitutionmutations Y34W and P195L. In another embodiment of the present disclosure, theidentity is at least 95%, 96%, 97%, 98%, 99%, 99.5% to the amino acid sequenceas set forth in SEQ ID NO: 3or SEQ ID NO: 61. In an embodiment of the presentdisclosure, there is provided an immunogenic polypeptide comprising apolypeptide having an amino acid sequence as set forth in SEQ ID NO: 3 or SEQID NO: 61.

[0043] In an embodiment of the present disclosure, there is provided animmunogenic polypeptide comprising a polypeptide having an amino acidsequence of at least 95% sequence identity to the sequence selected from SEQ IDNO: 5 or SEQ ID NO: 63, wherein the polypeptide comprises substitutionmutations A17P, Y34W, and P196L. In another embodiment of the presentdisclosure, the identity is at least 95%, 97%, 98%, 99%, 99.5% to the amino acidsequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 63. In an embodiment ofthe present disclosure, there is provided an immunogenic polypeptide comprisinga polypeptide having an amino acid sequence as set forth in SEQ ID NO: 5 or SEQID NO: 63.

[0044] In an embodiment of the present disclosure, there is provided animmunogenic polypeptide comprising a polypeptide having an amino acidsequence of at least 95% sequence identity to the sequence selected from SEQ IDNO: 7 or SEQ ID NO: 65, wherein the polypeptide comprises substitutionmutations Y34W and P177L. In another embodiment of the present disclosure, theidentity is at least 95%, 96%, 97%, 98%, 99%, 99.5% to the amino acid sequenceselected from SEQ ID NO: 7 or SEQ ID NO: 65. In an embodiment of the presentdisclosure, there is provided an immunogenic polypeptide comprising apolypeptide having an amino acid sequence as set forth in SEQ ID NO: 7 or SEQID NO: 65.

[0045] In an embodiment of the present disclosure, there is provided animmunogenic polypeptide comprising a polypeptide having an amino acidsequence of at least 95% sequence identity to the sequence selected from SEQ IDNO: 9 or SEQ ID NO: 67, wherein the polypeptide comprises substitutionmutations Y34W and P195L. In another embodiment of the present disclosure, theidentity is at least 95%, 96%, 97%, 98%, 99%, 99.5% to the amino acid sequenceselected from SEQ ID NO: 9 or SEQ ID NO: 67. In an embodiment of the presentdisclosure, there is provided an immunogenic polypeptide comprising apolypeptide having an amino acid sequence selected from SEQ ID NO: 9 or SEQID NO: 67.

[0046] In an embodiment of the present disclosure, there is provided animmunogenic polypeptide comprising a polypeptide having an amino acidsequence of at least 95% sequence identity to the sequence as set forth in SEQ IDNO: 85 or SEQ ID NO: 69, wherein the polypeptide comprises substitutionmutations A17P, Y34W, and P196L. In another embodiment of the presentdisclosure, the identity is at least 95%, 96%, 97%, 98%, 99%, 99.5% to the aminoacid sequence selected from SEQ ID NO: 85 or SEQ ID NO: 69. In an embodimentof the present disclosure, there is provided an immunogenic polypeptidecomprising a polypeptide having an amino acid sequence selected from SEQ IDNO: 85 or SEQ ID NO: 69.

[0047] In an embodiment of the present disclosure, there is provided animmunogenic polypeptide comprising a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69.Full length Immunogenic polypeptide

[0048] The immunogenic polypeptide, according to embodiments herein,may further comprise one or more peptides such as TPA signal sequence,oligomerization domain, His-tag, etc. In an embodiment, there is provided animmunogenic polypeptide comprising a polypeptide having at least 95% sequenceidentity to an amino acid sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, andSEQ ID NO: 69, wherein the immunogenic polypeptide further comprises apeptide selected from TPA signal peptide; oligomerization domain; HRV3Cprotease cleavage site or a portion thereof; His tag; one or more linkers; orcombinations thereof.

[0049] In an embodiment, there is provided an immunogenic polypeptidecomprising a polypeptide having at least 95% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69, whereinthe polypeptide is further attached to a oligomerization domain, preferably via alinker.

[0050] Various oligomerization domains are known and may be used inembodiments herein. Examples of oligomerization domains includes humancartilage matrix protein (hCMP), chicken CMP (cCMP), fish cartilage matrixprotein (F1CMP), fish isoform 2 cartilage matrix protein (F2-CMP), leucineZipper with double cysteine (CCIZ), Synthetic oligomerization domain (cCMPIZm), foldon, or glycosylated leucine zipper sequence (Gly IZ). In an embodiment,the oligomerization domain is a synthetic oligomerization domain (cCMP-IZm)having an amino acid sequence as set forth in SEQ ID NO. 38.

[0051] In an embodiment, there is provided an immunogenic polypeptidecomprising a polypeptide having at least 95% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69, whereinthe polypeptide is further attached, at the N-terminal end, to a TPA signal peptidehaving an amino acid sequence as set forth in SEQ ID NO. 36.

[0052] In an embodiment, there is provided an immunogenic polypeptidecomprising a polypeptide having at least 95% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69, whereinthe polypeptide is further attached at the C-terminal end to a HRV3C proteasecleavage site having an amino acid sequence as set forth in SEQ ID NO. 40.

[0053] The TPA signal peptide; oligomerization domain; HRV3C proteasecleavage site or a portion thereof; and / or the His tag, may be attached to thepolypeptide having at least 95% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69 by one or more linkers.

[0054] Various linkers are known and may be used in embodiments herein.In an embodiment, the linker is having an amino acid sequence selected from"AAS", "S", "AS", "GT", "GSAGS" (SEQ IDNO.52), "ASSEGTMMRGELKN"(SEQ ID NO.53), and / or "GS".

[0055] In an embodiment, the immunogenic polypeptide further comprisesa peptide "EIS" attached at the N-terminal end. The peptide "EIS" is a vectorderived peptide and is attached to the N-terminal end of the immunogenicpolypeptide. In an embodiment, the immunogenic polypeptide comprises TPAsignal peptide at the N-terminal end of the immunogenic polypeptide attached bythe peptide "EIS".

[0056] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "GS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "AAS" followed by a His-tag using a linker "S",wherein the polypeptide is attached to a peptide of sequence "EIS" at the Nterminal end which is attached to TPA signal peptide, wherein the oligomerizationdomain has an amino acid sequence as set forth in SEQ ID NO. 38. In anembodiment of the present disclosure, there is provided an immunogenic peptidecomprising a polypeptide having an amino acid sequence of at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identityto a sequence as set forth in SEQ ID NO: 13, wherein the polypeptide comprisessubstitution mutations Y60W and P221L. In an embodiment of the presentdisclosure, there is provided an immunogenic peptide comprising a polypeptidehaving an amino acid sequence selected from the group consisting of SEQ ID NO: 13.

[0057] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "GS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "AAS", wherein the polypeptide is attached to apeptide of sequence "EIS" at the N-terminal end, wherein the oligomerizationdomain has an amino acid sequence as set forth in SEQ ID NO. 38. In anembodiment of the present disclosure, there is provided an immunogenic peptidecomprising a polypeptide having an amino acid sequence of at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identityto a sequence as set forth in SEQ ID NO: 15, wherein the polypeptide comprisessubstitution mutations Y37W and P198L. In an embodiment of the presentdisclosure, there is provided an immunogenic peptide comprising a polypeptidehaving an amino acid sequence selected from the group consisting of SEQ ID NO:15.

