WO2015048129A1 - Avian influenza virus polypeptides and methods of their use - Google Patents

Abstract

The present invention is directed to an isolated avian influenza virus hemaglutinin (HA) polypeptide which comprises a native cleavage site corresponding to about amino acids 316 to about 345 of HA and further comprises at least one mutation as specified in Table 1. In one embodiment, the polypeptide comprises a native avian influenza HA amino acid sequence at amino acids corresponding to about 6 to about 18. In another embodiment, the polypeptide comprises a native influenza HA amino acid sequence at amino acids corresponding to about 20 to about 28. In a further embodiment, the polypeptide comprises a native influenza HA amino acid sequence at amino acids corresponding to about 367 to about 377.

Description

AVIAN INFLUENZA VIRUS POLYPEPTIDES AND METHODS OF THEIR USE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/881,739, filed September 24, 2014, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The field of this invention generally relates to polypeptides that can elicit immune responses against avian influenza, as well as methods of using the polypeptides for the prevention and/or treatment of avian influenza infection.

BACKGROUND OF THE INVENTION

[0003] Globally, influenza is the most economically significant respiratory disease in humans, pigs, horses and poultry (Wright et al., Orthomyxoviruses. In: Fields Virology. Knipe et al., eds. Lippincott Williams & Wilkins, Philadelphia, 2001. pp. 1533-1579.). Influenza virus is known for its continuous genetic and antigenic changes, which impede effective control of the virus (Wright et al. (2001); Webster et al., Microbiol. Rev. 56: 152-179 (1992)).

[0004] The influenza viruses are known to be classifiable in the various A, B, C topologies, according to the group antigen the viruses carry. The influenza viruses of the A, B, C types can be distinguished from one another on the basis of the antigen differences that can be found in the viral nucleocapsid (NP) and matrix (M) proteins. In particular, the A-type influenza viruses can be classified into subtypes on the basis of antigenic differences in the HA and NA molecules. Presently nine subtypes of the neuraminidase NA proteins, designated Nl to N9, and sixteen different subtypes of the hemagglutinin proteins, designated HI to H16, have been identified. In birds, viruses carrying any of the various HA and NA subtypes have also been isolated.

[0005] HA is a viral surface glycoprotein comprising approximately 560 amino acids. It is chiefly responsible for the adhesion of the viral particle to the host cell and for its penetration into the latter in the early stages of the infection. HA is the major viral protein that is most subject to post-translational rearrangements. After the synthesis of HA has been completed, the polypeptide follows the exocytotic pathway of the host cell, in the course of which HA is folded, assembled in trimers and glycosylated. Finally it is cleaved into two subunits HA1 and HA2; this cleavage is the key step in the activation of the molecule and in the acquisition of the infective capacity by the virion.

[0006] It is well known that the different composition of the cleavage site, concerning the basic amino acid residues, translates into the capacity of the avian influenza virus to produce localized, or symptomatic infections, or, vice versa, generalized infections having a lethal outcome for many avian species. It has therefore been suggested that this fact might influence the organ- tropism, the host specificity, as well as the pathogenicity of the virus. With respect to the pathogenicity of the virus, strains with multibase-site HA find proteases that cleave the polypeptide and give rise to multiple infection cycles with a massive production of infectious viral particles and causing a generalization of the infections in all of the districts within a short time period. The infection will consequently turn out to have an acute-hyperacute course, with very high mortality.

[0007] Avian Influenza is an acute and highly contagious viral infection of chickens and other fowl. As an influenza virus, it is classified in subtypes on the basis of antigen differences in the hemagglutinin (HA) and neuraminidase (NA) molecules, which reassort or mutate from season to season. Because it constantly mutates, vaccine preparation is difficult due to the unpredictability as to which strain will reappear in subsequent seasons. The strains used for vaccine preparation often do not reproduce under manufacturing conditions at a very fast rate, so that waiting for an appearance of a particular strain, and then manufacturing the correct vaccine to protect against the strain does not provide a viable option. Typically, the epidemic of the particular strain will last for several months, and then perhaps disappear for several years.

[0008] Eradication is the principal method for controlling the disease in avians, without obvious economic disadvantages, but if a vaccine with a fast onset of immunity could be produced, such a product would offer a viable alternative to mass slaughter of entire flocks.

[0009] There currently exists a need in the art for influenza vaccines, especially avian influenza vaccines which would provide a useful alternative to eradication of infected flocks. Such vaccines would need to elicit a quick immune response in the vaccinated birds.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to an isolated avian influenza virus hemaglutinin

(HA) polypeptide which comprises a native cleavage site corresponding to about amino acids 316 to about 345 of HA and further comprises at least one mutation as specified in Table 1. In one embodiment, the polypeptide comprises a native avian influenza HA amino acid sequence at amino acids corresponding to about 6 to about 18. In another embodiment, the polypeptide comprises a native influenza HA amino acid sequence at amino acids corresponding to about 20 to about about 28. In a further embodiment, the polypeptide comprises a native influenza HA amino acid sequence at amino acids corresponding to about 367 to about 377.

[0011] The present invention is also directed to an isolated avian influenza virus HA polypeptide which comprises a native cleavage site corresponding to about amino acids 6 to about 18, about 20 to about 28, about 316 to about 345, and about 367 to about 377, wherein the polypeptide further comprises at least one mutation as specified in Table 1.

[0012] The present invention is also directed to an isolated avian influenza HA polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to a mutated polypeptide shown in any of Tables 6-26.

[0013] The present invention is also directed to an isolated avian influenza HA polypeptide, wherein the polyeptide comprises an amino acid sequence having at least 80%, 90%, 95%, or 100%) identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 64, and 65. In another embodiment, the invention is directed to an isolated HA polypeptide fragment encompassing at least 100, at least 200, at least 300, at least 400, or at least 500 amino acids of the polypeptide described herein.

[0014] The present invention is also directed to an isolated polynucleotide encoding a polypeptide of the invention. In one embodiment, the invention is directed to a vector comprising the isolated polynucleotide. In another embodiment, the vector is a recombinant influenza virus. In another embodiment, the vector is comprised in a host cell. The invention is also directed to a method of producing an avian influenza HA polypeptide comprising culturing the host cell under conditions that result in expression of the polypeptide.

[0015] The present invention is also directed to an avian influenza vaccine composition comprising a polypeptide, polynucleotide, or vector of the invention. In one embodiment, the vaccine composition further comprises an adjuvant.

[0016] The present invention is also directed to a method of vaccinating a subject susceptible to influenza infection comprising administering an effective amount of a polypeptide, polynucleotide, vector, or vaccine composition of the invention, wherein the subject is an avian species. In another embodiment, the avian is a chicken, turkey, ostrich, pigeon, game hen, squab, guinea fowl, pheasant, quail, duck, goose, or emu. In a further embodiment, the avian is a chicken. [0017] In one embodiment, the polypeptide, polynucleotide, vector, or vaccine composition is administered via drinking water or spraying. In another embodiment, the dose administered is within the range of about 0.25 mL to 2.0 mL per avian member. In one embodiment, the vaccine is administered in no more than one dose. In one embodiment, the method comprises a prime -boost administration regime.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0018] Figure 1. Antigenicity plots of wild-type and modified AAT73266 Influenza HA.

[0019] Figure 2. Protein expression of ABW73807 mut2 wrt Arg326 (MB1343-1) or

ABW73807 target-less mut2 wrt Arg326 (MB1343-2)-expressing cells. Lane 1. Pre-Stained Protein Standard. Lane 2. MB1343-1, total protein, un-induced. Lane 3. MB1343-1, total protein, induced. Lane 4. MB1343-1, soluble protein, induced. Lane 5. MB1343-1, insoluble protein, induced. Lane 6. MB1343-2, total protein, un-induced. Lane 7. MB1343-2, total protein, induced. Lane 8. MB1343-2, soluble protein, induced. Lane 9. MB1343-2, insoluble protein, induced.

[0020] Figure 3. Western blot of ABW73807 mut2 wrt Arg326 or ABW73807 target-less mut2 wrt Arg326-expressing cells. Lane 1. Pre-Stained Protein Standard. Lane 2. MB1343-1, total protein, un-induced. Lane 3. MB1343-1, total protein, induced. Lane 4. MB1343-1, soluble protein, induced. Lane 5. MB1343-1, insoluble protein, induced. Lane 6. MB1343-2, total protein, un- induced. Lane 7. MB1343-2, total protein, induced. Lane 8. MB1343-2, soluble protein, induced. Lane 9. MB1343-2, insoluble protein, induced.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides novel avian influenza polypeptides, including, variant influenza HA polypeptides. Related polynucleotides, compositions comprising the HA polypeptides, and methods of making the polynucleotide and polypeptides are also provided. Methods of using the polynucleotides, polypeptides, and vaccine compositions, such as methods of preventing and/or treating avian influenza infection, are further provided.

I. Definitions

[0022] To facilitate an understanding of the present invention, a number of terms and phrases are defined below. [0023] The polypeptides and polynucleotides of the invention are variants of influenza HA sequences. In general, "influenza viruses" are made up of an internal ribonucleoprotein core containing a segmented single-stranded NA genome and an outer lipoprotein envelope lined by a matrix protein. The genome of influenza viruses is composed of eight segments of linear (-) strand ribonucleic acid (RNA), encoding the immunogenic hemagglutinin (HA) and neuraminidase (NA) proteins, and six internal core polypeptides: the nucleocapsid nucleoprotein (NP); matrix proteins (M); non-structural proteins (NS); and 3 RNA polymerase (PA, PB1, PB2) proteins. During replication, the genomic viral RNA is transcribed into (+) strand messenger RNA and (-) strand genomic cRNA in the nucleus of the host cell. Each of the eight genomic segments is packaged into ribonucleoprotein complexes that contain, in addition to the RNA, NP and a polymerase complex (PB1, PB2, and PA). The hemagglutinin molecule consists of a surface glycoprotein and acts to bind to N-AcetyiNeuraminic acid (NeuNAc), also known as sialic acid, on host cell surface receptors. In some embodiments herein, the polypeptides of the invention (and polypeptides encoded by the polynucleotides of the invention) can act to bind NeuNAc whether in vitro or in vivo. Such action can in some embodiments also be done by fragments of hemagglutinin which retain hemagglutinin activity. Hemagglutinin is made up of two subunits, HA1 and HA2 and the entire structure is about 550 amino acids in length and about 220 kD. Neuraminidase molecules cleave terminal sialic acid residues from cell surface receptors of influenza virus, thereby releasing virions from infected cells. Neuraminidase also removes sialic acid from newly made hemagglutinin and neuraminidase molecules. The hemagglutinin polypeptides of the invention show immunological cross -reactivity with one or more known hemagglutinin molecule from an influenza virus. The literature is replete with examples of such known hemagglutinin molecules.

[0024] Influenza is commonly grouped into influenza A and influenza B categories, as well as a typically less important C category. Influenza A and influenza B viruses each contain eight segments of single stranded RNA with negative polarity. The influenza A genome encodes eleven polypeptides. Segments 1-3 encode three polypeptides, making up a RNA-dependent RNA polymerase. Segment 1 encodes the polymerase complex protein PB2. The remaining polymerase proteins PB1 and PA are encoded by segment 2 and segment 3, respectively. In addition, segment 1 of some influenza strains encodes a small protein, PB1-F2, produced from an alternative reading frame within the PB1 coding region. Segment 4 encodes the hemagglutinin (HA) surface glycoprotein involved in cell attachment and entry during infection. Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, the major structural component associated with viral RNA. Segment 6 encodes a neuraminidase (NA) envelope glycoprotein. Segment 7 encodes two matrix proteins, designated Ml and M2, which are translated from differentially spliced mRNAs. Segment 8 encodes NS1 and NS2, two nonstructural proteins, which are translated from alternatively spliced mRNA variants. The eight genome segments of influenza B encode 11 proteins. The three largest genes code for components of the RNA polymerase, PB1, PB2 and PA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6 encodes the NA protein and the NB protein. Both proteins, NB and NA, are translated from overlapping reading frames of a bicistronic mRNA. Segment 7 of influenza B also encodes two proteins: Ml and BM2. The smallest segment encodes two products: NS1 is translated from the full length RNA, while NS2 is translated from a spliced mRNA variant.

