A universal influenza vaccine candidate and its uses thereof

Abstract

The Invention provides a vaccine candidate for universal Influenza vaccine and its uses thereof. Particularly, the invention provides a modified Influenza soluble HA (Hemagglutinin) monomer, trimer and a combination of monomer and trimer as a subunit vaccine candidate towards universal Influenza vaccine development and its uses and methods thereof.

Description

[0001] A UNIVERSAL INFLUENZA VACCINE CANDIDATE AND ITS USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of biotechnology, particularly the present invention relates to an Influenza HA (Hemagglutinin) vaccine and its uses thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Vaccination is considered as most effective way of treatment or prevention of disease. The available influenza vaccines tend to generate antibodies against circulating strains of viruses and as the virus replicates it undergoes random copying of errors, known as genetic mutations. Due to these genetic mutations the virus surface proteins or antigens are altered, which renders the pre-existing influenza vaccines ineffective overtime. The Hemagglutinin (HA) influenza virus consist of a globular head and stalk domain in trimeric form which generates subtype specific response and provides protection against limited subset of virus. The epitopes present on the outer / exposed surface of virus in trimeric form are not conserved across the strain and keeps on changing with time. Hence the vaccines using the trimeric form HA influenza virus are ineffective for treating a broad range of strains and requires constant updation over the time.

[0006] The HA influenza virus shows a breathing conformation, in which the HA subunits of virus opens up displaying the conserved epitopes at the trimeric interface of influenza virus, often remains occluded during the trimeric display of HA. The epitopes are capable of stimulating an immune response and lies at an interface between two protomers on an intact trimer. The epitopes lying inside the structure of the virus are conserved across various strains. Hence, a monomer based stable vaccine candidates displaying the trimeric interface region and occluded epitopes is a promising candidate.

[0007] In prior art, monomer A / Thailand / CU44 / 2006 developed from seasonal strain produced through baculovirus expression system using Spodoptera frugiperda (Sf-9, insect cells) with gp67 secretion signal sequence is present. The monomer developed, upon immunization as compared with the trimer was found to elicit low antibody titers in sera. The monomer showed protection only against PR8; A / PR8(HINI) virus, a homologous virus (H1N1). The monomers present in prior art were found to be less effective in comparison to the trimeric vaccine candidates. Hence, an effective universal, broadly protective influenza vaccine which is capable of eliciting broadly cross-reactive and hetero-subtype antibodies against different circulating strains are required. There is a need for a vaccine candidate, which emphasize on an open trimer and a stabilized monomer based HA protein immunogen displaying structurally occluded conserved epitopes acting as a universal vaccine candidate against a broad range of viruses thereby eliminating the need of constant updation.

[0008] OBJECT OF THE INVENTION

[0009] An object of the invention is to provide an engineered monomer or trimer, or a combination of monomer and trimer based immunogen displaying structurally occluded conserved epitopes of HA (Hemagglutinin) of influenza virus acting as a universal influenza vaccine candidate, a method, a composition and its utility.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention discloses an engineered pCDNA-HA open trimer of influenza Hemagglutinin (HA) protein immunogen of amino acid SEQ ID No. 2 and nucleotide SEQ ID No. 5, wherein the said immunogen is modified by the introduction of F88E and I91W mutations in the head region of pCDNA-HA trimer of amino acid SEQ ID No. 1 and nucleotide SEQ ID No. 4 .

[0012] The present invention also discloses an engineered codon optimized monomer of influenza Hemagglutinin (HA) protein immunogen of amino acid SEQ ID No. 3 and nucleotide SEQ ID No. 6.

[0013] The present invention further discloses a combination of pCDNA-HA open trimer and monomer of influenza Hemagglutinin (HA) protein immunogen in the range of (0 -100)% to (100 - 0) % of the said combination .

[0014] The present invention discloses a method for developing an open trimer and monomer based hemagglutinin (HA) immunogens displaying structurally occluded conserved epitopes of HA influenza virus. The method comprising:

[0015] 1. Designing of DNA Expression cassette; 2. Transient expression and purification of protein generated through DNA expression cassette.

[0016] The present invention discloses an immunogenic vaccine composition of a pCDNA-HA open trimer or a monomer or a combination of a pCDNA-HA open trimer and monomer along with adjuvants for vaccine formulation.

[0017] The present invention further discloses the use of the immunogenic composition of monomer or a trimer or a combination of monomer and a trimer based hemagglutinin (HA) immunogen displaying structurally occluded conserved epitopes of HA influenza virus as a universal vaccine candidate acting against broad range of virus strains.

[0018] The following brief description of the figures of the drawings exemplify the invention to understand the invention clearly and easily and it should not be construed to limit / restrict the scope of the invention.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 depicts domain organization and concept of design of; a. pCDNA-HA trimer (HA trimer) construct; b. modified pCDNA-HA trimer (HA trimer*, Open trimer) construct and c. monomeric soluble HA protein immunogen.

[0021] Figure 2 depicts a gel filtration profile of pCDNA-HA trimer construct; a. shows the pCDNA-HA trimer construct purified through Ni-NTA; b. shows the Western blot of HA trimer purified fraction from Ni NTA and c. shows the Western blot of HA trimer purified fraction from Ni NTA and gel filtration chromatography.

[0022] Figure 3 depicts the gel filtration profile of pCDNA-HA trimer construct (black) and monomeric soluble HA protein immunogen (Red); Blue native page in inset show the oligomeric status of the two protein. Western blot of purified HA trimer, modified HA trimer and HA monomer, Influenza A virus H1N1 HA antibody (GTX-127357), C102 monoclonal (RBS directed, GTX - 28262) and FR-496 (Influenza reagent research).

[0023] Figure 4A depicts the binding profile of pCDNA-HA trimer construct and monomeric soluble HA protein immunogen with broadly neutralizing antibody CR 6261 and a table showing kinetic parameter measured on Biolayer Interferometry (BLI).

[0024] Figure 4B depicts the binding profile of pCDNA-HA trimer and modified pCDNA-HA trimer protein immunogen showing binding with monoclonal antibody FluA-20 and table showing kinetic parameter measured using BLI.

[0025] Figure 5 depicts the immunization and challenge schedule, groups, antigen, antigen dose administered, adjuvant and route of administration for pCDNA-HA trimer construct; monomeric soluble HA protein immunogen and control PBS group.

[0026] Figure 6 depicts a humoral response post immunization from the three groups: pCDNA- HA trimer construct; monomeric soluble HA protein immunogen and control PBS group. Figure 7 depicts the immunization schedule, groups, antigen, antigen dose administered, adjuvant and route of administration for pCDNA-HA trimer* construct, also called as open trimer; and control PBS group.