[0058] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "AS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "GT" followed by a His-tag using a linker "S",wherein the polypeptide is attached to a peptide of sequence "EIS" at the Nterminal end which is attached to TPA signal peptide, wherein the oligomerizationdomain has an amino acid sequence as set forth in SEQ ID NO. 38. In anembodiment of the present disclosure, there is provided an immunogenic peptidecomprising a polypeptide having an amino acid sequence of at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identityto a sequence as set forth in SEQ ID NO: 18, wherein the polypeptide comprisessubstitution mutations Y60W and P221L. In an embodiment of the presentdisclosure, there is provided an immunogenic peptide comprising a polypeptidehaving an amino acid sequence selected from the group consisting of SEQ ID NO: 18.

[0059] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "AS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "GT", wherein the polypeptide is attached to apeptide of sequence "EIS" at the N-terminal end which is attached to TPA signalpeptide, wherein the oligomerization domain has an amino acid sequence as setforth in SEQ ID NO. 38. In an embodiment of the present disclosure, there isprovided an immunogenic peptide comprising a polypeptide having an amino acidsequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orat least 99.5% sequence identity to a sequence as set forth in SEQ ID NO: 20,wherein the polypeptide comprises substitution mutations Y37W and P198L. Inan embodiment of the present disclosure, there is provided an immunogenicpeptide comprising a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 20.

[0060] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 5attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "AS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "GT" followed by a His-tag using a linker "S",wherein the polypeptide is attached to a peptide of sequence "EIS" at the Nterminal end which is attached to TPA signal peptide, wherein the oligomerizationdomain has an amino acid sequence as set forth in SEQ ID NO. 38. In anembodiment of the present disclosure, there is provided an immunogenic peptidecomprising a polypeptide having an amino acid sequence of at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identityto a sequence as set forth in SEQ ID NO: 23, wherein the polypeptide comprisessubstitution mutations A43P, Y60W, and P222L. In an embodiment of the presentdisclosure, there is provided an immunogenic peptide comprising a polypeptidehaving an amino acid sequence selected from the group consisting of SEQ ID NO: 23.

[0061] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 5attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "AS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "GT", wherein the polypeptide is attached to apeptide of sequence "EIS" at the N-terminal end which is attached to TPA signalpeptide, wherein the oligomerization domain has an amino acid sequence as setforth in SEQ ID NO. 38. In an embodiment of the present disclosure, there isprovided an immunogenic peptide comprising a polypeptide having an amino acidsequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orat least 99.5% sequence identity to a sequence as set forth in SEQ ID NO: 25,wherein the polypeptide comprises substitution mutations A20P, Y37W andP199L. In an embodiment of the present disclosure, there isprovided an immunogenic peptide comprising a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 25.

[0062] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 7attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "AS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "GT" followed by a His-tag using a linker "S",wherein the polypeptide is attached to a peptide of sequence "EIS" at the Nterminal end which is attached to TPA signal peptide, wherein the oligomerizationdomain has an amino acid sequence as set forth in SEQ ID NO. 38. In anembodiment of the present disclosure, there is provided an immunogenic peptidecomprising a polypeptide having an amino acid sequence of at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identityto a sequence as set forth in SEQ ID NO: 28, wherein the polypeptide comprisessubstitution mutations Y60W and P203L. In an embodiment of the presentdisclosure, there is provided an immunogenic peptide comprising a polypeptidehaving an amino acid sequence selected from the group consisting of SEQ ID NO: 28.

[0063] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 7attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "AS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "GT", wherein the polypeptide is attached to apeptide of sequence "EIS" at the N-terminal end which is attached to TPA signalpeptide, wherein the oligomerization domain has an amino acid sequence as setforth in SEQ ID NO. 38. In an embodiment of the present disclosure, there isprovided an immunogenic peptide comprising a polypeptide having an amino acidsequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orat least 99.5% sequence identity to a sequence as set forth in SEQ ID NO: 30,wherein the polypeptide comprises substitution mutations Y37W and P180L. Inan embodiment of the present disclosure, there is provided an immunogenicpeptide comprising a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 30.

[0064] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 9attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "AS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "GT" followed by a His-tag using a linker "S",wherein the polypeptide is attached to a peptide of sequence "EIS" at the Nterminal end which is attached to TPA signal peptide, wherein the oligomerizationdomain has an amino acid sequence as set forth in SEQID NO. 38. In anembodiment of the present disclosure, there is provided an immunogenic peptidecomprising a polypeptide having an amino acid sequence of at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identityto a sequence as set forth in SEQ ID NO: 33, wherein the polypeptide comprisessubstitution mutations Y60W, and P221L. In an embodiment of the presentdisclosure, there is provided an immunogenic peptide comprising a polypeptidehaving an amino acid sequence selected from the group consisting of SEQ ID NO: 33.

[0065] In an embodiment, the immunogenic polypeptide comprisesa polypeptide having an amino acid sequence as set forth in SEQ ID NO: 9attached to a oligomerization domain at C-terminal end of the polypeptide by alinker "AS", wherein the oligomerization domain is further attached to a HRV3Cprotease cleavage site by a linker "GT", wherein the polypeptide is attached to apeptide of sequence "EIS" at the N-terminal end which is attached to TPA signalpeptide, wherein the oligomerization domain has an amino acid sequence as setforth in SEQID NO. 38. In an embodiment of the present disclosure, there isprovided an immunogenic peptide comprising a polypeptide having an amino acidsequence of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orat least 99.5% sequence identity to a sequence as set forth in SEQ ID NO: 35,wherein the polypeptide comprises substitution mutations Y37W and P198L. Inan embodiment of the present disclosure, there is provided an immunogenicpeptide comprising a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 35.

[0066] In an embodiment of the present disclosure, there is provided animmunogenic peptide comprising a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81.

[0067] In an embodiment, there is provided a polynucleotide encoding theimmunogenic polypeptide as described herein.

[0068] In another embodiment, the polynucleotide has a nucleotide sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO:24, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO:82. The polynucleotide may be deoxy ribose nucleic acid (DNA) or ribose nucleicacid (RNA). In an embodiment, the polynucleotide is selected from DNA, RNA,or messenger RNA (mRNA).

[0069] In an embodiment, there is provided a recombinant vector containingthe polynucleotide as disclosed herein, operably linked to a promoter.

[0070] In an embodiment, there is provided a recombinant host cellcomprising the recombinant vector as described herein. The host cell is a bacterialcell, yeast cell, insect cell, or mammalian cell. The bacterial cell is Escherichiacoli, and the yeast cell is selected from the group consisting of Pichia X33, PichiaGlycoSwitch , DSMZ 70382, GS115, KM71, KM71H, BG09, GS190, GS200,JC220, JC254, JC227, JC300-JC308, YJN165, and CBS7435, and wherein theinsect 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 consistingof Expi293FExpi-CHO-S , CHO-K1, CHO-S, HEK293F , CHOBC,SLIM , SPOT , SP2 / 0 , Sp2 / 0- Ag14, CHO DG44, HEK 293S, HEK 293 Gnt1- / - ,HEK293-EBNA1, CHOL-NSO, and NSO. Further, the host cell may be selectedfrom OPENPichia (NCYC 2543 hoc1tr), OPENPichia his4, NCYC 2543 typestrain, OPENPichia pep4, OPENPichia yps1, OPENPichia pep4 yps1, andOPENPichia mutS strains.

[0071] Embodiments herein provide vaccine compositions. In anembodiment, there is provided a vaccine composition comprising theimmunogenic polypeptide as described herein, and a pharmaceutically acceptablecarrier. The pharmaceutically acceptable carrier is an adjuvant selected from anoil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, analuminum hydroxide adjuvant, and combinations thereof.

