Hemagglutinin based immunogenic polypeptides, compositions and methods thereof

Abstract

The present disclosure relates to immunogenic polypeptides for eliciting an immune response against influenza. These polypeptides are suitable for use in combination with hemagglutinin (HA)-containing influenza vaccine formulations. Also disclosed are polynucleotides encoding the polypeptides, DNA constructs, recombinant vectors, and recombinant bacterial host cells for expression. The disclosure further provides vaccine compositions comprising the immunogenic polypeptides and pharmaceutically acceptable carriers. Methods for producing the vaccine composition and eliciting an immune response against influenza infection in a subject are also described.

Description

[0001] HEMAGGLUTININ BASED IMMUNOGENIC POLYPEPTIDES, COMPOSITIONS AND METHODS THEREOF

[0002] FIELD OF INVENTION

[0003]

[0001] The present disclosure relates to novel hemagglutinin (HA)-based immunogens. Further, the present disclosure discloses immunogenic polypeptides, and vaccine compositions for eliciting immune response against influenza infection. It further relates to a method for eliciting an immune response against influenza infection in a subject.

[0004] BACKGROUND OF INVENTION

[0005]

[0002] The influenza virus, a versatile human pathogen from the Orthomyxoviridae family, is an enveloped virus that causes widespread respiratory illness (Girard et al., 2005). The infection typically presents various symptoms, including a rapid onset of high fever, muscle pain, dry cough, sore throat, and nasal irritation. There are around a billion cases of seasonal influenza annually, including 3-5 million cases of severe illness.

[0006]

[0003] Influenza viruses are classified into four types: A, B, C, and D, with Influenza A viruses (IAV) being particularly important due to their role in seasonal epidemics. The classification of lAVs is based on variations in these glycoproteins, which determine the unique subtypes and specific strains of the virus. While all subtypes are present in birds, H1N1 and H3N2 are the predominant strains in humans.

[0007]

[0004] Antibodies neutralising the influenza virus primarily target hemagglutinin (HA), a trimeric glycoprotein embedded in the viral envelope. HA plays a crucial role in the virus's infection process. HA has two main structural regions: a large head domain and a smaller stem domain. The protein is anchored to the viral membrane by a C-terminal sequence attached to the stem domain. After translation, HA undergoes a specific cleavage within a loop region, producing two polypeptides: HA1, which largely forms the head domain, and HA2, which predominantly constitutes the stem region. The uncleaved precursor protein is known as HAO.

[0008]

[0005] The need to update the seasonal influenza vaccine annually arises from the high variability of the virus. This variability is especially pronounced in the HA molecule, specifically in its head domain, where antigenic drift and shift give rise to numerous variants. The head domain is immunodominant, meaning it is the primary target for most neutralizing antibodies, blocking the virus's ability to bind to host cell receptors. However, due to the significant variation in this region, immunity developed from exposure to one strain does not protect against other strains. The antibodies generated during the initial infection are typically effective only against strains closely related to the virus encountered first. However, targeting the HA stem has been challenging, as the immunodominant HA head domain often directs the immune response away from the conserved stem. The fusogenic HA-stem is meta-stable at neutral pH (Bullough et al., 1994). Early efforts to express the HA2-subunit in isolation at neutral pH led to the formation of the stable, extended coiled- coil structure characteristic of the low pH conformation of HA (Chen et al., 1995), which does not elicit a functional or protective immune response. Hence, a need remains to design a universal vaccine against multiple influenza strains.

[0009] SUMMARY OF THE INVENTION

[0010]

[0006] In an aspect of the present disclosure, there is provided an immunogenic polypeptide that has amino acid sequence of at least 95% to 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NO: 51.

[0011]

[0007] In another aspect of the present disclosure, there is provided an immunogenic polypeptide in combination with any other HA containing influenza vaccine formulation.

[0012]

[0008] In an aspect of the present disclosure, there is provided a polynucleotide encoding the immunogenic polypeptide.

[0013]

[0009] In an aspect of the present disclosure, there is provided a DNA construct comprising the polynucleotide encoding the immunogenic polypeptide.

[0014]

[0010] In an aspect of the present disclosure, there is provided a recombinant vector containing the polynucleotide encoding the immunogenic polypeptide.

[0015] [OH] In an aspect of the present disclosure, there is provided a recombinant bacterial host cell containing the recombinant vector.

[0016]

[0012] In an aspect of the present disclosure, there is provided a vaccine composition comprising the immunogenic polypeptide, and a pharmaceutically acceptable carrier.

[0017]

[0013] In an aspect of the present disclosure, there is provided a method for producing the vaccine composition, wherein the method comprises: (a) culturing the recombinant bacterial host cell under suitable conditions to obtain the immunogenic polypeptide; (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.

[0018]

[0014] In an aspect of the present disclosure, there is provided a method for eliciting an immune response against influenza infection in a subject, the method comprising administering the subject with an effective amount of the vaccine composition.

[0019]

[0015] 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.

[0020] BRIEF DESCRIPTION OF DRAWINGS

[0021]

[0016] 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.

[0022]

[0017] Figure 1 depicts schematic representation of immunogenic polypeptides which are hemagglutinin (HA) Stem based immunogen Designs, having HA stem fragments from influenza subtypes Hl, H3, H5 and B. These designs show the arrangement of key regions (i.e. Fl, F2 and F3 fragments) and linkers that stabilize the conserved HA stem while removing the variable head, in accordance with the embodiments herein.

[0023]

[0018] Figure 2 illustrates results of SDS-PAGE analysis, wherein (A) represents the SDS-PAGE analysis results of purified immunogenic polypeptide (Left panel): MW marker in Lane 1, 10 pg of H3HA2.1 N22. WB analysis (Right panel): MW marker in Lane 1, 10 pg of H3HA2.1 N22 probed with 1 :250 dilution of Anti-H3 virus goat sera; (B) represents SDS-PAGE analysis of purified immunogenic polypeptide (Left panel): MW marker in Lane 1, 10 pg of H3HA2.1_N22_vl. WB analysis (Right panel): MW marker in Lane 1, 10 pg of H3HA2.1_N22_vl probed with 1 :250 dilution of Anti-H3 virus goat sera; and (C) represents SDS-PAGE analysis of purified immunogenic polypeptides (Left panel): MW marker in Lane 1, 10 pg of HlHA2.1_N22_v2. WB analysis (Right panel): MW marker in Lane 1, 10 pg of HlHA2.1_N22_v2 probed with 1 :250 dilution of CR9114, in accordance with the embodiments herein.

[0019] Figure 3 shows characterization data for H3HA2.1_N22, wherein (A) represents CD spectra; and B represents fluorescence spectra, for refolded and purified immunogenic polypeptide, H3HA2.1 N22, in accordance with the embodiments herein.

[0024]

[0020] Figure 4 illustrates the results of immunization study, wherein (A) represents immunization schedule; (B) represents comparison of ELISA endpoint titers of MynfluOOl with Mynflu002 using Hl, H3, HV, HY delCD (MynfluOOl components) and H3HA2.1 N22 as coating antigens, in accordance with the embodiments herein.

[0025]

[0021] Figure 5 depicts HAI titers against matched (Hl, H3, HV, and HY) viruses, wherein (A) illustrates HAI titers for MynfluOOl immunized groups and (B) illustrates HAI titers for Mynflu002 immunized groups and (C) represents comparison of HAI titers of MynfluOOl with Mynflu002 immunized groups against matched (Hl, H3, HV, HY) and mismatched challenge viruses (X31, this is a chimeric virus that harbors HA and NA from A / Aichi / 2 / 1968 (H3N2), while having the internal genes of PR8), in accordance with the embodiments herein.

[0026]

[0022] Figure 6 illustrates MNT titers of MynfluOO 1 and Mynflu002 immunized groups for antigen matched virus strains (Hl, H3, HV, HY), in accordance with the embodiments herein.

[0027]

[0023] Figure 7 illustrates mice immunization studies, wherein (A) represents average body weight reduction post 10MLD50 X31 challenge; and (B) represents survival (%) post 10MLD50 X31 challenge, in accordance with the embodiments herein.

[0028]

[0024] Figure 8 illustrates that the designed immunogenic polypeptides were purified from the insoluble fraction of the cell-culture lysate wherein (A) represents the gel image: ImM IPTG was used to induce the cells; 1 ml of the induced whole cell culture (lane 1) was removed for SDS-PAGE; and the remaining cells were lysed by sonication; Cell pellets (lane 2) and supernatant (lane 3) were separated by centrifugation and lane 4 is the marker. The arrow indicates the expected band of each construct, which is present in the insoluble fraction of the cell lysate; (B) represents nano-DSF equilibrium thermal unfolding wherein the normalized first derivative of the fluorescence intensity ratio (350 nm / 330 nm) is plotted as a function of temperature (°C); and (C) represents binding of BHA21F to CR9114 in a concentration series 100 nM, 500 nM, 1000 nM, and 2000 nM, in accordance with the embodiments herein.

[0029]

[0025] Figure 9 illustrates the results of immunization studies, wherein (A) represents schematic representation of immunization schedule; (B) represents prime immunization (day 0) and boost immunization (day 21), serum titers were obtained using ELISA Endpoint titers against BHA21F immunogenic polypeptide: A two-tailed Student’s t-test was performed for pairwise ELISA endpoint titer comparison (** indicates p < 0.01). Mice (n = 6 / group) immunized at day 0 (prime) and day 21 (boose) with SWE-adjuvanted immunogenic polypeptides, 21 days post-boost, animals were challenged intranasally with 10 MLD50 of the B / Brisbane / 60 / 2008 virus; and further post challenge up to 14 days (C) survival and (D) percentage weight change was monitored and for control, SWE-adjuvant- treated mice were used, in accordance with the embodiments herein

[0030]

[0026] Figure 10 depicts characterization of HlHA2.1_N22_v2, wherein (A) represents intrinsic tryptophan fluorescence spectra, for refolded and purified immunogenic polypeptide; and (B) represents binding kinetics of HlHA2.1_N22_v2 to the broadly neutralizing antibody CR9114, in accordance with the embodiments herein.

[0031]

[0027] Figure 11 depicts characterization of H3HA2.1_N22_vl, wherein (A) represents intrinsic Tryptophan fluorescence spectra, for refolded and purified immunogenic polypeptide, H3HA2.1_N22_vl; and (B) represents binding kinetics of H3HA2.1_N22_vl to the broadly neutralizing antibody CR9114, in accordance with the embodiments herein.

[0032]

[0028] Figure 12 depicts results of immunization and challenge studies in mice, wherein (A) is a schematic representation of immunization schedule; (B) represents boost sera HAI titers against matched seasonal strains and challenge virus shown for each vaccine group; and (C) represents average body weight loss, in accordance with the embodiments herein.

[0033]

[0029] Figure 13 depicts results of expression, characterization, and immunization and challenge studies for H5HA2.1_vl, wherein (A) represents SDS-PAGE analysis results- left panel comprising MW marker in Lane 1, 10 pg of H5HA2.1_vl. Right panel comprising: MW marker in Lane 1, 10 pg of H5HA2.1_vl probed with 1 :20000 dilution of Anti-H5 stem mice sera; and (B) represents intrinsic Tryptophan fluorescence spectra, for refolded and purified immunogenic polypeptide, H5HA2. l_vl, in accordance with the embodiments herein.

[0034]

[0030] Figure 14 shows data for characterization of H5HA2.1_vl binding to conformation-specific broadly neutralizing antibodies; wherein (A) shows data for CR6261, and (B) shows data for CR9114, using Surface Plasmon Resonance (SPR), in accordance with the embodiments herein.

[0035]

[0031] Figure 15 depicts data for assign stability of H5HA2.1_vl across broad spectrum of lyophilization buffer conditions, wherein (A) represents protein recovery postlyophilization and reconstitution, as measured by a BCA assay; (B) represents SDS-PAGE and Western blot analysis of liquid and lyophilized samples, probed with specific mouse sera, and (D) represents ELISA results using antigen-specific sera for both liquid and lyophilized proteins, in accordance with the embodiments herein.

[0036]

[0032] Figure 16 depicts results of immunization and challenge studies in mice, wherein (A) represents schematic representation of immunization schedule; (B) represents boost sera HAI titers against matched seasonal strains and challenge virus are shown for each vaccine group, Dotted lines indicate the HAI titer of 40; and (C) represents average body weight loss, in accordance with the embodiments herein.

[0037] DESCRIPTION OF THE INVENTION

[0038]

[0033] 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.

[0039] Definitions

[0040]

[0034] 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 by a person of skill in the art. The terms used 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.

[0041]

[0035] 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 objects of the article.

[0042]

[0036] Throughout this specification, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” are used in the inclusive, open sense and will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. It is not intended to be construed as “consists of only”.

[0043]

[0037] The term “including”, as used herein, means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0038] The term "at least one" is used to mean one or more and thus includes individual components as well as mixtures / combinations.

[0044]

[0039] The term “pharmaceutically acceptable carrier” refers to substance such as a carrier, excipient, adjuvant, etc. known to a person skilled in the art, which can be used for preparing vaccines. The term “pharmaceutically effective amount” refers to an amount that is capable of eliciting an immune response.

[0045]

[0040] As used herein, the term “adjuvant” refers to a compound that, when used in combination with an immunogen, augments or otherwise alters or modifies the immune response induced against the immunogen. Modification of the immune response may include intensification or broadening the specificity of either or both antibody and cellular immune responses.

[0046]

[0041] As used herein, the term “excipient” refers to the component that is known to a person skilled in the art that can be added as a vehicle alongside an immunogenic polypeptide in an immunogenic composition.

[0047]

[0042] The term “immunogen”, as used herein, refers to a substance capable of eliciting an immune response. The substance includes polypeptide fragment(s) capable of inducing the immune response including production of antibody, activation of immune cells, etc, in a subject.

[0048]

[0043] The term “immunogenicity”, as used herein, refers to the ability of the immunogen to elicit an immune response.

[0049]

[0044] The term “immunogenic composition” refers to a composition comprising the polypeptide fragment along with a pharmaceutically acceptable carrier that elicits a prophylactic or therapeutic immune response in a subj ect. In the present disclosure, the terms “immunogenic composition”, “vaccine composition”, and “vaccine” are used interchangeably. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition, infection, disease, etc.

[0050]

[0045] 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.

[0051]

[0046] The term “polynucleotide” refers to a molecule having a combination of nucleotide monomers which are connected to each other through covalent bonds. The term includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and mRNA (messenger RNA). Accordingly, the polynucleotide according to embodiments herein may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and mRNA (messenger RNA).

