TECHNICAL FIELD

The present disclosure relates to polypeptide sequences and its corresponding polynucleotide sequence. It also relates to a recombinant vector and a recombinant host cell. Further, it also presents a method of obtaining the polypeptide, an immunogen, a method of obtaining an immunogen and a kit thereof. More specifically the instant disclosure relates to recombinant polynucleotide encoding recombinant Influenza polypeptides and its use in inducing immune response.

BACKGROUND

Influenza (flu) virus is the cause of major respiratory illness in humans resulting in 20,000 - 40,000 deaths annually in the U.S. alone and killing millions in pandemic years. Viral subtypes are classified on the basis of the sequences of hemagglutinin (HA) and neuraminidase (NA) surface proteins. Currently only strains of the H1N1, H3N2 subtypes and the B type viruses circulate in human population. In recent years however, certain strains of highly pathogenic avian influenza (H5N1) have been identified as the causative agents of a severe form of flu in humans and it has been suggested that these have the potential to cause a pandemic.

Existing vaccines are mainly composed of the HA and NA from inactivated virus grown mainly in chicken eggs or the recently approved cold adapted live virus. However, due to antigenic shift and drift, the trivalent vaccine (designed against the circulating H1N1, H3N2 and B type viral strains) has to be regularly modified based on the newly emergent strains of the virus. A major limitation with current influenza vaccines is that growth of the virus in chicken eggs is time consuming and the generation of a new vaccine takes 6-8 months. Hence, production of large amounts of vaccine at short notice during an epidemic/pandemic is difficult. A recombinant subunit vaccine that is easy to manipulate, produce, scale up and provides long lasting protection would be the ideal choice for controlling outbreaks like the current swine flu pandemic.

HA is the most abundant protein on the viral coat and is highly immunogenic. HA as a stand-alone vaccine candidate has been investigated in several studies. HA is synthesized as a precursor (HAO) that trimerizes in the ER (endoplasmic reticulum) and is transferred to the cell surface via the Golgi apparatus. HA is cleaved by cellular proteases into HA1 and HA2 subunits and this cleavage converts the protein into a fusion-active form. Entry of virions into target cells is mediated by binding of HA1 to sialic acid on the cell surface. The virion enters the cell by endocytosis and is transported to the endosome, where the resulting acidic pH induces major conformational changes in the HA molecule, leading to exposure of the fusion peptide on the HA2 subunit and subsequent fusion of viral and endosomal membranes. The crystal structures of HA in the precursor, neutral pH and low pH forms have been solved (5-8) and reveal the changes that occur upon cleavage and after the low pH conformational switch.

Although HA2 is considerably more conserved than HA1, early mapping studies of antigenic regions on HA revealed that neutralizing antibodies are directed only against the receptor binding HA1 subunit. Other studies have demonstrated that HA2 directed antibodies can provide protection in mice. Recently, several neutralizing monoclonal antibodies have been isolated that bind the stem region of HA. These Abs have been shown to cross react and neutralize several subtypes of viruses across clades and thus provide broad range protection. These Abs act by targeting the HA2 region of the molecule and presumably prevent the conformational change of HA at low pH, thus blocking fusion of viral and host membranes. It is therefore speculated that an engineered antigen that could focus the immune response to these epitopes and elicit protection against viral infection could serve as the basis for a more universal vaccine.

Previous attempts of recombinantly expressing soluble HA2 in the absence of HA1 have exclusively produced the low pH conformation of the protein even though expression and refolding were carried out at a neutral pH. However, very recently the stalk region of HA was successfully expressed on the surface of mammalian cells and HIV gag viral-like-particles.

The currently used trivalent vaccines for flu are either the inactivated virus or the live attenuated virus which are both produced in chicken eggs. New vaccines have to be formulated every six months to match the then circulating viruses. Once new vaccine strains have been identified, it takes nearly six months for the manufacture of the vaccine. This is a severe limitation when large doses of the vaccine have to be made in a short span of time as in the case of pandemics or epidemics.

Recombinant vaccines that contain the entire hemagglutinin molecule (produced from animal cell culture) can be made in a relatively short period of time. However, these techniques are expensive and difficult to scale up. The yield of the recombinant proteins produced in animal cells is also much lower as compared to bacterially expressed proteins.

The influenza virus constantly mutates its envelope proteins HA and NA in order to evade the immune system. Most of the neutralizing Abs that are elicited upon vaccination or exposure to the virus are directed to the head region of the HA molecule (HA1) that contains the receptor binding region. Hence the HA1 subunit is highly variable. However, the long stalk like C-terminal domain called HA2 is highly conserved within a subtype and also within a clade. A vaccine based on the HA2 domain is therefore likely to confer broad range protection and alleviate the need for frequent vaccination. However, HA is a metastable protein at neutral pH and the HA2 region adopts a non-native, low pH like conformation when expressed independently.

The designed immunogen (HA6) is recombinantly produced in bacteria and can be scaled up for large scale production in a very little span of time unlike the conventional egg based vaccines. The production of the protein in bacteria can be easily scaled up in an economical fashion unlike animal cell culture based vaccines. Owing to the high degree of conservation of the HA2 subunit, the designed immunogen is capable of providing broad range protection. A vaccine made from HA6 like constructs is likely to provide protection for longer than the conventional seasonal vaccines. This approach is also

easily extendable to other subtypes of influenza (A type) HA including HI, H2, H5, H7 and H9.

STATEMENT OF THE DISCLOSURE

Accordingly, the present disclosure relates to nucleotide sequence set forth in Seq Id No.

1; peptide sequences set forth in Seq Id Nos. 2 and 3, wherein the peptide sequence set forth as Seq Id No. 3 corresponds to the nucleotide sequence set forth in the Seq Id No; a method of obtaining a peptide sequence set forth as Seq Id No. 2, said method comprising acts of: a.) identifying conserved regions of HA1 and HA2 subunits of HA peptide of Influenza virus b.) combining the identified conserved regions to obtain the peptide sequence set forth as Seq Id No. 2; a method of obtaining a recombinant peptide sequence set forth as Seq Id No. 3, said method comprising acts of: a.) identifying conserved regions of HA1 and HA2 subunits of HA peptide of Influenza virus b.) combining the identified conserved regions to obtain a peptide sequence set forth as Seq Id No. 2 c.) introducing mutations in the conserved regions of the peptide sequence of step (b), to obtain the recombinant peptide sequence set forth as Seq Id No. 3; a recombinant vector having accession number MTCC 5631, said vector comprising nucleotide sequence set forth as Seq Id No.l; a recombinant cell, comprising the vector as stated above; a method of obtaining the recombinant cell, said method comprising acts of: a.) obtaining vector comprising nucleotide sequence set forth as Seq ID No. 1 b.) transforming a host cell with the vector to obtain the recombinant cell; a method of obtaining protein comprising peptide sequence set forth as Seq Id No. 3, said method comprising acts of: a.) inserting nucleotide sequence set forth as Seq Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell; b.) expressing the nucleotide within the cell for obtaining the protein comprising the peptide sequence set forth as Seq Id No. 3 c.) purifying the protein of step b); immunogen comprising a protein with peptide sequence set forth as Seq Id No. 3, optionally along with adjuvant(s) or pharmaceutically acceptable additive(s) or combination thereof; a method of obtaining immunogen comprising protein with peptide sequence set forth as Seq Id No. 3, optionally along with adjuvant(s) or pharmaceutically acceptable additive(s) or any combination thereof, said method comprising acts of: a.) inserting the nucleotide sequence set forth as Seq Id No. 1

into a vector and transforming a host cell with the vector to obtain a recombinant cell b.) expressing the nucleotide within the cell for obtaining and purifying the protein, to obtain the immunogen and c.) optionally adding adjuvant(s) or pharmaceutically acceptable additive(s) or combination thereof, to the immunogen of step b.); a kit having component selected from group comprising nucleotide sequence set forth as Seq Id No. 1 or peptide sequence set forth as Seq Id Nos. 2 or 3 or vector or cell or immunogen, adjuvant(s), pharmaceutically acceptable additive(s) or any combinations thereof along with instruction manual.