[0027] Figure 8 depicts a humoral response post immunization from: pCDNA-HA trimer* and control PBS group.

[0028] Figure 9 depicts the IgG Isotyping of the sera from the two immunized groups; pCDNA- HA trimer construct and monomeric soluble HA protein immunogen.

[0029] Figure 10 depicts the IgG Isotyping of the sera from the pCDNA-HA trimer* construct and protein immunogen.

[0030] Figure 11 depicts the cellular response of the splenocytes of the immunized animals under unstimulated condition.

[0031] Figure 12 depicts the cellular response of the splenocytes of the immunized animals stimulated with monomeric soluble HA protein immunogen.

[0032] Figure 13 depicts the cellular response of the splenocytes of the immunized animals stimulated with pCDNA-HA trimer construct.

[0033] Figure 14 depicts the effector versus memory response of unstimulated splenocytes.

[0034] Figure 15 depicts the effector versus memory response of splenocytes stimulated with monomeric soluble HA protein immunogen.

[0035] Figure 16 depicts the effector versus memory response of splenocytes stimulated with HA Trimer.

[0036] Figure 17 depicts the weight profile of the immunized and control animals post challenge with PR8 and X31 virus.

[0037] Figure 18 depicts histopathological score in lymphocytes, Neutrophils and Inflammation.

[0038] Figure 19 depicts the image of LUNG of HA Trimer Immunized Group.

[0039] Figure 20 depicts the image of LUNG of HA Monomer Immunized Group.

[0040] Figure 21 depicts the image of LUNG of Control Group.

[0041] Figure 22 depicts the schematic of serum depletion assay for pCDNA- HA trimer construct and monomeric soluble HA protein immunogen. Figure 23 depicts ELISA performed with plates coated with pCDNA- HA trimer construct and monomeric soluble HA protein immunogen of the depleted sera after each round of depletion and Western blot showing lot of 3 proteins; pCDNA-HA trimer construct; modified pCDNA-HA trimer construct and monomeric soluble HA protein immunogen with 3 different sera.

[0042] Figure 24 depicts the confocal images of the A549 cells infected with PR8 (A / Puerto Rico / 8-MC / 1934 (H1N1)) virus with immunized and depleted sera.

[0043] Figure 25 depicts the confocal images of the A549 cells infected with X31 (A / X-31(H3N2)) virus with immunized and depleted sera.

[0044] Figure 26 depicts the confocal images of the A549 cells infected with A / California / 04 / 2009 (HlNl)pdmO9 with immunized and depleted sera.

[0045] Figure 27 depicts the confocal images of the A549 cells infected with A / Hong Kong / 1 / 1968 (H3N2) virus with immunized and depleted sera.

[0046] Figure 28 depicts the Fab of 8H10 antibody docked with HA monomer sequences.

[0047] Figure 29 depicts the FL-1056 Fab antibody docked with HA monomer sequences.

[0048] Figure 30 depicts 1066 Fab antibody docked with HA monomer sequences.

[0049] Figure 31 depicts CR9114 Fab antibody docked with HA monomer sequences.

[0050] Figure 32 depicts FluA-20 Fab antibody docked with HA monomer sequences.

[0051] Figure 33 depicts S5V2-29 Fab antibody docked with HA monomer sequences.

[0052] Figure 34 depicts SAXS structure solution raw file of the Complex (Pymol) through the DAMMFF bead model.

[0053] Figure 35 depicts SAXS structure showing fitted model of the Complex.

[0054] Figure 36 depicts SAXS structure solution of the complex.

[0055] DETAILED DESCRIPTION

[0056] The present invention discloses an engineered pCDNA-HA open trimer of influenza Hemagglutinin (HA) protein immunogen of amino acid SEQ ID No. 2 and nucleotide SEQ ID No. 5, wherein the said immunogen is modified by the introduction of F88E and I91W mutations in the head region of pCDNA-HA trimer of amino acid SEQ ID No. 1 and nucleotide SEQ ID No. 4 .

[0057] The present invention also discloses an engineered codon optimized monomer of influenza Hemagglutinin (HA) protein immunogen of amino acid SEQ ID No. 3 and nucleotide SEQ ID No. 6.

[0058] The present invention discloses a combination of pCDNA-HA open trimer and monomer of influenza Hemagglutinin (HA) protein immunogen in the range of (0 -100) % to (100 - 0)% of the said combination.

[0059] The present invention discloses a method for developing a monomer based hemagglutinin (HA) immunogen displaying structurally occluded conserved epitopes of HA influenza virus. The method comprises the steps of :

[0060] 1. Designing of DNA Expression cassette;

[0061] 2. Transient expression and purification of DNA expression cassette.

[0062] 1. Designing of DNA Expression cassette The DNA Expression cassette designing further comprising: a. preparing a trimer construct as set forth in SEQ ID No. 1, b. modifying the pCDNA-HA trimer construct of step (a) by the introduction of mutations in the head region of the HA protein, resulting in an open trimer represented by SEQ ID No. 2, c. cleaving the modified pCDNA-HA open trimer of step (b) to obtain the engineered codon optimized influenza HA protein immunogen.

[0063] A pCDNA-HA trimer construct as set forth in SEQ ID No. 1 was prepared using a codon optimized (optimized for expression in mammalian host) HA expressing 504 amino acid sequences of A / California / 07 / 2009(H1N1) strain (NCBI Reference Sequence: YP 009118626.1) comprising N terminal starting from DTLCIGYHANNSTD and C terminal ending at YSEEAKLNREEIDGVK of HA protein. Two amino acid mutations using site directed mutagenesis in the 504 amino acid sequences of HA protein was introduced in pCDNA-HA trimer to form a modified pCDNA-HA trimer by using the codon optimized HA expressing 504 amino acid sequences. The two mutations are F88E and I91W, wherein the two mutations comprise an amino acid sequence set forth in SEQ ID No. 2. The monomer HA protein immunogen comprises amino acid sequence set forth in SEQ ID No. 3, wherein the HA protein immunogen is stabilized by the introduction of mutations in the HA protein, maintains monomeric conformation in solution and the SAXS structure. As used herein, monomeric expression construct of hemagglutinin (HA) protein of Influenza A virus or a monomeric soluble HA protein immunogen herein after used interchangeably. Transient expression and purification of DNA expression cassette

[0064] The pCDNA-HA trimer construct and the modified pCDNA-HA trimer construct was transformed into E.coli strain based on DH5a competent cells to collect purified plasmid DNA . The purified plasmid of pCDNA-HA trimer construct and modified pCDNA-HA trimer construct was transfected into Expi293 Cells , producing the secretory protein in the transfected media. The purified pCDNA-HA trimer was then concentrated and loaded on gel filtration column equilibrated in PBS, wherein the protein fractions of the purified pCDNA- HA trimer was eluted from column, wherein the purified Ni-NTA purified fractions of the pCDNA-HA trimer was dialyzed, and wherein Histag and foldon domain was cleaved from the dialyzed protein by Thrombin protease by overnight incubation in the presence of CaCh to form the digested protein, and purifying the digested protein by loading on the gel filtration column to form stabilized HA monomer as set forth in SEQ ID No. 3.