[0025] Influenza types A and B are typically associated with influenza outbreaks in human populations. However, type A influenza also infects other creatures as well, e.g., birds, pigs, and other animals. The type A viruses are categorized into subtypes based upon differences within their hemagglutinin and neuraminidase surface glycoprotein antigens. Hemagglutinin in type A viruses has 14 known subtypes and neuraminidase has 9 known subtypes. In humans, currently only about 3 different hemagglutinin and 2 different neuraminidase subtypes are known, e.g., HI, H2, H3, Nl, and N2. In particular, two major subtypes of influenza A have been active in humans, namely, H1N1 and H3N2. H1N2, however has recently been of concern. Influenza B viruses are not divided into subtypes based upon their hemagglutinin and neuraminidase proteins.

[0026] As used herein, "avian influenza virus" refers to any influenza virus that may infect birds. "Highly pathogenic avian influenza virus (HPAI)" refers to an avian influenza virus which is highly virulent and characterized by high mortality. In one embodiment, the avian influenza virus is of the H5 subtype. In another embodiment, the avian influenza virus is of the H7 subtype. In another embodiment, the avian influenza virus is of the H5N1 subtype. In one embodiment, the avian influenza virus is ABW73807 A/chicken/Korea IS/2006 (H5N1). In another embodiment, the avian influenza virus is AEJ90156 A/Mallard duck/Korea/W401/2011 (H5N1).

[0027] The name "hemagglutinin" is derived from the viruses' ability to agglutinate red blood cells. The envelope glycoprotein HA is a rod-like shaped trimer of identical monomers. The HA protein is synthesised in the infected cell as a single polypeptide chain, HA0. This initial molecule has to be cleaved by the host cell proteases into disulfide linked HA1 (47 kDa) and HA2 (29 kDa) subunits in order for the virus to mediate membrane fusion and subsequent infection. The HAl subunit is the globular domain of the HA molecule which comprises the receptor binding site, responsible for virus attachment to sialic acid receptors on the host cell. The five antigenic sites A, B, C, D and E at the globular head direct the host antibody response. The HA is the primary viral antigen and the only antigen inducing a virus neutralising response in the host. The HA main functions are virion-to-host cell membrane fusion and fusion of the endocytosed virion with the endosomal membrane allowing release of the genome into the cytoplasm. HA is a prototype 1 integral membrane protein that is targeted to the ER membrane through an N-terminal signal peptide sequence and cleaved by signal peptidase. The HA2 subunit forms the stem of the molecule. The N-terminus of HA2 (fusion peptide) is hydrophobic and is highly conserved in the HAs of different influenza virus strains, and it is essential in HA fusion activity. The HA is post translationally modified by addition of N-linked carbohydrates at asparagine residues (N) on each monomer and palmitic acid to cysteine (C) residues in the cytoplasmic tail region. HA binds to 5- N-acetyl neuramic acid (sialic acid) on the host cell surface and positions and are essential in determining preferred host cell tropism. Human infectious strains preferentially bind to sialic acid with a-(2,6) linkage to galactose, while avian influenza viruses (AIV) preferentially bind to a-(2,3),

[0028] By "animal," "subject," or "host" is intended to mean avians (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary). The term "animal" also includes an individual animal in all stages of development, including embryonic and fetal stages. In one embodiment, the animal or subject is a bird or other fowl. In one embodiment, the subject is a duck or chicken.

[0029] The terms "protein", "peptide", "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0030] The antigenic polypeptides of the invention are capable of protecting against or treating influenza infection. That is, they are capable of stimulating an immune response in an animal. By "antigen" or "immunogen" means a substance that induces a specific immune response in a host animal. The antigen may comprise a whole organism, such as an influenza virus, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof. [0031] The term "immunogenic or antigenic polypeptide" as used herein includes polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. Preferably the protein fragment is such that it has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the invention comprises or consists essentially of or consists of at least one epitope or antigenic determinant. An "immunogenic or antigenic" polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. By "immunogenic or antigenic fragment" is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al, 1984; Geysen et al., 1986. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.

[0032] As discussed, the invention encompasses active fragments and variants of the antigenic polypeptide. Thus, the term "immunogenic or antigenic polypeptide" further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein. In one embodiment, the polypeptides of the invention comprise amino acid substitutions as set forth in Table 1.

[0033] The term "conservative variation" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic-aspartate and glutamate; (2) basic-lysine, arginine, histidine; (3) non-polar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar-glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid that will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the definition of the reference polypeptide. All of the polypeptides produced by these modifications are included herein. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

[0034] Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al., 1993; Bergmann et al, 1996; Suhrbier, 1997; Gardner et al., 1998. Immunogenic fragments, for purposes of the present invention, will usually include at least about 10-15 amino acids, at least about 15-25 amino acids, at least about 25-50 amino acids, at least about 50-100 amino acids, at least about 100-150 amino acids, at least about 150-200 amino acids, at least about 200-250 amino acids, at least about 250-300 amino acids, at least about 300-350 amino acids, at least about 350- 400 amino acids, at least about 400-450 amino acids, or at least about 450-500 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.

[0035] Accordingly, a minimum structure of a polynucleotide expressing an epitope is that it comprises or consists essentially of or consists of nucleotides encoding an epitope or antigenic determinant of an influenza polypeptide. A polynucleotide encoding a fragment of an influenza polypeptide may comprise or consist essentially of or consist of a minimum of 15 nucleotides, or more generally at least about 150 consecutive or contiguous nucleotides of the sequence encoding the polypeptide or polypeptide fragment. Epitope determination procedures, such as, generating overlapping peptide libraries (Hemmer et al., 1998), Pepscan (Geysen et al., 1984; Geysen et al., 1985; Van der Zee R. et al., 1989; Geysen, 1990; Multipin^® Peptide Synthesis Kits de Chiron) and algorithms (De Groot et al, 1999; PCT/US2004/022605) can be used in the practice of the invention. [0036] The term "epitope" refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site". Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

[0037] An "immunological response" to a polypeptide, polynucleotide, composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a polypeptide, polynucleotide, composition or vaccine of the invention. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

[0038] A "protective immune response" against influenza virus refers to an immune response exhibited by an individual (e.g., a human) that is protective against disease when the individual is subsequently exposed to and/or infected with wild-type influenza virus. In some instances, the wild-type (e.g., naturally circulating) influenza virus can still cause infection, but it cannot cause a serious or life-threatening infection. Typically, the protective immune response results in detectable levels of host engendered serum and secretory antibodies that are capable of neutralizing virus of the same strain and/or subgroup (and possibly also of a different, non-vaccine strain and/or subgroup) in vitro and in vivo.

[0039] The term "adjuvant" is used herein to mean any molecule added to the polynucleotide or polypeptide vaccines described herein to enhance antigenicity of the influenza antigen described herein.

[0040] The term "nucleic acid" and "polynucleotide" refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rR A, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism.

[0041] The term "gene" is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

[0042] The invention further comprises a complementary strand to a polynucleotide encoding an influenza antigen, epitope or immunogen. The complementary strand can be polymeric and of any length, and can contain deoxyribonucleotides, ribonucleotides, and analogs in any combination.

[0043] An "isolated" biological component (such as a nucleic acid or protein or intact virus) refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.

[0044] The term "purified" as used herein does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment. That is the polypeptide is separated from cellular components. By "substantially purified" is intended such that at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or more of the cellular components or materials have been removed. Likewise, the polypeptide may be partially purified. By "partially purified" is intended that less than 60% of the cellular components or material is removed. The same applies to polynucleotides. The polypeptides disclosed herein can be purified by any of the means known in the art.

[0045] "Variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is, they have the ability to elicit an immune response.

[0046] Homologs of influenza polypeptides from avians are intended to be within the scope of the present invention. As used herein, the term "homologs" includes orthologs, analogs and paralogs. The term "analogs" refers to two polynucleotides or polypeptides that have the same or similar function, but that have evolved separately in unrelated organisms. The term "orthologs" refers to two polynucleotides or polypeptides from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode polypeptides having the same or similar functions. The term "paralogs" refers to two polynucleotides or polypeptides that are related by duplication within a genome. Paralogs usually have different functions, but these functions may be related. Analogs, orthologs, and paralogs of a wild-type influenza polypeptide can differ from the wild-type influenza polypeptide by post-translational modifications, by amino acid sequence differences, or by both. In particular, homologs of the invention will generally exhibit at least 80- 85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98%, 99% sequence identity, with all or part of the wild-type influenza polypeptide or polynucleotide sequences, and will exhibit a similar function. Variants include allelic variants. The term "allelic variant" refers to a polynucleotide or a polypeptide containing polymorphisms that lead to changes in the amino acid sequences of a protein and that exist within a natural population (e.g., a virus species or variety). Such natural allelic variations can typically result in 1-5% variance in a polynucleotide or a polypeptide. Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different species, which can be readily carried out by using hybridization probes to identify the same gene or genetic locus in those species. Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity of gene of interest, are intended to be within the scope of the invention. [0047] The sequence identity between two amino acid sequences may be established by the

NCBI (National Center for Biotechnology Information) pairwise blast and the blosum62 matrix, using the standard parameters (see, e.g., the BLAST or BLASTX algorithm available on the "National Center for Biotechnology Information" (NCBI, Bethesda, Md., USA) server, as well as in Altschul et al.; and thus, this document speaks of using the algorithm or the BLAST or BLASTX and BLOSUM62 matrix by the term "blasts").

[0048] The "identity" with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics.TM. Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.

[0049] The sequence identity or sequence similarity of two amino acid sequences, or the sequence identity between two nucleotide sequences can be determined using Vector NTI software package (Invitrogen, 1600 Faraday Ave., Carlsbad, Calif.).

[0050] The following documents provide algorithms for comparing the relative identity or homology of sequences, and additionally or alternatively with respect to the foregoing, the teachings in these references can be used for determining percent homology or identity: Needleman S B and Wunsch C D; Smith T F and Waterman M S; Smith T F, Waterman M S and Sadler J R; Feng D F and Dolittle R F; Higgins D G and Sharp P M; Thompson J D, Higgins D G and Gibson T J; and, Devereux J, Haeberlie P and Smithies O. And, without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.

[0051] A "vector" refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo. The heterologous polynucleotide may comprise a sequence of interest for purposes of prevention or therapy, and may optionally be in the form of an expression cassette. As used herein, a vector needs not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.

[0052] The term "recombinant" means a polynucleotide of semisynthetic, or synthetic, origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.

[0053] "Heterologous" means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide, may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.

[0054] The term "vaccine," as that term is used herein, is a composition that elicits an immune response (cellular and/or humoral) in a subject. A vaccine may reduce the risk of infection but does not necessarily prevent infection. In specific, non-limiting embodiments, a vaccine increases the level of cellular and/or humoral immunity by at least 30 percent, 50 percent, or 100 percent of baseline levels.

[0055] In some embodiments, a "priming" or first administration of a modified influenza

HA protein, or polynucleotide encoding said protein, is followed by one or more "boosting" or subsequent administrations of a modified influenza HA protein or polynucleotide encoding said protein ("prime and boost" method). For instance, a first administration with a modified HA polypeptide or polynucleotide encoding said protein is followed by one or more subsequent administrations of a modified HA polypeptide or polynucleotide encoding said polypeptide.

II. Polypeptides of the invention

[0056] The present invention relates to avian influenza vaccine or pharmaceutical or immunological compositions which may comprise an effective amount of a recombinant avian influenza polypeptide and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle.

[0057] Compositions comprising an influenza antigen and fragments and variants thereof that elicit an immunogenic response in an animal are provided. The antigenic polypeptides or fragments or variants may be formulated into vaccines, pharmaceutical, or immunological compositions and used to elicit or stimulate a protective or therapeutic response in an animal. In one embodiment the polypeptide antigen is a hemagglutinin polypeptide or active fragment or variant thereof. [0058] It is recognized that the antigenic polypeptides or antigens of the invention may be full length polypeptides or active fragments or variants thereof. By "active fragments" or "active variants" is intended that the fragments or variants retain the antigenic nature of the polypeptide. Thus, the present invention encompasses any influenza polypeptide, antigen, epitope or immunogen that elicits an immunogenic response in an animal. The influenza polypeptide, antigen, epitope or immunogen may be any influenza polypeptide, antigen, epitope or immunogen, such as, but not limited to, a protein, peptide or fragment or variant thereof, that elicits, induces or stimulates a response in an animal.