[0052]

[0047] 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.

[0053]

[0048] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0054]

[0049] Within each group of influenza virus, the stalk domain of hemagglutinin (HA) shares antigenic similarity, and varying combinations of HA and neuraminidase (NA) lead to different Influenza A virus subtypes. Conversely, Influenza B (IBV) and C (ICV) viruses are not classified into subtypes and are largely confined to humans. Influenza B viruses have recently diverged into two distinct lineages: B / Victoria-like and B / Yamagata-like, both of which circulate among humans, though B / Yamagata seems to have recently gone extinct (Ferguson et al., 2003; Koutsakos et al., 2021). Influenza A and B viruses are prevalent in human populations and are the primary cause of seasonal epidemics. In contrast, Influenza C viruses typically result in mild infections in children and are thus considered less clinically significant. Influenza D viruses predominantly affect cattle and occasionally other animals, with no recorded cases of human infection (Krammer et al., 2018).

[0055]

[0050] The subdominant HA stem domain is a target for developing a 'universal' flu vaccine due to its more conserved antigenic regions across different HA subtypes, compared to the highly variable antigenic sites in the globular head domain of HA (Ellebedy & Ahmed, 2012; Pica & Palese, 2013). The reduced surface accessibility and structural limitations needed to maintain fusion competence contribute to the significant residue conservation within the HA-stem (Harris et al., 2013). However, targeting the HA stem has been challenging, as the immunodominant HA head domain often directs the immune response away from the conserved stem. The fusogenic HA-stem is meta-stable at neutral pH (Bullough et al., 1994). Early efforts to express the HA2-subunit in isolation at neutral pH led to the formation of the stable, extended coiled-coil structure characteristic of the low pH conformation of HA (Chen et al., 1995), which does not elicit a functional or protective immune response. Embodiments herein provide novel designs of HA stems and demonstrate the additional benefit of adding HA stem components to the existing influenza vaccine formulations. The present inventors have developed new HA stem-based designs from mutated HA stem polypeptides derived from Hl, H3, and H5 influenza A, and from influenza B strains, to present conserved epitopes from these subtypes. The designed polypeptides are intended to develop a universal, epitope-based vaccine that provides broad protection against multiple influenza strains.

[0056]

[0051] The present disclosure provides immunogenic polypeptides (also interchangeably referred to herein as “immunogens”) comprising mutant polypeptides derived from HA stem domain of Influenza viruses. The immunogenic polypeptides, according to embodiments herein, are engineered to enhance cross-reactive antibody responses, offering broader protection against divergent influenza strains. By preserving the structural integrity of the HA protein while improving its immunogenicity, the present disclosure provides a solution for developing a more effective, universal influenza vaccine. Further, combining the immunogenic polypeptides, of the present disclosure, with existing influenza vaccines provides an added layer of protection, potentially enhancing the breadth of immune responses against a wider range of influenza strains.

[0057]

[0052] Embodiments herein provide immunogenic polypeptides and compositions capable of eliciting an immune response against influenza infection in a subject. In an embodiment, the immunogenic polypeptide comprises a first polypeptide fragment (Fl) linked to a second polypeptide fragment (F2) and the second polypeptide fragment linked to a third polypeptide fragment (F3). The fragments may be linked with or without a linker. In an example, Fl may be linked to F2 by a linker LI, and F2 may be linked to F3 by way of a linker L2. In another example, Fl may be linked to F2 by a linker LI, and F2 may directly be linked to F3 without a linker.

[0058]

[0053] In some embodiments, the immunogenic polypeptide comprises a first polypeptide fragment (Fl) linked to a second polypeptide fragment (F2) by a linker LI, and the second polypeptide fragment linked to a third polypeptide fragment (F3) by a linker L2. In other embodiments, the immunogenic polypeptide comprises a first polypeptide fragment (Fl) linked to a second polypeptide fragment (F2) by a linker LI and the second polypeptide fragment linked to a third polypeptide fragment (F3) without a linker.

[0054] Accordingly, the immunogenic polypeptide as described herein may be in one or more configurations. In some embodiments, the immunogenic polypeptide has a formula of Formula I:

[0059] F1-L1-F2-L2-F3 ... .Formula I.

[0060]

[0055] In other embodiments, the immunogenic polypeptide has a formula of Formula II:

[0061] Fl-Ll-F2-F3-L2-oligomerization domain . . . .Formula II

[0062]

[0056] Accordingly, in an embodiment, the immunogenic polypeptide has a formula selected from the group consisting of a. F1-L1-F2-L2-F3 (Formula I); and b. Fl-Ll-F2-F3-L2-oligomerizati on domain (Formula II).

[0063]

[0057] In some embodiments, the immunogenic polypeptide comprising the first polypeptide fragment (Fl), second polypeptide fragment (F2) and the third polypeptide fragment (F3) achieve a linear stem configuration.

[0064]

[0058] The polypeptide fragments Fl, F2, and F3, incorporate several key substitution mutations which include: (i) amino acid substitution of unpaired cysteine (Cys) residues with serine (Ser) to prevent unwanted disulfide bond formation; (ii) amino acid substitutions destabilizing low-pH conformations, and preventing premature structural changes that could impact vaccine efficacy; and (iii) amino acid substitutions of hydrophobic amino acid residues that are newly exposed in the stem constructs after comparison with full-length HA, with polar residues to improve stability and stabilize the stem in a conformation that preserves known neutralization epitope(s).

[0065]

[0059] The substitution mutations (interchangeably referred to as “substitution” or “mutation”), according to embodiments herein, are also described herein by use of the following notations: “amino acid residue substituted: amino acid position in the sequence: amino acid residue substitute”, or “amino acid position in the sequence: amino acid residue substitute”. For example, the notation “11 IP” indicates an amino acid mutation with proline (P), at position 111 in a sequence. Similarly, an amino acid substitution where, for instance, if phenylalanine (F) is substituted with proline (P), at position 111 in a sequence, it may be indicated as “Fl 1 IP”. Such notations are generally known to a person skilled in the art and generally used in representing amino acid mutations / substitutions in a given 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”. 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.

[0066]

[0060] The first polypeptide fragment (Fl), according to embodiments herein, may be selected from a group consisting of (a) a polypeptide corresponding to amino acid position 1-35 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 7; (b) a polypeptide corresponding to amino acid position 1-37 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 7; (c) a polypeptide corresponding to amino acid position 1-45 of a hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at position A19S of the hemagglutinin polypeptide; and (d) a polypeptide corresponding to amino acid position 1-36 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11.

[0067]

[0061] In an embodiment, the first polypeptide fragment (Fl) is selected from a group consisting of: (a) a polypeptide corresponding to amino acid position 1-35 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 7; (b) a polypeptide corresponding to amino acid position 1-37 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 7; (c) a polypeptide corresponding to amino acid position 1-45 of a hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at position A19S of SEQ ID NO: 3 or SEQ ID NO: 5; and (d) a polypeptide corresponding to amino acid position 1-36 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11.

[0068]

[0062] Accordingly, in some embodiments, Fl is a polypeptide having a sequence of at least 95% identity to a sequence selected from the group consisting of : a. a sequence corresponding to positions 1-35 of SEQ ID NO: 1 or SEQ ID NO: 7; b. a sequence corresponding to positions 1-37 of SEQ ID NO: 1 or SEQ ID NO: 7; c. a sequence corresponding to positions 1-45 of SEQ ID NO: 3 or SEQ ID NO: 5; and d. a sequence corresponding to positions 1-36 of SEQ ID NO: 9 or SEQ ID NO: 11,

[0069]

[0063] The second polypeptide fragment (F2), according to embodiments herein, may be selected from a group consisting of: (e) a polypeptide corresponding to amino acid position 272-319 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at position V272T, substitution at position C275S, and substitution at position BOOT of the hemagglutinin polypeptide; (f) a polypeptide corresponding to amino acid position 287-319 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at position BOOT, and a substitution at position C303S of the hemagglutinin polypeptide; (g) a polypeptide corresponding to amino acid position 271-318 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions I294T and I299T, and a substitution at position C274S of the hemagglutinin polypeptide; (h) a polypeptide corresponding to amino acid position 286-318 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a substitution at position I294T, and a substitution at position C302S of the hemagglutinin polypeptide; (i) a polypeptide corresponding to amino acid position 307-321 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3, wherein the polypeptide comprises a substitution at position Y308H of the hemagglutinin polypeptide; (j) a polypeptide corresponding to amino acid position 274-321 of a hemagglutinin polypeptide having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position Y302H of the hemagglutinin polypeptide, and optionally a substitution at position C277S of the hemagglutinin polypeptide; and (k) a polypeptide corresponding to amino acid position 305- 347 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises substitution at positions A315S and I318N, and a substitution at position C321S of the hemagglutinin polypeptide.

[0070]

[0064] In an embodiment, the second polypeptide fragment (F2) is selected from a group consisting of: (e) a polypeptide corresponding to amino acid position 272-319 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at position V272T, substitution at position C275S, and substitution at position I300T of SEQ ID NO: 1; (f) a polypeptide corresponding to amino acid position 287-319 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at position BOOT, and a substitution at position C303S of SEQ ID NO: 1; (g) a polypeptide corresponding to amino acid position 271-318 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions I294T and I299T, and a substitution at position C274S of SEQ ID NO: 7; (h) a polypeptide corresponding to amino acid position 286-318 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a substitution at position I294T, and a substitution at position C302S of SEQ ID NO: 7; (i) a polypeptide corresponding to amino acid position 307-321 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3, wherein the polypeptide comprises a substitution at position Y308H of SEQ ID NO: 3; (j) a polypeptide corresponding to amino acid position 274-321 of a hemagglutinin polypeptide having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position Y302H of the hemagglutinin polypeptide, and optionally a substitution at position C277S of SEQ ID NO: 5; and (k) a polypeptide corresponding to amino acid position 305- 347 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises substitution at positions A315S and I318N, and a substitution at position C321S of SEQ ID NO: 9 or SEQ ID NO: 11.

[0071]

[0065] In some embodiments, F2 is a polypeptide having a sequence of at least 95% identity to a sequence selected from the group consisting of : a. a sequence corresponding to positions 272-319 of SEQ ID NO: 1, and comprising amino acid substitutions at position V272T, C275S, and BOOT of SEQ ID NO: 1; b. a sequence corresponding to positions 287-319 of SEQ ID NO: 1; and comprising amino acid substitutions at position BOOT, and C303S of SEQ ID NO: 1; c. a sequence corresponding to positions 271-318 of SEQ ID NO: 7; and comprising amino acid substitutions at position I294T, I299T, and C274S of SEQ ID NO: 7; d. a sequence corresponding to positions 286-318 of SEQ ID NO: 7; and comprising amino acid substitutions at position I294T, and C302S of SEQ ID NO: 7; e. a sequence corresponding to positions 307-321 of SEQ ID NO: 3; and comprising amino acid substitution at position Y308H of SEQ ID NO: 3; f. a sequence corresponding to positions 274-321 of SEQ ID NO: 5; and comprising amino acid substitution at position Y302H, and optionally C277S of SEQ ID NO: 5; and g. a sequence corresponding to positions 305- 347 of SEQ ID NO: 9 or SEQ ID NO: 11; and comprising amino acid substitution at position A315S, I318N, and C321S, and optionally C277S of SEQ ID NO: 9 or SEQ ID NO: 11;

[0072]

[0066] The third polypeptide fragment (F3), according to embodiments herein, may be selected from a group consisting of: (1) a polypeptide corresponding to position 328-502 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises substitution at positions F390D and L400D of the hemagglutinin polypeptide; (m) a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position F392D and V402D of the hemagglutinin polypeptide; (n) a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position F408D and V419D of the hemagglutinin polypeptide; (o) a polypeptide corresponding to amino acid position 331-505 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions F393D and L403D, and a substitution at position V396T of the hemagglutinin polypeptide; and (p) a polypeptide corresponding to amino acid position 348-510 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises substitution at positions M417D and L420D of the hemagglutinin polypeptide.

[0073]

[0067] In an embodiment, the third polypeptide fragment (F3) is selected from a group consisting of: (1) a polypeptide corresponding to position 328-502 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises substitution at positions F390D and L400D of SEQ ID NO: 1; (m) a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position F392D and V402D of SEQ ID NO: 3 or SEQ ID NO: 5; (n) a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position F408D and V419D of SEQ ID NO: 3 or SEQ ID NO: 5; (o) a polypeptide corresponding to amino acid position 331-505 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions F393D and L403D, and a substitution at position V396T of SEQ ID NO: 7; and (p) a polypeptide corresponding to amino acid position 348-510 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises substitution at positions M417D and L420D of SEQ ID NO: 9 or SEQ ID NO: 11.

[0074]

[0068] In some embodiments, F3 is a polypeptide having a sequence of at least 95% identity to a sequence selected from the group consisting of: a. a sequence corresponding to positions 328-502 of SEQ ID NO: 1; and comprising amino acid substitution at position F390D and L400D of SEQ ID NO: 1; b. a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5; and comprising amino acid substitution at position F392D and V402D of SEQ ID NO: 3 or SEQ ID NO: 5; c. a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5; and comprising amino acid substitution at position F408D and V419D of SEQ ID NO: 3 or SEQ ID NO: 5; d. a sequence corresponding to positions 331-505 of SEQ ID NO: 7; and comprising amino acid substitution at position F393D, L403D, and V396T of SEQ ID NO: 7; and e. a sequence corresponding to positions 348-510 of SEQ ID NO: 9 or SEQ ID NO: 11; and comprising amino acid substitution at position M417D and L420D of SEQ ID NO: 9 or SEQ ID NO: 11,

[0075]

[0069] In an embodiment, LI and L2 are linkers, wherein LI has a sequence selected from SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27; and L2 has a sequence selected from SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27.

[0076]

[0070] Embodiments herein provide an immunogenic polypeptide in a linear stem design. In an embodiment, the immunogenic polypeptide comprises a first polypeptide fragment (Fl) linked to a second polypeptide fragment (F2) and the second polypeptide fragment linked to a third polypeptide fragment (F3).