[0065] The SEQ ID No. 4 represents nucleotide sequence for pCDNA-HA trimer construct; SEQ ID No. 5 represents nucleotide sequence for modified pCDNA-HA trimer construct and SEQ ID No. 6 represents nucleotide sequence for HA monomer.

[0066] The engineered codon optimized monomer soluble influenza HA protein immunogen is generated through protease cleavage reaction of a modified pCDNA-HA trimer as set forth in SEQ ID No. 5 to form an immunogenic composition.

[0067] The method of preparing a pCDNA-HA trimer construct comprising the steps of: forming an expression cassette, wherein a designed construct comprising the codon optimized HA protein sequences of A / Califomia / 07 / 2009(H1N1) influenza virus strain cloned between BamHI and Xhol of pCDNA 3.1+ expression vector, wherein N terminal signal sequences “MKAILVVLLYTFATANA” of HA protein is removed from the amino acid sequence and replaced with CD5 leader sequences “MPMGSLQPLATLYLLGMLVASVLA” as set forth in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3; and wherein the expression construct comprises 2 amino acid linker VE, following CD5 sequences; thrombin cleavage site, LVPRGS; 3 amino acids linker GSG after thrombin cleavage site; foldon trimerization motif sequences, “YIPEAPRDGQAYVRKDGEWVLLSTFL”; single amino acid G after foldon domain; and 6 Histidine residues. The N terminal signal sequences are removed from the designed construct and replaced with CD5 leader sequences. As used herein, the N terminal sequences are “MKAILVVLLYTFATANA” and the CD5 leader sequences are MPMGSLQPLATLYLLGMLVASVLA. The codon optimized (optimized for expression in mammalian host) 504 amino acid sequences selected from the amino acid number 18 to 521 of A / Califomia / 07 / 2009(H1N1) HA protein; N terminal starting from DTLCIGYHANNSTD and C terminal ending at YSEEAKLNREEIDGVK were used in this design. The expression construct contains 2 amino acid linker VE. VE is added after CD5 leader sequences and amino acids for the thrombin cleavage site LVPRGS added after the coding sequences of HA protein. A linker of 3 amino acids GSG is added after the thrombin cleavage site, followed by foldon trimerization motif sequences are added. As used herein the foldon trimerization motif sequences are YIPEAPRDGQAYVRKDGEWVLLSTFL. Thereafter, a single amino acid G added after the Foldon motif, followed by 6 Histidine residues. The resultant expression cassette is cloned into pCDNA 3.1+ expression vector in BamHI and Xhol site. The designed construct is first sequence verified by DNA sequencing (with T7 forward and BGH reverse primer). This designed construct is referred as pCDNA-HA trimer.

[0068] The preparation of the modified pCDNA-HA trimer construct comprising identifying F88 and 191 residue into the A / Califomia / 07 / 2009(HlNl)sequence of the HA protein; and mutating the F88 and 191 residue of the amino acid sequence of the HA protein to generate F88E and 191W mutations as the modified pCDNA-HA trimer construct.

[0069] The binding kinetic assays of monoclonal antibody FluA-20 with modified pCDNA-HA Trimer (Open Trimer), monomer or a combination of pCDNA-HA open trimer and monomer are performed using Octet Red96 instrument (ForteBio, Inc., USA). The modified pCDNA- HA trimer, monomer or a combination of pCDNA-HA open trimer and monomer binds to the monoclonal antibody FluA-20 with nano-molar affinity. The binding of monoclonal antibody CR6261 to HA trimer, HA monomer or a combination of pCDNA-HA open trimer and monomer are performed using anti-human Fc sensors (AHC). The modified pCDNA-HA trimer, monomer or a combination of pCDNA-HA open trimer and monomer, binds to neutralizing antibody CR6261 in nano-molar affinity.

[0070] The present invention further discloses a highly synergistic vaccine composition of a pCDNA- HA open trimer or a monomer or a combination of a pCDNA-HA open trimer and monomer along with adjuvants for vaccine formulation. The adjuvant according to the present invention is an emulsion, in particular an oil-in-water emulsion, and may optionally contain other immunostimulants. The oil-in-water emulsion comprises a metabolizable non-toxic oil such as squalene, and optionally an emulsifier (i.e., a surfactant) such as poly-oxyethylene sorbitan monooleate (TWEEN-80 ™ or POLYSORBATE 80 ™) which is a non-ionic surfactant. More preferably the adjuvant is a squalene-based oil-in- water nano- emulsion. The squalene-based oil-in-water nano-emulsion is selected from a group comprising C30H50 (USP), AS150, AS110, AS40 (SDA BIO), Squalene (CD creative Diagnostics), MF-59 (MedchemExpress.com), AddaVax (Invivogen). Most preferably the squalene-based oil-in-water nano-emulsion is AddaVax.

[0071] The purified proteins in PBS (phosphate buffered saline) is formulated with a squalene-based oil-in-water nano-emulsion, in a ratio of 1 : 1 (protein in PBS : Adda Vax) to form a vaccine candidate.

[0072] The purified proteins is dissolved in a buffer and formulated with a squalene-based oil-in- water nano-emulsion, in a ratio of 1 : 1 (protein in buffer: adjuvant) to form a vaccine candidate.

[0073] The buffers can be selected from the group comprising phosphate, citrate, acetate, histidine, succinate, gluconate, glycine and borate , more preferably the buffer used is a phosphate buffer, most preferably the buffer used is PBS (phosphate buffer saline).

[0074] The modified pCDNA-HA Trimer (Open Trimer), monomer or a combination of pCDNA- HA open trimer and monomer as disclosed in present invention shows very high efficacy in the treatment of disease caused by HA influenza virus and the composition is highly synergistic in nature.

[0075] The present invention further discloses a method of treating or preventing an infection caused by the HA influenza virus in a living host having or susceptible to such infection, wherein the said method comprises administering the immunogenic composition intramuscularly, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intraci stemally, or rectally, in liquid dosage forms. The present invention also discloses the use of immunogenic composition in preventing disease or infections caused by the different subtypes of influenza viruses such as respiratory tract infection of the nose, throat and lungs, bacterial pneumonia, ear infections, sinus infections and worsening of chronic medical conditions, such as congestive heart failure, asthma, or diabetes.