[0059] A particular antigenic polypeptide of interest is hemagglutinin (HA). It is an antigenic glycoprotein and is responsible for binding the virus to the cell that is being infected. There are different HA antigens, any of which can be used in the practice of the invention. Of interest is the HA from H5N1, a highly pathogenic avian flu virus. More particularly, the HA can be isolated from H5N1 isolated from the A/chicken/Korea/IS/2006 (H5N1) or A/Mallard duck/ orea/W401/2011 (H5N1) strains. However, HA from other influenza viruses (i.e. H1-H16) can be used in the practice of the invention including HI, H3, H5, H6, H7, H9 and the like. It is further recognized that HA precursors of any of the HA proteins can be used.

[0060] HA is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem region. Both regions carry N-linked oligosaccharides, which play an important role in the biological function of HA (Schulze, I. T., J Infect Dis, 1997. 176 Suppl 1 : p. S24-8; Deshpande, K. L., et al., PNAS USA, 1987, 84(1): p. 36-40). Among different subtypes of influenza A viruses, there is significant variation in the glycosylation sites of the head region, whereas the stem oligosaccharides are more conserved and required for fusion activity (Ohuchi, R., et al, J Virol, 1997, 71(5): p. 3719-25). Glycans near antigenic peptide epitopes interfere with antibody recognition (Skehel, J. J., et al, PNAS USA, 1984, 81(6): p. 1779-83), and glycans near the proteolytic site modulate cleavage and influence the infectivity of influenza virus (Deshpande, K. L., et al, 1987). Nucleotide sequence analysis of 62 H5 genes support the hypothesis that additional glycosylation near the receptor binding site within the HA globular head is an adaptation of the virus following interspecies transmission from wild birds, particularly waterfowl, to poultry (Banks, J., et al., Avian Dis, 2003, 47(3 Suppl): p. 942-50).

[0061] Over 150 B cell epitopes as well as 113 CD4+ and 35 CD8+ T cell epitopes have been identified for HA protein of influenza virus, however, only a limited number of epitopes have been reported for avian influenza strains/subtypes (Bui, H. H., et al, PNAS USA, 2007, 104(1): p. 246-51). Examination of the sites of amino acid substitutions in natural and monoclonal antibody- selected antigenic variants indicated that all antigenic sites are on the surface of the membrane distal HA1 domain predominantly surrounding the receptor-binding sites. There are two notable features of the antigenic sites: the loop like structure of several of them and the incidence of carbohydrate side chains (Skehel, J. J., et al., Annu Rev Biochem, 2000, 69: p. 531-69). The localization and fine structure of two H5 antigenic sites have been described (Kaverin, N. V., et al, J Gen Virol, 2002. 83(Pt 10): p. 2497-505). Site 1 is an exposed loop comprising HA1 residues 140-145 that corresponds to antigenic sites A of H3 and Ca2 of HI, and site 2 comprised two subsites, one (HA1 residues 156 and 157) that corresponds to site B in the H3 subtype and one (HA1 residues 129 to 133) that corresponds to site Sa in the HI subtype. An epitope mapping study suggested that HA antigenic structure of recent H5N1 isolated differs substantially from that of a low-pathogencity H5 strain and is rapidly evolving (Kaverin, N. V., et al., J Virol, 2007. 81(23): p. 12911-7). An epitope conservancy analysis suggests significant levels of interstrain cross-reactivity are likely for T cell epitopes, but much less so for antibody epitopes. Using an overlapping peptide library, a T cell epitope of avian influenza virus was identified for the first time, which is a 15-mer peptide, H246-260 within the HA1 domain which induced action of T cells in chickens immunized against H5 HA (Haghighi, H. R., et al., PLoS ONE, 2009. 4(11): p. e7772).

[0062] The polypeptide design of the molecules of the invention focuses the antibody response to a chosen epitope of a protein antigen. The site is generally one that is of critical importance to the function of the molecule, e.g., a site that it uses to interact with other molecules, its active site if it is an enzyme, or a part that needs to be modified to enable the molecule to perform its essential function. A site that could also be a good target for a focused antibody response would be the epitope of a neutralizing antibody. The design strategy is by the general reduction of the antigenicity of all parts of the antigen, except that of the chosen site. This is accomplished by the judicious replacement of all the exposed residues of the antigen, except those in the chosen site, with amino acids whose physicochemical properties render them less reactive, while at the same time preserving the overall structure of the molecule. The attempt to preserve structure is made by replacing the exposed residue with one that has the same propensity for secondary structure. In addition, all the buried residues, as well as those which probably play crucial roles in maintaining tertiary structure, e.g., prolines and cysteines, are preserved. Preservation of the overall structure of the antigen is critical since antibody epitopes are generally conformational, i.e., they are constructed from several parts of an antigen that lie adjacent to each other in the three-dimensional structure. [0063] The polypeptides described herein target the cleavage site of HPAI H5N1.

Antibodies bound to the cleavage site can interfere with the action of the proteolytic enzymes and prevent the formation of the infectious form of the virus. The importance of eliciting antibodies specific for the cleavage site region of hemagglutinin has been recognized and attempts to develop vaccines using peptides containing this region have been made (Horvath et al. 1998, Bianchi et al. 2005). The use of immunogens that present epitopes in correct context and structure, is better than the use of peptides, since unconstrained peptides can assume many different structures in solution. HA is modified according to the algorithm shown in Table 1.

I f in

ififiTO Helix She Coi l Tufri aeid Change to ;

A 1 T hr Ala

¾sn Ala Thr Ser Gly

¾¾p Ala Thr S-er

Ala T hr l Thr

Sin JK. \-L-3i T hr Thr

His Ala T hr Thr Th

Lys Ala Thr Thr hr

■Ala T hr Ala Ala

¾rp^' Ala Thr Ala Vsl

Thr hr

Cys , Giy.,. lie .,. Leu , Met , Sex , Thi^

:. iid &l. a & iiiot . re l ced ;

Table 1. The amino acid replacement rules designed to reduce the antigenicity of protein epitopes. (Padlan EA. Phil Sci Letts 2010; 3(2):36-47.)

[0064] The HA peptides may also comprise isosteres of two or more residues in the immunogenic peptide. An isostere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol. VII (Weinstein ed., 1983).

[0065] Modifications of peptides with various amino acid mimetics or unnatural amino acids are particularly useful in increasing the stability of the peptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al, Eur. J. Drug Metab. Pharmacokin. 11 :291-302 (1986). Half-life of the peptides of the present invention is conveniently determined using a 25% human serum (v/v) assay. The protocol is generally as follows. Pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture media and used to test peptide stability. At predetermined time intervals a small amount of reaction solution is removed and added to either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled (4°C) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.

[0066] The HA peptides of the present invention or analogs thereof which have immune stimulating activity may be modified to provide desired attributes other than improved serum half- life. For instance, the ability of the peptides to induce CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Particularly preferred immunogenic peptides/T helper conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the CTL peptide may be linked to a T helper peptide without a spacer.

[0067] The immunogenic peptides may be linked to a T helper peptide either directly or via a spacer either at the amino- or carboxy-terminus of the HA peptide. The amino-terminus of either the immunogenic peptide or the T helper peptide may be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389. [0068] In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes CTL. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the alpha and epsilon amino groups of a Lys residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be injected directly in a micellar form, incorporated into a liposome or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment a particularly effective immunogen comprises palmitic acid attached to alpha and epsilon amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

[0069] As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide. See, Deres et al, Nature 342:561-564 (1989), incorporated herein by reference. Peptides of the invention can be coupled to P₃CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Further, as the induction of neutralizing antibodies can also be primed with P₃CSS conjugated to a peptide which displays an appropriate epitope, the two compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection.

[0070] In addition, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support, or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide. Modification at the C-terminus in some cases may alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH₂ acylation, e.g., by alkanoyl (C1-C20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.

III. Polynucleotides, Vectors, Expression Systems, and Recombinant Viruses

[0071] The present invention includes polynucleotides encoding any of the polypeptides, or polypeptide fragments described herein. [0072] The present invention includes recombinant constructs incorporating one or more of the nucleic acid sequences described herein. Such constructs optionally include a vector, for example, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), etc., into which one or more of the polynucleotide sequences of the invention, e.g., comprising an avian HA comprising at least one amino acid substitution, or a subsequence thereof etc.. For example, the inserted nucleic acid can include a viral chromosomal sequence or cDNA including all or part of at least one of the polynucleotide sequences of the invention. In one embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.

[0073] Polynucleotides encoding the polypeptides of the present invention can be included in any one of a variety of vectors suitable for generating sense or antisense RNA, and optionally, polypeptide (or peptide) expression products (e.g., a hemagglutinin molecule of the invention, or fragments thereof). Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses and many others (e.g., pCMV/R) (Barouch et al. 2005 J Virol 79:8828-8834). Any vector that is capable of introducing genetic material into a cell, and, if replication is desired, which is replicable in the relevant host, can be used.

[0074] In an expression vector, the HA polynucleotide sequence of interest is physically arranged in proximity and orientation to an appropriate transcription control sequence (e.g., promoter, and optionally, one or more enhancers) to direct mRNA synthesis. That is, the polynucleotide sequence of interest is operably linked to an appropriate transcription control sequence. Examples of such promoters include: LTR or SV40 promoter, E. coli lac or trp promoter, phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.

[0075] A variety of promoters are suitable for use in expression vectors for regulating transcription of influenza virus genome segment sequences. In certain embodiments, the cytomegalovirus (CMV) DNA dependent RNA Polymerase II (Pol II) promoter is utilized. If desired, e.g., for regulating conditional expression, other promoters can be substituted which induce RNA transcription under the specified conditions, or in the specified tissues or cells. Numerous viral and mammalian promoters are available, or can be isolated according to the specific application contemplated. For example, alternative promoters obtained from the genomes of animal and human viruses include such promoters as the adenovirus (such as Adenovirus 2), papilloma virus, hepatitis-B virus, polyoma virus, and Simian Virus 40 (SV40), and various retroviral promoters. Mammalian promoters include, among many others, the actin promoter, immunoglobulin promoters, heat-shock promoters, and the like.

[0076] Transcription is optionally increased by including an enhancer sequence. Enhancers are typically short, e.g., 10-500 bp, cis-acting DNA elements that act in concert with a promoter to increase transcription. Many enhancer sequences have been isolated from mammalian genes (hemoglobin, elastase, albumin, alpha-fetoprotein, and insulin), and eukaryotic cell viruses. The enhancer can be spliced into the vector at a position 5' or 3' to the heterologous coding sequence, but is typically inserted at a site 5' to the promoter. Typically, the promoter, and if desired, additional transcription enhancing sequences are chosen to optimize expression in the host cell type into which the heterologous DNA is to be introduced. Optionally, the amplicon can also contain a ribosome binding site or an internal ribosome entry site (IRES) for translation initiation.

[0077] The vectors of the invention also can include sequences necessary for the termination of transcription and for stabilizing the mRNA, such as a polyadenylation site or a terminator sequence. Such sequences are commonly available from the 3' and, occasionally 5', untranslated regions of eukaryotic or viral DNAs or cDNAs. In one embodiment, the bovine growth hormone terminator can provide a polyadenylation signal sequence.

[0078] The vector containing the appropriate nucleic acid sequence as described above, as well as the appropriate regulatory sequences, can be employed to transform a host cell permitting expression of the protein. While the vectors of the invention can be replicated in bacterial cells, frequently it will be desirable to introduce them into mammalian cells, e.g., Vero cells, BHK cells, MDCK cells, 293 cells, COS cells, or the like, for the purpose of expression.

[0079] The invention also relates to recombinant influenza viruses comprising a modified avian influenza virus HA gene segment described herein. In accordance with such an aspect, the virus further comprises gene segments to complete the full set of gene segments found in a genome of an influenza virus (i.e., complementing influenza virus gene segments). In certain embodiments, the complementing influenza virus gene segments may all be derived from the same type or subtype of an influenza virus. In other embodiments, the complementing influenza virus gene segments may be derived from one, two or more different types or subtypes of an influenza virus. In some embodiments, the complementing influenza virus gene segments may all be derived from the same strain of an influenza virus. In other embodiments, the complementing influenza virus gene segments may be derived from one, two or more different strains of an influenza virus. In certain embodiments, the complementing influenza virus gene segments can be derived from an attenuated influenza virus strain.