[0077]

[0071] In an embodiment, the immunogenic polypeptide comprises a first polypeptide fragment (Fl) linked to a second polypeptide fragment (F2) by a linker LI and the second polypeptide fragment linked to a third polypeptide fragment (F3) with or without a linker L2, wherein (i) the first polypeptide fragment (Fl) is selected from a group consisting of: a polypeptide corresponding to amino acid position 1-35 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 7; a polypeptide corresponding to amino acid position 1-37 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 7; a polypeptide corresponding to amino acid position 1-45 of a hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at position A19S of the hemagglutinin polypeptide; and a polypeptide corresponding to amino acid position 1-36 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11; (ii) the second polypeptide fragment (F2) is selected from the group consisting of: a polypeptide corresponding to amino acid position 272-319 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at position V272T, substitution at position C275S, and substitution at position BOOT of the hemagglutinin polypeptide; a polypeptide corresponding to amino acid position 287-319 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at position BOOT, and a substitution at position C303S of the hemagglutinin polypeptide; a polypeptide corresponding to amino acid position 271-318 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions I294T and I299T, and a substitution at position C274S of the hemagglutinin polypeptide; a polypeptide corresponding to amino acid position 286- 318 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a substitution at position I294T, and a substitution at position C302S of the hemagglutinin polypeptide; a polypeptide corresponding to amino acid position 307-321 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3, wherein the polypeptide comprises a substitution at position Y308H of the hemagglutinin polypeptide; a polypeptide corresponding to amino acid position 274-321 of a hemagglutinin polypeptide having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position Y302H of the hemagglutinin polypeptide, and optionally a substitution at position C277S of the hemagglutinin polypeptide; and a polypeptide corresponding to amino acid position 305- 347 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises substitution at positions A315S and I318N, and a substitution at C321S of the hemagglutinin polypeptide; and (iii) the third polypeptide fragment (F3) selected from the group consisting of: a polypeptide corresponding to position 328-502 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at positions F390D and L400D of the hemagglutinin polypeptide; a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at positions F392D and V402D of the hemagglutinin polypeptide; a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position F408D and V419D of the hemagglutinin polypeptide; a polypeptide corresponding to amino acid position 331-505 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions F393D and L403D, and a substitution at position V396T of the hemagglutinin polypeptide; and a polypeptide corresponding to amino acid position 348-510 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises substitution at positions M417D and L420D of the hemagglutinin polypeptide, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, and the linker L2 is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27.

[0078]

[0072] In an embodiment, there is provided an immunogenic polypeptide having a formula selected from the group consisting of: (a) F1-L1-F2-L2-F3 (Formula I); and (b) FILI -F2-F3-L2-oligomerizati on domain (Formula II), wherein Fl is a polypeptide having a sequence of at least 95% identity to a sequence selected from the group consisting of : (i) a sequence corresponding to positions 1-35 of SEQ ID NO: 1 or SEQ ID NO: 7; (ii) a sequence corresponding to positions 1-37 of SEQ ID NO: 1 or SEQ ID NO: 7; (iii) a sequence corresponding to positions 1-45 of SEQ ID NO: 3 or SEQ ID NO: 5; and (iv) a sequence corresponding to positions 1-36 of SEQ ID NO: 9 or SEQ ID NO: 11, wherein F2 is a polypeptide having a sequence of at least 95% identity to a sequence selected from the group consisting of : (v) a sequence corresponding to positions 272-319 of SEQ ID NO: 1, and comprising amino acid substitutions at position V272T, C275S, and BOOT of SEQ ID NO: 1; (vi) a sequence corresponding to positions 287-319 of SEQ ID NO: 1; and comprising amino acid substitutions at position BOOT, and C303S of SEQ ID NO: 1; (vii) a sequence corresponding to positions 271-318 of SEQ ID NO: 7; and comprising amino acid substitutions at position I294T, I299T, and C274S of SEQ ID NO: 7; (viii) a sequence corresponding to positions 286-318 of SEQ ID NO: 7; and comprising amino acid substitutions at position I294T, and C302S of SEQ ID NO: 7; (ix) a sequence corresponding to positions 307-321 of SEQ ID NO: 3; and comprising amino acid substitution at position Y308H of SEQ ID NO: 3; (x) a sequence corresponding to positions 274-321 of SEQ ID NO: 5; and comprising amino acid substitution at position Y302H, and optionally C277S of SEQ ID NO: 5; and (xi) a sequence corresponding to positions 305- 347 of SEQ ID NO: 9 or SEQ ID NO: 11; and comprising amino acid substitution at position A315S, B 18N, and C321S, and optionally C277S of SEQ ID NO: 9 or SEQ ID NO: 11; wherein F3 is a polypeptide having a sequence of at least 95% identity to a sequence selected from the group consisting of : (xii) a sequence corresponding to positions 328-502 of SEQ ID NO: 1; and comprising amino acid substitution at position F390D and L400D of SEQ ID NO: 1; (xiii) a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5; and comprising amino acid substitution at position F392D and V402D of SEQ ID NO: 3 or SEQ ID NO: 5; (xiv) a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5; and comprising amino acid substitution at position F408D and V419D of SEQ ID NO: 3 or SEQ ID NO: 5; (xv) a sequence corresponding to positions 331-505 of SEQ ID NO: 7; and comprising amino acid substitution at position F393D, L403D, and V396T of SEQ ID NO: 7; and (xvi) a sequence corresponding to positions 348-510 of SEQ ID NO: 9 or SEQ ID NO: 11; and comprising amino acid substitution at position M417D and L420D of SEQ ID NO: 9 or SEQ ID NO: 11, wherein LI and L2 are linkers, wherein LI has a sequence selected from SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27; and L2 has a sequence selected from SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27.

[0079]

[0073] In an embodiment, there is provided an immunogenic polypeptide, wherein the Fl is linked to F2 by way of a linker LI, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, and wherein said F2 is linked to F3 by way of a linker L2, wherein the linker is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27.

[0080]

[0074] In an embodiment, there is provided an immunogenic polypeptide, further comprising an oligomerization domain at the N-terminal or C-terminal of the immunogenic polypeptide.

[0081]

[0075] In an embodiment, there is provided an immunogenic polypeptide, wherein the oligomerization domain is a foldon having an amino acid sequence as set forth in SEQ ID NO: 33, linked to the third polypeptide fragment (F3) by a linker.

[0082]

[0076] In an embodiment, there is provided an immunogenic polypeptide, wherein the Fl is linked to F2 by a linker LI, and wherein said F2 is linked to F3 which is further linked to an oligomerization domain by a linker L2, preferably the oligomerization domain is a foldon having an amino acid sequence as set forth in SEQ ID NO: 33. In an embodiment, there is provided an immunogenic polypeptide having a formula selected from the group consisting of: a. F1-L1-F2-L2-F3 (Formula I); and b. Fl-Ll-F2-F3-L2-oligomerizati on domain (Formula II), wherein

[0083] (a) Fl has a sequence corresponding to positions 1-35 of SEQ ID NO: 1 or SEQ ID NO: 7, F2 has a sequence corresponding to positions 272-319 of SEQ ID NO: 1, and comprising amino acid substitutions at position V272T, C275S, and BOOT of SEQ ID NO: 1, and F3 has a sequence corresponding to positions 328-502 of SEQ ID NO: 1; and comprising amino acid substitution at position F390D and L400D of SEQ ID NO: 1, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 13 and L2 has a sequence of SEQ ID NO. 18;

[0084] (b) Fl has a sequence corresponding to positions 1-37 of SEQ ID NO: 1 or SEQ ID NO: 7, F2 has a sequence corresponding to positions 287-319 of SEQ ID NO: 1 and comprising amino acid substitutions at position BOOT and C303S of SEQ ID NO: 1, and F3 has a sequence corresponding to positions 328-502 of SEQ ID NO: 1 and comprising amino acid substitution at position F390D and L400D of SEQ ID NO: 1, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 22 and L2 has a sequence of SEQ ID NO. 18;

[0085] (c) Fl has a sequence corresponding to positions 1-45 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitutions at position A19S of SEQ ID NO: 3 or SEQ ID NO: 5, F2 has a sequence corresponding to positions 307-321 of SEQ ID NO: 3 and comprising amino acid substitutions at position Y308H of SEQ ID NO: 3, and F3 has a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitution at position F392D and V402D of SEQ ID NO: 3 or SEQ ID NO: 5, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 25 and L2 has a sequence of SEQ ID NO. 27;

[0086] (d) Fl has a sequence corresponding to positions 1-45 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitutions at position A19S of SEQ ID NO: 3 or SEQ ID NO: 5, F2 has a sequence corresponding to positions 274-321 of SEQ ID NO: 5 and comprising amino acid substitutions at position Y302H of SEQ ID NO: 5, and F3 has a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitution at position F408D and V419D of SEQ ID NO: 3 or SEQ ID NO: 5, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 13 and L2 has a sequence of SEQ ID NO. 18;

[0087] (e) Fl has a sequence corresponding to positions 1-45 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitutions at position A19S of SEQ ID NO: 3 or SEQ ID NO: 5, F2 has a sequence corresponding to positions 274-321 of SEQ ID NO: 5 and comprising amino acid substitutions at position Y302H and C277S of SEQ ID NO: 5, and F3 has a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitution at position F408D and V419D of SEQ ID NO: 3 or SEQ ID NO: 5, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 13 and L2 has a sequence of SEQ ID NO. 18; (f) Fl has a sequence corresponding to positions 1-35 of SEQ ID NO: 1 or SEQ ID NO: 7, F2 has a sequence corresponding to positions 271-318 of SEQ ID NO: 7 and comprising amino acid substitutions at position I294T, I299T, and C274S of SEQ ID NO: 7, and F3 has a sequence corresponding to positions 331-505 of SEQ ID NO: 7 and comprising amino acid substitution at position F393D, L403D, and V396T of SEQ ID NO: 7, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 13 and L2 has a sequence of SEQ ID NO. 18;

[0088] (g) Fl has a sequence corresponding to positions 1-37 of SEQ ID NO: 1 or SEQ ID NO: 7, F2 has a sequence corresponding to positions 286-318 of SEQ ID NO: 7 and comprising amino acid substitutions at position I294T and C302S of SEQ ID NO: 7, and F3 has a sequence corresponding to positions 331-505 of SEQ ID NO: 7 and comprising amino acid substitution at position F393D, L403D, and V396T of SEQ ID NO: 7, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 22 and L2 has a sequence of SEQ ID NO. 18; or

[0089] (h) Fl has a sequence corresponding to positions 1-36 of SEQ ID NO: 9 or SEQ ID NO: 11, F2 has a sequence corresponding to positions 305- 347 of SEQ ID NO: 9 or SEQ ID NO: 11 and comprising amino acid substitutions at position A315S, I318N, and C321S SEQ ID NO: 9 or SEQ ID NO: 11, and F3 has a sequence corresponding to positions 348-510 of SEQ ID NO: 9 or SEQ ID NO: 11 and comprising amino acid substitution at position M417D and L420D of SEQ ID NO: 9 or SEQ ID NO: 11, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 27 and L2 has a sequence of SEQ ID NO. 13.

[0090]

[0077] In an embodiment, there is provided an immunogenic polypeptide, wherein (i) the first polypeptide fragment (Fl) is a polypeptide corresponding to amino acid position 1- 35 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 (ii) the second polypeptide fragment (F2) is a polypeptide corresponding to amino acid position 272-319 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at position V272T, substitution at position C275S, and substitution at position BOOT of the hemagglutinin polypeptide; and (iii) the third polypeptide fragment (F3) is a polypeptide corresponding to position 328-502 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at positions F390D and L400D of the hemagglutinin polypeptide, wherein the polypeptide comprises substitution at positions M417D and L420D of the hemagglutinin polypeptide, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 13; and the linker L2 is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27; preferably SEQ ID NO. 18.

[0091]

[0078] In an embodiment, the immunogenic polypeptide is having an amino acid sequence of 95% to 100% sequence identity to a sequence as set forth in SEQ ID NO: 37, and wherein the immunogenic polypeptide comprises amino acids 44T, 47S, 72T, 158D, and 168D at positions corresponding to SEQ ID NO: 37.

[0092]

[0079] In an embodiment, there is provided an immunogenic polypeptide, wherein (i) the first polypeptide fragment (Fl) a polypeptide corresponding to amino acid position 1-37 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1; (ii) the second polypeptide fragment (F2) is a polypeptide corresponding to amino acid position 287-319 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at position BOOT, and a substitution at position C303S of the hemagglutinin polypeptide; and (iii) the third polypeptide fragment (F3) is a polypeptide corresponding to position 328-502 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises a substitution at positions F390D and L400D of the hemagglutinin polypeptide, wherein the polypeptide comprises substitution at positions M417D and L420D of the hemagglutinin polypeptide, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 22, and the linker L2 is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 18.

[0093]

[0080] In an embodiment, the immunogenic polypeptide is having an amino acid sequence of 95% to 100% sequence identity to a sequence as set forth in SEQ ID NO: 39, and wherein the immunogenic polypeptide comprises amino acids 56T, 59S, 142D, and 152D at positions corresponding to SEQ ID NO: 39.

[0081] In an embodiment, there is provided an immunogenic polypeptide, wherein (i) the first polypeptide fragment (Fl) is a polypeptide corresponding to amino acid position 1- 45 of a hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at position A19S of the hemagglutinin polypeptide; (ii) the second polypeptide fragment (F2) is a polypeptide corresponding to amino acid position 307-328 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3, wherein the polypeptide comprises a substitution at position Y308H of the hemagglutinin polypeptide; and (iii) the third polypeptide fragment (F3) is a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at positions F392D and V402D of the hemagglutinin polypeptide, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 25, and the linker L2 is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 27.

[0094]

[0082] In an embodiment, the immunogenic polypeptide is having an amino acid sequence of 95% to 100% sequence identity to a sequence as set forth in SEQ ID NO: 41, and wherein the immunogenic polypeptide comprises amino acids 40S, 52H, 153D, and 163D at positions corresponding to SEQ ID NO: 41.

[0095]

[0083] In an embodiment, there is provided an immunogenic polypeptide, wherein (i) the first polypeptide fragment (Fl) is a polypeptide corresponding to amino acid position 1- 45 of a hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at position A19S of the hemagglutinin polypeptide; (ii) the second polypeptide fragment (F2) is a polypeptide corresponding to amino acid position 274-321 of a hemagglutinin polypeptide having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 5, wherein the polypeptide comprises a substitution at position Y302H of the hemagglutinin polypeptide, and optionally a substitution at position C277S of the hemagglutinin polypeptide; and (iii) the third polypeptide fragment (F3) is a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at positions F408D and V419D of the hemagglutinin polypeptide, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 13, and the linker L2 is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 18. In an embodiment, the immunogenic polypeptide is having an amino acid sequence of 95% to 100% sequence identity to a sequence as set forth in SEQ ID NO: 43, and wherein the immunogenic polypeptide comprises amino acids 2 IS, 82H, 184D, and 195D at positions corresponding to SEQ ID NO: 43.