BRIEF DESCIPTIQN OF ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Figure 1 shows structures of the HA ectodomain; (A) Regions that are included in HA6 are shown in either maroon (HA1) or yellow (HA2). The rest of the molecule is shown in cyan. This figure was derived from the X-ray structure of HA from the (H3N2) isolate A/HK/68 (PDB ID 1HGD) and was drawn using the program Rasmol. Conformation of residues (45-110) HA2 in the neutral pH (B) and low pH (C) structures of HA. The stretch 57-98 is shown in pink and the HA2 residues 63F and 73V which are buried in the low pH form but are exposed in the neutral pH form are shown in green and blue respectively. Only a portion of the HA2 trimer (residues 45-110) is shown here for clarity.

Figure 2 shows sequence of the designed construct HA6. Mutated residues are underlined and linkers and vector derived sequences are shown in italics.

Figure 3 shows gel picture of SDS gel showing the fusion protein cytochromeb5_(57-
98)HA2 before (A) and after cleavage (B) with TEV protease

Figure 4 shows CD studies on the Wt and mutant (57-98) HA2 peptides; (A) Far UV CD spectra of Wt (57-98) HA2 peptide at pH 7.0 (•) and 4.5 (o), 25°C, depicting the random coil to helix transition upon acidification. (B) pH titration of Wt and mutant (57-98) HA2 peptides. The 6222 of the Wt (•), 63D (o), 73D (T) and 63D, 73D (A) mutant peptides are plotted as a function of pH. 73D mutant and 63D, 73D double mutant peptides are highly destabilized and do not form a coiled coil at any pH. Lines through the points are only for
visual clarity.

Figure 5 shows spectroscopic analysis of HA6; (A) Far UV CD spectrum of 5uM HA6 in PBS, pH 7.4 at 25°C. (B) Mean residue ellipticity of HA6 as a function of pH in 5mM CGH buffer, 25°C. The molecule loses its helicity as the pH is lowered confirming that HA6 does not form the extended coiled coil structure observed at the low pH for Wt HA2. (C) Isothermal Chemical Denaturation curve at pH 8.0, 25°C for 2uM HA6. The denaturation of the protein was monitored by the fluorescence intensity at 338 nm as a function of denaturant concentration.

Figure 6 shows fluorescence studies of HA6; (A) Fluorescence Emission spectra of 2uM HA6 under native conditions (20mM Tris pH 8.0, solid line) or denaturing conditions (6M GdnCl, 20mM Tris, pH 8.0, dashed line) at 25°C. (B) Binding of ANS to luM HA6 in a buffer containing lOOuM ANS. The protein does not bind ANS to a significant extent (sample 3) as compared to a control molten globule of CcdB (sample 2) indicating that the molecule is quite compact and does not have large, exposed hydrophobic patches.

Figure 7 shows the use of engineered disulfides to confirm that HA6 adopts the neutral pH conformation of HA. Monomers from trimeric HA2 at neutral pH (A) and low pH (B). Residues that were mutated to Cysteines are shown in stick representation. In red the (3F,116N) pair and in blue the (40S,118L) pair. The introduced Cysteine pairs (3C,116C) and (40C,118C) can form disulfides only if the molecule is in the neutral pH conformation. The low pH structure does not have the coordinates of residue F3 (HA2)

but this residue is expected to be in the top part of the figure, over 90A away from residue 116. Only a monomer of HA2 is shown in the figure for clarity.

Figure 8 shows characterization of (3C,116C) HA6 and (40C,118C) HA6 proteins; (A) Far UV CD spectra of (3C, 116C) HA6 (black) and (40C, 118C) HA6 (red) in PBS, pH 7.4 at 25°C. Both the mutant proteins are well folded and have CD spectra similar to Wt HA6 (Fig. 3A). (B) Reverse Phase HPLC profiles of Wt, (3C, 116C) and (40C, 118C) mutants. The differences in the retention times of the reduced proteins in comparison with the native proteins and with each other indicate formation of the expected disulfide bonds. Wt and (40C, 118C) HA6 show single peaks on RP-HPLC indicating that they are well folded with only a single combination of disulfides. (3C, 116C) HA6 shows one major and one minor conformer in the oxidized state.

Figure 9 shows gel filtration of H3HA6 on a Superdex 200 analytical column.

Figure 10 shows immunogenicity and protection against lethal challenge (A/HK/68) by HA6 in mice. Mice of 10 per group were immunized with indicated vaccines either twice (x2) or once (xl) and challenged intranasally with an LD90 of mouse adapted A/HK/68. Weight changes (A) and mortality (B) were monitored for 20 days post challenge. Convalescent mice from intranasal A/HK/68 inoculation (A/HK68) were included as the positive control, and mice receiving CpG7909 alone as adjuvant control.

Figure 11 shows binding of the anti-HA6 sera to recombinant H3 HA proteins. Anti-HA6 sera from study group 3 in Table 1 (Fig. 9A) and convalescent sera from A/HK/68 infected mice (Fig. 9B) were tested for binding activities to recombinant H3 A/Brisbane/16/07 HA by ELISA assays. HA6 was included as a control. The data represent the mean OD450nm values of 10 mouse sera from each respective group. Convalescent sera bind both HA6 and A/Brisbane/07 poorly confirming that antibodies in these sera are largely directed to the globular head of HA. In contrast, anti-HA6 sera bind well to both HA6 and A/Brisbane/07. Anti-HA6 sera have an approximately 100 fold higher half-maximal binding titer for A/Brisbane/07 than convalescent sera.

Figure 12 shows binding of mAbl2Dl to immobilized HA6 and competition binding in the presence of anti-HA6 serum.(A) ELISA binding of mAb 12D1 to HA6, recombinant HA proteins (A/Brisbane/10/07, A/Wyoming/03), seasonal vaccine and peptide (57-98) HA2 (250ng per well in each case). The antibody does not bind to a linear peptide (57-98) HA2 containing the putative epitope residues but binds well to HA6 and the other full length, soluble recombinant HA proteins. (B) Competition binding of 12D1 (lOng/ml) to recombinant A/Brisbane/10/07 HA (lOng per well) in the presence of various dilutions of anti-HA6 guinea pig serum and naive guinea pig serum. Anti-HA6 serum competes with 12D1 indicating the presence of 12D1 like Abs in the serum. In all cases binding of 12D1 to immobilized protein was detected by an appropriately tagged anti-mouse IgG.