[0080] In certain embodiments, the modified avian influenza virus HA gene segment and one, two or more of the complementing influenza virus gene segments may be derived from the same type or subtype of an influenza virus. In other embodiments, the modified influenza virus HA gene segment and one, two or more of the complementing influenza virus gene segments may be derived from different types or subtypes of an influenza virus. In some embodiments, the modified influenza virus HA gene segment and one, two or more of the complementing influenza virus gene segments may be derived from the same strain of an influenza virus. In other embodiments, the modified influenza virus HA gene segment and one, two or more of the complementing influenza virus gene segments may be derived from different strains of an influenza virus.

[0081] In certain embodiments, a recombinant influenza virus described herein comprises at least one complementing influenza virus gene segment that encodes a fusion protein. A fusion protein can be a fusion of an influenza virus protein or a fragment thereof with a heterologous protein (such as a viral antigen, a bacterial antigen, a parasitic antigen, a fungal antigen, a tumor antigen, a tumor associated antigen, a cytokine, a growth factor, a peptide tag, or a detectable substance.

[0082] In certain embodiments, a recombinant influenza virus described herein comprises at least one complementing influenza virus gene segment that encodes a bicistronic mRNA. Techniques for creating an influenza virus gene segment that encodes a bicistronic mRNA are known in the art. Bicistronic techniques allow the engineering of coding sequences of multiple proteins into a single mRNA through the use of internal ribosome entry site (IRES) sequences. Briefly a coding region of one protein is inserted into the open reading frame of a second protein. The insertion is flanked by an IRES and any untranslated signal sequences necessary for proper expression and/or function. The insertion must not disrupt the open reading frame, polyadenylation or transcriptional promoters of the second protein (see, e.g., Garcia-Sastre et al., 1994, J. Virol. 68:6254-6261 and Garcia-Sastre et al, 1994 Dev. Biol. Stand. 82:237-246, each of which is hereby incorporated by reference in its entirety). See also, e.g., U.S. Pat. No. 6,887,699, U.S. Pat. No. 6,001,634, U.S. Pat. No. 5,854,037 and U.S. Pat. No. 5,820,871, each of which is incorporated herein by reference in its entirety. Any IRES known in the art or described herein may be used in accordance with the invention (e.g., the IRES of BiP gene, nucleotides 372 to 592 of GenBank database entry HUMGRP78; or the IRES of encephalomyocarditis virus (EMCV), nucleotides 1430-2115 of GenBank database entry CQ867238.). One of the open reading frames of the bicistronic mRNA may encode an influenza virus protein or a fragment thereof and the other open reading frame of the bicistronic mRNA may encode a heterologous protein (such as a viral antigen, a bacterial antigen, a parasitic antigen, a fungal antigen, a tumor antigen, a tumor associated antigen, a cytokine, a growth factor, a peptide tag, or a detectable substance (see Section 5.1.3 for examples of such antigens, cytokines, growth factors, peptide tags, and detectable substances).

[0083] In specific embodiments, a recombinant influenza virus described herein is attenuated. In a particular embodiment, the recombinant influenza virus is attenuated such that the virus remains, at least partially, infectious and can replicate in vivo, but only generate low titers resulting in subclinical levels of infection that are non-pathogenic. Such attenuated viruses are especially suited for embodiments described herein wherein the virus or an immunogenic composition thereof is administered to a subject to induce an immune response.

[0084] In some embodiments, a recombinant influenza virus described herein comprises one or more attenuating mutations in a modified influenza virus HA gene segment. In some embodiments, a recombinant influenza virus described herein comprises one or more attenuating mutations in a complementing influenza virus gene segment. In certain embodiments, a recombinant influenza virus described herein comprises one or more attenuating mutations in two, three or more complementing influenza virus gene segments. In some embodiments, a recombinant influenza virus described herein comprises one or more attenuating mutations in a modified influenza virus HA gene segment and one or more attenuating mutations in a complementing influenza virus gene segment.

[0085] In certain embodiments, the one or more attenuating mutations may be in the open reading frame of a gene segment encoding one or more of the following: NS1, NS2, NP, NA, PB1, PB2 and/or PA. In a specific embodiment, the one or more attenuating mutations may be in the open reading frame of a NA gene segment. In another specific embodiment, the one or more attenuating mutations may be in the open reading of an NP gene segment. In another embodiment, the one or more attenuating mutations may be in the open reading frame of a PB1 gene segment. In another embodiment, the one or more attenuating mutations may be in the open reading frame of a PB2 gene segment. In certain embodiments, the one or more attenuating mutations in a gene segment of an influenza virus can be accomplished according to any method known in the art, such as, e.g., selecting viral mutants generated by chemical mutagenesis, mutation of the genome by genetic engineering, selecting reassortant viruses that contain segments with attenuated function, or selecting for conditional virus mutants (e.g., cold-adapted viruses). In a specific embodiment, one or more temperature sensitive mutations that are attenuating may be introduced in an open reading frame of a gene segment. In some embodiments, the one or more temperature sensitive mutations include one or more of the following: PB1 (K391E, E581G, A661T), PB2 (N265S), and NP (D34G).

[0086] In a specific embodiment, an attenuated recombinant influenza virus comprises a complementing influenza virus gene segment encoding an HA from a pandemic or seasonal influenza virus and a second complementing influenza virus gene segment encoding a viral polymerase subunit (i.e., e.g., PA, PB1 or PB2) with one or more attenuating mutations.

IV. Cell Culture and Expression Hosts

[0087] The present invention also relates to host cells that are introduced (transduced, transformed or transfected) with polynucleotides, or vectors of the invention, and the production of polypeptides of the invention by recombinant techniques. Host cells are genetically engineered (i.e., transduced, transformed or transfected) with a vector, such as an expression vector, of this invention. As described above, the vector can be in the form of a plasmid, a viral particle, a phage, etc. Examples of appropriate expression hosts include: bacterial cells, such as E. coli, Streptomyces , and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; or insect cells such as Drosophila and Spodoptera frugiperda.

[0088] Commonly, mammalian cells are used to culture the HA polypeptides of the invention. Suitable host cells for the replication of the HA sequences herein include, e.g., Vero cells, BHK cells, MDCK cells, 293 cells and COS cells, including 293T cells, COS7 cells or the like. Typically, cells are cultured in a standard commercial culture medium, such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10% fetal bovine serum), or in serum free medium, under controlled humidity and C0₂ concentration suitable for maintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2). Optionally, the medium contains antibiotics to prevent bacterial growth, e.g., penicillin, streptomycin, etc., and/or additional nutrients, such as L- glutamine, sodium pyruvate, non-essential amino acids, additional supplements to promote favorable growth characteristics, e.g., trypsin, β-mercaptoethanol, and the like.

[0089] The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the inserted polynucleotide sequences. The culture conditions, such as temperature, pH and the like, are typically those previously used with the particular host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including Sambrook et al, Molecular Cloning-A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. ("Ausubel") Additionally, variations in such procedures adapted to the present invention are readily determined through routine experimentation and will be familiar to those skilled in the art.

[0090] In mammalian host cells, a number of expression systems, such as viral-based systems, can be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence is optionally ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing the polypeptides of interest in infected host cells. In addition, transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

[0091] A host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves a precursor form into a mature form, of the protein is sometimes important for correct insertion, folding and/or function. Additionally proper location within a host cell (e.g., on the cell surface) is also important. Different host cells such as COS, CHO, BHK, MDCK, 293, 293T, COS7, etc. have specific cellular machinery and characteristic mechanisms for such post translational activities and can be chosen to ensure the correct modification and processing of the current introduced, foreign protein.

[0092] For long-term, high-yield production of recombinant proteins encoded by, or having subsequences encoded by, the polynucleotides of the invention, stable expression systems are optionally used. For example, cell lines, stably expressing a polypeptide of the invention, are transfected using expression vectors that contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells are allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Thus, resistant clumps of stably transformed cells, e.g., derived from single cell type, can be proliferated using tissue culture techniques appropriate to the cell type. [0093] Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The cells expressing said protein can be sorted, isolated and/or purified. The protein or fragment thereof produced by a recombinant cell can be secreted, membrane-bound, or retained intracellularly, depending on the sequence (e.g., depending upon fusion proteins encoding a membrane retention signal or the like) and/or the vector used. Expression products corresponding to the nucleic acids of the invention can also be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like. Refer to Sambrook and Ausubel, supra.

[0094] In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the expressed product. For example, when large quantities of a polypeptide or fragments thereof are needed, vectors that direct high-level expression of fusion proteins that are readily purified are favorably employed. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the coding sequence of interest, e.g., sequences comprising those found herein, etc., can be ligated into the vector in-frame with sequences for the amino-terminal translation initiating methionine and the subsequent 7 residues of beta-galactosidase producing a catalytically active beta galactosidase fusion protein; ρΓΝ vectors; pET vectors; and the like. Similarly, in the yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used for production of the desired expression products.

V. Methods for therapeutic and preventative administration of vaccines

[0095] The term "immunogenic" refers to the ability of a substance to cause a humoral and/or cellular response, whether alone or when linked to a carrier, in the presence or absence of an adjuvant. "Neutralization" refers to an immune response that blocks the infectivity, either partially or fully, of an infectious agent. A "vaccine" is an immunogenic composition capable of eliciting protection against disease, whether partial or complete. A vaccine may also be useful for treatment of an infected individual, in which case it is called a therapeutic vaccine. A vaccine, as referred to herein, encompasses any of the polynucleotides or polypeptides described herein.

[0096] The term "therapeutic" refers to the ability of treating influenza virus infection. The term "effective amount" for a therapeutic or prophylactic treatment refers to an amount of polypeptide or polynucleotide sufficient to induce an immunogenic response in the subject to which it is administered, or to otherwise detectably immunoreact in its intended system (e.g., immunoassay). Preferably, the effective amount is sufficient to effect treatment, as defined above. The exact amount necessary will vary according to the application. For vaccine applications or for the generation of polyclonal antiserum/antibodies, for example, the effective amount may vary depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular polypeptide selected and its mode of administration, etc. It is also believed that effective amounts will be found within a relatively large, non-critical range. An appropriate effective amount can be readily determined using only routine experimentation.

[0097] Vaccines of the invention can be administered prophylactically in an appropriate carrier or excipient to stimulate an immune response specific for one or more strains of influenza virus. Typically, the carrier or excipient is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, allantoic fluid from uninfected Hens' eggs (i.e., normal allantoic fluid "NAF") or combinations thereof. The preparation of such solutions insuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, etc.

[0098] Generally, the vaccines of the invention are administered in a quantity sufficient to stimulate an immune response specific for one or more strains of influenza virus. Preferably, administration of the influenza viruses elicits a protective immune response. Dosages and methods for eliciting a protective immune response against one or more influenza strains are known to those of skill in the art. For example, live attenuated influenza virus vaccines are provided in the range of

5 8 3 7

about 10 -10 pfu (plaque forming units/ml) or about 10 to 10 egg infectious dose per dose administered. Typically, the dose will be adjusted within this range based on, e.g., species, age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The vaccine formulation can be systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needleless injection device. Alternatively, the vaccine formulation can be administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract. While any of the above routes of delivery results in a protective systemic immune response, intranasal administration confers the added benefit of eliciting mucosal immunity at the site of entry of the influenza virus. For intranasal administration, attenuated live virus vaccines are often preferred, e.g., an attenuated, cold adapted and/or temperature sensitive recombinant or reassortant influenza virus. While stimulation of a protective/therapeutic immune response with a single dose is preferred, additional dosages can be administered, by the same or different route, to achieve the desired prophylactic effect.

[0099] The polynucleotides, polypeptides, or recombinant viruses of the invention can be administered by the oral, ocular, nasal, intradermal, intramuscular, in ovo or any other appropriate route which is shown to elicit an appropriate protective response in the vaccinated recipients. The polynucleotides, polypeptides, or recombinant viruses of the invention can also be administered using a prime and boost regime if deemed necessary.

[00100] Optionally, the formulation for prophylactic administration of polynucleotides, polypeptides, or recombinant viruses of the invention also contains one or more adjuvants for enhancing the immune response to the influenza antigens. Suitable adjuvants include: saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59 or any other adjuvant deemed suitable for poultry and livestock.

[00101] If desired, polynucleotides, administration of polynucleotides, polypeptides, or recombinant viruses of the invention can be performed in conjunction with administration of one or more immunostimulatory molecules. Immunostimulatory molecules include various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1 ; B7.2, etc. The immunostimulatory molecules can be administered in the same formulation as the influenza viruses, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.