[0096]

[0084] In another embodiment, the immunogenic polypeptide is having an amino acid sequence of 95% to 100% sequence identity to a sequence as set forth in SEQ ID NO: 45, and wherein the immunogenic polypeptide comprises amino acids 21S, 57S, 82H, 184D, and 195D at positions corresponding to SEQ ID NO: 45.

[0097]

[0085] In an embodiment, there is provided an immunogenic polypeptide, wherein (i) the first polypeptide fragment (Fl) is a polypeptide corresponding to amino acid position 1- 35 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7; (ii) the second polypeptide fragment (F2) is a polypeptide corresponding to amino acid position 271-318 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions I294T and I299T, and a substitution at position C274S of the hemagglutinin polypeptide; and (iii) the third polypeptide fragment (F3) is a polypeptide corresponding to amino acid position 331- 505 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions F393D and L403D, and a substitution at position V396T of the hemagglutinin polypeptide, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 13, and the linker L2 is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 18.

[0098]

[0086] In an embodiment, the immunogenic polypeptide is having an amino acid sequence of 95% to 100% sequence identity to a sequence as set forth in SEQ ID NO: 47, and wherein the immunogenic polypeptide comprises amino acids 46S, 66T, 7 IT, 157D, 160T, and 167D at positions corresponding to SEQ ID NO: 47.

[0099]

[0087] In an embodiment, there is provided an immunogenic polypeptide, wherein (i) the first polypeptide fragment (Fl) is a polypeptide corresponding to amino acid position 1- 37 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth SEQ ID NO: 7; (ii) the second polypeptide fragment (F2) is a polypeptide corresponding to amino acid position 286-318 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises a substitution at position I294T, and a substitution at position C302S of the hemagglutinin polypeptide; and (iii) the third polypeptide fragment (F3) is a polypeptide corresponding to amino acid position 331-505 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises substitution at positions F393D and L403D, and a substitution at position V396T of the hemagglutinin polypeptide, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 22, and the linker L2 is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 18. In an embodiment, the immunogenic polypeptide is having an amino acid sequence of 95% to 100% sequence identity to a sequence as set forth in SEQ ID NO: 49, and wherein the immunogenic polypeptide comprises amino acids 50T, 58S, 141D, 144T, and 151D at positions corresponding to SEQ ID NO: 49.

[0100]

[0088] In an embodiment, there is provided an immunogenic polypeptide comprising a first polypeptide fragment (Fl) linked to a second polypeptide fragment (F2) by a linker LI and the second polypeptide fragment linked to a third polypeptide fragment (F3), wherein (i) the first polypeptide fragment (Fl) is a polypeptide corresponding to amino acid position 1-36 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11; (ii) the second polypeptide fragment (F2) is a polypeptide corresponding to amino acid position 305- 347 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises substitution at positions A315S and I318N, and a substitution at C321S of the hemagglutinin polypeptide; and (iii) the third polypeptide fragment (F3) is a polypeptide corresponding to amino acid position 348-510 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises substitution at positions M417D and L420D of the hemagglutinin polypeptide, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 27.

[0101]

[0089] In an embodiment, the immunogenic polypeptide comprises an oligomerization domain at the N-terminal or C-terminal of the immunogenic polypeptide, wherein the oligomerization domain is a foldon having amino acid sequence as set forth in SEQ ID NO: 33. In another embodiment, the foldon is attached to the third polypeptide fragment (F3) by a linker L2, wherein the linker L2 is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27, preferably SEQ ID NO. 13.

[0102]

[0090] In an embodiment, the immunogenic polypeptide is having an amino acid sequence of 95% to 100% sequence identity to a sequence as set forth in SEQ ID NO: 51, and wherein the immunogenic polypeptide comprises amino acids 52S, 55N, 58S, 154D, and 157D at positions corresponding to SEQ ID NO: 51.

[0103]

[0091] In an embodiment, the immunogenic polypeptide has amino acid sequence of at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51. In another embodiment, the immunogenic polypeptide has amino acid sequence of at least 96% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51. In another embodiment, the immunogenic polypeptide has amino acid sequence of at least 97% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51. In yet another embodiment, the immunogenic polypeptide has amino acid sequence of at least 98% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51. In another embodiment, the immunogenic polypeptide has amino acid sequence of at least 99.5% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID N0:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51.

[0104]

[0092] In another embodiment, the immunogenic polypeptide is selected from:

[0105] (a) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 37, and comprising amino acid substitutions 44T, 47S, 72T, 158D, and 168D at positions corresponding to SEQ ID NO: 37;

[0106] (b) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO:39, and comprising amino acid substitutions 56T, 59S, 142D, and 152D at positions corresponding to SEQ ID NO: 39;

[0107] (c) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO:41, and comprising amino acid substitutions 40S, 73H, 153D, and 163D at positions corresponding to SEQ ID NO: 41;

[0108] (d) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO:43, and comprising amino acid substitution 21 S, 82H, 184D, and 195D at positions corresponding to SEQ ID NO: 43;

[0109] (e) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 45, and comprising amino acid substitution 21 S, 57S, 82H, 184D, and 195D at positions corresponding to SEQ ID NO: 45;

[0110] (f) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 47, and comprising amino acid substitutions 46S, 66T, 71T, 157D, 160T, and 167D at positions corresponding to SEQ ID NO: 47;

[0111] (g) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO; 49, and comprising amino acid substitutions 50T, 58S, 141D, 144T, and 151D at positions corresponding to SEQ ID NO: 49; and

[0112] (h) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in and SEQ ID NO: 51, and comprising amino acid substitutions 52S, 55N, 58S, 154D, and 157D at positions corresponding to SEQ ID NO: 51.

[0113]

[0093] In another embodiment of the present disclosure, the immunogenic polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO;

[0114] 49, and SEQ ID NO: 51.

[0115]

[0094] The immunogenic polypeptide, according to embodiments herein, may further be combined to achieve a multimeric protein. In an embodiment of the present disclosure, the immunogenic polypeptide comprises a combination of at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 immunogenic polypeptides of amino acid sequences selected from SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51. In another embodiment of the present disclosure, the immunogenic polypeptide comprises a combination of the immunogenic polypeptides of amino acid sequences selected from SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51.

[0116]

[0095] The immunogenic polypeptide, according to embodiments herein, may further comprise one or more peptides such as TPA signal sequence, HRV3C protease cleavage site, oligomerization domain, Histidine tag, etc. In an embodiment, there is provided an immunogenic polypeptide comprising a polypeptide having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51, wherein the immunogenic polypeptide further comprises a peptide selected from TPA signal peptide; oligomerization domain; HRV3C protease cleavage site or a portion thereof; Histidine tag; one or more linkers; or combinations thereof at the N- terminal or C-terminal end.

[0117]

[0096] The immunogenic polypeptides, according to embodiments herein, may further be attached to an oligomerization domain at the N-terminal or C-terminal end of the immunogenic polypeptide. In an embodiment of the present disclosure, the immunogenic polypeptide further comprises an oligomerization domain at the N-terminal or C-terminal end of the immunogenic polypeptide. Various oligomerization domains are known and may be used in embodiments herein. Examples of oligomerization domains includes human cartilage matrix protein (hCMP), chicken CMP (cCMP), fish cartilage matrix protein (Fl- CMP), fish isoform 2 cartilage matrix protein (F2-CMP), leucine Zipper with double cysteine (CCIZ), Synthetic oligomerization domain (cCMP-IZm), foldon, or glycosylated leucine zipper sequence (Gly IZ). In an embodiment of the present disclosure, there is provided an immunogenic polypeptide, wherein the oligomerization domain is a foldon having an amino acid sequence as set forth in SEQ ID NO: 33, attached to the immunogenic polypeptide by a linker L2 having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27. In an embodiment, the Histidine tag has a sequence as set forth in SEQ ID NO: 36.

[0118]

[0097] In an embodiment of the present disclosure, there is provided an immunogenic polypeptide in combination with any other HA containing influenza vaccine formulation. The term “other HA containing influenza vaccine formulation” or “other HA containing influenza vaccine composition”, as used herein, refers to an influenza vaccine formulation having one or more HA antigens. In an example, other HA containing influenza vaccine formulation is an immunogenic composition as described in IN202141062288, which is herein incorporated by reference in its entirety

[0119]

[0098] In an embodiment of the present disclosure, there are provided polynucleotides encoding the immunogenic polypeptide described herein. The polynucleotides, according to embodiments herein, may be DNA, RNA, or mRNA fragment. In an embodiment, the polynucleotide is a DNA fragment encoding the immunogenic polypeptide. In another embodiment, the polynucleotide is an RNA fragment encoding the immunogenic polypeptide. In yet another embodiment, the polynucleotide is an mRNA fragment encoding the immunogenic polypeptide.

[0120]

[0099] In an embodiment of the present disclosure, there is a 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. 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, and SEQ ID NO. 52.

[0121]

[0100] In another embodiment of the present disclosure, there is a polynucleotide having a sequence selected from the group consisting of 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, and SEQ ID NO. 52.

[0122]

[0101] In an embodiment of the present disclosure, there is provided a DNA construct comprising the polynucleotide described herein. In another embodiment, the DNA construct optionally comprises a polynucleotide encoding a TPA signal peptide; an oligomerization domain; a HRV3C protease cleavage site or a portion thereof; a Histidine tag; one or more linkers; or combinations thereof. In yet another embodiment, the DNA construct optionally comprises a polynucleotide encoding a His-tag, at the N-terminal or C-terminal end.

[0123]

[0102] In an embodiment, the polynucleotide encoding the Histidine tag has a sequence selected from SEQ ID NO: 36 or SEQ ID NO: 53.

[0124]

[0103] In an embodiment of the present disclosure, there is provided a recombinant vector having the polynucleotide described herein, operably linked to a promoter.

[0125]

[0104] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising the recombinant construct as described herein or the recombinant vector as described herein. The recombinant host cell may be selected from a bacterial cell, yeast cell, insect cell, or mammalian cell. In an embodiment, the recombinant host cell is a bacterial cell, preferably Escherichia coli. In another embodiment, the recombinant host cell is a yeast cell, wherein the yeast cell is selected from Pichia X33, Pichia GlycoSwitch® , DSMZ 70382, GS115, KM71, KM71H, BG09, GS190, GS200, JC220, JC254, JC227, JC300-JC308, YJN165, or CBS7435. In another embodiment, the recombinant host cell is an insect cell, wherein the insect cell is selected from the group consisting of Expi-Sf9®, Sf9, High Five®, Sf21, Sf-RVN and S2. In another embodiment, the recombinant host cell is a mammalian cell, wherein the mammalian cell is selected from the group consisting of Expi293F®Expi-CHO-S ® , CH0-K1, CHO-S, Flp-In CHO HEK293F® , CHOBC™, SLIM™ , SPOT™ , SP2 / 0 , Sp2 / 0- Agl4, CHO DG44, HEK 293S, HEK 293 Gntl- / - ,HEK293-EBNA1, CHOL-NSO, and NSO. Further, the recombinant host cell may be selected from OPENPichia (NCYC 2543 hocltr), OPENPichia his4, NCYC 2543 type strain, OPENPichia pep4, OPENPichia ypsl, OPENPichia pep4 ypsl, and OPENPichia mutS strains.

[0126]

[0105] Embodiments herein provide a vaccine composition. In an embodiment, there is provided a vaccine composition comprising the immunogenic polypeptide as described herein, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be 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. 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.

[0127]

[0106] The vaccine composition, according to embodiments herein may be lyophilized. In an embodiment, the vaccine composition is lyophilized composition.

[0128]

[0107] The vaccine composition may comprise an excipient selected from buffer, sugar, alcohol, sugar alcohol, salt, aldehyde, amino acid and their derivatives, or combination thereof. In an embodiment, the vaccine composition is a lyophilized composition comprising the immunogenic polypeptide, and at least one excipient, preferably selected from buffer, sugar, sugar alcohol, salt, amino acid salt, or combination thereof. In an embodiment, the buffer is 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; amino acid sugars and their derivatives are selected from arginine hydrochloride (ArgHCl), Histidine, Arginine, Glycine, alanine, proline, glutamate, sarcosine, trimethylamine N-oxide (TMAO). In an embodiment, the lyophilized composition has a pH in the range of 6 to 8, preferably 7.4.

[0129]

[0108] In another embodiment of the present disclosure, there is provided a vaccine composition, wherein the immunogenic polypeptide is present in combination with other HA containing influenza antigens or compositions. In an example, there is provided a vaccine composition, wherein the immunogenic polypeptide as described herein is combined with the immunogenic compositions as described in IN202141062288, which is herein incorporated by reference in its entirety.

[0130]

[0109] In another embodiment of the present disclosure, there is provided a vaccine composition as described herein, wherein the vaccine composition comprises a combination of at least 2, at least 3, at least 4, or at least 5 immunogenic polypeptides having amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51.

[0131] [HO] In an embodiment of the present disclosure, there is provided a vaccine composition comprising the immunogenic polypeptides as disclosed herein, in combination with any other HA containing influenza vaccine composition.

[0132] [Hl] In an embodiment of the present disclosure, there is provided a vaccine composition comprising comprising an adjuvant.

[0133]

[0112] In an embodiment of the present disclosure, there is provided a method for producing the vaccine composition as described herein, wherein the method comprises: (a) culturing the recombinant host cell as described herein under suitable conditions to obtain the immunogenic polypeptide as described herein; (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.

[0134]

[0113] In an embodiment of the present disclosure, there is provided a method for eliciting an immune response against influenza infection in a subject, the method comprising administering the subject with an effective amount of the vaccine composition as described herein. In an embodiment, the influenza infection is selected from an Influenza A infection caused by Influenza A viruses, Influenza B infection caused by Influenza B viruses, or Influenza C infection caused by Influenza C viruses.

[0135]

[0114] In an embodiment of the present disclosure, there is provided a method as described herein, wherein the vaccine composition is administered by a mode selected from the group consisting of intranasal, parenteral, subcutaneous, intramuscular and intradermal.