DETAILED DESCRIPTION

The present disclosure relates to a nucleotide sequence set forth in Seq Id No. 1.
The present disclosure also relates to a peptide sequences set forth in Seq Id Nos. 2 and 3,
wherein the peptide sequence set forth as Seq Id No. 3 corresponds to the nucleotide
sequence set forth in the Seq Id No. 1.

The present disclosure also relates to a method of obtaining a peptide sequence set forth
as Seq Id No. 2, said method comprising acts of:
a.) identifying conserved regions of HA1 and HA2 subunits of HA peptide of

Influenza virus; and b.) combining the identified conserved regions to obtain the peptide sequence set forth as Seq Id No. 2. The present disclosure also relates to a method of obtaining a recombinant peptide sequence set forth as Seq Id No. 3, said method comprising acts of:
a.) identifying conserved regions of HA1 and HA2 subunits of HA peptide of

Influenza virus; b.) combining the identified conserved regions to obtain a peptide sequence

set forth as Seq Id No. 2; and c.) introducing mutations in the conserved regions of the peptide sequence of step (b), to obtain the recombinant peptide sequence set forth as Seq Id No. 3.

In an embodiment of the present disclosure, the peptide sequence set forth in Seq Id No. 3
corresponds to the peptide sequence of step (b) having mutations at positions 63 or 73
within the HA2 subunit or at positions 297, 300, 302, and 305 within HA1 subunit or any
combination of mutations thereof.

In another embodiment of the present disclosure, the mutations are selected from group
comprising point mutation, insertion, deletion, substitution and frameshift mutation or
any combination thereof.

The present disclosure also relates to a recombinant vector having accession number
MTCC 5631, said vector comprising nucleotide sequence set forth as Seq Id No.l.
The present disclosure also relates to a recombinant cell, comprising the vector as stated
above.

The present disclosure also relates to a method of obtaining the recombinant cell as stated
above, said method comprising acts of:
a. obtaining vector comprising nucleotide sequence set forth as Seq ID No.
1; and

b. transforming a host cell with the vector to obtain the recombinant cell.
The present disclosure also relates to a method of obtaining protein comprising peptide sequence set forth as Seq Id No. 3, said method comprising acts of:

a.) inserting nucleotide sequence set forth as Seq Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell;

b.) expressing the nucleotide within the cell for obtaining the protein comprising the peptide sequence set forth as Seq Id No. 3; and

c.) purifying the protein of step b). The present disclosure also relates to an immunogen comprising a protein with peptide sequence set forth as Seq Id No. 3, optionally along with adjuvant(s) or pharmaceutically acceptable additive(s) or combination thereof.

The present disclosure also relates to a method of obtaining immunogen comprising protein with peptide sequence set forth as Seq Id No. 3, optionally along with adjuvant(s) or pharmaceutically acceptable additive(s) or any combination thereof, said method comprising acts of:

a.) inserting the nucleotide sequence set forth as Seq Id No. 1 into a vector
and transforming a host cell with the vector to obtain a recombinant
cell; b.) expressing the nucleotide within the cell for obtaining and purifying
the protein, to obtain the immunogen; and c.) optionally adding adjuvant(s) or pharmaceutically acceptable
additive(s) or combination thereof, to the immunogen of step b.)

In an embodiment of the present disclosure, the vector is selected from group comprising bacterial expression vectors, yeast expression vectors and animal cell expression vectors; and the host cell is selected from group comprising bacteria, yeast and animal cells.

In another embodiment of the present disclosure, the adjuvant is selected from group comprising CpG7909, IMX, MAA, Freund's adjuvant, Mycobacterium w (Mw) and Adjuplex LAP or any combination thereof; and the pharmaceutically acceptable additive is selected from group comprising excipients, gums, sweeteners, coatings, binders, disintegrants, lubricants, disintegration agents, suspending agents, granulating agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavoring agents, viscosity enhancers and antioxidants or any combination thereof. The present disclosure also relates to a kit having component selected from group comprising nucleotide sequence set forth as Seq Id No. 1 or peptide sequence set forth as Seq Id Nos. 2 or 3 or vector as stated above or cell as stated above or immunogen as stated above, adjuvant(s), pharmaceutically acceptable additive(s) or any combinations thereof along with instruction manual.

The present disclosure relates to Influenza which is an Orthomyxovirus that causes respiratory infection in humans leading to morbidity and sometimes mortality. The mature virion has an envelope that contains two major proteins - hemagglutinin (HA) and neuraminidase (NA) that are highly antigenic molecules. However, due to frequent

antigenic shift and drift, the antibodies to the original strain poorly neutralize the newer strains of virus.

In an embodiment of the present disclosure, the influenza protein hemagglutinin (HA), being highly antigenic, is used for vaccine design. HA is a trimeric protein synthesized as a precursor (HAO) and cleaved endoproteolytically into HA1 and HA2, this cleavage being essential for its functioning. When the virus infects a new cell, it is taken up by endocytosis. Inside the endosomes, the acidic environment triggers a conformational change in the protein HA which then brings about the fusion of viral and endosomal
membranes to release the viral nucleocapsid into the cytoplasm.

In an embodiment of the present disclosure, the HA2 subunit of Hemagglutinin is highly conserved within a subtype and even within a clade. A vaccine derived from the HA2 subunit provides intra-subtypic and intra-clade protection. When recombinant HA or the virus is used as an immunogen, the majority of the neutralizing Abs are elicited to the HA1 subunit. An HA2 immunogen (without the head domain) would elicit Abs directed against the HA2. However, earlier attempts to express the HA2 subunit independently in bacteria resulted in protein that was folded into the low pH conformation and not the desired neutral pH form. Hence, the present disclosure is directed to the inclusion of HA1 residues that interact with HA2 and destabilization of the low pH form by introduction of mutations in order to stabilize the HA2 fragment to be expressed independently.

In an embodiment of the present disclosure, influenza HA is the primary target of neutralizing antibodies during infection and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA 1 subunit of HA shows much higher sequence variability relative to the metastable, fusion active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. In the instant disclosure designing of a HA2 based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2 is carried out. The resulting construct (HA6) is expressed in E.coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation

competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 is highly immunogenic in mice and the mice are protected against lethal challenge by the homologous A/HK/68 mouse adapted virus. An HA6 like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy which plays a role in pandemic preparedness.

The present disclosure is further described with the help of the following examples and figures. However, these examples should not be construed to limit the scope of the disclosure.
The Biological material present in the instant disclosure in the form of vectors comprising the genes of interest are deposited at the International Depository - Microbial

Type Culture Collection & Gene Bank, Chandigarh. The deposited vector is assigned the following MTCC Numbers: pET 26 b (+) HA6- MTCC 5631.

EXAMPLE 1: DESIGN OF THE IMMUNOGEN

The HA2 subunit is considerably more conserved than HA1 (Table 1, approximately 90% vs. 67% for Influenza A HI and H3 subtypes in the surface exposed regions) and serve as a "universal" influenza vaccine candidate if it is to provide sufficient immunogenicity and protection. Furthermore, since HA2 is not involved in receptor binding, it is much more likely that epidemic or pandemic strains will arise through mutations in the HA1 rather than the HA2 subunit. However, the conformation of HA2 found in the neutral pH HA structure is believed to be metastable. It is known that the cleavage of HAO and the disassembly of HA1 and HA2 subunits are essential for the low pH induced conformational change. It is therefore reasoned that HA2 is expressed in its neutral pH conformation if regions of HA1 that are in close spatial proximity to HA2 are included in the designed molecule.