[0076] Without being limited by scope, the identification of conserved epitopes of HA influenza virus or epitopes with limited variability offers a mechanism towards universal influenza vaccine development. Such vaccines would also have the potential to protect against newly emerging influenza strains. The immunogenic composition is a candidate for an influenza vaccine, hence forth the immunogen and its uses as vaccine candidates thereof.

[0077] EXAMPLES:

[0078] Example 1: DNA expression cassette designing

[0079] The hemagglutinin (HA) protein; Influenza A virus; (A / California / 07 / 2009(H1N1)), NCBI Reference Sequence: YP 009118626.1, was selected for the studies. The expression cassette was designed by removing N terminal signal sequences “MKAILVVLLYTFATANA” and were replaced with CD5 leader sequences; MPMGSLQPLATLYLLGMLVASVLA. The codon optimized (optimized for expression in mammalian host) 504 amino acid sequences selected from the amino acid number 18 to 521 of A / California / 07 / 2009(H1N1) HA protein; N terminal starting from DTLCIGYHANNSTD and C terminal ending at YSEEAKLNREEIDGVK were used in this design. The expression construct contains 2 amino acid linker; VE adder after CD5 leader sequences and amino acids for the thrombin cleavage site; LVPRGS were added after the coding sequences of HA protein. A linker of 3 amino acids GSG was added after the thrombin cleavage site, followed by foldon trimerization motif sequences; YIPEAPRDGQAYVRKDGEWVLLSTFL were added. A single amino acid G was added after the Foldon motif, followed by 6 Histidine residues. The resultant expression cassette was cloned into pCDNA 3.1+ expression vector in BamHI and Xhol site. The designed construct was first sequence verified by using T7 forward and BGH reverse primer. This designed construct is called as HA trimer herein after.

[0080] The HA monomer expression cassette was designed by introducing F88E and 191W mutation in the HA trimer expression cassette site directed mutagenesis using primers; F88E F 5'- CCT GAA CAA GAA GGT GGA CGA CGG CGA GCT GGA CAT CTG GAC CTA TAA TGC C -3'

[0081] F88E R 5'- GGT CCA GAT GTC CAG CTC GCC GTC GTC CAC CTT CTT GTT CAG GTT CTC -3'

[0082] DM9 1WF 5'- GAA CAA GAA GGT GGA CGA CGG CGA GCT GGA CTG GTG GAC

[0083] CTA TAA TGC CGA G -3'

[0084] DM9 1WR 5'- CAG CTC GGC ATT ATA GGT CCA CCA GTC CAG CTC GCC GTC GTC CAC CTT CTT G -3'

[0085] Two mutations were introduced sequentially; the mutated sequences were verified using DNA sequencing after each mutation using BGH reverse primer, the resultant expression cassette is called here as HA trimer*or modified HA Trimer.(Figure 1)

[0086] Example 2: Transient expression and purification of DNA expression cassette

[0087] Following sequence conformation, the pCDNA-HA Trimer and modified pCDNA-HA Trimer constructs were transformed in E. Coli strain based DH5a competent cells and plated on LB Agar plate with Ampicillin as resistant marker. Single colony for each respective expression construct was picked from the plate and sub-cultured in LB broth for overnight. 500ml SB culture was inoculated with 1% of the overnight grown primary culture and was further cultured for overnight. Following morning the harvested culture was processed for the purification of plasmids (pCDNA-HA Trimer and modified pCDNA-HA Trimer) through cesium preparation methodology and the purified plasmid DNA was further used for transfection and protein production.

[0088] For transfection and protein production Expi 293 cells sub-cultured by seeding in shaker flasks at 1 x 106viable cells / ml in 20 ml of pre-warmed Expi293 expression medium and incubated the culture flasks in a 37°C incubator containing a humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 110 rpm. The Expi293 were cultured till it reached the density of approximately 4-5 x 106viable cells / ml. For transfection, cells were diluted to 2.8 x 106cells / ml of density with total volume of 300ml in 1000ml capacity flasks and were seeded in the flask for Ihrs before transfection in fresh pre warmed media. 300 pg of plasmid DNA mixed in 5 ml DMEM® media in 50 ml falcon tube and in another tube 750pl of Expifectamine™ Transfection Reagent in 5ml of DMEM® in 50 ml falcon tube (with 1 :2.5, DNA to plasmid ratio). It was then mixed gently and incubated for 25 minutes at room temperature. After 25 minutes of incubation at room temperature the DNA- Expifectamine™ complex was added into the prepared cell suspension. 5 day Post incubation, the transfected media / supematant was harvested from the culture by spinning at 9000RPM for lOMins and used for the purification of protein.

[0089] The harvested supernatant was loaded on to Ni-NTA column (5ml column resin) equilibrated with 50mM Tris pH 7.4, 500mM NaCl, lOmM Imidazole, through gravitational flow. The column was washed with approximately 200ml of 50mM Tris pH 7.4, 500mM NaCl and lOmM Imidazole followed by 50mM Tris, 500mM NaCl and lOmM Imidazole for another 100ml and subsequently eluted from the column with 50mM Tris pH 7.4, 500mM NaCl and 500mM Imidazole. The purity of purified protein was verified and checked using SDS PAGE (Figure 2). The fraction of HA trimer purified from the Ni-NTA columns were further concentrated and loaded on the gel filtration column equilibrated in PBS, the protein fractions eluted from 9.8ml to 15ml of column volume. HA trimer*(HA*) or modified HA trimer (HA*) once purified from the Ni-NTA column, dialyzed against the PBS, the foldon trimerization domain from the dialyzed protein was Histag cleaved from the purified protein using Thrombin protease in an overnight incubation in presence of CaCE. The overnight digested protein was further loaded onto gel filtration column, this purification generated the stabilized monomer that is called as HA monomer. The purified proteins were snap-frozen in liquid nitrogen and stored at -80 °C until further use. (Figure 3)

[0090] Example 3: Western blot analysis

[0091] The purified protein samples for analysis were run on 12 % of SDS-PAGE. The gel was run for 2 hrs at 120 Volts for proper resolution of protein. Resolved proteins in gel were then transferred to PVDF membrane. The membrane was blocked with 5% skimmed milk, incubated with Anti-His antibody (SAB1306082, Sigma-Aldrich, ANTI-HIS TAG antibody produced in rabbit, dilution 1 : 1000), Influenza A virus H1N1 HA antibody (Rabbit Polyclonal antibody, GeneTex: GTX-127357, 1 : 1000 dilution), C102 monoclonal (RBS directed, Mouse Monoclonal, GeneTex: GTX - 28262) and FR-496 (Mouse Monoclonal Antibody to Recombinant Hl HA from Influenza A / Brisbane / 59 / 2007 (H1N1), Clone AT163.333.93, Influenza reagent research, 1 : 1000 dilution) for overnight at 4°C. The membrane was incubated with HRP conjugated anti-mouse (1 :2000) and anti-rabbit secondary (1 :2000) and developed using Femtolucent Plus HRP (G Biosciences).