[0136] EXAMPLES

[0137]

[0115] The disclosure will now be illustrated with working examples, which are 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 to 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 apply.

[0138] Example 1: Construct generation and Cloning of the Immunogenic polypeptides.

[0139]

[0116] The gene of interest for H3HA2.1 N22 stem was synthesized and cloned into pET-28a(+) intermediate vector (between Ncol and Nhel) by Genscript, USA. The intermediate vector and vector backbone, eMS3_N21_HRV3C_F-pET28a(+), were digested by Ncol and Nhel restriction enzymes to generate the insert and vector backbone, respectively. Using H3HA2.1_N22-pET28a(+) as template, site-directed mutagenesis was done to generate H3HA2.1_N22_vl insert with Ndel and Hindlll overhangs. The gene of interest and the vector backbone eMSl_HlHA10F_N22-pET26(b+) were both digested using Ndel and Hindlll restriction enzymes. The genes of interest for HlHA2.1_N22_vl, HlHA2.1_N22_v2, HlHA6_N22_v3 were synthesized at Twist Biosciences, USA. The gene fragments were amplified using PCR to generate inserts with overhangs. The respective inserts and the vector backbone were digested using Ncol and Hindlll restriction enzymes. The HlHA6_N22_v2 gene was synthesized at Twist and the insert was PCR amplified. The resultant PCR fragment with overhangs and vector backbone eMSl_HlHA10F_N22-pET26(b+) were both digested using Ndel and Hindlll restriction enzymes. Using HlHA6_N22_v2 as template, the HlHA6_N22_v4 insert was amplified by PCR. The PCR amplified gene of interest and the vector backbone eMSl_HlHA10F_N22- pET26(b+) were both digested using Ndel and Hindlll restriction enzymes. The gene of interest for H5HA2.1_vl, and H5HA2.1_v2 were synthesized at Twist Biosciences, USA. The inserts were amplified with overhangs containing Ndel and Hindlll restriction sites using PCR. The amplified inserts and the vector backbone were both digested using Ndel and Hindlll restriction enzymes. The respective, digested, inserts and vector backbones were ligated and transformed into XLIO-Gold cells and plated on LB Agar + kanamycin plates and incubated at 37°C in the incubator, overnight. Positive colonies were confirmed by sequencing followed by transformation into the BL21(DE3) competent cells for expression studies.

[0140] LINEAR STEM DESIGNS

[0141] Hl Immunogen Design:

[0142]

[0117] Based on the sequence of the H1N1 isolate A / Wisconsin / 588 / 2019 (GISAID Isolate ID: EPI ISL 404460; SEQ ID NO: 1).

[0143] H1HA2.1 N22 vl:

[0144]

[0118] The accessible surface area (ASA) of each residue in HA was analyzed, both with and without residue stretches from the HA1 and HA2 subunits (using PDB ID 3LZG (Xu et al., 2010)). Residue numbering in HA1 and HA2 subunits follows the Burke-Smith scheme (Burke & Smith, 2014). Hydrophobic residues with a side-chain ASA difference of > 10 A2were analyzed. The following mutations were introduced to mask hydrophobic patches: V272T and BOOT. In full-length HA, Cys50 and Cys275 form an intramolecular disulfide bond; however, since Cys50 was excluded from the present design, Cys275 was mutated to Ser to prevent improper intermolecular disulfide bonding. Additionally, low-pH conformation-destabilizing mutations F390D and L400D, as characterized in a previous study (Bommakanti et al., 2012), were incorporated. The Asp substitution was chosen because it has been previously shown that Asp substitutions at buried hydrophobic residues are more destabilizing than substitutions with other charged amino acids. The first HA1 fragment (residues 1-35) was linked to residues 272-319 of HA1 with a 6-residue GSAGSA linker, and the HA1 (272-319) and HA2 (328-502) fragments were joined by a 4-residue GSAG linker to produce HlHA2.1_N22_vl as set forth in SEQ ID NO: 37. H1HA2.1 N22 v2:

[0145]

[0119] The first HA1 fragment (residues 1-37) was linked to residues 287-319 using a 3-residue GSA linker, and the HA1 (287-319) and HA2 (328-502) fragments were connected with a 4-residue GSAG linker. Hydrophobic residues were analyzed, with a total side-chain ASA difference of > 10 A2using PDB ID 3LZG. In the second design of HlHA2.1_N22_v2, the BOOT mutation was introduced to mask the exposed hydrophobic patch. In full-length HA, Cys279 and Cys303 form an intramolecular disulfide bond, but since Cys279 was omitted from the present design, Cys303 was mutated to Ser to prevent unwanted intermolecular disulfide bond formation. Additionally, low-pH conformationdestabilizing mutations F390D and L400D, previously characterized by Bommakanti et al. (2012), were incorporated into the design. The HlHA2.1_N22_v2 produced is as set forth in SEQ ID NO: 39. These design elements, including the linker strategies and residue mutations, aim to stabilize the HA stem domain and mask hydrophobic patches for better solubility and folding.

[0146] H3 Immunogen Design:

[0147]

[0120] H3HA1.1 design based on A / HK / 1 / 68 sequence: Uniprot ID: Q91MA7. H3HA2.1 designs based on the sequence of the H3N2 isolate A / Darwin.6 / 2021 (GISAID Isolate ID: EPI ISL 2233238).

[0148] H3HA1.1

[0149]

[0121] HA1 fragment 1 (1-45) was linked to HA1 fragment 2 (307-321) and HA2 residues fragment 3 (330-504). The accessible surface area (ASA) of every residue in HA was calculated in the absence and presence of residue stretches in the HA1 and HA2 subunits (PDB ID used for analysis is 1HGD (Sauter et al., 1992)). All H3 HA hydrophobic residues with a total side-chain ASA difference of > 10 A2in the calculations were analyzed. The following mutations were incorporated to mask the hydrophobic patch: A19S and Y308H in the HA1 subunit. Low-pH conformation destabilizing mutations: F392D and V402D, previously characterized (Bommakanti et al., 2012), were also incorporated in the design. The linkers used in the construct are GSAGS and GGGG for joining fragments 1-2 and fragments 2-3, respectively to produce H3HA1.1 as set forth in SEQ ID NO: 41.

[0150] H3HA2.1 N22

[0122] The first fragment, 1-45, was connected to 274-321 by a 6-residue GSAGSA linker and further, the HA1 (274-321) and HA2 (330-504) fragments were connected by a 4-residue linker (GSAG). The mutations are the same as those in the H3HA1.1 design, which is A19S, Y302H, F408D and V419D. The H3HA2.1 N22 produced is as set forth in SEQ ID NO: 43

[0151] H3HA2.1 N22 vl

[0152]

[0123] Based on the H3HA2.1 N22 design, as mentioned above, the residue boundaries were HA1 (1-45), connected to HA1 (274-321) by a 6-residue GSAGSA linker. The HA1 (274-321) and HA2 (330-504) fragments were connected by a 4-residue linker (GSAG). Along with the mutations in the H3HA2.1 N22 design, A19S and Y302H and the low-pH destabilizing mutations, F408D and V419D, the free Cys277 was mutated to Ser. Cys52 and Cys277 form an intramolecular disulfide bond in full-length HA. Since Cys52 was not incorporated in the present design, the inventors mutated Cys277 to Ser to prevent incorrect intermolecular disulfide bond formation. The H3HA2.1_N22_vl, thus produced is as set forth in SEQ ID NO: 45.

[0153] H5 Immunogen Design:

[0154]

[0124] Based on the sequence of the H5N1 isolate A / dairy cattle / Texas / 24-008749- 001-original / 2024 (GISAID Isolate ID: EPI ISL 19014384; SEQ ID NO: 7).

[0155] H5HA2.1 vl

[0156]

[0125] The accessible surface area (ASA) of every residue in HA was calculated in the absence and presence of residue stretches in the HA1 and HA2 subunits (PDB ID used for analysis is 5HUF (Yang et al., 2016)). All H5HA hydrophobic residues with a total sidechain ASA difference of > 10 A2in the calculations were analyzed. The following mutations were incorporated to mask the hydrophobic patch: I294T and I299T in the HA1 subunit and V396T in the HA2 subunit. Cys42 and Cys274 form an intramolecular disulfide bond in full-length HA. Since Cys42 was not incorporated in the present design, the inventors mutated Cys274 to Ser to prevent incorrect intermolecular disulfide bond formation. Low- pH conformation destabilizing mutations: F393D and L403D, previously characterized (Bommakanti et al., 2012), were also incorporated in the design. The first fragment, 1-35, was connected to 271-318 by a 6-residue GSAGSA linker. The HA1 (271-318) and HA2 (331-505) fragments were connected by a 4-residue linker (GSAG) to produce H5HA2. l_vl as set forth in SEQ ID NO: 47.

[0157] H5HA2.1 v2

[0158]

[0126] The accessible surface area (ASA) of every residue in HA was calculated in the absence and presence of residue stretches in the HA1 and HA2 subunits (PDB ID used for analysis is 5HUF). All H5 HA hydrophobic residues with a total side-chain ASA difference of > 10 A2were analyzed. I294T was incorporated to mask the hydrophobic patch in the HA1 subunit and V396T in the HA2 subunit. Cys278 and Cys302 form an intramolecular disulfide bond in full-length HA. Since Cys278 was not incorporated in the present design, the inventors mutated Cys302 to Ser to prevent incorrect intermolecular disulfide bond formation. Low-pH conformation destabilizing mutations: F393D and L403D, previously characterized (Bommakanti et al, 2012), were also incorporated in the design. The first fragment, 1-37, was connected to 286-318 by a 3-residue GSA linker. The HA1 (286-318) and HA2 (331-505) fragments were connected by a 4-residue linker (GSAG) to produce H5HA2.1_v2 as set forth in SEQ ID NO: 49.

[0159] Influenza B Immunogen Design

[0160]

[0127] Based on the sequence of B / Brisbane / 60 / 2008 (GISAID Isolate ID: EPI ISL 183972; SEQ ID NO: 9).

[0161] BHA21F

[0162]

[0128] The accessible surface area (ASA) of every residue in HA was calculated in the absence and presence of residue stretches in the HA1 and HA2 subunits (PDB ID used for analysis is 4FQM). The exposed hydrophobic residues A315S and I318N have been mutated. Residues in the B loop M417D and L420D were mutated to Aspartate to destabilize the low-pH conformation of HA. Cysteine 321 has been mutated to Serine to prevent intermolecular disulfide bond formation. The first fragment HA1 (1-36), was attached to HA1 (305-347) by a 4-residue GGGG linker. Further, the HA1 (305-347) was directly attached to HA2 (348-510) fragments without a linker. The HA2 (348-510) fragment was attached to a Foldon by a 6-residue GSAGSA linker. The mutations were A315S, I318N, M417D and L420D, and C321S. The BHA21F produced is as set forth in SEQ ID NO: 51.

[0129] The schematic representation of all the linear stem designs is depicted in Figure 1.

[0163] Example 2: Expression and purification of H3HA2.1 N22, H3HA2.1_N22_vl and H1HA2.1 N22 v2.

[0164]

[0130] The expression vectors with the gene of interest, expressing the immunogenic polypeptides, were transformed into E. coli BL21(DE3). A single colony was inoculated into lOmL of Terrific Veg broth supplemented with 50pg / mL kanamycin. The primary culture was grown overnight at 37°C, with continuous shaking at 180 rpm. The primary culture was then used to inoculate the secondary culture (500 ml scale), which was grown until an O.D 600nm of ~0.6 was reached. The culture was induced with 0.5 mM IPTG, and expression was carried out for 3 hours, 180 rpm at 37°C. The H3HA2.1_N22, H3HA2.1_N22_vl and HlHA2.1_N22_v2 proteins expressed in the pellet as inclusion bodies. Inclusion bodies were solubilized in 6M GdnHCl, and refolding was carried out using the rapid dilution method. Refolded proteins were buffer exchanged into Tris buffer and AEX chromatography was carried out with Q-Sepharose. Flow through was finally dialyzed into IX PBS, pH 7.4. Refolding methods were optimized with yields of ~26mg / L forH3HA2.1_N22, ~100mg / L forH3HA2.1_N22_vl and ~23mg / L for HlHA2.1_N22_v2. The H3 HA proteins were >95% pure and were detected on a western blot when probed with H3 strain-specific sera ((A)- (B), Figure 2). The H1HA2. l_N22_v2 was >85% pure and was detected on a western blot when probed with CR9114, a universal HA stem-specific monoclonal antibody ((C), Figure 2). Similar expression and purification techniques were employed for other variants.

[0165] Example 2.1: Characterization studies

[0166] Circular Dichroism and Florescence spectroscopy

[0167] H3HA2.1 N22

[0168]

[0131] The CD (circular dichroism) spectra for H3HA2.1 N22 stem in IX PBS, pH 7.4 was recorded on a Jasco J-715C spectropolarimeter flushed with nitrogen gas. The concentration of the purified protein was ~5-10pM. Measurements were done at 25°C in a 1mm path length quartz cuvette with a scan rate of 100 nm / min, response time of 4s, and a bandwidth of 0.5 nm. Each spectrum was an average of three scans. The protein spectrum was corrected for buffer (PBS, pH 7.4) signals. The fluorescence emission spectra were recorded on a Jasco FP-6300 Spectrofluorometer. The concentration of the protein was ~lpM. Measurements were done at 25°C with an excitation wavelength of 280 nm, scan rate of 100 nm / min, a bandwidth 2 nm (bandwidth Ex. - 5 nm, Em. - 5 nm) with a data pitch of 1 nm. Each spectrum was an average of three scans. CD and Fluorescence spectra indicate that stems are well folded (Figure 3).

[0169] HlHA2.1_N22_v2

[0170]

[0132] Intrinsic tryptophan fluorescence spectroscopy was also conducted to further assess the polypeptide's folded state. The data is an average of three scans, with an excitation wavelength of 280nm. The emission spectrum was recorded from 280-400 nm. The emission maximum wavelength was found to be 399nm, which was below 340 nm ((A), Figure 10). This blue-shifted emission indicates that the tryptophan residues are predominantly located in a hydrophobic, non-polar environment, suggesting a well-folded and compact protein structure.

[0171] H3HA2.1 N22 vl

[0172]

[0133] Intrinsic tryptophan fluorescence spectroscopy was also conducted to further assess the polypeptide's folded state. The data is an average of three scans, with an excitation wavelength of 280nm. The emission spectrum was recorded from 280-400 nm. The emission maximum wavelength was found to be 399nm, which was below 340 nm ((A), Figure 11). This blue-shifted emission indicates that the tryptophan residues are predominantly located in a hydrophobic, non-polar environment, suggesting a well-folded and compact protein structure.