The accessibilities of all residues in the HA neutral pH structure (PDB ID:1HGD) are calculated and HA1 fragments that have interactions with HA2 are identified using the program PREDBURASA. The program PREDBURASA calculates accessibilities of all the residues in the protein in presence and absence of the interacting partner. The program is run using the coordinates from the pdb file lhgd and the accessibilities of all residues in the HA1 chains in the presence and absence of the HA2 chains are calculated. HA] residues that have a difference of 5A2 or more in absolute accessibility in the above calculation and a 10% or higher accessibility difference are considered to be interacting with HA2. Similar cutoffs and approach have been previously used to stabilize domain Dl of human CD4. HA2 interacting residues consisted largely of residues (7-46) and (290-321) of HA 1 (Fig. 1 A).

Using the software RosettaDesign, mutations are chosen to remove exposed hydrophobic patches generated due to interactions lost with the rest of HA. These mutations are V297T, I300E, Y302T, and C305T in HAL
Finally flexible soluble linkers of appropriate length, similar in sequence to a linker used previously in HIV immunogen designs, are designed to link the three fragments into a single polypeptide chain. The length of the linker is chosen by taking into account the distance between the C-alpha atoms of the terminal residues of the fragments to be connected in the target structure. The designed protein contains residues (1-172) of HA2, a 7 amino acid linker (GSAGSAG),(7-46) of HA 1, a 6 amino acid linker (GSAGSA) followed by residues (290-321) of HA1 with the above-mentioned mutations incorporated in it. The design is based on the sequence of H3 HA (A/HK/68) because only for this strain the crystal structure of both the neutral pH and low pH forms of HA are available. In addition, H3 viruses show a greater degree of genetic diversity than HI and B as inferred from the HA sequences of the viruses in the past 10 years (Table 1). Table 1: Fraction of exposed residues that are highly conserved in different HA fragments. Residues that are at least 95% conserved and have accessibilities greater than 20% (exposed) are identified by mapping the % conservation of the residues onto the structure of HA (PDB ID: 1HGD).

Fraction of exposed residues which are >95% conserved. The total number of exposed residues in HA1, HA2 and HA6 is 146,74 and 105 respectively.

b Sequences of the vaccine straim of the past ten years were obtained from the NCBI Influenza Virus resource (6).

c All the non-identical full length HA sequences available at the NCBI flu resource were used for the analysis. The average conservation (in the exposed reaioas of HA) for HI and H3 strains is 67%, 90% and 89% for HAL HA2 and HA6 respectively.

The residues 63F and 73 V in the HA2 subunit are buried in the coiled coil of the low pH structure (positions a and d respectively in the helical wheel). In the neutral pH conformation, these residues form a part of the loop connecting helices 4 and 5 and are exposed (Fig. IB and Fig. 1C). It is reasoned that mutation of these hydrophobic residues to Asp would destabilize the coiled coil at neutral pH and introduced two additional mutations, F63D and V73D, in the HA2 chain. The Asp substitution is 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 resulting construct, inclusive of all mutations, is denoted as HA6. The final sequence of the protein encoded by the cloned gene is shown in Fig. 2.

A similar immunogen is also designed based on the sequence of a different H3 strain (A/ Phil/2/82) that has an overall sequence identity of 85% at exposed residues to HK68 HA. The sequence of HA from A/Phil/2/82 is mapped onto the structure of HA from A/HK/68 and regions corresponding to HA6 are identified. Mutations that remove hydrophobic

patches and destabilize the low pH form are also included in this construct as in the case
ofHA6.

The sequence of the immunogen HA6a including the vector derived sequence is shown in
sequence listing as sequence Id No 4.
An alignment of HA6 with HA6a (protein sequence including vector derived residue) is
given below:

EXAMPLE 2; STUDIES ON PEPTIDE SYSTEM

In order to test if the above Asp mutations are capable of destabilizing the extended HA2 coiled coil that is formed at low pH, a small 42-residue peptide (57-98 of HA2) that includes this region is chosen for further study.
Previous studies have shown that a similar peptide undergoes a random coil to triple helix transition at low pH, similar to that observed in native HA. The gene for this peptide is cloned as a fusion to cytochrome b5 and the mutations 63D and/or 73D are introduced into the gene using megaprimer PCR. Details of megaprimer PCR are described in: Wei D, Li M, Zhang X, Xing L. An improvement of the site-directed mutagenesis method by combination of megaprimer, one-side PCR and Dpnl treatment. Anal Biochem. 2004 Aug 15;331(2):401-3. PubMed PMID: 15265749. A modified version of the same is used. One mutant primer and one vector specific primer are used in the first round of PCR to generate a megaprimer. The megaprimer (product of first round PCR) is gel purified and

used as primer for the second round of PCR without an annealing step. Vector containing the Wt gene is used as template in both the PCR reactions. After amplification of the entire vector in the second round of PCR, the parent plasmid is digested using Dpnl and the resultant PCR product transformed into competent DH5a cells. Plasmid was isolated from transformed cells and clones were verified by sequencing.

The resulting fusion proteins are expressed in E.coli and purified. The peptides are derived from the fusion proteins described above by cleavage with TEV protease and further purified using reverse phase HPLC.

TEV protease cleavage: The fusion proteins are cleaved using a 1:100 molar ratio of TEV to protein in 50mM Tris (pH 8.0), 2mM DTT and 0.5mM EDTA at 22°C overnight. About 90% of the protein is cleaved under these conditions ( Fig 3). The cleaved peptides are purified from the mixture using a semipreparative RP C-18 column (10 mm * 250 mm) on a Shimadzu HPLC system using a gradient of water and acetonitrile (going from 55% to 75% acetonitrile at 1%/min using a flow rate of 5 ml/min). The peptides obtained are greater than 95% pure.

Conformation of wild type (Wt) and mutant peptides is assessed using circular dichroism

(CD) spectroscopy. The molar ellipticity at 222nm is monitored as a function of pH to estimate alpha helical content. The Wt peptide is a random coil at neutral pH and formed a helical coiled coil at pH 4.5 as depicted in Fig. 4A. The decrease in ellipticity observed for 63D and/or 73D in Fig. 4B indicate that the introduced mutations have indeed destabilized the coiled coil. The apparent midpoints of the random coil to helix transitions are 5.3, 4.7 and 3.65 for the Wt, F63D and V73D mutants. Hence the V73D mutation is more destabilizing than the F63D mutation. The F63D, V73D double mutant is highly destabilized and did not form a coiled coil throughout the pH range 2.0-7.0. Both these mutations are therefore included in the designed HA6 molecule as described above with the aim of destabilizing the low pH conformation of HA.