[0092] Example 4: Binding kinetic Assay Binding kinetics assays were done using Octet Red96 instrument (ForteBio, Inc., USA). The binding of monoclonal antibody CR6261 to HA trimer and HA Monomer was performed using anti-human Fc sensors (AHC). CR6261 at a concentration of I Opg / ml were captured on AHC sensors for 150sec. HA protein as analyte was 3 fold serially diluted in kinetic buffer (PBS with 0.02 % Tween 20 and 0.1 % BSA) and 200ul of diluted analyte was dispensed into 96-well microtiter plates per well. Antibodies immobilized sensors were immersed in diluted analytes for 300sec to record association. The dissociation was recorded by transferring the sensors to wells containing buffer for 600 sec. Data were collected was analyzed using ForteBio Data Analysis software, 9.0 (ForteB ioInc). 1 : 1 global curve-fitting model was used for the analysis of antibodies with one to one binding stoichiometry. Whole experimental procedures were performed at RT. The fittings were considered to be satisfactory if %2 < 0.5. Standard deviation was calculated from three independently performed experiments. The HA trimer and mHA binds to the bnAb CR6261 with 0.48 x 10'12M and 0.71 x 10'9M affinity respectively (Figure 4A).

[0093] The binding kinetics of monoclonal antibody FluA-20 with modified Trimer (Open Trimer) was performed using Octet Red96 instrument (ForteBio, Inc., USA). The binding of FluA-20 with HA trimer and modified HA trimer (HA Trimer* also called as open trimer) was performed using anti-human Fc sensors (AHC). FluA-20 at a concentration of lOpg / ml were captured on AHC sensors for 120sec. The HA trimeric and modified HA trimeric protein as analyte was 3 fold serially diluted in kinetic buffer (PBS with 0.02 % Tween 20 and 0.1 % BSA) and 200ul of diluted analyte was dispensed into 96-well microtiter plates per well. Antibodies immobilized sensors was immersed in wells containing HA protein to record the association. The dissociation was performed the kinetic buffer. Data were analyzed using ForteBio Data Analysis software, 9.0 (ForteB ioInc). 1 : 1 global curve-fitting model was used for the analysis. Whole experimental procedures were performed at RT, the fitting was considered as satisfactory with %2 < 0.5. Standard deviation was calculated from three independently performed experiments. The HA trimer did not show any binding, the modified HA trimer (open trimer) binds to the monoclonal antibody FluA-20 with 0.564 x 10'9M respectively (Figure 4B)

[0094] EXAMPLE 5: VACCINE COMPOSITION OF THE PRESENT INVENTION WITH MONOMERIC HA PROTEIN

[0095] A protein in PBS solution was obtained by dissolving 30pg protein in lOOpl of PBS. Further, a synergistic vaccine formulation was prepared by using the protein in PBS solution and lOOpl of adjuvant, preferably AddaVax. The purified proteins solution in PBS as vaccine candidate were mixed with adjuvant (Protein in PBS AddaVax) at 1 :1 ratio.

[0096] EXAMPLE 6: EXAMINATION OF THE IMMUNOGENIC POTENTIAL OF THE HA MONOMER OF THE PRESENT INVENTION

[0097] Example 6.1: Evaluation of immunogenicity in animals.

[0098] Animal studies were performed as per Institutional Animal Ethics Committee (IAEC) approval. The mice immunization studies were performed under the approval number lAEC / THSTUlOO; entitling project “Immunogenicity assessment of H1N1 soluble HA candidates and protection studies of BALB / c mice” following protocols for care and use of laboratory animals. The animal immunization studies were performed at Small Animal Facility (SAF), Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India. Registration number: 1685 / GO / ReBi / S / 2013 / CPCSEA

[0099] For animal immunization studies, 7-8 week old, BALB / c (male) mice inbred in THSTI Small animal facility (SAF), twelve animals per group were immunized with different formulation following prime / boost immunization regimen, whereas 4 animals were taken as healthy group where no treatment given. Each experimental group of animals was immunized thrice with 30 pg of purified HA trimer and HA monomer at 21 days apart in AddaVax as adjuvant in a formulation with at antigen-adjuvant ratio as 1 : 1 ratio except the animals in control groups. The control groups 12 in numbers; were immunized with same volume of PBS and adjuvant as used in experimental groups. The control group received PBS, in 200ul formulation (100 ul PBS+100 ul AddaVax). For the HA trimer and HA monomer group protein, 30 pg of purified protein in PBS was mixed with AddaVax before immunization. The animals were bled 2 weeks after each immunization (Figure 5). The sera were collected and stored at -80 °C for future use. Each protein immunized group shows very high antigenic titer upon single boost, the response of HA monomer immunized group was found to be similar to HA trimeric group. As expected, no HA protein specific binding (HA monomer and trimer) was shown by animals of the control group in antigen ELISA. (Figure 6). In another immunogenicity experiment testing the open trimer, 7-8 week old, BALB / c (male) mice inbred in THSTI Small animal facility (SAF) were taken as 5 animal per groups. Here control group received PBS along with adjuvant Addavax, the open trimer group animals were immunized with 30pg of purified protein following prime boost immunization regimen as discussed above. The immunization scheme shown in Figure 7 and the immunogenicity as well as the IgG responses are shown in figure 8. The data shows high titer immune response post booster dose with end point titer of 7.33 X 106. (Figure 8).

[0100] Example 6.2. Anti-HA serum ELISA

[0101] The binding-antibody response to HA trimer and HA monomeric protein was measured using ELISA as described earlier (Vaccine 36, 1627-1636.). Serially diluted antisera (1 :3 times) with 1 :500 as starting dilution in dilution buffer (1 :5 times dilution of blocking buffer) were added to wells of the plates. The plates were incubated at room temperature (RT) for Ih. The plates were then washed four times with washing buffer (PBS + 0.1 % tween 20) and incubated with Peroxidase AffiniPure Goat Anti-Mouse IgG (1 :2000 dilutions, Jackson Immuno Research, USA) for 45mins. Subsequently washed with the washing buffer for four times, lOOul of TMB substrate (Thermo Fisher Scientific) was added to the washed wells. The reaction was stopped by adding 100 pl of 1 N H2SO4 and the plates were read at 450 nm on a 96-well microtiter plate reader.