[0072] In an exemplary embodiment of the present disclosure, thepharmaceutically acceptable carrier is selected from the group consisting ofalhydrogel (aluminium hydroxide adjuvant), Alhydrogel CpG, Addavax (oil-inwater adjuvant), SWE (squalene-in-water emulsion adjuvant), and MF59.

[0073] In an embodiment, the vaccine composition comprises animmunogenic polypeptide of amino acid sequence of at least 95% sequenceidentity to amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO: 79, and SEQ ID NO: 81. In an embodiment, the vaccinecomposition comprises an immunogenic polypeptide of amino acid sequence ofamino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ IDNO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQID NO: 79, and SEQ ID NO: 81.

[0074] In an embodiment, the vaccine composition comprises animmunogenic polypeptide of amino acid sequence of at least 95% sequenceidentity to amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,SEQ ID NO: 79, and SEQ ID NO: 81. In an embodiment, the vaccine compositioncomprises an immunogenic polypeptide of amino acid sequence of amino acidsequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 20,SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ IDNO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, andSEQ ID NO: 81.

[0075] In another embodiment, the vaccine composition is a cocktailformulation comprising a combination of at least 2, at least 3, at least 4 or 5immunogenic polypeptides of amino acid sequence having at least 95% identity toa sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9. In another embodiment, the vaccinecomposition is a cocktail formulation comprising a combination of theimmunogenic polypeptides of amino acid sequence having at least 95% identity toa sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 59, SEQ IDNO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,and SEQ ID NO: 81. In another embodiment, the vaccine composition is a cocktailformulation comprising a combination of the immunogenic polypeptides of aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 59, SEQID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO:79, and SEQ ID NO: 81.

[0076] In yet another embodiment, the vaccine composition is a cocktailformulation comprising a combination of at least 2, at least 3, at least 4 or 5immunogenic polypeptides of amino acid sequence having at least 95% identity toa sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO:20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ IDNO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,and SEQ ID NO: 81. In yet another embodiment, the vaccine composition is acocktail formulation comprising a combination immunogenic polypeptides ofamino acid sequence having at least 95% identity to a sequence selected from thegroup consisting of SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ IDNO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81. In yetanother embodiment, the vaccine composition is a cocktail formulation comprisinga combination immunogenic polypeptides of amino acid sequence selected fromthe group consisting of SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQID NO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81.

[0077] In an embodiment of the present disclosure, there is provided methodfor producing the vaccine composition as disclosed herein, said methodcomprising: (a) culturing the recombinant host cell as described herein, to expressthe immunogenic polypeptide as described herein; (b) subjecting the immunogenicpolypeptide to purification; and (c) contacting the immunogenic polypeptide ofstep (b) with a pharmaceutically acceptable carrier, to obtain the vaccinecomposition. The vector, according to embodiments herein, comprising thenucleotide encoding the immunogenic polypeptide may be introduced into the hostcell by transfection, to obtain the recombinant host cell which may then be culturedto express the immunogenic polypeptide as described herein. Various transfectionmethods are known for eg: lipofection, electroporation, etc which may be used toachieve the recombinant host cell as described herein. The expressedimmunogenic polypeptide may then be purified by using protein purificationmethods generally known in field (for eg: Ni-NTA affinity chromatography) toobtain the purified immunogenic polypeptide. The immunogenic polypeptide maythen be mixed with a suitable pharmaceutically acceptable carrier to obtain thevaccine composition. One or more immunogenic polypeptides may be mixed withthe pharmaceutically acceptable carrier.

[0078] In an embodiment of the present disclosure, there is provided methodfor producing the vaccine composition as disclosed herein, said methodcomprising: (a) culturing the recombinant host cell as described herein, undersuitable conditions to express the immunogenic polypeptide, wherein theimmunogenic polypeptide has an amino acid sequence of at least 95% sequenceidentity to amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,SEQ ID NO: 79, and SEQ ID NO: 81; (b) subjecting the immunogenic polypeptideto purification; and (c) contacting the immunogenic polypeptide of step (b) with apharmaceutically acceptable carrier, to obtain the vaccine composition.

[0079] In another embodiment, there is provided a method of eliciting animmune response against diverse Sarbecovirus in a subject, the method comprisingadministering the subject with an effective amount of the vaccine composition asdisclosed herein. In another embodiment, the Sarbecovirus is selected from clade1a, clade 1b, clade 2, or clade 3 virus. In another embodiment, the Sarbecovirusesis selected from the group consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade1a), SARS-CoV-2(clade 1b), RaTG13 (clade 1b), RmYNO2 (clade 2), andBtKY72 (clade 3).

[0080] In another embodiment, the Sarbecovirus is selected from the groupconsisting of SARS-CoV-1 (clade 1a), WIV-1 (clade 1a), SARS-CoV-2(clade 1b),RaTG13 (clade 1b), RmYNO2 (clade 2), BtKY72 (clade 3), Pangolin_GD,RaTG13 (MN996532), Pangolin_GX-P2V (EPI_ISL_410542), SARS-CoV1_Urbani_HP03 (AY278741), SARS-CoV-1_BJ02_HP03M (AY278487), SARSCoV-1_HGZ8L1-A_HP03E (AY394981), SARS-CoV-1_GD01_HP03L(AY278489), SARS-CoV-1_GZ-C_HP03L (AY394979), SARS-CoV-1_Sino1-11_HP03L (AY485277), SARS-CoV-1_Sin852_HP03L (AY559082), SARSCoV-1_SZ3_PC03 (AY304486), SARS-CoV-1_SZ13_PC03 (AY304487),SARS-CoV-1_SZ1_PC03 (AY304489), SARS-CoV-1_GD03T0013_HP04(AY525636), SARS-CoV-1_GZ0402_HP04 (AY613947), SARS-CoV-1_PC4-127_PC04 (AY613951), SARS-CoV-1_PC4-137_PC04 (AY627045), SARSCoV-1_PC4-13_PC04 (AY613948), WIV16 (KT444582), WIV1 (KF367457),Rs7327 (KY417151), LYRa11 (KF569996), Rs4231 (KY417146), RsSHC014(KC881005), Rs4084 (KY417144), BM48-31 (NC014470), BtKY72(KY352407), ZXC21 (MG772934), ZC45 (MG772933), JL2012 (KJ473811), Rf1(DQ412042), HeB2013 (KJ473812), 273-2005 (DQ648856), Rf4092(KY417145), YN2013 (KJ473816), RmYNO2 (EPI_ISL_412977), As6526(KY417142), Rs4237 (KY417147), Rs4081 (KY417143), Rp3 (DQ071615), 279-2005 (DQ648857), Shaanxi2011 (JX993987), Yunnan2011 (JX993988), Rs4247(KY417148), HKU3-13 (GQ153548), HKU3-1 (DQ022305), GX2013(KJ473815), Longquan-140 (KF294457), HKU3-8 (GQ153543), HuB2013(KJ473814), Hp-BCoV_Zhejiang_2013 (KF636752), HKU1 (KF686346), OC43(KX344031), MERS-CoV (NC_019843), Bat-CoV-GCCDC1 (MT350598),Rousettus-CoV_HKU9 (MG762674), Rc-o319 (LC556375), RacCS203(MW251308), RshSTT182 (EPI_ISL_852604), PDF-2370, PRD-0038, RsYN04(EPI_ISL_1699444), BB9904 (KR559017), Khosta-1 (MZ190137), Khosta-2(MZ190138), and RhGB01 (MW719567).

[0081] In an embodiment, the vaccine composition is administered by amode selected from the group consisting of intranasal, subcutaneous, intravenous,intra-arterial, intra-peritoneal, intramuscular, intradermal, oral, dermal, andbuccal.