[0173] Biolayer Interferometry (BLI) studies

[0174]

[0134] The binding of flu proteins with mAbs CR8020 and CR9114 was assessed through BLI studies. BLI measurements were made using ForteBio biosensors (Fortebio - Sartorius).

[0175] HlHA2.1_N22_v2

[0176]

[0135] The interaction between the analyte (HlHA2.1_N22_v2) and the pan-influenza neutralizing antibody (CR9114) was characterized using bio-layer interferometry (BLI). Octet Red96 (Pall ForteBio) was used to check the binding affinity of immunogens at 25°C. CR9114 (10 pg / ml) was immobilised on ProteinG tips. After immobilisation, the tips were dipped in varying concentrations of analytes (62.5 - 1000 nM) in 1XPBS buffer (pH 7.4). The association was recorded for 200s, and dissociation for 200s. Regeneration of tips was carried out using 0.1 M glycine (pH 2.5). The representative binding curves show a high- affinity interaction, with a dissociation constant (KD) of 0.3 nM, confirming the ability of this designed antigen to bind conformation-specific antibody CR9114 ((B), Figure 10).

[0177]

[0136] Table 1: Binding parameters of HlHA2.1_N22_v2.

[0178] H3HA2.1 N22 vl

[0179]

[0137] The interaction between analyte (H3HA2.1_N22_vl) and the pan-influenza neutralizing antibody (CR9114), was characterized using bio-layer interferometry (BLI). Octet Red96 (Pall ForteBio) was used to check the binding affinity at 25°C. CR9114 (10 pg / ml) was immobilised on ProteinG tips. After immobilisation of CR9114, the tips were dipped in varying concentrations of analytes (62.5 - 1000 nM) in IX PBS buffer (pH 7.4). The association was recorded for 200s, and dissociation for 200s. Regeneration of tips was carried out using 0.1 M glycine (pH 2.5). The representative binding curves show a high- affinity interaction, with a dissociation constant (KD) of 0.25 nM, confirming the ability of this designed antigen to bind conformation-specific antibody CR9114 ((B), Figure 11).

[0180]

[0138] Table 2: Binding parameters of H3HA2.1_N22_vl.

[0181] Example 2.2: Immunization and challenge studies in mice

[0182] H3HA2.1 N22

[0183]

[0139] 6 to 8 weeks mice were immunized intramuscularly with a total of 15 pg of Sepivac SWE (Squalene in Water Emulsion) adjuvanted MynfluOOl and Mynflu002 (i.e. MynfluOOl + H3HA2.1_N22) vaccine formulations in a prime-boost regimen with a three- week interval Adjuvant-treated mice were used as controls ((A), Figure 4). MynfluOOl near full length HA antigens of Hl, H3, HV and HY.

[0184]

[0140] Hl, H3, HV, and HY, HA-specific antibodies in sera samples, were measured 14 days post-boost using ELISA (Enzyme-Linked Immunosorbent assay), HI (Hemagglutination Inhibition), and MN (Microneutralization) assays.

[0185]

[0141] MynfluOOl and Mnyflu002 formulations both elicited high ELISA endpoint titers in a range from 25,600-1024,00 against Hl, H3, HV and HY proteins ((B), Figure 5). High HAI titers were seen against NH23-24 matched viruses, including H1N1 (A / Wisconsin / 67 / 22), H3N2 (A / Massachusetts / 18 / 22), HV (B / Austria / 1359417 / 21) and HY (B / Phuket / 3073 / 2013) (Figure 6). Similarly, microneutralization titers were high against Hl and H3 virus (160-640) and low for HV and HY virus (40-160) for both vaccines against matched and mismatched viruses (Figure 6).

[0186]

[0142] Additional protection provided upon adding stems to the MynfluOOl vaccine was evaluated in female BALB / c mice in an immunization followed by a viral challenge study. Two intramuscular immunizations with low doses (3.75 pg per antigen / dose / animal) of SWE adjuvanted proteins, spaced three weeks apart, were administered. Marginally better protection was seen in Mynflu002 group than in MynfluOOl alone. Lower body weight loss seen in Mynflu002 group than MynfluOOl alone, clearly indicates that H3HA2.1_N22 is adding to the protection (Figure 7).

[0187]

[0143] Table 3: Immunization group, antigen dose and survival (%) for X31 challenge.

[0188] Combination ofH3HA2.1 N22 vl and H 1HA2.1 N22 v2

[0189]

[0144] Female BALB / c mice (6-8 weeks old) were immunized with 18 pg of MynfluOOl and 18 pg of Mynflu002 (MynfluOOl + HlHA2.1_N22_v2 + H3HA2. l_N22_vl ) in SWE adjuvant (squalene water emulsion, Sepivac) in a total volume of 100 pL (50 pL / hind quarter) (1 : 1, protein: adjuvant). The mice were boosted with the same vaccine composition and with the same dosage at three weeks post-prime vaccination. Adjuvant-treated mice were used as controls. Sera samples were isolated from the bleeds drawn before prime (day -3), post-prime (day 14), and post-boost (day 35) ((A), Figure 12). Hl, H3, HV, and HY, HA-specific antibodies in sera samples, were measured 14 days postboost HI (Hemagglutination Inhibition) assay.

[0190]

[0145] Comparable HAI titers were seen in MynfluOOl and Mynflu002 against NH23- 24 matched viruses, including H1N1 (A / Wisconsin / 67 / 22), H3N2 (A / Massachusetts / 18 / 22), HV (B / Austria / 1359417 / 21), and HY (B / Phuket / 3073 / 2013). Addition of immunogenic polypeptides does not seem to diminish head titers ((B), Figure 12). One week after boost bleed, mice were anesthetized and intranasally challenged with 10 MLD50 mouse-adapted X31 virus. Survival and weight loss of the challenged and control mice groups were monitored daily for 14 days post-challenge. The weight of individual mice (surviving) was recorded at each time point. Mynflu002 fully protects against the X31 challenge, whereas MynfluOOl gave only 57.1 % protection (C), Figure 12). Additionally, lower body weight loss was seen in the Mynflu002 group than in the MynfluOOl alone, clearly indicating that the addition of H3HA2.1_N22_vl stem to MynfluOOl significantly enhances the breadth of protection to a highly mismatched challenge strain, with over fifty years of evolutionary divergence from the sequence of the vaccine antigen.

[0191]

[0146] Table 4: Antigens and % protection

[0192] ELISA- Endpoint titers of serum-binding antibodies

[0193]

[0147] Briefly, 96 well ELISA plates were coated with 4 pg / mL Hl, H3, HV, HY delCD or H3HA2.1_N22_vl immunogens and incubated at 25 °C for 2-3 h. Plates were washed with PBST, followed by incubation with blocking with 3 % skimmed milk (in PBST) at 25°C for 1 h. Four-fold serially diluted antisera raised against immunogens were added to wells and incubated at 25 °C for 1 h. Following three washes with PBST, plates were incubated with 1 : 10,000 diluted goat ALP-conjugated anti -mouse IgG secondary antibody at 25 °C for 1 h. Plates were washed thrice with PBST and incubated with pNPP liquid substrate at 37 °C for 30 min. Optical density (O.D.) was measured at 405 nm. The ELISA endpoint titers were determined as the highest sera dilution with an O.D. signal above 0.2 at 405 nm.

[0194] HAI assay:

[0195]

[0148] Human erythrocytes were separated from whole blood (Lampire Biologicals). After isolation and washing, 30 ml of 1% human RBC suspension (vol / vol in 1% BSA-PBS) was added to 30 ml serial dilutions of HA protein or influenza virus in 1% BSA, PBS in a U-bottom 96-well plate (total volume, 60 ml). Agglutination was read after incubation for 30 minutes at room temperature.

[0196] MNT assay :

[0197]

[0149] Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay for the presence of antibodies that neutralized the infectivity of 100 TCID50 (50% tissue culture infectious dose) of 1918 (H1N1) viruses on MDCK cell monolayers by using two wells per dilution on a 96-well plate as described (18). After 2 days of incubation, cells were fixed, and ELISA was performed to detect the presence of viral nucleoprotein (NP) and determine the neutralization activity.

[0198] Statistical Analysis:

[0150] Data analysis was performed using GraphPad Prism software 9.0.0. The ELISA binding, neutralization titers, and pseudoviral virus neutralization titer data were analysed with a two-tailed Mann-Whitney test and non-parametric Kruskal-Wallis with Dunn’s multiple, respectively. Weight changes in mice and hamsters were analysed with a two- tailed Student’s t-test. (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001).

[0199]

[0151] All mice immunization studies were approved by the Institutional Animal Ethics Committee (CAF / ETHICS / 847 / 2021, CAF / ETHICS / 8 / 2022). These were carried out at the Central Animal Facility (CAF), Indian Institute of Science, according to CPC SEA and ARRIVE guidelines.

[0200] Example 3: Expression and purification of BHA21F:

[0201]

[0152] In E. coli BL21(DE3*) cells, BHA21F were overexpressed, separated from the inclusion bodies, and refolded in vitro. In summary, the Tartoff-Hobbs HiVegTM media (HiMedia) primary culture was grown at 37°C for an entire night. Subsequently, 1% of the primary inoculum was added to the secondary culture, which was then grown at 37°C until an OD600 of approximately 0.7 was achieved. The cells were induced with 1 mM isopropyl- P-thiogalactopyranoside (IPTG) and grown for the entire night at 20°C. Harvested cells at 6000*g was then re-suspended in 50ml of lx PBS (pH 7.4), which was enhanced with 1 mM PMSF, a protease inhibitor. After sonicating the cells to lyse them, they were centrifuged for 30 minutes at 14,000*g at 4°C. The cell pellets were left to solubilize overnight at room temperature in ~6M GdnCl in lx PBS (pH 7.4). Following centrifugation of the solubilized cell pellets, the supernatant was run through Ni-NTA resin (GE Healthcare Life Sciences, Amersham) at room temperature. The resin had been pre-equilibrated with 6M GdnCl (in lx PBS, pH 7.4), had been twice washed with 80mM imidazole in 6M GdnCl (in lx PBS), and had been eluted with 300mM imidazole in 6M GdnCl (in lx PBS, pH 7.4) as well. Ultimately, the protein that had been eluted was refolded in 1.5M ArgHCl, and GdnCl was eliminated through four dialysis cycles at 4°C against lx PBS (pH 7.4). The proteins were then concentrated to 1 mg / ml and stored at -80°C.

[0202]

[0153] BHA21F was purified using Ni-affinity chromatography from the inclusion bodies under denaturing conditions ((A), Figure 8). The whole cell lysate was sonicated in a lysis buffer. Proteins were refolded using 1.5M ArgHCl, then serially diluting the denaturant GdnCl through dialysis against lx PBS at 4 °C. All the proteins were finally concentrated to ~1 mg / ml and stored at -80 °C. The total yield was 11.2 mg per litre of culture for BHA21F construct.

[0203] Example 3.1: Nano-Differential Scanning Fluorimetry

[0204]

[0154] The thermal stability of the designed BHA21F immunogens was determined by using nano-DSF (nano-differential scanning fluorimetry) on a Prometheus NT.48 instrument (Nano Temper). Thermal unfolding of protein samples at a concentration of 5 pM in lx PBS, pH 7.4, was monitored from 20 °C to 90 °C with a 1 °C / min rise. The normalized ratio of the first derivative of fluorescence (350 nm / 330 nm) was plotted against temperature.

[0205]

[0155] Nano-DSF data revealed that the immunogen BHA21F is thermostable and has an apparent melting temperature (Tm) of 74.2 °C((B), Figure 8).

[0206] Example 3.2: Surface Plasmon Resonance to check binding affinity

[0207]

[0156] Biacore2000 optical biosensor (Biacore, Uppsala, Sweden) was used to check the binding affinity of immunogen BHA21F to the stem directed bnAb CR9114 at 25°C. 30 pl / min flow rate was maintained. Eight hundred response units (RU) of CR9114 were immobilized on a research-grade CM5 sensor chip (GE Healthcare, Uppsala, Sweden) using amine coupling. For the negative control, an ovalbumin-immobilized sensor channel was used. The various concentrations of analytes in PBS (pH 7.4) with 0.05% Tween 20 surfactant were passed over the chip surface. The association was recorded for 100s and dissociation for 200s. Regeneration of the chip was done using 4M MgCL. The negative control flow channel signals were subtracted to correct the nonspecific binding for each binding curve. The monomeric concentration of all the proteins was used to get the kinetic parameters. The data were fitted with a 1 : 1 Langmuir interaction model using Bia Evaluation version 3.1 to obtain the kinetic parameters.

[0208]

[0157] CR9114 is a pan Influenza; HA stem-directed neutralizing antibody (bnAb); BHA21F construct showed binding with a KD in the nanomolar range, indicating that this immunogen-bound to CR9114 with very high affinity (0.35X10'9M). The ability of the stem construct to bind these bnAb CR9114 with high affinity means that the structural integrity of designed stem immunogens was maintained ((C), Figure 8).

[0209] Example 3.3: Mice Immunization and Challenge Studies

[0158] For prime immunization (day 0) and boost immunization (day 21), 6-8 weeks BALB / c mice in a group of 6 were intramuscularly immunized with 20 pg of BHA21F immunogen along with SWE (Squalene-in water emulsion) adjuvant. 1 : 1 v / v immunogen: SWE ratio per animal per dose was used. Adjuvant-treated mice with PBS were used as controls only. Sera samples were isolated from the bleeds at day 14 and day 35 for prime and post-boost immunization, respectively.

[0210]

[0159] ELISA was done to determine serum antibody titers against test immunogens. Briefly, at room temperature, in 96 well Nunc plates, 4 pg / ml of test immunogens (50 pL / well) were coated for 2 h by shaking at 300 rpm. For 1 hr, the blocking was done with 3% BSA in PBST after washing with IX PBS containing 0.05% Tween 20 (PBST) four times. Four-fold serially diluted antisera raised against test immunogens were added to wells for an hour at 300rpm shaking. Wells were washed with PBST thrice, 50 pL of HRP- conjugated goat anti-mouse IgG secondary antibody (1 :8000) was added, and plates were incubated for an hour. This was followed by washing with PBST four times. TMB liquid substrate was added to each well and then incubated at 37 0C for 30 min. Optical density was measured at 450 nm. The highest serum dilution possessing a signal above 0.2 O. D at 450 nm was taken for the endpoint titer for ELISA. Data were plotted using GraphPad Prism v8.4.3. A two-tailed Student’s t-test was performed for pairwise ELISA endpoint titer comparisons. BHA21F immunogens elicited high humoral immune responses ((B), Figure 9).