EXAMPLE 3: Cloning and Purification of HA6
E.coli codon optimized genes corresponding to the designed proteins (HA6 and HA6a) are synthesized and cloned into the bacterial expression vector pET-26b(+) between the

Ndel and Hindlll sites of the MCS to obtain a recombinant vector. Host cells are transformed with the obtained recombinant vector by following method:

a. Inoculating a single colony of host cell from a rich plate (Luria, Luria-Bertani)
into about 2 ml of rich broth (Luria-Bertani; RB) in a plating tube. Shake
overnight at 37°C.

b. Subculturing the overnight 1:100 broth in 1 Volume Unit of RB+20 mM MgS04
(typically 250 ml). Grow to OD590=0.4-0.6 or Klett=60 (-2-3 h).

c. Centrifuging at about 5,000 rpm for about 5 min at about 4 C.

d. Gently resuspending the pellet in about 1/2.5 Volume Unit ice cold TFBI. For
about 250 ml subculture, about 100 ml TFBI is used; 50 ml/bottle. The
resuspended cells is combined in one bottle. Further steps are carried out on ice.

e. Incubating on ice for about 5 min.

f. Centrifuging at about 5,000 rpm for about 5 min at about 4°C.

g. Resuspending the pellet in about 1/25 original volume cold TFB2. For about 250
ml of original subculture, about 10 ml TFB2 is used.

h. Incubating on ice for about 15-60 min. Before aliquoting about 100 ul/tube for storage at about -70°C. The tubes are quick freezed. A convenient way to do this is to use ice-bath racks. These have a movable "lid" rack, with an ice compartment bottom (American Scientific Products, cat # S9233-1); the top-labeled tubes (open) are set up on a rack with ice in the bottom compartment; the cells are distributed; then the tubes are closed. In another bottom compartment, a dry ice/ethanol (or isopropanol) bath is set up, then the "lid" rack (which carries the tubes) is transferred to the dry ice bath bottom compartment for ~15 sec; isopropanol is drained, transferred to an empty bottom compartment and the whole thing is put in about -70 freezer. After the tubes are well-frozen they can be dumped loose into a box or ice-cream carton, or transferred to slots in a storage box.

Liquid nitrogen can also be used, but not with these racks.
i. To transform, an aliquot is thawed on ice; DNA is added; incubated for about 1 h on ice; heat shock about 45 seconds at about 37°C; incubated on ice for about 2 min; dilute about 15-fold into RB with no drug (for phenotypic expression); grow

with vigorous aeration at about 37°C for about 20 min.; plate on selective medium.
LB (Luria-Bertani medium)
per liter:
lOgTryptone(Difco)
5 g Yeast Extract (Difco)
5gNaCl
2 ml IN NaOH

TFBI
30 mM KOAc (potassium acetate)
lOOmMRbCl
lOmMCaCb
50 mM MnCl2
15% glycerol

Adjust to pH about 5.8 with acetic acid and filter (about 0.45 urn, Nalgene units or Millipore filters) to sterilize.
In a preferred embodiment, this is made in the following manner:

Make up to about 410 ml; distribute in about 100 ml sterile aliquots; and use about 1
aliquot/250 ml culture.

TFBII
10 mM MOPS or PIPES 75 mM CaC12 lOmMRbCl 15% glycerol
Adjust pH to about 6.5 with KOH and filter to sterilize Make up as
make to about 150 ml; filter; use about 10 ml per original about 250 ml culture.
The transformed cells are allowed to express the protein. The purification of the proteins after expression in bacteria is done as indicated below. E.coli BL21(DE3) cells transformed with the plasmid are grown in Terrific Broth at 37°C to an A600 of 0.8 before inducing the culture with ImM IPTG. The cells are grown for another 6 hours before harvesting them by centrifugation at 3,500g. The cells are resuspended in 20mM Tris (pH 8.0) and lysed by sonication. The cell lysate is separated from the inclusion bodies by centrifugation at 18,500g. The inclusion bodies are washed in about 0.05% Triton X-100, about 20mM Tris (about pH 8.0) and subjected to centrifugation at about 18,500g. The inclusion bodies (insoluble fraction) are then solubilized in about 8M GdnCl, about ImM DTT, about 20mM Tris (about pH 8.0). The protein is purified from clarified, solubilized inclusion bodies by binding and refolding on a Ni-NTA column (Amersham). The protein is eluted in about 1M GdnCl, about 500mM Imidazole, about 20mM Tris (about pH 8.0). The eluted protein is desalted into de-ionized water on an (Amersham) Hitrap Desalting column and stored in aliquots at about -80°C.

Broadly, the protein is expressed in E.coli BL21(DE3) cells and purified by immobilized metal affinity chromatography after resolubilization from inclusion bodies. The yield is about 2mg/ L of culture. SDS-PAGE with Coomassie staining confirmed that the protein is at least 95% pure.

EXAMPLE 4: CD and Fluorescence Spectroscopy

All spectra are acquired at 298K. The CD spectrum of the protein at a concentration of about 5uM is recorded in 1XPBS on a JASCO J- 175 Spectropolarimeter. The spectra are recorded using a 0.1cm path length cuvette by scanning from about 250nm to about 195nm at a rate of about 50nm/min. The helix content of the molecule is determined. To study the effect of pH on the structure, the 6222 of the protein/peptide in about 5mM CGH buffer is monitored as a function of pH.
CD spectroscopy of the protein revealed a largely helical structure consistent with the designed target structure Fig. 5A. In intact HA, each monomer of HA2 has an 18 amino acid helix (residues 38-55) packed against a longer helix (residues 76-125) in an antiparallel fashion. The trimer is stabilized by coiled coil interactions involving the N-terminal region of the longer helix (Fig.lA). The ellipticity at 222nm in Fig. 5A corresponds to -40% a-helix content, which is consistent with the predicted value of 34% in the target structure.

The intrinsic fluorescence emission spectrum of the protein at a concentration of about 2uM is recorded using a 1cm path length cuvette under native (about 20mM Tris, about pH 8.0) or denaturing conditions (about 6M GdnCl, about 20mM Tris, about pH 8.0) after excitation at about 280nm on a Fluoromax-3 Fluorimeter. Equilibrium unfolding studies are carried out on the molecule using the denaturant GdnCl. The unfolding of about 2uM HA6 in Tris buffer (about pH 8.0) is monitored by measuring the intrinsic Trp fluorescence emission at about 338nm after exciting the molecule at about 280nm in the presence of increasing amounts of denaturant.

Intrinsic fluorescence emission spectra under native and denaturing conditions also indicate that the molecule is well folded (Fig. 6A) as the peak of the emission spectrum shows the expected red shift upon denaturation.

In order to test if the molecule is well folded and compact, ANS binding studies are also carried out. To test for binding to ANS (l-anilino-8-napthalene-sulphonate), about luM of the protein is incubated in the presence of about lOOuM ANS in about 20mM Tris (about pH 8.0) for about 30 minutes and the fluorescence emission at about 482nm after excitation at about 388nm is monitored. In comparison to a molten globule control, HA6 did not bind ANS significantly (Fig. 6B) indicating that it does not have exposed hydrophobic patches. A previously characterized molten globule of luM Controller of Cell Division B protein (CcdB) at about pH 4.0 (4) is also tested for ANS binding as a control.

An isothermal denaturation melt using GdnCl revealed a cooperative unfolding transition characteristic of a well folded protein (Fig. 5C).

The four Cys residues in the molecule (residues 14 HA1, 137 HA2, 144 HA2 and 148 HA2) appeared to be oxidized as judged from a DTNB assay. These residues are involved in two disulfide bridges (14 HA 1-137 HA2 and 144 HA2-148 HA2) in the native HA molecule. The lack of free thiols in the protein suggests that it is properly folded.