[0102] Example 6.3. Anti-HA serum ELISA FOR ISOTYPING

[0103] The sera from protein boost in all the immunized groups were further used studies for its polarization towards TH1 / TH2 immune response as well as towards isotype class switching important for virus clearance and vaccine efficacy. To evaluate IgG isotype specific immune response of the HA Trimer and HA monomer immunized sera, the antigen coated plates were incubated with 1 :500 times diluted anti sera as initial dilution and titrated further with 1 :3 times sera dilution. After Ihrs of incubation the plates were washed and 1 :2000 times diluted Peroxidase conjugated IgG isotype specific antibodies were added to the plates; (IgGl; 115- 035-205, 115-035-206 ; IgG2a, 115-035-207 ; IgG2b, 115-035-208 IgG2c, 115-035-209 ; IgG3 and 115-035-003 ; IgG were obtained from Jackson Immuno Research (USA)). Post incubation the plates were washed and processed further as described above. The IgG isotype response was dominated by IgGl antibodies followed by IgG2a and further IgG2b responses. The antibody response of monomer immunized group was more focused towards IgGl antibodies with less immune response towards IgG2a as compared to Trimeric immunized group, with no IgG2c immune response observed from either group (Figure 9). The IgG isotype analysis of open timer also suggest the primary IgG isotype response generated from this group is IgGl followed by IgG2A followed by IgG2B (Figure 10). Example 6.4: In vitro stimulation of splenocytes

[0104] Stimulation of splenocytes was carried out in 96 well plate in vitro in presence of Concanavalin A at 37 °C for 6 h or protein antigens (50 pg / ml) at 37 °C for 72 h in a 5 % CO2 incubator. Thereafter, the cells were pelleted down in 96 well plates and the culture soup were aspirated, and the stimulated cells were used for intracellular cytokine staining as mentioned below

[0105] Example 6.5 Intracellular cytokine staining

[0106] Splenocytes stimulated in vitro by respective proteins and positive control were stained for surface CD4-PerCp, CD8-FITC markers in dark for 20 min at room temperature (RT). The cells were then permeabilized and fixed with BD Cytofix / Cyto Perm according to the manufacturers ’s manual. Thereafter, fFNy-Alexa flour 647, IL17A-PE-Cy7 and IL2-PE staining was carried out in Cytofix buffer at RT for 20 min in dark. The stained cells were then washed and acquired on BD FACS Canto II and were analysed on FlowJo software (Tree star).

[0107] To understand the immunogenicity of the monomer or trimer immunized mouse, antigen specific restimulation assay of the splenocytes was carried out. Single cells from spleen, isolated from immunized mice, were re-stimulated with either HA monomer or trimeric protein and their intracellular cytokine secretion was accessed through flow cytometry (Figure 11). The results indicated 2 fold increase in Thl response and 2-4 fold increase in cytotoxic T cell response upon monomer specific stimulation in the group immunized with monomer antigen as compared to the group immunized with trimer antigen (Figure 12). On the other hand, when splenocytes were re-stimulated with trimer we found 3-4 fold increase in Thl response and cytotoxic T cell response in the monomer immunized group as compared to the trimer immunized group. (Figure 13)

[0108] Further, the effector vs memory response was evaluated in these immunized groups through antigen specific re-stimulation. The data showed elevated CM and EM response in the CD4+ T cell compartment in the trimer immunized group as compared to the monomer immunized group upon re-stimulation with the monomer. Similarly EM and CM response in the CD8+ T cell compartment was also seen in the Trimer immunized group as compared to the monomer immunized group (Figure 14). In addition, there was also 4-5 fold increase in activated CD8+ T cell memory response in Trimer immunized group as compared to the monomer immunized group (Figure 15). In line with the above observation, when splenocytes were restimulated with trimer, the antigen specific activated memory CD4+ T cell response and CD8+ T cell response in monomer immunized group was found. Increasing trend in EM response and CM response was also seen in CD4+ and CD8+ compartments (Figure 16)

[0109] Example 6.6 Challenge Studies of Immunized animals

[0110] The Immunized animals from each group were divided into 3 subgroups of 4 animals each, 2 subgroups from each groups; HA trimer, HA monomer immunized, and control group were challenged with mouse adapted strain of H1N1 (PR8) and H3N2 (X31) virus and third subgroup kept unchallenged. Here an addition group of 4 animals were also considered in the experimental plan which were given no treatment and treated as healthy and non-vaccinated group. Mice were challenged using 50ul of 10 MLD50 (50% mouse lethal dose) of A / PR8 (H1N1) and X31 (H3N2), where 25pl of virus was given to each nostril of the animals. Animals post challenge were monitored daily for changes in body weight and survival.

[0111] The control group (PBS + adjuvant) animals showed rapid loss of body weight with both viruses; X31 and PR8. However, no weight loss was observed in animals immunized with HA monomer or HA trimer as compared to the healthy untreated mice. The control group animals which were challenged with PR8 and X31 started dying from the 6thday post infection, on 8thday each remaining animals lost more than 35% of the body weight, hence were sacrificed, and their organs were harvested to evaluate pathological abnormalities. Whereas the HA monomer or HA trimer group animals remained healthy and were monitored for 14 days. (Figure 17)

[0112] Example 6.7: Histopathology studies

[0113] Histopathological studies performed with the harvested lungs of the animals showed show no infiltration of neutrophils or any sign of fibrosis. In the scale of 0-3 for the low to high infiltration score lymphocytes and inflammation score were assign as 1 for both monomer and trimer group which were 3 for control group showing no lung pathology (Figures 18-21)

[0114] Example 6.8: Depletion of HA trimer specific antibodies from HA monomer immunized sera

[0115] HA monomer serum depletion assay was performed in order to characterize the antibodies which are potential contribution of HA monomers from the structurally occluded surface ( Figure 22). Here Tysol activated Dyna beads (Dynabeads™ MyOne™ Tosylactivated) were coated with HA trimeric protein following manufacturers protocol and was used to perform a magnetic bead based depletion of Monomeric sera. The HA trimer coated beads were incubated with HA monomer sera for Ihr, post incubation subjected to magnetic separation of beads from the sera. The depleted sera after first round of depletion incubated further with fresh coated beads to perform 2ndround of depletion. The process was continued for 7 rounds of depletion. (Figure 23)

[0116] After 7 rounds of depletion the depleted sera was obtained which contains the antibodies which are targeted towards structurally occluded epitopes. To estimate the extent of depletion after each round, ELISA was performed following methodology similar to “Example 4.2. Anti-HA serum ELISA”, here the ELISA plates were coated with HA trimer and HA monomer protein and Binding of depleted sera after each rounds of depletion was analyzed by incubating the depleted sera in 1 :500 ration and further diluted sequentially one third. The plates were incubated at room temperature and washed four times with washing buffer and incubated with Peroxidase AffiniPure Goat Anti-Mouse IgG (1 :2000 dilutions, Jackson Immuno Research, USA). It was then further washed and incubated with lOOul of TMB substrate (Thermo Fisher Scientific). The reaction was stopped by adding 100 pl of 1 NH2SO4 and the plates were read at 450 nm on a 96-well microtiter plate reader.