[0211]

[0160] Mice were intranasally challenged with 10MLD50 mice with B / Brisbane / 60 / 2008 virus after twenty-one days of the second immunization. At least 14 days post-challenge, the challenged and control mice groups were monitored daily for survival and weight loss.

[0212]

[0161] Weight loss was observed till day 7 for all the immunized mice, which recovered slowly after the initial weight loss. ((C), Figure 9). BHA21F showed 83% protection against the B / Brisbane / 60 / 2008) Virus (homologous challenge) ((D), Figure 9).

[0213] Example 4: Expression and purification of H5HA2.1_vl:

[0214]

[0162] The expression vector containing the gene encoding H5HA2.1_vl stem was transformed into E. coli BL21(DE3). A single colony was inoculated into lOmL of Terrific Veg broth supplemented with 50pg / mL kanamycin. The primary culture was grown overnight at 37°C, with continuous shaking at 180 rpm. 1% of the primary culture was then used to inoculate the secondary culture (500 ml scale), which was grown until an O.D. 600nm of ~0.6 was reached. The culture was induced with 1 mM IPTG, and expression was carried out for 3 hours, 180 rpm at 37°C. The H5HA2.1_vl protein was expressed in the pellet as inclusion bodies.

[0215]

[0163] Inclusion bodies were solubilized in 6M GdnHCl, and refolding was carried out using the rapid dilution method. Refolded proteins were buffer exchanged into Tris buffer (30 mM) and AEX chromatography was carried out with Q-Sepharose. Flow through was finally dialyzed into IX PBS buffer, pH 7.4. Refolding methods were optimized with a yield of -70 mg / L for H5HA2.1_vl. The H5HA2.1_vl was 97% pure and was detected on a western blot when probed with H5 stem-specific mice sera (1 :20000 dilution) ((A), Figure 13).

[0216] Example 4.1: Characterization studies

[0217] Circular Dichroism and Fluorescence spectroscopy

[0218]

[0164] Intrinsic tryptophan fluorescence spectroscopy was conducted to assess the polypeptide's folded state. The data is an average of three scans, with an excitation wavelength of 280nm. The emission spectrum was recorded from 280-400 nm. The emission maximum wavelength was found to be 399nm, which was below 340 nm ((B), Figure 13). This blue-shifted emission indicates that the tryptophan residues are predominantly located in a hydrophobic, non-polar environment, suggesting a well-folded and compact structure.

[0219] Example 4.2: Surface Plasmon Resonance to check binding affinity

[0220]

[0165] Binding interactions between H5HA2.1_vl and the broadly neutralizing antibodies CR6261 and CR9114 were analyzed using Biacore T200 instrument (Cytiva) (Figure 14). Purified antibodies were immobilized on a CM5 sensor chip, and serial dilutions of analyte (H5HA2.1_vl) were injected at a flow rate of [20 pL / min], Association (200s) and dissociation phases (500s) were monitored in real time, and sensorgrams were reference-subtracted and fitted using a 1 : 1 Langmuir binding model. Both CR6261 and CR9114 displayed robust binding to H5HA2. l_vl, with distinct association and dissociation kinetics, confirming high-affinity recognition of conserved epitopes within the H5HA2.1_vl stem region.

[0221] Example 4.3: Assessment of stability of H5HA2.1 across broad spectrum of lyophilization buffer conditions.

[0166] H5HA2. l_vl was lyophilized using IX PBS buffers pH 7.4 containing different excipients (Table 5).

[0222]

[0167] Table 5: Exemplary Lyophilization compositions having H5HA2.1.

[0223]

[0168] Protein recovery post-lyophilization and reconstitution, as measured by a BCA assay, showed minimal protein loss ((A), Figure 15). SDS-PAGE and western blot analysis of liquid and lyophilized compositions, probed with anti H5HA2.1_vl mice sera (1 :5000 dilution), demonstrated that the immunogen's profile remained consistent after lyophilization ((B), Figure 15). Indirect ELISA was performed using the H5HA2. l_vl stem antigen, either in liquid form or reconstituted after lyophilization. The antigen was serially diluted from 1000 ng / mL to 8 ng / mL and immobilized onto ELISA plates. Bound stem proteins were further probed with the primary antibody CR9114-hFc, followed by detection with a goat anti-human IgG alkaline phosphatase (ALP) conjugate. Signal was developed at 405nm using BCIP / NBT liquid substrate, resulting in a blue colour reaction. ELISA results using antigen-specific sera for both liquid and lyophilized proteins further confirmed the stability of the protein. These findings collectively demonstrate that the H5HA2.1_vl stem protein maintains its stability across a broad range of lyophilization buffer conditions ((C), Figure 15).

[0224] Example 4.4: Mice Immunization and Challenge Studies

[0225]

[0169] Female BALB / c mice (6-8 weeks old) were immunized with MynfluOOl, commercial IIV (Fluarix, a quadrivalent seasonal influenza vaccine), or their combinations with H5HA2.1_vl immunogen. The total antigenic dose for MynfluOOl and Fluarix (when combined with H5HA2.1_vl individually) was kept to 15 pg, while Fluarix alone was administered at 12 pg. MynfluOOl was formulated with SWE adjuvant at a 1 : 1 ratio (antigen: SWE), whereas Fluarix alone was delivered without adjuvant. All formulations were injected in a total volume of 100 pL (50 pL per hind limb). Booster doses with the same formulations and dosages were given three weeks after the prime immunization. Adjuvant- only groups served as controls ((A), Figure 16).

[0226]

[0170] Sera were collected at baseline (day -1), post-prime (day 14), and post-boost (day 35). HA-specific antibody responses to Hl, H3, HV, and HY were assessed 14 days post-boost using hemagglutination inhibition (HI) assay. MynfluOOl in combination with H5 stem elicited significantly higher HAI titers compared to Fluarix in combination with H5 stem against NH22-23 matched strains, including H1N1 (A / Wisconsin / 67 / 22), H3N2 (A / Darwin / 6 / 21), HV (B / Austria / 1359417 / 21), and HY (B / Phuket / 3073 / 2013) ((B), Figure 16).

[0227]

[0171] One week after boost bleed, mice were anesthetized and intranasally challenged with 100 MLDso of reverse genetics (rg) H5N1 (A / dairy cattle / Texas / 2024). Survival and body weight were monitored daily for 14 days post-challenge, with weights of surviving mice recorded at each time point. The results demonstrated that combining MynfluOOl with H5HA2.1_vl provided the strongest protection, with no weight loss observed against the high dose rgH5Nl challenge. In contrast, Fluarix alone offered poor protection. Remarkably, supplementation of Fluarix with a small amount of H5HA2.1_vl (3 pg) significantly improved protection and reduced morbidity, while a higher dose of H5HA2. l_vl alone (15 pg) conferred complete protection against high-dose challenge ((C), Figure 16).

[0228]

[0172] Table 6: Antigens and % protection

[0229] Advantages of the Present Disclosure

[0230]

[0173] The present disclosure provides immunogenic polypeptide comprising hemagglutinin (HA) stem peptides that shift the immune response toward the conserved stem rather than the highly variable globular head. While previous efforts have aimed at redirecting immunity to this conserved region through protein minimization and stabilization techniques, the present approach introduces novel HA stem designs that can be incorporated into existing influenza vaccine formulations, providing an added advantage. The immunogens of the present disclosure are engineered to enhance cross-reactive antibody responses, offering broader protection against divergent influenza strains. By preserving the structural integrity of the HA protein while improving its immunogenicity, this approach represents a promising step toward developing a more effective, universal influenza vaccine.

[0231]

[0174] Furthermore, combining the designed HA stem with existing influenza vaccines provides an added layer of protection, potentially enhancing the breadth of immune responses against a wider range of influenza strains. The immunogenic polypeptides of the present disclosure are capable of expression in bacteria at low costs and in high yield.

[0232]

[0175] Substitution mutations, in accordance with the embodiments herein are depicted in Table 7.

[0233]

[0176] Table 7: depicts the substitution mutations, according to the present disclosure.

[0234]

[0177] Sequence description and SEQ IDs of all the exemplary sequences according to the present disclosure are depicted in Table 8 .

[0235]

[0178] Table 8:

[0236]

[0237] REFERENCES

[0238]

[0001] Bommakanti, G., Citron, M. P., Hepler, R. W., Callahan, C., Heidecker, G. J., Ahmad Najar, T., Lu, X., Joyce, J. G., Shiver, J. W., Casimiro, D. R., ter Meulen, J., Liang, X., & Varadarajan, R. (2010). Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. https: / / doi.Org / 10.1073 / pnas.1007465107 / - / DCSupplemental

[0239]

[0002] Bommakanti, G., Lu, X., Citron, M. P., Najar, T. A., Heidecker, G. J., ter Meulen, J., Varadarajan, R., & Liang, X. (2012). Design of Escherichia coli-Expressed Stalk Domain Immunogens of H1N1 Hemagglutinin That Protect Mice from Lethal Challenge. Journal of Virology, 56(24), 13434-13444. https: / / doi.org / 10.1128 / jvi.01429-12

[0240]

[0003] Bullough, P. A., Hughson, F. M., Skehel, J. J., & Wiley, D. C. (1994). Structure of influenza haemagglutinin at the pH of membrane fusion. Nature, 377(6492), 37- 43. https: / / doi.org / 10.1038 / 371037A0

[0241]

[0004] Burke, D. F., & Smith, D. J. (2014). A Recommended Numbering Scheme for Influenza A HA Subtypes. PLOS ONE, 9(H), el 12302. https: / / doi.org / 10.1371 / JOURNAL.PONE.0112302

[0242]

[0005] Chen, J., Wharton, S. A., Weissenhorn, W ., Calder, L. J., Hughson, F. M., Skehel, J. J., & Wiley, D. C. (1995). A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low- pH-induced conformation. Proceedings of the National Academy of Sciences of the United States of America, 92(26), 12205-12209. https: / / doi.org / 10.1073 / PNAS.92.26.12205

[0243]

[0006] Corti, D., & Lanzavecchia, A. (2013). Broadly neutralizing antiviral antibodies. Annual Review of Immunology, 31, 705-742. https: / / doi.org / 10.1146 / ANNUREV- IMMUNOL-032712-095916

[0244]

[0007] Ellebedy, A. H., & Ahmed, R. (2012). Re-engaging cross-reactive memory B cells: the influenza puzzle. Frontiers in Immunology, 3(MAR). https: / / doi.org / 10.3389 / FIMMU.2012.00053

[0245]

[0008] Ferguson, N. M., Galvani, A. P., & Bush, R. M. (2003). Ecological and immunological determinants of influenza evolution. Nature, -722(6930), 428-433. https: / / doi.org / 10.1038 / NATURE01509

[0246]

[0009] Fiore, A. E., Bridges, C. B., & Cox, N. J. (2009). Seasonal influenza vaccines. Current Topics in Microbiology and Immunology, 333( 1 ), 43-82. https: / / doi.org / 10.1007 / 978-3-540-92165-3_3

[0010] Girard, M. P., Cherian, T., Pervikov, Y., & Kieny, M. P. (2005). A review of vaccine research and development: human acute respiratory infections. Vaccine, 23(50), 5708-5724. https: / / doi.Org / 10.1016 / J.VACCINE.2005.07.046

[0247] [Oil] Kallewaard, N. L., Corti, D., Collins, P. J., Neu, U., McAuliffe, J. M., Benjamin, E., Wachter-Rosati, L., Palmer-Hill, F. J., Yuan, A. Q., Walker, P. A., Vorlaender, M. K., Bianchi, S., Guarino, B., De Marco, A., Vanzetta, F., Agatic, G., Foglierini, M., Pinna, D., Fernandez -Rodriguez, B., ... Skehel, J. J. (2016). Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes. Cell, 166(3), 596- 608. https: / / doi.Org / 10.1016 / J.CELL.2016.05.073

[0248]

[0012] Kirkpatrick, E., Qiu, X., Wilson, P. C., Bahl, J., & Krammer, F. (2018). The influenza virus hemagglutinin head evolves faster than the stalk domain. Scientific Reports, 5(1). https: / / doi.org / 10.1038 / S41598-018-28706-l

[0249]

[0013] Koutsakos, M., Wheatley, A. K., Laurie, K., Kent, S. J., & Rockman, S. (2021). Influenza lineage extinction during the COVID-19 pandemic? Nature Reviews. Microbiology, 79(12), 741-742. https: / / doi.org / 10.1038 / S41579-021-00642-4

[0250]

[0014] Krammer, F., Smith, G. J. D., Fouchier, R. A. M., Peiris, M., Kedzierska, K., Doherty, P. C., Palese, P., Shaw, M. L., Treanor, J., Webster, R. G., & Garcia-Sastre, A. (2018). Influenza. Nature Reviews. Disease Primers, 4(0), 1-21. https: / / doi.org / 10.1038 / S41572-018-0002-Y

[0251]

[0015] Lu, Y., Welsh, J. P., & Swartz, J. R. (2014). Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines. Proceedings of the National Academy of Sciences of the United States of America, 777(1), 125-130. https: / / doi.org / 10.1073 / PNAS.1308701110

[0252]

[0016] Mallajosyula, V. V. A., Citron, M., Ferrara, F., Temperton, N. J., Liang, X., Flynn, J. A., & Varadarajan, R. (2015). Hemagglutinin Sequence Conservation Guided Stem Immunogen Design from Influenza A H3 Subtype. Frontiers in Immunology, 6(JUN), 329. https: / / doi.org / 10.3389 / FIMMU.2015.00329

[0253]

[0017] Paules, C. I., Lakdawala, S., McAuliffe, J. M., Paskel, M., Vogel, L., Kallewaard, N. L., Zhu, Q., & Subbarao, K. (2017). The Hemagglutinin A Stem Antibody MEDI8852 Prevents and Controls Disease and Limits Transmission of Pandemic Influenza Viruses. The Journal of Infectious Diseases, 216(3), 356-365. https: / / doi.org / 10.1093 / INFDIS / JIX292