[0082] In another embodiment, there is provided a kit comprising theimmunogenic polypeptide as disclosed herein; or the vaccine composition asdescribed herein, and an instruction leaflet.

[0083] Although the subject matter has been described with reference tospecific embodiments, this description is not meant to be construed in a limitingsense. Various modifications of the disclosed embodiments, as well as alternateembodiments of the subject matter, will become apparent to persons skilled in theart upon reference to the description of the subject matter. It is thereforecontemplated that such modifications can be made without departing from thespirit or scope of the present subject matter as defined.EXAMPLES

[0084] The disclosure will now be illustrated with working examples, whichis intended to illustrate the working of disclosure and not intended to takerestrictively to imply any limitations on the scope of the present disclosure. Unlessdefined otherwise, all technical and scientific terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art to which thisdisclosure belongs. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the disclosed methods andcompositions, the exemplary methods, devices and materials are described herein.It is to be understood that this disclosure is not limited to particular methods, andexperimental conditions described, as such methods and conditions may vary.

[0085] Substitution mutations, in accordance with the embodimentsherein are depicted in Table 1.Table 1: depicts the substitution mutations, according to the present disclosure.Example 1: Stabilization of diverse Sarbecoviruses RBDs

[0087] The percentage amino acid identity of the specific RBDs wascompared with SARS-CoV2 RBD and with other members of the respectiveclades. Considering the conservation of RBD sequence within the clade, SARSCoV-1 and WIV-1 (clade 1a); SARS-CoV-2 B.1, SARS-CoV-2 XBB1.5 andRaTG13 (clade 1b); RmYNO2 (clade 2) and BtKY72 (clade 3) were selected asthe representatives for their respective clades. The sequence and structuralconservation of the target residues was analysed. While residues 365 and 527 werefully conserved in all the selected Sarbecoviruses, it was interesting to note thatone of the SARS CoV-2 stabilizing mutations A348P was naturally present inclade 1a, 2 and 3 viruses. A348P, Y365W and P527L mutations were introducedin the selected Sarbecoviruses in order to study the transferability of thesemutations in diverse sequence backgrounds.Methods

[0088] The polypeptide fragments were prepared using the immunogenicpolypeptide sequences depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, and SEQ ID NO: 9, and the oligomerization domain (SEQ IN NO:38). The polypeptide fragments were expressed and tested for stability andimmunogenicity. The polypeptide fragments had the sequences as set forth in SEQID NO: 13 without TPA signal, SEQ ID NO: 18 without TPA signal, SEQ ID NO:23 without TPA signal, SEQ ID NO: 28 without TPA signal, and SEQ ID NO: 33without TPA signal. The sequences as set forth in SEQ ID NO: 13 without TPAsignal, SEQ ID NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal,SEQ ID NO: 28 without TPA signal, and SEQ ID NO: 33 without TPA signal areas depicted in SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,and SEQ ID NO: 58, respectively.

[0089] Each of the polypeptides having sequences depicted in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, wereindependently fused with a fragment of S2 subunit (SEQ ID NO: 83) at the Cterminal end using a linker "AS" to obtain stabilized SARS CoV-1 RBD-S2 (SEQID NO: 59), Stabilized WIV-1 RBD-S2 (SEQ ID NO: 61); Stabilized RaTG13RBD-S2 (SEQ ID NO: 63); Stabilized RmYNO2 RBD-S2 (SEQ ID NO: 65); andStabilized BtKY72 RBD-S2 (SEQ ID NO: 67), respectively. The polypeptidehaving a sequence depicted in SEQ ID NO: 85 was fused with a fragment of S2subunit (SEQ ID NO: 83) at the C-terminal end using a linker "AS" to obtainstabilized SARS CoV-2 XBB1.5 RBD-S2 (SEQ ID NO: 69).

[0090] The cocktail formulations were prepared using the polypeptidefragments as depicted in SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 withoutTPA signal, and SEQ ID NO: 33 without TPA signal, combined with SWEadjuvant, at 1:1 v / v polypeptide fragments: adjuvant ratio, and neutralization titers.The 1:1 v / v polypeptide fragments: adjuvant ratio. The cocktail formulations mayalso be prepared using the polypeptide fragments selected from SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 15,SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQID NO: 81.Protein Expression and Purification

[0091] The genes encoding Sarbecoviruses RBD proteins and monoclonalantibodies (mAbs) were synthesized at GenScript (USA) and TWIST Biosciences(USA). All the RBD derivatives [ i.e. SEQ ID NO: 13 without TPA signal, SEQID NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO:28 without TPA signal, and SEQ ID NO: 33 without TPA signal] were purifiedfrom transiently transfected Expi293F cells following the manufacturer'sguidelines (Gibco, Thermo Fisher, Waltham, MA, USA). Briefly, Expi293F cellswere diluted to a density of 3 x 106 cells / mL. For transfection, the desired plasmid(pCDNA5FRTTO mammalian expression vector) was complexed withExpiFectamine293 according to the manufacturer's protocol and transientlytransfected into Expi293F cells. Enhancer 1 and Enhancer 2 were added 16 hourspost transfection. The culture supernatant was collected after 5 days, and proteinwas purified through Ni-NTA affinity chromatography using Ni Sepharose 6 fastflow resin (GE Healthcare, Chicago, IL, USA) for RBD derivatives. Thesupernatant was added to a pre-equilibrated Ni-NTA column. Following a 2-column wash with 1x PBS (pH 7.4) supplemented with 20 mM imidazole, theprotein was eluted in 1X PBS with 300mM imidazole (pH 7.4). Pooled elutedfractions were dialyzed thrice against 1x PBS (pH 7.4). For expression andpurification of mAbs, Expi293F cells were co-transfected by Heavy and Lightchain plasmids in 1:1 ratio by using polyethylenimine, and supernatants wereharvested after 5 days post-transfection. The antibodies were purified fromsupernatants by using Protein A / G beads, dialyzed and stored in phosphatebuffered saline (PBS) for further use. Purified protein samples were analyzed on12% SDS-PAGE gel and quantified using NanoDrop spectrophotometer (Figure1, Table 3). Table 3 depicts summary of purified yields of Sarbecoviruses RBDderivatives. Similarly, the immunogenic polypeptide selected from SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ IDNO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, andSEQ ID NO: 81 may also be expressed and purified.Table 3: Thermal Unfolding experiments

[0092] Thermal melting studies of the RBD derivatives [ i.e. SEQ ID NO:13 without TPA signal, SEQ ID NO: 18 without TPA signal, SEQ ID NO: 23without TPA signal, SEQ ID NO: 28 without TPA signal, and SEQ ID NO: 33without TPA signal] were performed using nanoDSF (Prometheus NT.48), asdescribed in Chattopadhyay, G. et.al, 2019, at a temperature range of 20 °C to 95°C. Experimental measurements were conducted at 100% LED intensity with aninitial discovery scan, and the scan counts (350 nm) ranged between 2000 and3000. For lyophilized protein, reconstitution was done using 1X PBS buffer beforethe DSF experiment. The results have been summarized in Figure 2.SPR binding studies