[00102] In another embodiment, the polynucleotides and vectors of the invention including influenza genome segments can be employed to introduce heterologous nucleic acids into a host organism or host cell, such as a mammalian cell, e.g., cells derived from a human subject, in combination with a suitable pharmaceutical carrier or excipient as described above. Typically, the heterologous nucleic acid is inserted into a non-essential region of a gene or gene segment, e.g., the M gene of segment 7. The heterologous polynucleotide sequence can encode a polypeptide or peptide, or an RNA such as an antisense RNA or ribozyme. The heterologous nucleic acid is then introduced into a host or host cells by producing recombinant viruses incorporating the heterologous nucleic acid, and the viruses are administered as described above. [00103] Alternatively, a vector of the invention including a heterologous nucleic acid can be introduced and expressed in a host cell by co-transfecting the vector into a cell infected with an influenza virus. Optionally, the cells are then returned or delivered to the subject, typically to the site from which they were obtained. In some applications, the cells are grafted onto a tissue, organ, or system site (as described above) of interest, using established cell transfer or grafting procedures. For example, stem cells of the hematopoietic lineage, such as bone marrow, cord blood, or peripheral blood derived hematopoietic stem cells can be delivered to a subject using standard delivery or transfusion techniques.

[00104] Alternatively, the viruses comprising a heterologous nucleic acid can be delivered to the cells of a subject in vivo. Typically, such methods involve the administration of vector particles to a target cell population. Administration can be either systemic, e.g., by intravenous administration of viral particles, or by delivering the viral particles directly to a site or sites of interest by a variety of methods, including injection (e.g., using a needle or syringe), needleless vaccine delivery, topical administration, or pushing into a tissue, organ or skin site. For example, the viral vector particles can be delivered by inhalation, orally, intravenously, subcutaneously, subdermally, intradermally, intramuscularly, intraperitoneally, intrathecally, by vaginal or rectal (cloacal in birds) administration, or by placing the viral particles within a cavity or other site of the body, e.g., during surgery.

[00105] The above described methods are useful for therapeutically and/or prophylactically treating a disease or disorder by introducing a vaccine of the invention comprising a heterologous polynucleotide encoding a therapeutically or prophylactically effective polypeptide (or peptide) or RNA (e.g., an antisense RNA or ribozyme) into a population of target cells in vitro, ex vivo or in vivo. Typically, the polynucleotide encoding the polypeptide (or peptide), or RNA, of interest is operably linked to appropriate regulatory sequences as described above in the sections entitled "Expression Vectors" and "Additional Expression Elements." Optionally, more than one heterologous coding sequence is incorporated into a single vector or virus. For example, in addition to a polynucleotide encoding a therapeutically or prophylactically active polypeptide or RNA, the vector can also include additional therapeutic or prophylactic polypeptides, e.g., antigens, co- stimulatory molecules, cytokines, antibodies, etc., and/or markers, and the like.

[00106] For therapeutic or immunization purposes, nucleic acids encoding one or more of the peptides of the invention can also be administered (DNA vaccines). A number of methods are conveniently used to deliver the nucleic acids to the subject. For instance, the nucleic acid can be delivered directly, as "naked DNA." This approach is described, for instance, in Wolff et. al, Science 247: 1465-1468 (1990) as well as U.S. Patent Nos. 5,580,859 and 5,589,466. The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles. The nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for instance, in WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691 ; Rose U.S. Pat No. 5,279,833; WO 91/06309; and Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414. The peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the polypeptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351 :456-460 (1991)) which is incorporated herein by reference. A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g., Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.

VI. Kits comprising HA compositions of the invention

[00107] The present invention provides kits that comprise the HA polynucleotides, polypeptides, or other agents described herein and that can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified mutant HA polypeptide in one or more containers. In some embodiments, the kits contain all of the components necessary for vaccine administration including directions for administering the polypeptides. One skilled in the art will readily recognize that the disclosed polynucleotides, polypeptides, and recombinant viruses of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

[00108] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure. EXAMPLES

[00109] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Example 1 - HA sequence identification

[00110] The avian influenza A H5N1 hemagglutinin sequences analyzed here were those which had been deposited in the Influenza Virus Database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) as of December 31, 2011. Only full-length sequences obtained from viruses isolated in Bangladesh, China, Egypt, Hong Kong, India, Indonesia, Thailand, and Viet Nam were considered. There were 42 full-length hemagglutinin sequences from Bangladesh, 443 from China, 410 from Egypt, 142 from Hong Kong, 47 from India, 259 from Indonesia, 186 from Thailand, and 335 from Viet Nam. Those, which do not have the additional basic residues in the cleavage site and which are probably not highly pathogenic, were not included in the analysis. Also not included were a few sequences that looked "strange", i.e., significantly different from the others. In terms of length, four HPAI H5N1 hemagglutinin types were observed. Representative sequences of the four types are presented in Table 2. There were a total of 1,271 sequences of the first type (represented by GenBank entry AAT73266), 415 of the second type (represented by ABJ96667), 153 of the third type (represented by AEQ72885), and 14 of the fourth type (represented by ACO07039). (Henceforth, the molecules will be identified simply by their GenBank Accession codes.) Sequences of the second type have a one-residue deletion in the cleavage site region; those of the third type have a deletion at position 129; those of the third type have a deletion at position 255 and three additional residues in the cleavage site region (Table 2). A vaccine for only the first type, the most numerous, is proposed here. Vaccines for the other types and for the HPAI H5N1 of other countries will be designed as soon as more sequences become available. Table 2 - Alignment of HPAI H5N1 hemagglutinins of various lengths

10 20 30 40 50 60

I I I I I I

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAG AAT73266 DQICIGYHAN STEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ABJ96667 DQICIGYHAN STEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILRDCSVAGW AEQ72885 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCNLDGVKPLILKDCSVAG ACO07039

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDF DYEELKHLLSRINHFEKIQIIPKS AAT73266 LLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKS ABJ96667 LLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKD AEQ72885 LLGNPMCDEFLNVSEWSYIVEKASPANGLCYPGDF DYEELKHLLNRITHLKKIKIIPKS ACO07039

130 140 150 160 170 180

I I I I I I

S SSHEASLGYSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHH AAT73266 SWSDHEASSGYSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLIL GIHH ABJ96667 S SDHEAS-GVSSACPYQGRSSFFRN LTKKNDAYPTIKKSYNNTNQEDLLVLWGIHH AEQ72885 YWSNHEASSGVSAACSYLGEPSFFRNVVWLIKKNNTYPPIKVNYTNTNQEDLLVLWGIHH ACO07039

190 200 210 220 230 240

I I I I I I

P DAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKP DAIN AAT73266 S DAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMDFF TILKPNDAIN ABJ96667 P DAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKS DAIN AEQ72885 PNNEAEQIQIYQNLITHISVGTSTLNQRLIPKIATRSKVNGQSGRMEFFWTILKP DSIN ACO07039

250 260 270 280 290 300

I I I I I I FESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFH IHPLTIG AAT73266 FESNGNFIAPEYAYKIVKKGDSAIMKSEVEYG CNTKCQTPIGAINSSMPFH IHPLTIG ABJ96667 FESNGNFIAPENAYKIVKKGDSTIMKSELEYSNCNTKCQTPIGAINSSMPFH IHPLTIG AEQ72885 FDSNGNFIAPEYAY-IVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFH IHPLTIG ACOO7039

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQRER RRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AAT73266

ECPKYVKSNKLVLATGLRNSPLRE RRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ ABJ96667

ECPKYVKSNRLVLATGLRNSPQGEK RRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AEQ72885

ECPKYVKSNRLVLATGLRNAPQREREGGRRRKRGLFGAIAGFIEGG QGI4VDG YGYHHSNEQ ACOO7039

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFN LERRIENLNKKMEDGFLD AAT73266 GSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFN LERRIENLNKKMEDGFLD ABJ96667 GSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFN LERRIENLNKKMEDGFLD AEQ72885 GSGYAADKES QKAIDGITNKVNS11DKMNTQFEAVGREFNNLERRIENLNKKMEDGFLD ACOO7039 430 440 450 460 470 480

I I I I I I

VWTY AELLVLME ERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME AAT73266 V TY AELLVLME ERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME ABJ96667 VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHRCD ECME AEQ72885 V TYNAELLVLMENERTLDFHDSNVKNLYEKVRLQLRD AKELGNGCFEFYHKCD ECME ACO07039

490 500

I I SVP.NGTYDYPQYSEEARLKREEISG AAT73266

SVRNGTYDYPQYSEEARLKREEISG ABJ96667

SVP.NGTYDYPQYSEEARLKREEISG AEQ72885

SVRNGTYDYPQYSEEARLSREEING ACOO 7039 The target epitope

[00111] The part of the hemagglutinin that was judged to be the best target for a focused antibody response is the one that contains the cleavage site, specifically the part that is centered at amino acid 326 (following the numbering scheme of Table 2), which is the middle residue in a string of reactive residues which includes the basic residues in the cleavage site region. No structure of an antibody-hemagglutinin complex that involves the cleavage site is currently available. An antibody epitope that contains the cleavage site was approximated by including all the amino acids that lie within 18 Angstroms of amino acid 326, henceforth designated the aa326 epitope. The choice of 18 Angstroms was arbitrary but is quite reasonable in view of the results of an analysis of the 14 structures of antibody-hemagglutinin complexes available in the Protein Data Bank as of April 5, 2012. That analysis showed that 241 of the 252 atoms in the 14 epitopes lie within 18 Angstroms of the epitope centers (5 of the 11 which lie outside the 18-Angstrom radius are main chain atoms).

Modeling of the structures of the various hemagglutinins:

[00112] A three-dimensional structure for the antigen is needed to know which residues are within the chosen target epitope and which residues are sufficiently exposed to be considered for replacement. There are no known three-dimensional structures of an uncleaved HPAI H5N1 hemagglutinin, so possible structures were obtained by homology modeling using the online facility, SWISS-MODEL (Arnold et al. 2006, Kiefer et al. 2009, Peitsch 1995) (implemented at http://swisEmodel.expasv.org ).

[00113] Comparison of all the hemagglutinin sequences of the first type showed that AAT73266, which was isolated from Thailand, is identical to 54 others - the most for any of the sequences. Further, AAT73277 from Viet Nam, which is identical to 23 others, differs from AAT73266 at only one position, a methionine-for-leucine substitution at position 175. Subsequent modeling showed that the residue at position 175 is completely buried and the methionine-for- leucine substitution will almost certainly have no effect on the overall structure, or the antigenicity, of the molecule. The AAT73266 sequence was therefore chosen as the representative hemagglutinin sequence for modeling purposes.

[00114] The template used for homology modeling was the crystal structure in the Protein Data Bank entry 2FK0, which is for a cleaved avian H5N1 hemagglutinin (Stevens et al. 2006). The 2FK0 and AAT73266 hemagglutinin sequences are compared in Table 3. Table 3 - Alignment of the 2FK0 and AAT73266 hemagglutinin sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHAN STEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGW 2FK0

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAG AAT73266

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS 2FK0

LLGNP CDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKS AAT73266

130 140 150 160 170 180

I I I I I I

SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHH 2FK0

SWSSHEASLGYSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHH AAT73266

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKV GQSGR EFFWTILKPNDAIN 2FK0

PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKV GQSGR EFFWTILKPNDAIN AAT73266

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSS PFHNIHPLTIG 2FK0

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFH IHPLTIG AAT73266

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPgre GLFGAIAGFIEGGWQG VDGWYGYHHSNEQ 2FK0

ECPKYVKSNRLVLATGLR SPQRE R KRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AAT73266

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFN LERRIENLNKKMEDGFLD 2FK0

GSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFN LERRIENLNKKMEDGFLD AAT73266

430 440 450 460 470 480

I I I I I I

VWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME 2FK0

V TYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCDNECME AAT73266

490 500

I I SVRNGTYDYPQYSEEARLKREEISS 2FK0

SVRNGTYDYPQYSEEARLKREEISG AAT73266

[00115] The nine residues normally present at positions 322 to 330 (Table 3) are missing in 2FK0 and those residues were modeled using SWISS-MODEL. The AAT73266 model based on the full AAT73266 sequence and the 2FK0 structure was used in all subsequent modeling. All the hemagglutinin sequences considered are highly homologous so that the three-dimensional structures of those molecules can be expected to be very similar. The delineation of the aa326 epitope was made using the AAT73266 model; this epitope was designated as the prototype for the entire set of sequences.