EXAMPLE 5: HA6 ADOPTS THE DESIRED NEUTRAL PH CONFORMATION

In order to examine if HA6 is indeed folded into the desired neutral pH conformation and not the low pH conformation, the following additional studies are carried out. Firstly, engineered disulfide bridges are used to confirm that the target fold is achieved. Comparing the structures of the neutral pH (PDB ID 1HGD) and low pH conformations of HA (PDB ID 1HTM) and using the program MODIP to pick possible positions for engineering disulfide bonds, two pairs of residues are chosen in the molecule (3,116 of HA2 and 40,118 of HA2). Residues in each pair are spatially close and predicted to form

disulfide bonds when mutated to Cys if the molecule is in the neutral pH like conformation. In the low pH conformation, these pairs of residues would be separated by greater than 90A (Fig. 7). The mutations are introduced into HA6 and the two mutant proteins are expressed and purified. Both these proteins are well folded and had CD spectra similar to that of the Wt protein (Fig. 8A). The proteins are tested for their free thiol content under denaturing conditions in a DTNB assay.

Determination of free thiol content: The oxidation state of the protein is determined by reacting about 5uM of the protein with about 500uM DTNB in about 4M GdnCl, about 50mM Tris (about pH 8.0) and monitoring the absorbance at about 412nm on a Varian Cary 100 Bio UV-Vis Spectrophotometer after about 15min of incubation. The free thiol content is calculated using an extinction coefficient of 13,700 M -1 for the TNB anion. The oxidation state of the proteins is also investigated using reverse phase HPLC. About 50 uM of each of the proteins in about 4M GdnCl, about 50mM Tris (about pH 8.0) either with or without about 500uM TCEP is injected onto an analytical RP C5 (15cm X 4.6mm) column and eluted using a gradient of water and acetonitrile at a flow rate of lml/min (30% acetonitrile to 45% acetonitrile).

In both the mutant proteins, the engineered disulfide bond is formed. There are no free Cys residues in the absence of DTT and the expected six Cys residues after reduction with DTT (and removal of DTT on a desalting column). WT and mutant HA6 proteins are characterized by RP-HPLC on a C5 HPLC column. Under reducing conditions, all three proteins eluted at a different acetonitrile concentration relative to the corresponding protein prior to reduction and as compared to each other (Fig. 8B). Formation of the additional disulfide bonds in the (3C, 116C) and (40C, 118C) mutants results in a more compact unfolded conformation as evidenced by the decreased retention times relative to Wt HA6.The formation of additional disulfide bonds in the (3C, 116C) and (40C, 118C) mutants indicates that HA6 is in the desired neutral pH like conformation and not the low pH conformation.

To further confirm this assertion, the helical content of the molecule is measured as a function of pH using CD spectroscopy. The 6222 of HA6 is plotted as a function of pH in Fig. 5B. The results show that the protein loses its helicity as the pH is lowered contrary to what would be expected if it were in the low pH conformation.

EXAMPLE 6: THE IMMUNOGEN PROTEIN DOES NOT AGGREGATE IN

SOLUTION

The experimental details and data for the gel filtration experiment are given below. Fig.9 shows that H3HA6 is not aggregated in solution as the elution volume of the major peak is greater than the void volume (~llml). The molecular weight of the protein is not calculated from this data due to the unavailability of suitable calibration markers (H3HA6 is expected to have an elongated shape as opposed to the calibration proteins which are globular proteins).
Size Exclusion Chromatography: About 250ug of the protein at a cone, of about 5mg/ml in water is loaded on an analytical Superdex 200 (10 X 300mm) column (Amersham) and eluted in about IX PBS (about pH 7.4) at a flow rate of about 0.5ml/min.

EXAMPLE 7: MOUSE IMMUNIZATION AND CHALLENGE

Female BALB/c mice, 10 per group are vaccinated intramuscularly with different doses - (dosage of the proteins used in the study is indicated in Table 2 below, lug, 5^g or 20ng of the proteins was used for immunizing mice) of the immunogen (HA6 or HA6a) at weeks 0 and 4. The naive control group is not vaccinated and a positive control group received a single dose of live virus either intramuscularly or intranasally at week 0. Sera are collected two weeks after the second immunization and all animals are challenged with either the homologous H3N2 strain (A/HK/68) or an H1N1 strain (A/PR/8/34) at a dose of 1LD90 (1.12*107 TCID 50/ml) a week later. The different proteins used for immunization and the dosage regimen used is shown in Table 2. The body mass of the mice is monitored for 20 days after the challenge by which time all the control mice had died.

More precisely, HA6 (Wt as well as (3C, 116C) and (40C, 118C) disulfide mutant proteins) are used to immunize mice before challenging them with one LD90 of homologous (A/HK/68 - H3N2) virus. The antibody titers in the immunized mice are determined by ELISA. It is found that all three immunogens tested are highly immunogenic and resulted in high antibody titers (Table 2). All the animals immunized with the HA6 proteins (Wt or disulfide mutants) are protected from a lethal challenge with the homologous virus (Fig. 10B and Table 2). The same results are reproduced with different adjuvants in a repeat study and protection is observed even with a single dose of lug of HA6 (Table 2). The disulfide mutants also conferred protection in mice. Weight measurement studies showed weight loss in the mice following pathogenic challenge before they fully recovered (Fig. 10A.).

Table 2: Dosage regime used for mice challenge studies. Each group consisted of 10 BALB/c mice. The survival of the mice after viral challenge is monitored for 20 days by which time the naive control mice died. All the immunogens tested (Wt, (3C,116C) &(40C,118C) mutants of HA6 & HA6a) are highly immunogenic and conferred protection against (H3N2) viral challenge (A/HK/68). However, there is no protection against an H1N1 virus challenge (A/PR/8/34).

the nuce were vaccinated at week 0 and given a booster at Week 4

b At week 6, bleeds were collected and the anri-HA6 antibody titers were determined by ELISA against Wt HA6 c At Week 7, the mice were challenged with 1LD90 of the homologous H3 virus (A'HK/68) and the survival was monitored up to 20 days.
Similar lmmunogenicity and protection was seen when MAA and IMX were used as adjuvants e Mice in which the FcR-y chain is knocked out. "n.d., not determined.
Guinea Pig immunizations: Female guinea pigs are immunized with about 100 ug of HA6 intramuscularly three times in 4-week intervals. Two weeks after the last immunization, sera are collected and used for the mAb 12D1 competition assay.

EXAMPLE 8: CROSS-STRAIN PROTECTION

Cross protection could not be tested using different strains of viruses due to lack of mouse adapted forms of these viruses. In order to test if HA6 or a similar molecule designed from another strain could provide cross-strain protection, an HA6 like construct (HA6a) is made from the HA sequence of A/Phil/2/82. The sequence of HA from A/Phil/2/82 is mapped onto the pdb lhgd and regions corresponding to the earlier HA6 design are identified. Mutations that are introduced in HA6 to destabilize the low pH form and to remove exposed hydrophobic patches are introduced in this construct also. The protein is expressed and purified in a similar manner to that of HA6 protein. Mice that are immunized with HA6a generate high titers of anti-HA6 antibodies (Table 2) and are protected (80% protection) against an infection with A/HK/68 (Fig. 10) showing that stem domain HA6 like proteins are capable of providing protection against drifted strains of H3N2 viruses.