[0018] Pica, N., & Palese, P. (2013). Toward a universal influenza virus vaccine: prospects and challenges. Annual Review of Medicine, 64, 189-202. https: / / doi.org / 10.1146 / ANNUREV-MED- 120611-145115

[0254]

[0019] Rathore, U., Kesavardhana, S., Mallajosyula, V. V. A., & Varadarajan, R. (2014). Immunogen design for HIV-1 and influenza. Biochimica et Biophysica Acta, 1844(11), 1891-1906. https: / / doi.Org / 10.1016 / J.BBAPAP.2014.05.010

[0255]

[0020] Rockman, S., Laurie, K. L., Parkes, S., Wheatley, A., & Barr, I. G. (2020). New Technologies for Influenza Vaccines. Microorganisms, 5(11), 1-20. https: / / doi.org / 10.3390 / MICROORGANISMS8111745

[0256]

[0021] Sauter, N. K., Hanson, J. E., Glick, G. D., Brown, J. H., Crowther, R. L., Park, S. J., Skehel, J. J., & Wiley, D. C. (1992). Binding of Influenza Virus Hemagglutinin to Analogs of Its Cell-Surface Receptor, Sialic Acid: Analysis by Proton Nuclear Magnetic Resonance Spectroscopy and X-ray Crystallography. Biochemistry, 31(40), 9609-9621. https: / / doi.org / 10.1021 / BI00155A013

[0257]

[0022] Steel, J., Lowen, A. C., Wang, T. T., Yondola, M., Gao, Q., Haye, K., Garcia- Sastre, A., & Palesea, P. (2010). Influenza virus vaccine based on the conserved hemagglutinin stalk domain. MBio, 7(1). https: / / doi.org / 10.1128 / MBIO.00018-10

[0258]

[0023] Xu, R., Ekiert, D. C., Krause, J. C., Hai, R., Crowe, J. E., & Wilson, I. A. (2010). Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science, 325(5976), 357-360. https: / / doi.org / 10.1126 / SCIENCE.1186430 / SUPPL FILE / XU.SOM.PDF

[0259]

[0024] Yang, H., Carney, P. J., Mishin, V. P., Guo, Z., Chang, J. C., Wentworth, D. E., Gubareva, L. V., & Stevens, J. (2016). Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses. Journal of Virology, 90(12), 5770-5784. https: / / doi.org / 10.1128 / JVI.00180- 16 / AS SET / 777AB98D-EB 17-4 IDE-88 Al -

[0260] 344 A70FE 16F4 / ASSETS / GRAPHIC / Z JV9991817130009. JPEG

Claims

I / We Claim:

1. An immunogenic polypeptide comprising a first polypeptide fragment (Fl) linked to a second polypeptide fragment (F2), and the second polypeptide fragment linked to a third polypeptide fragment (F3), wherein(i) the first polypeptide fragment (Fl) is selected from a group consisting of(a) a polypeptide corresponding to amino acid position 1-35 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 7;(b) a polypeptide corresponding to amino acid position 1-37 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 7;(c) a polypeptide corresponding to amino acid position 1-45 of a hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises substitution at position A19S of the hemagglutinin polypeptide; and(d) a polypeptide corresponding to amino acid position 1-36 of hemagglutinin polypeptide having amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11; ii) the second polypeptide fragment (F2) is selected from the group consisting of:(e) a polypeptide corresponding to amino acid position 272-319 of hemagglutinin polypeptide having an amino acid sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises amino acid substitutions at positions V272T, C275S, and BOOT of the hemagglutinin polypeptide;(f) a polypeptide corresponding to amino acid position 287-319 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises amino acid substitutions at positions BOOT, and C303S of the hemagglutinin polypeptide;(g) a polypeptide corresponding to amino acid position 271-318 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises amino acid substitutions at positions I294T, I299T, and C274S of the hemagglutinin polypeptide;(h) a polypeptide corresponding to amino acid position 286-318 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises amino acid substitutions at positions I294T, and C302S of the hemagglutinin polypeptide;(i) a polypeptide corresponding to amino acid position 307-321 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3, wherein the polypeptide comprises an amino acid substitution at position Y308H of the hemagglutinin polypeptide;(j) a polypeptide corresponding to amino acid position 274-321 of a hemagglutinin polypeptide having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 5, wherein the polypeptide comprises an amino acid substitution at position Y302H of the hemagglutinin polypeptide, and optionally a substitution at position C277S of the hemagglutinin polypeptide; and(k) a polypeptide corresponding to amino acid position 305-347 of a hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises amino acid substitutions at positions A315 S, 1318N, and C321 S of the hemagglutinin polypeptide; and(iii) the third polypeptide fragment (F3) is selected from the group consisting of:(l) a polypeptide corresponding to position 328-502 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises amino acid substitutions at positions F390D and L400D of the hemagglutinin polypeptide;(m) a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises amino acid substitutions at positions F392D and V402D of the hemagglutinin polypeptide;(n) a polypeptide corresponding to amino acid position 330-504 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polypeptide comprises amino acid substitutions at positions F408D and V419D of the hemagglutinin polypeptide;(o) a polypeptide corresponding to amino acid position 331-505 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 7, wherein the polypeptide comprises amino acid substitutions at positions F393D, L403D, and V396T of the hemagglutinin polypeptide; and(p) a polypeptide corresponding to amino acid position 348-510 of hemagglutinin polypeptide having a sequence of at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 11, wherein the polypeptide comprises amino acid substitutions at positions M417D and L420D of the hemagglutinin polypeptide,2. The immunogenic polypeptide as claimed in claim 1, wherein said Fl is linked to F2 by a linker LI, wherein the linker LI is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 22, SEQ ID NO. 25, and SEQ ID NO. 27, and wherein said F2 is linked to F3 by a linker L2, wherein the linker is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 13, SEQ ID NO. 18, SEQ ID NO. 25, and SEQ ID NO. 27.

3. The immunogenic polypeptide as claimed in claim 1, said immunogenic polypeptide having a formula selected from the group consisting of: a. F1-L1-F2-L2-F3 (Formula I); and b. Fl-Ll-F2-F3-L2-oligomerizati on domain (Formula II), wherein i) Fl has a sequence corresponding to positions 1-35 of SEQ ID NO: 1 or SEQ ID NO: 7, F2 has a sequence corresponding to positions 272-319 of SEQ ID NO: 1, and comprising amino acid substitutions at position V272T, C275S, and BOOT of SEQ ID NO: 1, and F3 has a sequence corresponding to positions 328-502 of SEQ ID NO: 1; and comprising amino acid substitutions at positions F390D and L400D of SEQ ID NO: 1, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 13 and L2 has a sequence of SEQ ID NO. 18; ii) Fl has a sequence corresponding to positions 1-37 of SEQ ID NO: 1 or SEQ ID NO: 7, F2 has a sequence corresponding to positions 287-319 of SEQ ID NO: 1 and comprising amino acid substitutions at positions BOOT and C303S of SEQ ID NO: 1, and F3 has a sequence corresponding to positions 328-502 of SEQ ID NO: 1 and comprising amino acid substitutions at positions F390D and L400D of SEQ ID NO:1, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 22 and L2 has a sequence of SEQ ID NO. 18; iii)F 1 has a sequence corresponding to positions 1-45 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitution at position A19S of SEQ ID NO: 3 or SEQ ID NO: 5, F2 has a sequence corresponding to positions 307-321 of SEQ ID NO: 3 and comprising amino acid substitution at position Y308H of SEQ ID NO: 3, and F3 has a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitutions at positions F392D and V402D of SEQ ID NO: 3 or SEQ ID NO: 5, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 25 and L2 has a sequence of SEQ ID NO. 27; iv)Fl has a sequence corresponding to positions 1-45 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitution at position A19S of SEQ ID NO: 3 or SEQ ID NO: 5, F2 has a sequence corresponding to positions 274-321 of SEQ ID NO: 5 and comprising amino acid substitution at position Y302H of SEQ ID NO: 5, and F3 has a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitutions at positions F408D and V419D of SEQ ID NO: 3 or SEQ ID NO: 5, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 13 and L2 has a sequence of SEQ ID NO. 18; v) Fl has a sequence corresponding to positions 1-45 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitution at position A19S of SEQ ID NO: 3 or SEQ ID NO: 5, F2 has a sequence corresponding to positions 274-321 of SEQ ID NO: 5 and comprising amino acid substitutions at positions Y302H and C277S of SEQ ID NO: 5, and F3 has a sequence corresponding to positions 330-504 of SEQ ID NO: 3 or SEQ ID NO: 5 and comprising amino acid substitutions at positions F408D and V419D of SEQ ID NO: 3 or SEQ ID NO: 5, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 13 and L2 has a sequence of SEQ ID NO. 18; vi) Fl has a sequence corresponding to positions 1-35 of SEQ ID NO: 1 or SEQ ID NO: 7, F2 has a sequence corresponding to positions 271-318 of SEQ ID NO: 7 and comprising amino acid substitutions at positions I294T, I299T, and C274S of SEQ ID NO: 7, and F3 has a sequence corresponding to positions 331-505 of SEQ ID NO: 7 and comprising amino acid substitutions at positions F393D, L403D, and V396T of SEQ ID NO: 7, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 13 and L2 has a sequence of SEQ ID NO. 18;vii) Fl has a sequence corresponding to positions 1-37 of SEQ ID NO: 1 or SEQ ID NO: 7, F2 has a sequence corresponding to positions 286-318 of SEQ ID NO: 7 and comprising amino acid substitutions at positions I294T and C302S of SEQ ID NO: 7, and F3 has a sequence corresponding to positions 331-505 of SEQ ID NO: 7 and comprising amino acid substitutions at positions F393D, L403D, and V396T of SEQ ID NO: 7, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 22 and L2 has a sequence of SEQ ID NO. 18; or viii) Fl has a sequence corresponding to positions 1-36 of SEQ ID NO: 9 or SEQ ID NO: 11, F2 has a sequence corresponding to positions 305- 347 of SEQ ID NO: 9 or SEQ ID NO: 11 and comprising amino acid substitutions at positions A315S, 1318N, and C321S SEQ ID NO: 9 or SEQ ID NO: 11, and F3 has a sequence corresponding to positions 348-510 of SEQ ID NO: 9 or SEQ ID NO: 11 and comprising amino acid substitutions at positions M417D and L420D of SEQ ID NO: 9 or SEQ ID NO: 11, wherein LI and L2 are linkers, preferably LI has a sequence of SEQ ID NO. 27 and L2 has a sequence of SEQ ID NO. 13.

4. The immunogenic polypeptide as claimed in claim 1, wherein the immunogenic polypeptide is selected from:(a) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 37, and comprising amino acids 44T, 47S, 72T, 158D, and 168D at positions corresponding to SEQ ID NO: 37;(b) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO:39, and comprising amino acids 56T, 59S, 142D, and 152D at positions corresponding to SEQ ID NO: 39;(c) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO:41, and comprising amino acid substitutions 40S, 73H, 153D, and 163D at positions corresponding to SEQ ID NO: 41;(d) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO:43, and comprising amino acid substitution 21S, 82H, 184D, and 195D at positions corresponding to SEQ ID NO: 43;(e) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 45, and comprising amino acid substitution 21 S, 57S, 82H, 184D, and 195D at positions corresponding to SEQ ID NO: 45;(f) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO: 47, and comprising amino acid substitutions 46S, 66T, 71T, 157D, 160T, and 167D at positions corresponding to SEQ ID NO: 47;(g) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in SEQ ID NO; 49, and comprising amino acid substitution 50T, 58S, 141D, 144T, and 151D at positions corresponding to SEQ ID NO: 49; and(h) a polypeptide having an amino acid sequence of at least 95% sequence identity to a sequence as set forth in and SEQ ID NO: 51, and comprising amino acid substitution 52S, 55N, 58S, 154D, and 157D at positions corresponding to SEQ ID NO: 51.

5. The immunogenic polypeptide as claimed in claim 1, further comprising an oligomerization domain at the N-terminal or C-terminal of the immunogenic polypeptide.

6. The immunogenic polypeptide as claimed in claim 5, wherein the oligomerization domain is a foldon having an amino acid sequence as set forth in SEQ ID NO: 33, linked to the third polypeptide fragment (F3) by a linker.

7. The immunogenic polypeptide as claimed in claim 1, wherein the immunogenic polypeptide has an amino acid sequence of at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO:41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NO: 51.

8. The immunogenic polypeptide as claimed in claim 1, comprising a combination of at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 immunogenic polypeptides having amino acid sequence of at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO:41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NO: 51.

9. The immunogenic polypeptide as claimed in claim 1 or claim 8, in combination with other HA containing influenza vaccine formulation.

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

11. The polynucleotide as claimed in claim 10, wherein the polynucleotide is 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. 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, and SEQ ID NO. 52.

12. A DNA construct comprising the polynucleotide as claimed in any one of claims 10 to 11.

13. The DNA construct as claimed in claim 12, further comprising a polynucleotide encoding a TPA signal peptide; an oligomerization domain; an HRV3C protease cleavage site or a portion thereof; a Histidine tag; one or more linkers; or combinations thereof.

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

15. A recombinant bacterial host cell comprising the vector as claimed in claim 14.

16. The recombinant bacterial host cell as claimed in claim 15, wherein the bacterial host cell is E.coli.

17. A vaccine composition comprising the immunogenic polypeptide as claimed in anyone of the claims 1-8, and a pharmaceutically acceptable carrier.

18. The vaccine composition as claimed in claim 17, wherein the composition comprises a combination of at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 immunogenic polypeptides of having amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO; 49, and SEQ ID NO: 51.

19. The vaccine composition as claimed in claim 17 or 18, further combined with other HA containing influenza vaccine composition.

20. The vaccine composition as claimed in claim 17, comprising an adjuvant.

21. The vaccine composition as claimed in claim 17, wherein the vaccine composition is a lyophilized composition.

22. A method for producing the vaccine composition as claimed in claim 17, wherein the method comprises: (a) culturing the recombinant bacterial host cell as claimed in 15 or 16 under suitable conditions to obtain the immunogenic polypeptide as claimed in any one of the claims 1-8; (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.

23. A method for eliciting an immune response against influenza infection in a subject, the method comprising administering an effective amount of the vaccine composition as claimed in claim 17 to the subject.

24. The method as claimed in claim 23, wherein the vaccine composition is administered by a mode selected from the group consisting of intranasal, parenteral, subcutaneous, intramuscular, and intradermal.

Images