[0093] Kinetics titrations were performed using a CM5 sensor chip (Cytiva)at 25°C. The activation of the carboxymethylated-dextran gold surface wasachieved by injecting EDC / s-NHS (2 / 4 mM) solution in autoclaved water pH 7.0injected at 5 μL / min for 400 seconds. Following the activation step, a 10 μg / mLsolution of Protein-G in sodium acetate (NaOAc) pH 5.0 was injected over theactivated surface at 5 μL / min. After covalent modification of the sensor surface, aquenching solution of ethanolamine pH 8.5 (Cytiva) was injected over the surfacefor 600 seconds to cap any residual active NHS esters. PBS (1X) pH 7.4 was usedfor the running buffer during titration. During the kinetics assay, one flow cellchannel with only Protein G served as a reference channel to monitor and subtractbinding responses due to non-specific interactions. 200-300 RU of monoclonalantibodies at 1 μg / mL were captured onto the chip surface for each cycle at 5μL / min for 60 seconds, followed by injection of RBD derivatives for 200 seconds.Then a dissociation step was performed using an injection of running buffer for200 seconds. Following the dissociation step, regeneration of the Protein G surfacewas performed using 1 injection of 0.1M glycine-HCl, pH 2.0 at 30 μL / min for 40seconds. The flow rate for association and dissociation was 30 μL / min. Thekinetics traces were reference subtracted using the responses of the referencechannel in each cycle and blank subtracted using a zero-concentration cycle. Thenthe kinetics constants ka, kd and KD values were determined using Biacore T200evaluation software.Mice Immunizations

[0094] Female C57BL / 6 mice (6-8 weeks old, n = 5 / group) were immunizedintramuscularly with RBD derivatives [ i.e. SEQ ID NO: 13 without TPA signal,SEQ ID NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal, SEQID NO: 28 without TPA signal, and SEQ ID NO: 33 without TPA signal],(5μg / animal in 100 μL of 1x PBS, pH 7.4) adjuvanted with SWE (1:1 v / v antigen:adjuvant ratio) (Sepivac SWE Batch No. 200915012131, Cat. No. 80748J,SEPPIC SA) on days 0 (prime), and 21 (boost). RBD cocktail group wasimmunized with 5μg / antigen having RBD derivatives from different clades asmentioned. Sera were isolated from blood drawn on days prior to prime (day -1),post-prime (day 14), and post-boost (day 35) through retro-orbital puncture.

[0095] For the assessment of RBD cocktail efficacy in pre-immunized mice,female BALB / c mice (6-8 weeks old, n = 5 / group) were first immunized withstabilized monomeric SARS CoV-2 RBD (1μg / animal in 100 μL of 1x PBS, pH7.4) adjuvanted with SWE (1:1 v / v antigen: adjuvant ratio) (Sepivac SWE BatchNo. 200915012131, Cat. No. 80748J, SEPPIC SA) on days 0 (prime), and thenboosted with RBD cocktail formulation (having SEQ ID NO: 13 without TPAsignal, SEQ ID NO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal,SEQ ID NO: 28 without TPA signal, and SEQ ID NO: 33 without TPA signal)(5μg / antigen, total 30μg) on day 30 and day 51. Sera obtained before prime andpost boost immunizations was used to conduct ELISA and pseudoviralneutralization assays. These studies were performed at Central Animal Facility,Indian Institute of Science. The Institutional Animal Ethics committee approvedall animal studies (IAEC no. CAF / ETHICS / 002 / 2023). Similarly, studies may alsobe performed using cocktail formulations having SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 20,SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and / or SEQ ID NO: 81.Enzyme-linked immunosorbent assay (ELISA)

[0096] ELISA was used to quantify the Sarbecoviruses RBD-specific IgGtiters in serum as described in Ahmed, S. et al. 2021. Briefly, ELISA plates werecoated with 4 μg / mL His tagged RBD derivatives at 25 °C, 1h. The coated plateswere blocked using blocking buffer (PBS containing 3% skimmed milk) for 1 hourat 25 °C. The sera (maximum concentration, 1:80-1:1280) from mice, were seriallydiluted 1:4 in PBST (PBS containing 3% skimmed milk and 0.05% Tween-20) for8 dilutions, followed by incubation with the coated ELISA plates for 60 min at25 °C. After washing with PBST three times, HRP-conjugated secondary antibodywas added to the plates and incubated for 60 min at 25 °C. After incubation withthe secondary antibody, the plates were washed with PBST three times, followedby adding 3,3',5,5'-tetramethylbenzidine (TMB) to visualize the reaction. Finally,6N HCl was used to stop the reaction. The chromogenic signal was measured at405 nm using an ELISA plate reader (Maxome Labsciences Cat # P3-5x10NO).The serum dilution with a signal observed two-fold above the negative control(empty blocked wells) was considered the endpoint titer for ELISA.Sarbecoviruses Pseudovirus Preparation and Neutralization Assay

[0097] HIV-1-based pseudotyped viruses were employed in pseudoviralneutralization assays, following a method previously described [Malladi SK, et al.Immunogenicity and Protective Efficacy of a Highly Thermotolerant, TrimericSARS-CoV-2 Receptor Binding Domain Derivative. ACS Infect Dis. 2021 Aug13;7(8):2546-2564. doi: 10.1021 / acsinfecdis.1c00276. Epub 2021 Jul 14. PMID:34260218; PMCID: PMC8996237.]. Briefly, adherent HEK293T cells weretransiently transfected with plasmid DNA pHIV-1 NL4-3Δenv-Luc andSarbecoviruses spike plasmids, using the ProFection mammalian transfection kit(Cat# E1200, Promega Inc., Singapore) for pseudovirus production. The genesencoding Spike proteins from SARS CoV-2 VOCs were synthesized at GenScript(USA) while spike plasmids encoding Sarbecoviruses spikes were kindly gifted byDr. Pamela J. Bjorkman (Caltech, USA). The culture supernatant was harvested48 h post-transfection, filtered through a 0.22 μm filter, and stored at -80 °C.Adherent HEK293 cells expressing hACE-2 and TMPRSS2 receptors (BEIresources, NIH, Catalog No. NR-55293) were cultured in a growth mediumconsisting of DMEM with 5% Fetal Bovine Serum (Thermo Fisher) andpenicillin-streptomycin (100 U / mL). Mice serum samples were heat-inactivatedand then serially diluted in the growth medium, starting from 1:20 dilutions. In thenext step, the pseudotyped virus was incubated with the serially diluted sera in atotal volume of 100 μL for 1 h at 37 °C. The adherent cells were then trypsinized,and 1 x 104 cells / well were added to achieve a final volume of 200 μL / well. Theplates were further incubated for 48 h in a humidified CO2 incubator at 37 °C.After incubation, neutralization was measured as an indicator of luciferase activityin the cells (relative luminescence units) using Nano-Glo luciferase substrate (Cat# N1110, Promega). Luminescence was measured using a Cytation-5 multimodereader (Bio-Tech Inc., Oklahoma City, OK, USA). The luciferase activity,measured as relative luminescence units (RLU), in the absence of sera wasconsidered as 100% infection. The serum dilution resulting in half-maximalneutralization of the pseudovirus (ID50) relative to the no-serum control wasdetermined from neutralization curves.Biolayer Interferometry experiment

[0098] The long-term stability of lyophilized RBD cocktail the RBD cocktail(having SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18 without TPA signal,SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without TPA signal, andSEQ ID NO: 33 without TPA signal) was assessed through DSF and BLI bindingstudies. BLI measurements were made using ForteBio biosensors (Fortebio -Sartorius). All data collection were performed at 25°C using settings of StandardKinetics Acquisition rate at a sample plate shake speed of 1000 rpm. mAbs wereloaded onto Protein G sensors, subsequently they were dipped into 1x PBS bufferfor 60 seconds to obtain baseline and then dipped into wells containing RBDderivatives at different concentrations in 1X PBS to monitor antibody association.The dissociation step was monitored for 200 seconds by dipping Ab-bound sensorsinto buffer. Antigen specific binding responses were obtained by subtractingresponses of blank sensors tested in parallel with 1X kinetics buffer. The specificbinding responses were fitted using ForteBio Data Analysis 12.0 software tool.Statistical Analysis