[0117] Example 6.9: ELISA of depleted sera:

[0118] The immunized sera and the depleted sera in the western blot was used. The immunized sera recognized the HA trimer, modified HA trimer (open trimer) and HA monomer, whereas the depleted sera recognized only modified HA trimer (Open Trimer) and HA monomer, conforming that all the HA trimer directed antibodies were depleted from the sera.

[0119] Example 6.10: Confocal immunofluorescence microscopy

[0120] Immuno fluorescence staining was performed to detect the binding interaction between anti HA Trimer, HA monomer and Depleted HA Monomer sera with cell line A549 expressing A / X-31(H3N2), A / Puerto Rico / 8-MC / 1934 (H1N1), A / Hong Kong / 1 / 1968 (H3N2) and A / California / 04 / 2009 (H1N1) pdm09 viruses. Briefly, A549 cells were seeded to 6Well plate and infected with 0.1MOI of virus. 48hrs post infection the cells were treated with 4% paraformaldehyde for 15minutes, washed with PBS, blocked with 3% BSA and incubated overnight with 1 : 1000 dilution of immunized and depleted sera, following next day cells were washed and incubated with secondary antibody; Goat anti Mouse IgG Alexa Fluor plus 488, Cat No# A32723 and mounted with Prolong gold antifade mount with DAPI, P36941. The visualization shows that depleted antibody binds to the all the 4 viruses mentioned above, hence highlighting the possibility of recognition of breathing conformation of virus at the cell surface. Cells were mounted using ProLong Gold anti-fade reagent with DAPI (Invitrogen, USA). Images were acquired on an Olympus FV3000 confocal microscope with 60X (NA 1.4) (Figures 24, 25, 26, 27)

[0121] Example 7: Structural studies of HA Monomer

[0122] Example 7.1: Docking of Antibodies Fab with HA monomer structure

[0123] The glycoprotein hemagglutinin (HA) of the H1N1 influenza virus X-ray crystallographic structure (PDB ID: 3LZG) was obtained from the Protein Data Bank (Berman HM et al., 2000). Additionally, Fab antibody 3D structures (PDB ID: 6N5B; 6N5D; 6N5E; 6OCB, 6E4X and 5CJQ) were also downloaded from the Protein Data Bank. The Fab antibody structures underwent preparation, including the cleaning of epitope residues to prevent duplication errors in residues and atoms, followed by the merging of both heavy (H) and light (L) chains into a single file and residue renumbering using PDBTOOLS v2.3 (Rodrigues JPGLM et al., 2018). Identified paratope residues from the Fab Antibody (residues: 96, 98, 99, 100, 263, 268, 305) were chosen for docking at the HA binding site residues numbers are; 200, 201, 202, 204, 205, 206, 208, 209, 210, 211, except for 5CJQ using HADDOCK v2.4 (https: / / wenmr.science.uu.n1 / haddock2.4 / ). The resulting docked poses were assessed and selected based on their HADDOCK score within each cluster. Interaction analysis was carried out using LigPlot+ v.2.2 (Laskowski RA, Swindells MB, 2011) and ChimeraX vl .7 (Pettersen EF et al., 2021).

[0124] TABLE 1: DOCKING ENERGY PARAMETER Example 7.2: Small Angle X-Ray scattering structure of CD6261Fab and HA monomer Complex

[0125] Example 7.2.1: Sample preparation: SAXS experiments were performed at home source SAXSpace, Anton Paar (CDRI, Lucknow). SAXS measurements were carried out for protein complex. The protein complex was purified from gel filtration chromatography in phosphate buffer using Superdex 200 10 / 300 column (GE Healthcare). The eluted fraction containing complex was concentrated to Img / ml and centrifuged at 12000 rpm for 20 min to remove any aggregation before the SAXS experiments.

[0126] Example 7.2.2: Data collection: Data collection was performed at 10°C and 2 frames of 30 minutes were collected at home source. Buffer scattering was measured to generate buffer subtracted intensity profile. Data was processed using ATSAS 2.8.1 primusqt. The forward scattering intensity 1(0) and radius of gyration R(g) were determined using AUTOGNOM which was also used to determine the molecule size of macromolecule by plotting pairdistribution functions (PDDF). P(r) approaches zero at its maximum distance Dmax. P(r) is a histogram of frequency of vector lengths connecting small volume elements inside the entire volume of scattering particle. AUTOGNOM results were processed by DAMMIF to generate 10 ab initio models. Multiple models were aligned and averaged using SUPCOMB and DAMAVER (Volkov and Syergun 2003). The unanimity of calculated models was represented by the lowest Normalized Spatial Discrepancy (NSD), which measure the average distance between nearest neighbor atoms in superimposed solution, which was determined by DAMSEL. The analogy of models and superimposition was performed using GASBOR. For visualization PyMOL and Chimera was used, the position of averaged atoms was represented as a sphere

[0127] Example 7.2.3: Structure analysis: Broadly neutralizing monoclonal antibody CR6261which neutralizes 12 out of 16 subtypes of influenza (except the 4 subtypes; H3, H7, H10 and Hl 5, which contain Asn38 Asn-Xaa-Ser / Thr consensus glycosylation motif) was isolated from the immune repertoire of vaccinated healthy individual. CR6261 Fab-HA crystal structure shows that it interacts with membrane proximal region end of the HA, primarily A-helix of HA2 and some HA1 residues at the stem region. mAb Fab binds to HA at 1 : 1 ratio per protomer of HA trimer, with relatively small, buried surface area (680A of HA; 70% HA2 and 30% HA1) highlighting the conserved residue involve in this interaction with fewer possibility of escape mutants. The interactions with HA were exclusively through heavy chain mediated hydrogen bonds and Vander wall interaction, no symmetry related contacts were seen, hence we used CR6261 to validate- is HA monomer is sufficient enough to engage CR6261 to form stable complex.