EXAMPLE 9: ANTIBODY BINDING TO RECOMBINANT HA PROTEIN

In order to test if HA6 induced antibodies could bind to HA from a recent H3 strain that is very different from A/HK/68 (75% identity in exposed regions), binding studies are carried out with recombinant HA of A/Brisbane/16/07. ELISA studies using anti-HA6 sera and convalescent sera from A/HK/68 infected mice showed that anti-HA6 sera bound immobilized recombinant HA from A/Brisbane/16/07 with approximately 100 fold greater half maximal titers than convalescent sera (Fig. 11). The latter contain Abs primarily directed against the exposed, globular head of HA. A sequence analysis revealed that the identity in exposed regions in the HA6 fragment of the two HA molecules (A/HK/68 and A/Brisbane/10/07) is 91% while in the HA1 fragment it is 66%. These results indicate that HA6 designed from HA of HK/68 strain provide protection against recent strains of H3 viruses also owing to the higher degree of conservation in this region of the molecule. This is confirmed by the protection conferred by HA6a (based on A/Phil/2/82) against HK68 pathogenic challenge.

Antibody titers: Serum antibody titers are determined by Enzyme-Linked Immuno Sorbent Assay (ELISA). 96-well plates are coated with 50 ul per well of a test antigen at
a concentration of 4 ug per ml in phosphate buffered saline (PBS) at 4°C overnight. Plates are washed six times with PBS containing 0.05% Tween-20 (PBST) and blocked with 3% skim milk in PBST (milk-PBST). Sera are diluted in a 4-fold series in milk-PBST and added in a volume of about 100(4.1 per well. Plates are incubated for about 2 hours at room temperature followed by six washes with PBST. About 50 ul of predetermined dilution (1:5000) of HRP-conjugated goat anti-mouse secondary antibodies in milk-PBST is added per well and incubated at room temperature for about 1 hour. Plates are washed six times followed by addition of about 100 ul per well of substrate TMB and stopped after about 3-5 minutes of development. The antibody titer is defined as the reciprocal of the highest dilution that gave an OD 450nm value above the mean plus two standard deviations of the conjugate control wells.

EXAMPLE 10: BINDING OF HA6 TO THE BROADLY NEUTRALIZING MAB 12D1

250ng per well of each of HA6, peptide (57- 98)HA2, recombinant A/Brisbane/10/07 HA, recombinant A/Wyoming/03 HA and Vaxigrip seasonal flu vaccine (for 2009-2010) are coated onto Nunc 96 well plates. Recombinant HA protein is obtained from Protein Science Corporation. The wells are blocked with about 1%BSA in PBS (about 0.05% Tween 20) and different concentrations of mAbl2Dl are added. After about 2 hours incubation at RT, the plates are washed and about 50 ul of 1:10,000 ALP conjugated anti-mouse Ab is added. After about 2 hours incubation at RT and washing, about 50ul ALP substrate is added. The absorbance at about 405nm is measured after about lOmin using a

Tecan microplate reader.

Binding of HA6 to a recently isolated, broadly neutralizing mAb 12D1 that cross reacts with several H3 viruses that span approximately 40 drift years 1968-2003 is tested by ELISA. The epitope to this mAb is believed to be in the region (76-106) of HA2 from binding studies to various truncated HA proteins. However, residues (99-106) of HA2 are largely buried in the neutral pH structure of HA. Hence, as a control, a peptide consisting of residues (57-98) of HA2 is used. This peptide is unstructured at neutral pH (Fig. 3A). While the mAb failed to bind to the peptide, it bound well to HA6 as well as to other

recombinant HA proteins expressed in baculovirus (Fig. 12A). These data demonstrate that 12D1 recognizes a conformational epitope that is well presented on the HA6 immunogen and that the HA2 region in HA6 is folded in a similar fashion to the corresponding region in the neutral pH structure of HA.

EXAMPLE 11: COMPETITION BINDING OF 12D1 TO RECOMBINANT

A/BRISBANE/10/07 HA IN THE PRESENCE OF ANTI-HA6 SERA
Sector(tm) Imager 6000 96-well plates are coated with about 10 ng of recombinant HA (A/Brisbane/10/07) per well. The plates are blocked with about 3% dry milk in PBS containing about 0.05% Tween 20. The test guinea pig serum is serially diluted two folds using PBST (Phosphate buffered saline containing about 0.05% Tween 20) MAb 12D1 at about lOng/ml is mixed with serial dilutions of the test guinea pig serum. About 25 ul of the mixture is added to each well and incubated at room temperature for one hour. Plates are washed with PBST, added with about 25ul of MSD Sulfo-tag labeled goat anti-mouse IgG at about 1 u.g/ml in PBST per well and incubated at room temperature for about one hour, which is followed with additional wash and addition of about 125 u.1 of substrate per well. The plates are read with Sector(tm) Imager 6000. All samples are run in duplicate.

Anti-HA6 antisera compete with 12D1 binding to HA (Fig. 12B) indicating that 12D1 like Abs are elicited by HA6.

EXAMPLE 12: SEQUENCE ANALYSIS

The HA protein sequences for vaccine strains (HI, H3 and B type) for all the flu seasons over the past ten years (recommended by WHO) are obtained from the Influenza Virus Resource at NCBI. All non-identical, full length HA sequences of HI, H3 and B type strains are also obtained from the NBCI resource. The HA sequences of each subtype are multiply aligned using ClustalX. The % conservation of residues at each position is mapped onto the HA neutral pH structure (PDB ID: 1HGD) for the individual subtypes. Conserved, exposed regions of the molecule are identified by calculating the accessibilities of the residues and using an accessibility cutoff of >20% to identify

exposed residues. The analysis is repeated for an HA6 version using a model of HA6 (derived from the structure of neutral pH HA).

In an embodiment of the present disclosure, a sequence analysis of vaccine strains of influenza virus used over the past decade revealed very few changes in the region of the molecule included in the present HA6 immunogen. The present immunogen is based on an H3 (A/HK/68) HA sequence. Phylogenetic analysis of HA sequences from various subtypes has revealed two groups (HI and H3 groups) and four clades (HI, H9, H3 and H7 - the former two clades belong to the HI group and the latter two clades belong to the H3 group). HI, H2 and H5 HAs belong to the HI clade and are closely related. A monoclonal antibody CI79 has previously been shown to neutralize viruses from these three subtypes. mAbs directed to closely related epitopes on the stem of HA have been shown to neutralize several viruses belonging to the homologous HI clade and other Abs have recently been shown to neutralize several H3 subtype viruses indicating the possibility that an HA2 based antigen might elicit broadly neutralizing Abs. Binding studies of HA6 with a broadly neutralizing mAb 12D1 indicate that epitope(s) that are capable of eliciting a broadly neutralizing Ab response are present on the HA6 immunogen. 12D1 binds to both HA6 and recombinant HA expressed in insect cells with similar affinities (Fig. 10A.). Further, competition binding experiments indicate that 12D1 like Abs are present in anti-HA6 guinea pig serum.