[0099] The p values for ELISA binding and neutralization titres wereanalyzed with a two-tailed Mann-Whitney test using the GraphPad Prism software9.0.0 (* indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001). VOCpseudoviral neutralization titre data were analyzed with non-parametric Kruskal-Wallis with Dunn's multiple-comparison tests using the GraphPad Prism software9.0.0 (* indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001).Example 2: Thermal stabilization and yield enhancement by transfer ofstabilizing mutations to diverse Sarbecoviruses RBDs without affectingconformational integrity

[00100] SARS-CoV-1 shares 74% amino acid identity with SARS CoV-2RBD and is closely related to another clade 1a member, WIV-1 (96% identical)(Table 4). Table 4 depicts percentage amino acid identity of the polypeptide ofreceptor binding domain (RBD) of Sarbecoviruses from different clades with thatof SARS CoV-2.Table 4:

[00101] WIV-1 is found in horseshoe bats (Rhinolophus sinicus) in China; itsusage of ACE2 as the receptor highlights the potential for zoonotic transmissionto humans. RaTG13 exhibited high genetic similarity with SARS-CoV-2, it wasalso identified in horseshoe bats (Rhinolophus affinis) in Yunnan Province, China.Similar to SARS-CoV-2, RaTG13 is capable of utilizing the angiotensinconverting enzyme 2 (ACE2) receptor for cell entry. RmYNO2 is a recentlydiscovered clade-2 virus which contains two deletions in the RBD that prevents itfrom using ACE2. Another SARS-related CoV that was included by the inventors,is BtKY72 (clade-3) which was identified in Kenyan Rhinolophus bats and showshuman ACE2-dependent entry. As oligomerization of antigen results in enhancedimmunogenicity, the inventors expressed the wild type (WT) and stabilized (St)derivatives of these Sarbecoviruses RBDs in Expi-293F cells (Recombinant hostcell) as oligomers through genetic fusion of a disulfide linked oligomerizationmotif at the respective C termini. For comparison, monomeric WT and Stabilizedderivatives of SARS-CoV-1 RBD were also expressed and purified.

[00102] Purification of these mammalian cell expressed proteins wasperformed via Ni-affinity chromatography. A significant enhancement in the yield(~4-23-fold increase) of the RBD derivatives (Immunogenic polypeptides) wasobserved upon introduction of the stabilizing mutations. The effect of thesemutations on protein thermal stability was probed by assessing the apparentthermal melting temperature (Tm) of the WT and stabilized derivatives throughnano-DSF. A notable increase (~7°C) in the Tm of the stabilized derivativesrelative to the wild type RBDs was observed. To verify proper folding of theseRBD derivatives, the binding was examined with a selected panel of broadlyneutralizing antibodies (bNAbs) that bind to different epitopes on RBD by usingSPR. Due to the lack of conservation in class 1 and class 2 RBD epitopes, it isimprobable for antibodies within these categories to exhibit significant crossreactivity towards Sarbecoviruses RBDs. However, class 3 and class 4 RBDbinding antibodies present more promising opportunities for neutralization acrossvarious clades, thereby offering potential protection against emergingSarbecoviruses. The epitope of bnAb10-40 is similar to the previously defined'class 4' antibody epitope, it makes polar contacts and hydrophobic interactionswith RBD residues (377-385). ADG-20 binds to a class 1 / 4 epitope that overlapswith the ACE2 binding site. This bnAb binds to RBDs from clade 1a, 1b and 3;however, there was no binding observed for the clade 2 RmYNO2 RBD which alsodoes not bind ACE2. S2X259 targets the conserved antigenic site II within theRBD. It interacts with amino acid residues 369-386, 404-411 and 499-50836. Allthe RBD derivatives (i.e. SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 withoutTPA signal, and SEQ ID NO: 33 without TPA signal) except the stabilizedRmYNO2 oligomer bound to this bnAb, this could be due to loss of conservationof residue D405 and G504 in RmYNO2 RBD (i.e. SEQ ID NO: 28 without TPAsignal) which are crucial for interaction with S2X259. All the stabilized RBDderivatives also bound to class 4 bnAb CR3022.

[00103] To probe the thermal tolerance conferred by these mutations, the WTand St RBD derivatives were incubated at different temperatures ranging from 4°Cto 70°C for 2 hours. The proteins were cooled to room temperature and subjectedto DSF. The thermal melt curves clearly demonstrated the extended thermaltolerance of the stabilized derivatives relative to the corresponding WT RBDs(Figure 3). Collectively, these results illustrate that mutations are stabilizing in allthe Sarbecoviruses included in this study; they not only result in enhanced thermalstability and thermal tolerance but also increase the yield of the purified proteinssignificantly. Binding experiments confirm that the stabilized RBD derivatives areproperly folded.Example 3: Eliciting neutralizing antibody responses in mice against diverseSarbecoviruses using stabilized RBD derivatives and a correspondingcocktail formulation.

[00104] To assess the immunogenicity of the stabilized Sarbecoviruses RBDderivatives, female C57BL / 6 mice (n=5 / group) were immunized intramuscularlywith individual RBD derivatives (5mg of stabilized RBDs from SARS-CoV-1 andWIV-1 (clade 1a); SARS CoV-2 and RaTG13 (clade 1b); RmYNO2 (clade 2) andBtKY72 (clade 3), individually, (i.e. SEQ ID NO: 13 without TPA signal, SEQ IDNO: 18 without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28without TPA signal, and SEQ ID NO: 33 without TPA signal) adjuvanted withSWE (pharmaceutically acceptable carrier) in a prime-boost regimen.

[00105] The immunogenicity of a Sarbecoviruses RBD cocktail (Vaccinecomposition) having 5mg of each of the stabilized RBDs from SARS-CoV-1 andWIV-1 (clade 1a); SARS CoV-2 and RaTG13 (clade 1b); RmYNO2 (clade 2) andBtKY72 (clade 3) (i.e. SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 withoutTPA signal, and SEQ ID NO: 33 without TPA signal) formulated with SWE as theadjuvant was evaluated.

[00106] Post boost immunization, ELISA against respective RBDs was doneto determine the serum IgG titres (Figure 4). High antibody titres were observedupon immunization with individual RBD derivatives, as well as with the RBDcocktail. In the context of SARS CoV-1 immunogens, enhancement in theimmunogenicity of the monomeric RBD derivative upon stabilization; however,in trimeric format this difference was insignificant. The stabilized monomericSARS CoV-1 RBD derivative elicited higher neutralizing antibody (NAb) titresagainst homologous (SARS-CoV-1, WIV-1) and heterologous (LYRa3, SHC014)clade1a pseudoviruses related to WT RBD. In contrast, the WT and mutanttrimeric derivatives demonstrated comparable neutralizing titres. The RBDcocktail elicited good neutralization against homologous SARS CoV-1, WIV-1(clade 1a); SARS CoV-2 B.1 (clade 1b) and BtKY72 (clade 3) and heterologouspseudoviruses (clade1a: LYRa3, SHC014; clade3: Khosta-2) (Figure 5).Importantly with a single RBD immunization, clade matched pseudoviruses wereeffectively neutralized but no cross neutralization of mismatched pseudoviruseswas observed. This emphasizes the need for incorporating RBDs from all theclades to achieve a broadly neutralizing pan-sarbecoviruses response.Unfortunately, the neutralization titres against clade 2 viruses could not beassessed as they do not bind ACE2 and thus a suitable neutralization assay was notavailable. Overall, the RBD cocktail formulation elicited good neutralizing titresagainst all the RBDs incorporated in the formulation confirming theimmunogenicity of the RBD derivatives in individual as well as cocktail format.It also elicited neutralization against all the heterologous pseudoviruses tested,indicating elicitation of a broad neutralization response in the immunized animals.Example 4: Induction of an immunogenic response in pre-immunized miceusing stabilized RBD cocktail (vaccine composition).