[0128] Here the solution structure of mHA-CR6261-Fab complex by SAXS was determined. Data collection was done in-house SAXSpace (Anton Paar) source at CDRI, Lucknow, India. SAXS data was collected at 1 mg / ml of complex sample in PBS. The buffer scattering was subtracted to obtain intensity profile of proteins sample using SAXS Analysis software (Anton Paar). Data collection, processing and analysis are shown in table 1. 3D, ab initio models were generated from SAXS data by using DAMMIF analysis (ATSAS package). Chimera vl.15 was used for fitting model in SAXS generated envelop; the complex (mHA- CR6261-Fab) structure was fitted in DAMMIF bead model (Figure 34). From pairwise distance distribution function we found that the maximum dimension (Dmax) is 12 nm (Figure 35). From the Kratky plot we found that complex exists in globular form as shown in figure 36. Guinier analysis shows that the sample is homogenous and without aggregation. The % for the DAMMIF analysis was 0.552 and NSD mean value of 10 generated models of complex is 0.645.

Claims

WE CLAIM:

1. An engineered pCDNA-HA open trimer of influenza Hemagglutinin (HA) protein immunogen of amino acid SEQ ID No. 2 and nucleotide SEQ ID No. 5, wherein the said immunogen is modified by the introduction of F88E and 191W mutations in the head region of pCDNA-HA trimer of amino acid SEQ ID No. 1.

2. The amino acid SEQ ID No. 1 as claimed in claim 1 is represented by nucleotide SEQ ID No. 4 .

3. An engineered codon optimized monomer of influenza Hemagglutinin (HA) protein immunogen of amino acid SEQ ID No. 3 and nucleotide SEQ ID No. 6.

4. A combination of pCDNA-HA open trimer as claimed in claim 1 and monomer as claimed in claim 3 in the range of (0 -100)% to (100 - 0)% of said combination .

5. A method of engineering HA protein immunogen, comprising the steps of :(i.) designing of DNA expression cassette; and(ii.) transient expression and purification of protein generated through DNA expression cassette.

6. The method as claimed in claim 5, wherein designing of DNA expression cassette comprising the steps of:(i.) preparing a pCDNA-HA trimer construct represented by amino acid SEQ ID No. 1;(ii.) modifying the pCDNA-HA trimer construct of step (i) by the introduction of mutations in the head region of the HA protein, resulting in an open trimer represented by SEQ ID No. 2; and(iii.) cleaving the modified pCDNA-HA open trimer of step (ii) to obtain the engineered codon optimized influenza HA monomer protein immunogen.

7. The method of preparing the pCDNA-HA trimer construct as claimed in claim 6, comprising the steps of :(i.) forming an expression cassette, wherein a designed construct comprising the codon optimized HA protein expressing 504 amino acid sequences of influenza virus strain is cloned between BamHI and Xhol of pCDAN 3.1+ expression vector; and(ii.) removing the N terminal signal sequences “MKAILVVLLYTFATANA” of HA protein from the amino acid sequence of step (i) and replacing with CD5 leader sequences “MPMGSLQPLATLYLLGMLVASVLA” in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3.

8. The expression cassette as claimed in claim 7, comprises: i. two amino acid linker VE, following CD5 sequences; ii. thrombin cleavage site, LVPRGS; iii. amino acids linker GSG after thrombin cleavage site; iv. foldon trimerization motif sequences, “YIPEAPRDGQAYVRKDGEWVLLSTFL”; v. single amino acid G after foldon domain; and vi. six histidine residues.

9. The method as claimed in claim 5, wherein the 504 amino acid sequences of the HA protein is selected from amino acid number 18 to 521 of A / California / 07 / 2009(H1N1) strain.

10. The method as claimed in claim 6, wherein modifying pCDNA-HA trimer construct comprising the steps of: i. identifying F88 and 191 residue into the amino acid sequence of the HA protein; and ii. mutating the F88 and 191 residue of the amino acid sequence of the HA protein as identified in step i, to generate F88E and 191W mutations as the modified pCDNA-HA trimer construct.

11. The method as claimed in claim 6, wherein the amino acid sequences of the HA protein comprise N terminal starting from “DTLCIGYHANNSTD” and C terminal ending at “YSEEAKLNREEIDGVK” as set forth in SEQ ID No. 1, SEQ ID No. 2, and SEQ IDNo. 3.

12. The modified pCDNA-HA trimer as claimed in claim 1, binds to the monoclonal antibody FluA-20 with nano-molar affinity.

13. The monomer of HA protein immunogen as claimed in claim 3, binds to the monoclonal antibody FluA-20 with nano-molar affinity.

14. The combination of pCDNA-HA open trimer and monomer as claimed in claim 4, binds to the monoclonal antibody FluA-20 with nano-molar affinity.

15. The modified pCDNA-HA trimer as claimed in claim 1, binds to neutralizing antibody CR6261 in nano-molar affinity.

16. The monomer of HA protein immunogen as claimed in claim 3, binds to neutralizing antibody CR6261 in nano-molar affinity.

17. The combination of pCDNA-HA open trimer and monomer as claimed in claim 4, binds to neutralizing antibody CR6261 in nano-molar affinity.

18. An immunogenic composition of pCDNA-HA open trimer of HA protein immunogen as claimed in claim 1, comprises purified protein in buffer along with the adjuvant in 1: 1 ratio, wherein the buffer is a phosphate buffer , most preferably PBS and the adjuvant is a squalene-based oil-in-water nano-emulsion, most preferably the adjuvant is AddaVax.

19. An immunogenic composition of monomer of HA protein immunogen as claimed in claim 3, comprises purified protein in buffer along with the adjuvant in 1: 1 ratio, wherein the buffer is a phosphate buffer , most preferably PBS and the adjuvant is a squalene-based oil-in-water nano-emulsion, most preferably the adjuvant is AddaVax.

20. An immunogenic composition of a combination of pCDNA-HA open trimer and monomer, as claimed in claim 4 comprises purified protein in buffer along with the adjuvant in 1: 1 ratio, wherein the buffer is a phosphate buffer , most preferably PBS and the adjuvant is a squalene-based oil-in-water nano-emulsion, most preferably the adjuvant is AddaVax.

21. The immunogenic composition of pCDNA-HA open trimer or a monomer or a combination of pCDNA-HA open trimer and monomer as claimed in claim 18-20, along with adjuvant as a universal influenza vaccine candidate for the prevention of disease or infection caused by different subtypes of influenza viruses.

22. The use of immunogenic composition as claimed in claim 18-20, for preventing disease or infections caused by the different subtypes of influenza viruses such as respiratory tract infection of the nose, throat and lungs, bacterial pneumonia, ear infections, sinus infections and worsening of chronic medical conditions, such as congestive heart failure, asthma, or diabetes.

23. A method of treating or preventing an infection caused by the HA influenza virus in a living host having or susceptible to such infection, wherein the said method comprises administering the immunogen as claimed in claim 1-4 and immunogenic composition as claimed in claim 18-20, intramuscularly, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally, in liquid dosage forms.

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