Design of the vaccines

[00116] Models based on the most common hemagglutinin sequences from each of the eight countries named above were generated using SWISS-MODEL. The solvent exposures of the amino-acid side chains were computed by the method of Connolly (1983), using programs developed by Sheriff et al. (1985) and adapted by the author. The secondary structure of each model was predicted using the program DSSP (Kabsch and Sander 1983). Amino-acid replacements designed to reduce the antigenicity of the residues outside the aa326 epitope were then made on the basis of solvent exposure and secondary structure using the rules presented in Table 1.

[00117] Two exposure levels were chosen for deciding which residues to replace. In the first case, residues whose side chains are at least 40% exposed to solvent were replaced; in the second case, at least 25% exposure was required for replacement. The mutated sequences represent vaccines against the HPAI H5N1 being analyzed.

Results and Discussion

[00118] A portrayal of the consequences of the amino-acid replacements is presented in Figure 1, where the "relative antigenicity", which is equated with the cumulative reactivity (Sandberg et al. 1998, De Genst et al. 2002) of all the residues within 18 Angstroms of a residue position (Padlan 2010), is plotted for AAT73266. Figure 1 shows the antigenicity plots before and after "de-Antigenization", during which the residues in the aa326 epitope were not replaced. The plots for the other hemagglutinins (not shown) are essentially indistinguishable, attesting to the close similarity of the sequences and of the three- dimensional structures of those molecules.

[00119] The residues in the aa326 epitope of AAT73266 are listed in Table 4. Included in Table 4 are the solvent exposures of those residues in the AAT73266 model. Also included are the identity and occurrence of the corresponding residues found in the sequences from the eight countries selected for this study. Sequence variation is noted at various positions, even those in the cleavage site region. Some of this variation may simply be due to errors in the sequence determination. Table 4 - The residues in the aa326 epitopes of hemagglutinins from countries with high incidence HFAI H5N1

sition Exposure Bangladesh China Egypt Hong Kong

6 0 00 G(36) G (226) G (250) G(93) ,V(1)

7 0 19 Y(36) Y (226) Y (249) ,F(1) Y(93 ,C(1)

8 0 27 H(36) H (226) H (249) ,Y(D H(94

9 0 98 A(36) A(225) ,T(1) A(249) ,G(1) A(94

10 0 36 N(36) N (224) ,Y(1) ,D(1) N (250) N(94

11 0 86 N(36) N (223) ,Y(3) N (249) ,D(1) N(94

12 0 32 S (36) S (225) ,W(1) S (250) S (94

13 1 00 T (36) T (226) (250) T (94

14 0 62 E(35) ,K(1) E (213) ,V(13) E (250) E (90) ,V(4)

15 0 42 Q(36) Q (226) Q (246) ,K<2) ,H(2) Q(93) ,L(1)

16 0 00 V(36) V(224) _rA(l) ,G(1) V (250) V(94

17 0 55 D(36) D (226) D (250) D(94

18 0 20 T (36) T (226) T (248) ,P(D ,A(1) T (94

20 0 23 M(36) M (226) M (249) ,KD M(94

21 0 19 E (36) E (225) ,Q(D E (250) E (94

22 0 78 K(36) K(226) K (250) K(94

23 0 76 N(36) N (226) N (250) N(94

24 0 23 V(36) V (225) ,1(1) V (247) ,1(3) V(94

25 0 53 T (36) T (224 ) ,P(1) ,A(1) (249) ,N(1) T (94

26 0 00 V(36) V (225) ,A(1) V (250) V(94)

27 0 00 T (36) T (226) (250) T (94

316 0 00 G(36) G (226) G (250) G(94

317 0 00 L(36) L (226) L (250) L(94

318 0 22 R(36) R (226) R (250) R(93) ,T(1)

319 0 17 N(36) (225) ,D(1) N (250) N (94

320 1 00 S (36) S (131) ,T(71) ,A(24) S (250) T (60) ,S(33) ,A(1)

321 0 60 P(36) P (220) ,L(6) P (250) P(94)

322 0 66 Q(35) ,K(1) Q (220) ,L(2) ,H(2) ,P(1) ,K|1) Q (248) ,R(D ,H(1) Q(94)

323 0 57 G(35) ,R(1) R (187) ,G (28) , I (9) , S (1) G (238) ,R(9) ,E (2) ,X(1) R(86) ,G(3) ,1(5)

324 0 49 E (36) E (223) _rG(l) ,K(1) ,X(1) E (241) ,K<8) ,D(1) E (94)

325 0 66 R(35) ,K(1) R (181) , I (15) ,G (23) ,E (5) , K(l) ,T (1) R (207) ,G(31) ,S(8) ,K(4) R(89) ,G(3) ,1(2)

326 0 61 R(36) R (218) ,G(7) ,1(1) R (250) R(94)

327 0 44 R(36) R (225) ,K(1) R (250) R(94)

328 0 28 K(36) K(211) ,R(15) K (249) ,R(D K(93) ,R(D

329 0 29 K(36) K(225) ,R(D K (250) K(93) ,R(D

330 0 30 R(36) R (226; R (250) R(94

331 0 00 G(36) G (226) G (250) G(94)

332 0 20 L(36) L (226) L (250) L(94)

333 0 00 F(36) F (226) F (250) F(94)

334 0 00 G(36) G (226) G (250) G(94)

335 0 00 A(36) A(226) A(250) A(94)

Table 4 cont'd - The residues in the aa326 epitopes of hemagglutinins from countries with high incidence of HFAI H5N1

Position Exposure India Indonesia Thailand Viet Nam

336 0 00 1(36) I 226) I 250) 1(94)

337 0 56 A(36) A 226) A 250) A(94)

338 0 00 G(36) G 222) ,R(3) ,S (1) G 250) G(94)

339 0 06 F(36) F 226) F 248) ,L<2) F(94)

340 0 00 1(36) I 226) I 250) I (94)

341 0 54 E (36) E 226) E 250) E (94)

342 0 00 G(36) G 226) G 250) G(94)

343 0 00 G(36) G 222) ,R(4) G 250) G(94)

344 0 40 (36) W 226) W 250) W(94) c/>

c 345 0 64 Q(36) Q 226) Q 249) ,H<1) Q(94)

CO 367* 0 43 D(36) D 226) D 250) D(94)

368* 0 34 (36) K 225) ,R(D K 243) ,R<6) ,T (1) (86) ,Q(8)

369* 0 82 E (36) E 226) E 248) ,V(1) ,K(1) E (94)

370* 0 60 S (36) S 226) S 250) S (94)

371* 0 34 T(36) T 226) T 250) T (94) m 372* 0 37 Q(36) Q 225) ,P(D Q 250) Q(94)

373* 0 56 K(36) K 216) ,R(10) K 238) ,R<12) K(94) c/> 374* 0 19 A(36) A (226) A 250) A(94)

X

m 375* 0 00 1(33) ,V(3) I (222) ,L(2) , (1) ,M(1) I 249) ,V(1) I (94) m 376* 0 61 D(35) ,N(1) D (226) D 250) D(91) ,N(3)

377* 0 68 G(36) G 226) G 250) G(94)

380* 0 90 N(36) N 226) N 250) N (94)

7J

c 435 0 07 E (36) E 226) E 250) E (94) r 438 0 22 L(36) L 226) L 250) L(94) m 439 0 20 D(36) D 224) ,N(1) ,Y(1) D 250) D(94) to 441 0 00 H(36) H (226) H 250) H(94)

442 0 00 D(36) D (226) D 249) ,G(1) D(94)

444* 0 00 N(36) N (226) N 250) N(94)

447* 0 00 N(36) N (223) ,H(1) , (1) ,D(1) N 250) N(94)

448* 0 30 L(36) L (226) 1 250) L<94)

451* 0 00 K(36) K (214; ,R(12) K 250) K<93) ,R<1)

465 0 73 N(36) N (225; ,D(1) N (250) N(94)

484* 0 54 N(36) N (226) N (250) N(94)

485* 0 66 G(36) G ;226) G (250) G(94)

Table 4 cont'd - The residues in the aa326 epitopes of hemagglutinins from countries with high incidence of HFAI H5N1

Position Exposure India Indonesia Thailand Viet Nam

6 0 00 G 47) G 255) G 182) G 174)

7 0 19 Y 47) Y 355) Y 182) Y 174)

8 0 27 H 47) H 255) H 182) H 174)

9 0 98 A 47) A 255) A 182) A 174)

10 0 36 N 47) N 255) N 182) N 174)

11 0 86 N 47) N 255) N 181) ,F(1) N 174)

12 0 32 S 47) S 255) S 182) S 174)

13 1 00 T 47) T 255) T 182) T 174)

14 0 62 E 47) E 255) E 182) E 174)

c/>

c 15 0 42 Q 46) ,R(1) Q 244) ,R(9) ,H(1) ,L(1) Q 182) Q 174)

CO 16 0 00 V 47) V 252) ,1 (3) V 180) ,1 (2) V 174)

c/> 17 0 55 D 47) D 255) D 182) D 174)

18 0 20 T 47) T 255) T 182) T 174)

20 0 23 M 47) M 255) M 182) M 174)

21 0 19 E 47) E 255) E 182) E 174)

m 22 0 78 K 47) K 255) K 174) , (8) K 172) ,R(2)

23 0 76 N 47) N 255) N 181) ,K(1) N 174)

c/> oo 24 0 23 V 47) V 255) V 182)

X ,1(1) V 173) ,1(1)

m 25 0 53 T 47) T 255) T 182) T 174)

m 26 0 00 V 47) V 255) V 182) V 174)

27 0 00 T 47) T 255) T 182) T 174)

316 0 00 G 47) G 255) G 182) G 174)

7J

c 317 0 00 L 47) L 255) L 182) L 174)

318 0 22 R 47) R 255) R 182) R 174)

m 319 0 17 N 47) N 255) N 182) N 174)

r 320 1 00 S 47) S 255) S 180) ,N(2) S 165) ,A(7) , (2)

321 0 60 P 46) ,H(1) P 255) P 182) P 174)

322 0 66 Q 47) Q 255) Q 182) Q 171) ,R(2) ,X(1)

323 0 57 G 47) R 251) ,K(2) ,1(2) R 170) ,1 (12) R 167) ,1 (7)

324 0 49 E 47) E 255) E 182) E 174)

325 0 66 R 47) R 129) ,S (126) R 158) ,K(24) R 121) ,G(50) ,1 (2)

326 0 61 R 47) R 255) R 175) ,K(7) R 173) ,1(1)

327 0 44 R 47) R 255) R 182) R 174)

328 0 28 K 46) ,R(1) K 252) ,R(2) ,E (1) K 182) K 152) ,R(22)

329 0 29 K 47) K 248) ,R(7) K 182) K 174)

330 0 30 R 47) R 255; R 182) R 17 ,K(1)

331 0 00 G 47) G 255) G 182) G 173) ,X(1)

332 0 20 L 47) L 255) L 182) L 174)

333 0 00 F 47) F 255) F 182) F 174)

334 0 00 G 47) G 255) G 182) G 174)

Table 4 cont'd - The residues in the aa326 epitopes of hemagglutinins from countries with high incidence of HFAI H5N1

Position Exposure India Indonesia Thailand Viet Nam

335 0 00 A 47) A 255) A 182) A 174)

336 0 00 I 47) I 255) I 182) I 174)

337 0 56 A 47) A 255) A 182) A 174)

338 0 00 G 47) G 255) G 182) G 174)

339 0 06 F 47) F 255) F 182) F 174)

340 0 00 I 47) I 255) I 181) ,L(1) I 174)

341 0 54 E 47) E 255) E 181) ,K(1) E 174)

342 0 00 G 47) G 255) G 182) G 174)

343 0 00 G 47) G 255) G 182) G 174)

c/>

c 344 0 40 W 47) W 255) W 182) 174)

CO 345 0 64 Q 47) Q 255) Q 182) Q 172) ,R(D

c/> 367* 0 43 D 47) D 255) D 181) ,N(1) D 172) ,N(1)

368* 0 34 K 47) K 254) ,R(1) K 182) K 172) ,Q(2)

369* 0 82 E 47) E 255) E 181) ,D(1) E 172) ,K(2)

370* 0 60 S 47) S 255) S 181) ,P(D S 174)

m 371* 0 34 T 47) T 255) T 178) ,S (4) T 173) ,S (1)

372* 0 37

c Q 47) Q 255) Q 182) Q 174)

/> 0 56 K 47)

X 373* K 255) K 182) K 174)

m 374* 0 19 A 47) A 255) A 182) A 174)

m 375* 0 00 I 47) I 255) I 182) I 173) ,L(D

376* 0 61 D 47) D 254) ,N(1) D 182) D 170) ,N(4)

377* 0 68 G 47) G 255) G 182) G 174)

7J

c 380* 0 90 N 47) N 255) N 182) N 172) ,1 (2)

435 0 07 E 47) E 255) E 182) E 174)

m 438 0 22 L 47) L 255) L 182) L 174)

r 439 0 20 D 47) D 255) D 182) D 173) ,N(1)

441 0 00 H 47) H 255) H 182) H 174)

442 0 00 D 47) D 255) D 182) D 173) ,E (1)

444* 0 00 N 47) N 255) N 182) N 173) ,K(1)

447* 0 00 N 47) N 255) N 182) N 174)

448* 0 30 L 47) L 255) L 182) L 174)

451* 0 00 K 47) K 254) ,R(1) K 182) K 173) ,Q(D

465 0 73 N 47) N 255) N 182) N 174)

484* 0 54 N 47) N 255) N 182) N 173) ,S (1)

485* 0 66 G 47) G 255) G 182) G 174)

[00120] The aa326 epitopes in the hemagglutinins that were analyzed are listed by country in Table 5. Only those epitopes which are present in at least 5% of the sequences in each country are included in the list. The vaccine designs based on each of the HPAI H5N1 hemagglutinin molecules listed in Table 5 are presented by country of origin in the following sections.