[00107] The inventors aimed to deduce whether the RBD cocktail formulation(vaccine composition) could induce comparable responses in pre-immunizedanimals and naive mice. Female BALB / c mice (n=5 / group) were immunized withSARS CoV-2 B.1 RBD (5mg) followed by two boost immunizations with thestabilized RBD cocktail formulation (30mg). Serum ELISA demonstrated thepresence of IgG titres against Sarbecoviruses RBDs after boost 1, boost 2 did notresult in further increase in antibody titres. However, with sera from the first boost,only weak and sporadic neutralization of homologous as well as heterologouspseudovirus was observed. Consistent, broad neutralization was observed onlyafter the second boost of the RBD cocktail. Consistent with the previous results innaive mice; the pan-Sarbecoviruses formulation was able to elicit broadneutralizing responses against Sarbecoviruses after two immunizations in preimmunized mice, confirming its relevance in the current scenario.Example 5: Analysing antigenicity retention of the lyophilized RBD cocktail(vaccine composition).

[00108] To assess the long-term stability of the RBD cocktail formulation(having SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18 without TPA signal,SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without TPA signal, andSEQ ID NO: 33 without TPA signal), it was subjected to lyophilization andincubated at either 4 or 37°C for a duration of one month. The stability of theformulations were confirmed through DSF and BLI experiments. As depicted inFigure 7, the lyophilized formulation stored at both 4 and 37°C exhibited similarmelting profiles up until day 15. However, a slight reduction in stability wasobserved on day 30. Additionally, the conformational integrity of the RBDcocktail was assessed by examining binding with the previously described bNAbs(CR3022, 10-40, ADG-20, S2X259). Consistent with the DSF analysis, thelyophilized formulation demonstrated appropriate binding curves with all theantibodies until day 15, with a slight decrease in binding signal observed on day30. These findings illustrate the long-term stability of the cocktail formulationwhen in a lyophilized state, an important factor to consider when evaluatingpotential vaccine candidate formulation.Results and Conclusion

[00109] The results demonstrate that the stabilizing mutations are effectivefor clade members: SARS CoV-1, WIV-1 (clade 1a); SARS CoV-2 B.1 (clade 1b)and BtKY72 (clade 3) and heterologous pseudoviruses (clade1a: LYRa3, SHC014;clade3: Khosta-2). The inventors have expressed the stabilized trimeric RBDderivatives (as depicted in SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 withoutTPA signal, and SEQ ID NO: 33 without TPA signal), in mammalian cells(recombinant host cell) and reported good purification yields. The stabilized RBDderivatives (as depicted in SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18without TPA signal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 withoutTPA signal, and SEQ ID NO: 33 without TPA signal) showed significantenhancement in the apparent melting temperature and short-term thermal tolerancethan WT RBDs. All the stabilized RBDs resulted in high IgG titres in mice inindividual and cocktail format and thus proved to be highly immunogenic.Stabilized trimeric RBD derivatives as depicted in SEQ ID NO: 15, SEQ ID NO:20, SEQ ID NO: 25, SEQ ID NO: 30, and SEQ ID NO: 35 would also achievesimilar results. The RBD cocktail formulation (having immunogenic polypeptidesas depicted in SEQ ID NO: 13 without TPA signal, SEQ ID NO: 18 without TPAsignal, SEQ ID NO: 23 without TPA signal, SEQ ID NO: 28 without TPA signal,and SEQ ID NO: 33 without TPA signal) performed comparable to the individualclade-matched immunogens and better in case of heterologous pseudoviralneutralization. Overall, the absence of cross-reactivity within clades appears to beevident based on the findings from pseudoviral neutralization assays followingvaccination with individual RBD derivatives, underscoring the significance ofexposure to a variety of antigens in order to develop a broad response, such as thecocktail formulation that the present inventors have employed. It was observedthat in addition to naive mice, the RBD cocktail formulation (vaccine composition)of the present disclosure demonstrates immunogenicity in SARS CoV-2 RBD preimmunized mice as well, mirroring the present situation after COVID-19vaccination drive. Further, the presently disclosed RBD cocktail (vaccinecomposition) exhibited remarkable thermal stability following lyophilization for aperiod of upto two weeks when stored at 37°C. In summary, the results show thatstabilization and production of diverse Sarbecoviruses RBD derivatives to be usedas vaccine antigens is a technically feasible and scalable process. The significantimprovement in yield and thermostability of immunogens by transfer of identifiedstabilizing mutations as described, would enhance the manufacturing efficiencyand distribution of RBD-based vaccines for Sarbecoviruses on a global scale,addressing the pressing need for quick, widespread and equitable vaccination.Example 6: Protective efficacy of Stabilized RBD trimer and RBD-S2monomer cocktail formulation (vaccine composition).

[00110] K18-hACE2 mice were immunized with SWE adjuvanted StabilizedRBD oligomer cocktail (1:1 v / v), and SWE adjuvanted RBD-S2 monomercocktail.

[00111] The stabilized RBD oligomer cocktail included WIV-1 (SEQ IDNO. 55), RaTG13 (SEQ ID NO. 56), RmYNO2 (SEQ ID NO. 57), BtKY72 (SEQID NO. 58); 2μg / antigen; total=8μg / mouse. Stabilized RBD-S2 monomer cocktailincluded WIV-1 (SEQ ID NO. 61), RaTG13 (SEQ ID NO. 63), RmYNO2 (SEQID NO. 65), BtKY72 (SEQ ID NO. 67); 2μg / antigen; total=8μg / mouse).

[00112]

[00102] To compare the protective efficacy of the stabilizedRBD oligomer cocktail and Stabilized RBD-S2 monomer cocktail; K18-hACE2mice (n=5 / group) were immunized intramuscularly with the cocktail formulations(8μg / mouse) adjuvanted with SWE in a prime-boost regimen. These mice werechallenged with heterologous SARS-CoV-1 and SARS-CoV-2 BA.5 viruses postboost immunization.

[00113] Figure 6 illustrates efficacy of stabilized RBD oligomer cocktail andStabilized RBD-S2 monomer cocktail, wherein A) is a schematic representationof the immunization protocol, and (B) depicts body weight change and lung viraltitres in mice post SARS-CoV-1 challenge and (C) depicts body weight changeand lung viral titres in mice post SARS-CoV-2 BA.5 challenge. Stabilized RBDS2 monomer cocktail showed enhanced protective efficacy against heterologousclade 1a and clade1b Sarbecoviruses.

Claims

1. An immunogenic polypeptide comprising a polypeptide selected from the group consisting of: a. a polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence selected from SEQ ID NO: 1 or SEQ ID NO: 59, wherein the polypeptide comprises substitution mutations Y34W and P195L; b. a polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as selected from SEQ ID NO: 3 or SEQ ID NO: 61, wherein the polypeptide comprises substitution mutations Y34W and P195L; c. a polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence selected from SEQ ID NO: 5 or SEQ ID NO: 63, wherein the polypeptide comprises substitution mutations A17P, Y34W, and P196L; d. a polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence selected from SEQ ID NO: 7 or SEQ ID NO: 65, wherein the polypeptide comprises substitution mutations Y34W and P177L; e. a polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence selected from SEQ ID NO: 9 or SEQ ID NO: 67, wherein the polypeptide comprises substitution mutations Y34W and P195L; and f. a polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence selected from SEQ ID NO: 85 or SEQ ID NO: 69, wherein the polypeptide comprises substitution mutations A17P, Y34W, and P196L.