Mice immunized with HA6a (based on A/Phil/2/82) are protected against a challenge with HA from HK/68 strain indicating successful cross protection. Binding studies with recombinant HA of a recently circulating H3 strain (A/Brisbane/16/07) that is closely related to the H3 strain in the 2009-2010 flu vaccine, showed good binding with HA6 antisera while convalescent sera showed 100 fold lower binding (Fig. 11). These results indicate that HA6 elicits high titer cross reactive Abs, by focusing the immune response to the conserved stem region of the molecule. Even relatively few mutations (one or two mutations) in the HA globular domain are known to abolish neutralization of the virus by antibodies. The A/Phil/2/82 HA that has been used in this study to show cross-strain protection has only 85% identity in the exposed regions with the challenge strain A/HK/68. Hence it is not possible that immunization with inactivated A/Phil/2/82 virus

would confer protection to an HK/68 challenge The HA6 protein is highly immunogenic in mice, provides protection against a homologous viral challenge, and provides cross strain protection within the subtype. It also elicits Abs that can compete with broadly neutralizing mAb 12D 1.

Natural influenza infection, neutralizing antibodies elicited against HA lead to clearance of the virus. Four major antigenic sites have been identified on the HA molecule all of which are on the receptor binding HA1 subunit. The HA2 subunit of influenza HA is typically considered not to play a major role in recovery from natural infection. However, recent studies have shown that antibodies against HA2 may help in improving recovery from viral infection. Several studies have also shown that crossreactive neutralizing antibodies are directed to HA2 (14-18, 32). Until the present work, production of a soluble, native (neutral pH like) HA2 immunogen has proved to be difficult, owing to the metastable nature of HA. The present disclosure reports, the successful design and characterization of a soluble immunogen that largely consists of HA2 in its neutral pH conformation. This is achieved by introducing regions of HA1 that are interacting with the HA2 subunit and by destabilizing the low pH conformation. Since this immunogen is expressed in E.coli, it can be rapidly and cheaply produced in large quantities.
Immunity to natural influenza infection is attributed to anti-HA antibodies and immunity is lost if Abs fail to recognize HA. HA directed Abs protect from infection primarily by preventing attachment of the virus to the host cell and also by preventing fusion of the viral and host cell membranes. The HA6 immunogen described here is devoid of the globular head domain of HA that contains the receptor binding site. Hence receptor mediated Abs (that would show HI inhibition activity) would not be elicited using this protein. The fact that HA6 is capable of eliciting mAbl2Dl like responses suggests that neutralizing Abs are also likely to contribute to protection by mechanisms similar to 12D1. The mAb 12D1 confers protection by inhibition of viral fusion to the host cell membrane, unlike HA1 directed Abs that prevent infection by blocking receptor binding.

Broadly neutralizing HA2 stem-specific monoclonal antibodies with neutralizing activity for HI clade viruses and recently for H3 subtype viruses have been isolated. These studies indicated that epitopes in the stem of HA are accessible to antibodies. Further improvements in the design of the HA6 immunogen and those of other subtypes can lead to constructs which possibly elicit high titers of cross-reactive antibodies capable of neutralizing multiple subtypes of the virus. The HI, H2, H5, H7 and H9 subtypes of influenza A (including pandemic H1N1) also contains hydrophobic residues in the loop contacting helices 4 and 5 that are exposed at neutral pH and buried in the low pH structure. Asp mutations at these sites (63, 66, 70, 73 of HA2) are likely to stabilize corresponding HA6 like constructs derived from viruses of these subtypes.

We Claim:

1. Nucleotide sequence set forth in Seq Id No. 1.

2. Peptide sequences set forth in Seq Id Nos. 2 and 3, wherein the peptide sequence set forth as Seq Id No. 3 corresponds to the nucleotide sequence set forth in the Seq Id No. 1.

3. A method of obtaining a peptide sequence set forth as Seq Id No. 2, said method comprising acts of:
a.) identifying conserved regions of HA1 and HA2 subunits of HA peptide of
Influenza virus; and b.) combining the identified conserved regions to obtain the peptide sequence
set forth as Seq Id No. 2.

4. A method of obtaining a recombinant peptide sequence set forth as Seq Id No. 3,
said method comprising acts of:
a.) identifying conserved regions of HA1 and HA2 subunits of HA peptide of
Influenza virus; b.) combining the identified conserved regions to obtain a peptide sequence
set forth as Seq Id No. 2; and c.) introducing mutations in the conserved regions of the peptide sequence of
step (b), to obtain the recombinant peptide sequence set forth as Seq Id
No. 3.

5. The method as claimed in claim 4, wherein the peptide sequence set forth in Seq Id No. 3 corresponds to the peptide sequence of step (b) having mutations at positions 63 or 73 within the HA2 subunit or at positions 297, 300, 302, and 305 within HA1 subunit or any combination of mutations thereof.

6. The method as claimed in claim 4, wherein the mutations are selected from group comprising point mutation, insertion, deletion, substitution and frameshift mutation or any combination thereof.

7. A recombinant vector having accession number MTCC 5631, said vector comprising nucleotide sequence set forth as Seq Id No.l.

8. A recombinant cell, comprising the vector as claimed in claim 7.

9. A method of obtaining the recombinant cell as claimed in claim 8, said method
comprising acts of:
a. obtaining vector comprising nucleotide sequence set forth as Seq ID No.
1; and
b. transforming a host cell with the vector to obtain the recombinant cell.

10. A method of obtaining protein comprising peptide sequence set forth as Seq Id
No. 3, said method comprising acts of:
a.) inserting nucleotide sequence set forth as Seq Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell;
b.) expressing the nucleotide within the cell for obtaining the protein comprising the peptide sequence set forth as Seq Id No. 3; and
c.) purifying the protein of step b).

11. Immunogen comprising a protein with peptide sequence set forth as Seq Id No. 3, optionally along with adjuvant(s) or pharmaceutically acceptable additive(s) or combination thereof.

12. A method of obtaining immunogen comprising protein with peptide sequence set forth as Seq Id No. 3, optionally along with adjuvant(s) or pharmaceutically acceptable additive(s) or any combination thereof, said method comprising acts of:
a.) inserting the nucleotide sequence set forth as Seq Id No. 1 into a vector
and transforming a host cell with the vector to obtain a recombinant
cell; b.) expressing the nucleotide within the cell for obtaining and purifying
the protein, to obtain the immunogen; and c.) optionally adding adjuvant(s) or pharmaceutically acceptable
additive(s) or combination thereof, to the immunogen of step b.)

13. The vector as claimed in claim 7, the recombinant cell as claimed in claim 8, and
the methods as claimed in claims 9, 10 and 12, wherein the vector is selected from
group comprising bacterial expression vectors, yeast expression vectors and

animal cell expression vectors; and the host cell is selected from group comprising bacteria, yeast and animal cells.

14. The immunogen as claimed in claim 11 and the method as claimed in claim 12, wherein the adjuvant is selected from group comprising CpG7909, IMX, MAA, Freund's adjuvant, Mycobacterium w (Mw) and Adjuplex LAP or any combination thereof; and the pharmaceutically acceptable additive is selected from group comprising excipients, gums, sweeteners, coatings, binders, disintegrants, lubricants, disintegration agents, suspending agents, granulating agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavoring agents, viscosity enhancers and antioxidants or any combination thereof.

15. A kit having component selected from group comprising nucleotide sequence set forth as Seq Id No. 1 or peptide sequence set forth as Seq Id Nos. 2 or 3 or vector as claimed in claim 7 or cell as claimed in claim 8 or immunogen as claimed in claim 11, adjuvant(s), pharmaceutically acceptable additive(s) or any combinations thereof along with instruction manual.