Table 5 - Residues in the most common aa326 epitopes

6 10 18 20 27 315 320 330 340 345

GYHANNSTEQVDT MEKNVTVT GLRNSPQRERRRKKRGLFGAIAGFIEGGWQ AAT73266 Thailand

GYHANNSTEQVDT MEKNVTVT GLRNSPQREKRRKKRGLFGAIAGFIEGGWQ BAG80800 Thailand

GYHANNSTEQVDT MEKNVTVT GLRNSPQIERRRKKRGLFGAIAGFIEGGWQ AB033748 Thailand

GYHANNSTEQVDT MEKNVTVT GLRNSPQGERRRKKRGLFGAIAGFIEGGWQ ACU24777 Bangladesl

GYHANNSTEQVDT MEKNVTVT GLRNSPQRERRRKKRGLFGAIAGFIEGGWQ AAX53505 China

GYHANNSTEQVDT MEKNVTVT GLRNTPQRERRRKKRGLFGAIAGFIEGGWQ AAY21163 China

GYHANNSTEQVDT MEKNVTVT GLRNSPQGERRRKKRGLFGAIAGFIEGGWQ ABE68921 China

GYHANNSTEQVDT MEKNVTVT GLRNSPQGERRRKKRGLFGAIAGFIEGGWQ ABY76247 Egypt

GYHANNSTEQVDT MEKNVTVT GLRNSPQGEGRRKKRGLFGAIAGFIEGGWQ ADD21363 Egypt

GYHANNSTEQVDT MEKNVTVT GLRN PQRERRRKKRGLFGAIAGFIEGGWQ AAL31381 Hong Kong

GYHANNSTEQVDT MEKNVTVT GLRNSPQRERRRKKRGLFGAIAGFIEGGWQ ABC66568 Hong Kong

GYHANNSTEQVDT MEKNVTVT GLRNSPQRERRREKRGLFGAIAGFIEGGWQ ACJ26330 Hong Kong

GYHANNSTEQVDT MEKNVTVT GLRNTPQRERRRKKRGLFGAIAGFIEGGWQ AAC32078 Hong Kong

GYHANNSTEQVDT MEKNVTVT GLRNSPQGERRRKKRGLFGAIAGFIEGGWQ ACZ58110 India

GYHANNSTEQVDT MEKNVTVT GLRNSPQRERRRKKRGLFGAIAGFIEGGWQ ABU99029 Indonesia

GYHANNSTEQVDT MEKNVTVT GLRNSPQRESRRKKRGLFGAIAGFIEGGWQ AEH59179 Indonesia

GYHANNSTEQVDT MEKNVTVT GLRNSPQRERRRKKRGLFGAIAGFIEGGWQ AAT73277 Viet Nam

GYHANNSTEQVDT MEKNVTVT GLRNSPQREGRRKKRGLFGAIAGFIEGGWQ ACB70548 Viet Nam

GYHANNSTEQVDT MEKNVTVT GLRNSPQRERRREKRGLFGAIAGFIEGGWQ AEI26176 Viet Nam

367 377 3 180 435 438 441 444 447 451 455 484

DKESTQKAIDG N Ξ LD HD N NL K N NG AAT73266 Thailand

DKESTQKAIDG N Ξ LD HD N NL R N NG ABC66525 China

DKESTQlAIDG N E LD HD N NL K N NG ACX31983 Egypt

DRESTQKAIDG N E LD HD N NL K N NG ADM85886 Egypt

DQESTQKAIDG N E LD HD N NL K N NG AAC32078 Hong Kong

Bangladesh

[00121] ACU24777 shares its aa326 epitope with 28 of the 35 other hemagglutinin sequences from Bangladesh. A model for ACU24777 was built with SWISS-MODEL using the AAT73266 model as template. The computed solvent exposures of the ACU24777 residues were then used to propose the appropriate amino-acid replacements to reduce its overall antigenicity while preserving its aa326 epitope. The sequence of ACU24777 and the mutated sequences are presented in Table 6. The sequence resulting from the replacement of the residues outside the aa326 epitope that are at least 40% exposed is labeled "mut" and that resulting from the replacement of the residues outside the epitope that are at least 25% exposed is labeled "mut2". The "mut" and "mut2" sequences represent the molecules which are vaccines against the HPAI H5N1 virus that is circulating in Bangladesh with the same aa326 epitope as ACU24777.

For each of Tables 6 to 26, the residues in the Arg326 epitope are shown bold and underlined. The replacements are shown in red. The "mut" sequence is the result of replacing the residues that are outside the Arg326 epitope whose side chains are at least 40%) exposed, while preserving the Arg326 epitope; the "mut2" sequence is the result of replacing the residues outside the Arg326 epitope which are at least 25% exposed.

Table 6 - Bangladesh ACU24777 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ACU24777 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFLNVPE SYIVEKINPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKS ACU24777 LLGNPMCSEALSVPAWSYIVE IGPA GLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTALSVPAWSYIVETIGPAT6LCYPGTFGGAAELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKNDAYPTIKISYNNTNQEDLLVL GIHH ACU24777 SWSGHTASSGVSSACPTTG&SSFFANWWLIKTGGAYPTITISYTNTNTEDLLVLWGIHH mut SWSGH ASSGVSSACP G SSFFANVVWLIK GGAYPTITIS NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISVGTSTLNLRLVPKIATRSKVNGQSGR EFFWTILKPNDAIN ACU24777 PNGAAEQT.&.LYQSPTTYISVGTSTLNLRLVPKIATRS VGGQSGRM FFWTIL PGDAI mut PGGAAAQTALY SPTTAISVGTSTL L LVPTIATRSTVGGTSGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPENAYKIVKKGDSTIMKSELEYG CNTKCQTPIGAINSSMPFH IHPLTIG ACU24777 FxSGGNFIAPENAYKIVKxGSSTIM SALAYGTCxT CQTPIGAISSSMPFHNIHPLTIG mut FTSGGNFIAPE AYKIVKTGSSTIM S L G C T CQTPIGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDG YGYHHSNEQ ACU24777 CPKYVTSG^LVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD ACU24777 GSGAAADKESTQKAIDGVTNKV&S 11AKMST&&AAVGAEFSGLERRIANLN KME GFLD mut GSGAAADKESTQKAIDGVT KVAS I IATMSTA&AAVG&EFSGLAARIA&LNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHRCD ECME ACU24777 V TY AELLVLMENERTMFroS VK LYDKVRLQLRG ATTLG GCF FYHACS.¾ACM& mut TYNAELLVLMENERTLDFHDS VK LYDKVRLALAGNAT LGNGCF FTHACSAACMft. mut2

490 500

I I SVR GTYDYPQYSEESRLKREEISG ACU24777

SVANGTYSYPAYSA&SRLAMAISG mut

SVANGTYSAPA&SAASALAAAAISG mu12 China

Three representative hemagglutinin sequences from China are listed in Table 5. AAX53505 shares its aa326 epitope with 60 of the 225 other HPAI H5N1 hemagglutinin sequences from China, AAY21163 shares its epitope with 35 others, and ABE68921 with 21. The sequence of AAX53505 and its mutated versions are presented in Table 7, those of AAY21163 are presented in Table 8, and those of ABE68921 are in Table 9. The "mut" and "mut2" sequences in these tables represent vaccines against HPAI H5N1 viruses circulating in China with similar sequences in their aa326 epitopes.

Table 7 - China AAX53505 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW AAX53505 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKASPANDLCYPGDF DYEELKHLLSRINHFEKIQIIPKS AAX53505 LLGNPMCSE&ISVPAWSYIVETASPATGLCYPGTFGGYAELKHLLSRIG^FETITIIPTS mut LLGNP CSTAISVPAWSYIVETASPATGLCYPGTFGG&AELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSNHEASSGVSSACPYLGKSSFFRNW LIKKNSTYPTIKRSYNNTNQEDLLVL GIHH AAX53505 S SGH ASSGVSSACP LG SSFFANWWLIKTGSTYPTI RSY NTNTEDLLVLWGIHH mut SWSGH ASSGVSSACP LG SSFFANWWLI GSTYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGR EFFWTILKPNDAIN AAX53505 PNGAAEQT.&.LYQSPTTYISVGTSTLNQRLVPKIATRS VGGQSGRM FFWTIL PGDAI mut PSGAAAQTALY SPTTAISVGTSTL Q LVPilATRSTVGGTSGRMTFT TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSAIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG AAX53505 F SNGNFIAPEYAYKIVKiGSSAIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut F SGGNFIAPETAYKIVK GSSAIMiTSALA GTC TTCQTPMGAISSSMPFHSITPLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AAX53505 CPKYVTSG^LVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD AAX53505 GSGAAADKESTQKAIDGVTNKVAS 1 IAKMST&&AAVGAEFSGLERRI&NLN&KME GFLD mut GSGAAADKESTQKAIDGVTNKVAS 11ATMSTAAAAVG&EFSGLARRI ALNAKMA&GFLD mut2

430 440 450 460 470 480

I I I I I I

VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME AAX53505 WTYNAELLVLMENERTMFmS^K LYDKVRLQLRGNATTLG GCFTFYH CSAACMA mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTFTH CSA&CKSS mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG AAX53505

SVANGTYSYPAYSAAARLA&ftAISG mut

SVANGTYSAP &SAAA&LAS& ISG mu12 Table 8 - China AAY21163 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW AAY21163 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPANDLCYPGDF DYEELKHLLSRINHFEKIQIIPKS AAY21163 LLGNPMCSEAISVPAWSYIVE AGPA GLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTAISVPAWSYIVETAGPATGLCYPGTFGG&AELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSNHEASSGVSSACPYNGKSSFFR W LIKKNSTYPTIKRSYNNTNQEDLLIL GIHH AAY21163 S SGH ASSGVSSACP GG SSFF.S.NWWLIKTGSTYPTI RSY NTNTEDLLILWGIHH mut SWSGH ASSGVSSACP GG SSFFANWWLI GSTYPTITTS NTG LLILWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGR EFFWTILKPNDTIN AAY21163 PNGAAEQT.&.LYQSPTTYISVGTSTLNQRLVPKIATRS VGGQSGRM FFWTIL PGDTI mut PSGAA¾QTALY SPTTAISVGTSTL Q LVPiIATRSTVGG SGRMTFi TILTPGDTI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSAIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG AAY21163 F SGGNFIAPEYAYKIVKiGSSAIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSAIM S LA G C T CQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AAY21163 CPKYVTSG^LVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLR TPQRERRRKKRGLFGAIAGFIEGGWQGMVGGVYGY SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD AAY21163 GSG¾AADKESTQK¾IDGVT KVASI IAK ST&&AAVG&EFSGLERRI&NLN&KMEDGFLD mut GSGAAADKESTQKAIDGVTNKVAS I IATMST.¾.a.¾AVGAEFSGLA&RI.MLN.¾.KMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHKCDNECME AAY21163 WTYNAELLVLMENERTMFmS^K LYDKVRLQLRGNATTLG GCFTFYH CSA¾CM& mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTF H CSA&CKSS mut2