2. The immunogenic polypeptide as claimed in claim 1, wherein the polypeptide is having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, and SEQ ID NO: 69.

3. The immunogenic polypeptide as claimed in claim 1, wherein the polypeptide further comprises a peptide selected from TPA signal peptide; oligomerization domain; HRV3C protease cleavage site or a portion thereof; His tag; one or more linkers; or combinations thereof.

4. The immunogenic polypeptide as claimed in claim 3, wherein the oligomerization domain has an amino acid sequence as set forth in SEQ ID NO. 38.

5. The immunogenic polypeptide as claimed in claim 3, wherein the TPA signal peptide is having an amino acid sequence as set forth in SEQ ID NO. 36, and wherein the HRV3C protease cleavage site is having an amino acid sequence as set forth in SEQ ID NO. 40.

6. The immunogenic polypeptide as claimed in claim 3, wherein the linker has an amino acid sequence selected from "AAS", "S", "AS", "GT","GSAGS", and / or "GS".

7. The immunogenic polypeptide as claimed in claim 3, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81.

8. A polynucleotide encoding the immunogenic polypeptide as claimed in any one of claims 1 to 7.

9. The polynucleotide as claimed in claim 8, wherein the polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO: 82.

10. The polynucleotide as claimed in claim 8, wherein the polynucleotide is selected from DNA, RNA, or mRNA.

11. A recombinant vector containing the polynucleotide as claimed in any one of claims 8 to 10 operably linked to a promoter.

12. A recombinant host cell comprising the recombinant vector as claimed in claim 11.

13. The recombinant host cell as claimed in claim 12, wherein the host cell is a bacterial cell, yeast cell, insect cell, and mammalian cell.

14. The recombinant host cell as claimed in claim 12, 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 ExpiSf9, Sf9, High Five , Sf21, and S2, and wherein the mammalian cell is selected from the group consisting of Expi293FExpi-CHO-S, CHO-K1, CHO-S, HEK293F , CHOBC, SLIM , SPOT , SP2 / 0 , Sp2 / 0- Ag14, CHO DG44, HEK 293S, HEK 293 Gnt1- / - ,HEK293-EBNA1, CHOL-NSO, and NSO.

15. A vaccine composition comprising the immunogenic polypeptide as claimed in anyone of the claims 1-7, and a pharmaceutically acceptable carrier.

16. The vaccine composition as claimed in claim 15, wherein the vaccine composition is a cocktail formulation comprising a combination of at least 2, at least 3, at least 4, or 5 immunogenic polypeptides of amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 81.

17. The vaccine composition as claimed in claim 15, wherein the pharmaceutically acceptable carrier is an adjuvant selected from an oil-in-water adjuvant, a polymer and water adjuvant, a water-in-oil adjuvant, an aluminum hydroxide adjuvant, and combinations thereof.

18. A method for producing the vaccine composition as claimed in claim 15, said method comprising: (a) culturing the recombinant host cell as claimed in any one of claims 12 to 14 to express the immunogenic polypeptide as claimed in any one of the claims 1-7; (b) subjecting the immunogenic polypeptide to purification; and (c) contacting the immunogenic polypeptide of step (b) with a pharmaceutically acceptable carrier, to obtain the vaccine composition.

19. A method of eliciting an immune response against Sarbecoviruses in a subject, the method comprising administering the subject with an effective amount of the vaccine composition as claimed in any one of claims 15-18.

20. The method as claimed in claim 19, wherein the Sarbecovirus is selected from clade 1a, clade 1b, clade 2, or clade 3 virus.

21. The method as claimed in claim 19, wherein the Sarbecovirus is selected from the group consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade 1a), SARSCoV-2(clade 1b), RaTG13 (clade 1b), RmYNO2 (clade 2), and BtKY72 (clade 3).

22. The method as claimed in claim 19, wherein the vaccine composition is administered by a mode selected from the group consisting of intranasal, subcutaneous, intravenous, intra-arterial, intra-peritoneal, intramuscular, intradermal, oral, dermal, and buccal.

23. A kit comprising the immunogenic polypeptide as claimed in any one of the claims 1-7; or the vaccine composition as claimed in anyone of the claims 15-18, and an instruction leaflet.

24. The method as claimed in claim 19, wherein the Sarbecovirus is selected from the group consisting of SARS-CoV-1 (clade 1a), WIV-1 (clade 1a), SARSCoV-2(clade 1b), RaTG13 (clade 1b), RmYNO2 (clade 2), BtKY72 (clade 3), Pangolin_GD, RaTG13 (MN996532), Pangolin_GX-P2V (EPI_ISL_410542), SARS-CoV-1_Urbani_HP03 (AY278741), SARS-CoV-1_BJ02_HP03M (AY278487), SARS-CoV-1_HGZ8L1-A_HP03E (AY394981), SARS-CoV1_GD01_HP03L (AY278489), SARS-CoV-1_GZ-C_HP03L (AY394979), SARS-CoV-1_Sino1-11_HP03L (AY485277), SARS-CoV-1_Sin852_HP03L (AY559082), SARS-CoV-1_SZ3_PC03 (AY304486), SARS-CoV1_SZ13_PC03 (AY304487), SARS-CoV-1_SZ1_PC03 (AY304489), SARSCoV-1_GD03T0013_HP04 (AY525636), SARS-CoV-1_GZ0402_HP04 (AY613947), SARS-CoV-1_PC4-127_PC04 (AY613951), SARS-CoV-1_PC4- 137_PC04 (AY627045), SARS-CoV-1_PC4-13_PC04 (AY613948), WIV16 (KT444582), WIV1 (KF367457), Rs7327 (KY417151), LYRa11 (KF569996), Rs4231 (KY417146), RsSHC014 (KC881005), Rs4084 (KY417144), BM48-31 (NC014470), BtKY72 (KY352407), ZXC21 (MG772934), ZC45 (MG772933), JL2012 (KJ473811), Rf1 (DQ412042), HeB2013 (KJ473812), 273-2005 (DQ648856), Rf4092 (KY417145), YN2013 (KJ473816), RmYNO2 (EPI_ISL_412977), As6526 (KY417142), Rs4237 (KY417147), Rs4081 (KY417143), Rp3 (DQ071615), 279-2005 (DQ648857), Shaanxi2011 (JX993987), Yunnan2011 (JX993988), Rs4247 (KY417148), HKU3-13 (GQ153548), HKU3-1 (DQ022305), GX2013 (KJ473815), Longquan-140 (KF294457), HKU3-8 (GQ153543), HuB2013 (KJ473814), HpBCoV_Zhejiang_2013 (KF636752), HKU1 (KF686346), OC43 (KX344031), MERS-CoV (NC_019843), Bat-CoV-GCCDC1 (MT350598), RousettusCoV_HKU9 (MG762674), Rc-o319 (LC556375), RacCS203 (MW251308), RshSTT182 (EPI_ISL_852604), PDF-2370, PRD-0038, RsYN04 (EPI_ISL_1699444), BB9904 (KR559017), Khosta-1 (MZ190137), Khosta-2 (MZ190138), and RhGB01 (MW719567).

25. The recombinant host cell as claimed in claim 12, wherein the host cell is selected from the group consisting of OPENPichia (NCYC 2543 hoc1tr), OPENPichia his4, NCYC 2543 type strain, OPENPichia pep4, OPENPichia yps1, OPENPichia pep4 yps1, and OPENPichia mutS strains.

26. The vaccine composition as claimed in claim 15, wherein the vaccine composition is a lyophilized composition.