490 500

I I SVK GTYDYPRYSEEARLNREEISG AAY21163

SV&NGTYSYPAYSJU^RLA AISG mut

SVANGTYSAPAJ-SAAA&LAAA&ISG mu12 Table 9 - China ABE68921 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ABE68921 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFLNVPE SYIVEKINPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKS ABE68921 LLGNPMCSEALSVPAWSYIVE IGPA GLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTALSVPAWSYIVETIGPATGLCYPGTFGGi¾AELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVL GIHH ABE68921 S SGH ASSGVSSACP TG&SSFF.S.NWWLIKTGGAYPTI RSY NTNTEDLLVLWGIHH mut SWSGH ASSGVSSACP G SSFFANVVWLIK GGAYPTITTS NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGR EFFWTILKPNDAIN ABE68921 PNGAAEQT.&.LYQSPTTYISVGTSTLNQRLVPKIATRS VGGQSGRM FFWTIL PGDAI mut PGGAAAQTALY SPTTAISVGTSTL Q LVPilATRSTVGGTSGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPENAYKIVKKGDSTIMKSELEYG CNTKCQTPIGAINSSMPFH IHPLTIG ABE68921 FxSGGNFIAPENAYKIVKxGSSTIM SALAYGTCxT CQTPIGAISSSMPFHNIHPLTIG mut F SGGNFIAPETAYKIVK GSSTIMiTSALA GTC TTCQTPIGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDG YGYHHSNEQ ABE68921 CPKYVTSG^LVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD ABE 68921 GSGAAADKESTQKAIDGVTNKVAS 11AKMST&&AAVGAEFSGLERRI&NLN&KMEAGFLD mut GSGAAADKESTQK¾IDGVTNKV¾S I IATMSTASAAVGAEFSGLAARIA&LNAKMAkGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHRCDNECME ABE68921 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYHACS.¾ACM& mut V TY AELLVLMENERTMFroSNVKNLYDKVRLALAG AT GNGCFTF HACSA&CKSs mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG ABE68921

SVANGTYSYPAYSAAARLA&ftAISG mut

SVANGTYSAPAJ-SA&AALAAAAISG mu12 Egypt

Two representative hemagglutinin sequences from Egypt are listed in Table 5. ABY76247 shares its aa326 epitope with 176 of the other 249 HPAI H5N1 hemagglutinin sequences from Egypt and ADD21363 shares its epitope with 17 others. The ABY76247 sequence and its mutated versions are presented in Table 10, while those of ADD21363 are in Table 11. The "mut" and "mut2" sequences in these tables represent vaccines against HPAI H5N1 viruses circulating in Egypt with similar sequences in their aa326 epitopes.

Table 10 - Egypt ABY76247 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ABY76247 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFLNVPE SYIVEKINPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKS ABY76247 LLGNPMCSEALSVPAWSYIVE IGPA GLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTALSVPAWSYIVETIGPATGLCYPGTFGGi¾AELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVL GIHH ABY76247 S SGH ASSGVSSACP TG&SSFF.S.NWWLIKTGGAYPTI RSY NTNTEDLLVLWGIHH mut SWSGH ASSGVSSACP G SSFFANVVWLIK GGAYPTITTS NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGR EFFWTILKSNDAIN ABY76247 PNGAAEQT.&.LYQSPTTYISVGTSTLNQRLVPKIATRS VGGQSGRM FFWTIL SGDAI mut PGSAA&QTALY SPTTAISVG STL Q LVPΐIATRSTVGGTSGRMTFT TILTSGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPENAYKIVKKGDSTIMKSELEYG CNTKCQTPIGAINSSMPFH IHPLTIG ABY76247 F SGGNFIAPENAYKIVKiGSSTIMTSALAYG CxT CQTPIGAISSSMPFH IHPLTIG mut F SGGNFIAPETAYKIVK GSSTIM5fSALA GTC TTCQTPIGAISSSMPFHSITPLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ ABY76247 CPKYVTSG^LVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD ABY76247 GSG¾AADKESTQK¾IDGVT KVAS11AKMST.¾.¾¾AVG.S.EFSGLERRIANLN&K EAGFLD mut GSGAAADKESTQKAIDGVTNKVAS I IATMST MAVGAEFS6LA&RI &LNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHRCDNECME ABY76247 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYHACS.¾ACM& mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTF HACSA&CKSS mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG ABY76247

SVANGTYSYPAYSA&ARLA&ftAISG mut

SVANGTYSAPAJ-SAAAALASAAISG mu12 Table 11 - Egypt ADD21363 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ADD21363 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFPNVSE SYIVEKINPANDLCYPGNF NYEELKHLLSRINRFEKIQIIPKS ADD21363 LLGNPMCSEAPSVSAWSYIVE IGPA GLCYPG FGGYAELKHLLSRIG F' I IIP'PS mut LLGNP CSTAPSVSAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWPDHEASLGVSSACPYQGGPSFYRNVVWLIKKNNTYPTIKESYHNTNQEDLLVL GIHH ADD21363 SWPGHEASLGVSSACPTTGGPSFYANWWLIKTGGTYPTITESYT T TEDLLVLWGIHH mut

SWPGH ASLGVSSACP GGPSFYANWWLIK GGTYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDEEEQTRIYKNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRVEFFWTILKSNDTIN ADD21363 PNGA¾,AQTAIY SPTTYISVGTSTLNQRLVPKIATRS VGGQSGRV FFWTIL SGDTI mut PSGAA¾QTAIY SPTTAISVGTSTL Q LVPiIATRSTVGG SGRVTFi TILTSSDTI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPENAYKIVKKGDSTIMKSELEYG CSTKCQTPIGAINSSMPFH IHPLTIG ADD21363 F SGGNFIAPENAYKIVK'IGSSTIMTSALAYG CST CQTPIGAISSSMPFHNIHPLTIG mut F SGGNFIAPETAYKIV GSSTIM SALA GTCSTTCQTPIGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQGEGRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ ADD21363 CPKYVTSG^LVLATGLRNSPQGEGRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLRNSPQGEGRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LEKRIEMLNKKMEDGFLD ADD21363 GSGAAADKESTQKAIDGVTNKVAS I IAK S A¾AAVGAEFSGLEKRIANLN¾KMEDGFLD mut GSGAAADKESTQKAIDGVTNKVAS 11ATMSTAAAAVGAEFSGLAKRIA&LNAKMAkGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHRCD ECME ADD21363 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYHACS.¾ACM& mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG AXTLGNGCFTF HACSA&CKA mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG ADD21363

SVANGTYSYPAYSA&ARLA&ftAISG mut

SVANGTYSAPA3-SA&A&LAAAAISG mu12 Hong Kong

Four representative hemagglutinin sequences from Hong Kong are listed in Table 5. AAL31381 shares its aa326 epitope with 37 of the other 93 HPAI H5N1 hemagglutinin sequences from Hong Kong, ABC66568 shares its epitope with 16 others, and both AAC32078 and ACJ26330 share their epitopes with 5 others. The AAL31381 sequence and its mutated versions are presented in Table 12, those of ABC66568 are in Table 13, those of AAC32078 are in Table 14, and those of ACJ26330 are in Table 15. The "mut" and "mut2" sequences in these tables represent vaccines against HPAI H5N1 viruses circulating in Hong Kong with similar sequences in their aa326 epitopes.

Table 12 - Hong Kong AAL31381 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW AAL31381 D ICIGYHA^STEQVDTII^KWTVT A ILE THSGTLCDLGGVTPLIL&GCSVAG mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKASPVNDLCYPGDF DYEELKHLLSRINHFEKIQIIPKS AAL31381 LLGNPMCSEAISVPAWSYIVE ASPVNGLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNPMCSmiSVPAWSYIVETASPV GLCYPGTFGGftftEIAHLLSAISTFXTITIIPTS mut2

130 140 150 160 170 180

I I I I I I

SWSNHEASSGVSSACPYHGKSSFFRN WLIKKNSAYPTIKRSYNNTNQEDLLVL GIHH AAL31381 S SGH ASSGVSSACP TG SSFFANWWLIKTGSAYPTI RSY NTNTEDLLVLWGIHH mut SWSGH ASSGVSSACP G SSFFANVVWLI GSAYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKP DAIN AAL31381 PNGAAEQT.a.LYQSPTTYISVGTSTLNQRLVPKIATRS VGGQSGRM FFWTIL PGDAIT mut PGGAA&Q ¾LY SPTTAISVG STL QTLVPTIATRSTVGGTSGRMTFT TILTPGDAIT mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSAIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG AAL31381 F SNGNFIAPEYAYKIVKiGSSAIM SALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSAIMTSAUWfGTCTTTCQTPMGAISSSMPFHSITPLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AAL31381 CPKYVTSG^LVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLR TPQRERRRKKRGLFGAIAGFIEGGWQGMVGGVYGY SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD AAL31381 GSGAAADKESTQKAIDGVT KVASI IAK STA&AAVG&EFSGLERRI&NLN&KMEAGFLD mut GSGAAADKESTQKAIDGVTNKV¾SIIA MST.¾ ¾AVGAEFSGLARRIA.¾.LN.¾.KMA¾GFLD mut2

430 440 450 460 470 480

I I I I I I

VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME AAL31381 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CSAACMA mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTF H CSA&CKSS mut2

490 500

I I SVK GTYDYPQYSEEARLKREEISG AAL31381

SV&TOTYSYPAYSA&ARLAAA&ISG mut

SVANGTYSAPAASAAA&LA&AAISG mu12 Table 13 - Hong Kong ABC66568 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ABC 66568 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPANDLCYPGDF DYEELKHLLSRINHFEKIQIIPKN ABC66568 LLGNPMCSEAISVPAWSYIVE AGPA GLCYPG FGGYAELKHLLSRIG FE I IIP A mut LLGNP CSTAISVPAWSYIVETAGPATGLCYPGTFGG¾AELAHLLSAIGTFTTITI IPAA mut2

130 140 150 160 170 180

I I I I I I

SWSSHEASLGVSSACPYQGKSSFFRNW LIKKNSAYPTIKRSYNNTNQEDLLVL GIHH ABC66568 S SSHEASLGYSSACP TG SSFF.S.NVYWLIK GSAYPTITRSY NTNTEDLLVLWGIHH mut SWSSH ASLGVSSACP G SSFFANWWLI GSAYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGR EFFWTILKPNDAIN ABC 66568 PNGAAEQT.&.LYQSPTTYISVGTSTLNQRLVPKIATRS VGGQSGRM FFWTIL PGDAI mut PSGAA¾QTALY SPTTAISVGTSTL Q LVPiIATRSTVGG SGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSAIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG ABC66568 F SGGNFIAPEYAYKIVKiGSSAIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut F SGGNFIAPETAYKIVK GSSAIM5fSALA GTC TTCQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ ABC66568 CPKYVTSG¾>LVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY ' SM mut TCPK VTSGALVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD ABC 66568 GSGAAADKESTQKAIDGVT KVAS11AKMST&&AAVGAEFSGLERRI¾NLN¾KMEAGFLD mut GSGAAADKESTQK¾IDGVTNKV¾SIIA MSTAAAAVGAEFSGI-¾ARIA¾,LNAKMA¾GFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHKCDNECME ABC66568 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CSAACMA mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTFTH CSA&CKSS mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG ABC66568

SVANGTYSYPAYSA&ARLA&ftAISG mut

SVA GTYSAPA&SA&AALA&&AISG mu12 Table 14 - Hong Kong AAC32078 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILERTHNGKLCDLNGVKPLILRDCSVAGW AAC32078 DTICIGYHANHSTEQVDTIME NVTVTTATTILEATHSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILEAT SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKASPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKS AAC32078 LLGNPMCSEAISVPAWSYIVE ASPA GLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTAISVPAWSYIVETASPATGLCYPGTFGG&AELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSNHDASSGVSSACPYLGRSSFFRNW LIKKNSTYPTIKRSYNNTNQEDLLVL GIHH AAC32078 S SGHDASSGVSSACP LG¾SSFFANWWLIKTGSTYPTI RSY NTNTEDLLVLWGIHH mut SWSGH ASSGVSSACP LG SSFFANVVWLI GSTYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPEIATRPKVNGQSGR EFFWTILKPNDAIN AAC32078 PNGAAEQTALYQSPTTYISVGTSTLNQRLVPEIATRP VGGQSGRM FFWTIL PGDAI mut PSGAAAQTALY SPTTAISVGTSTL Q LVPAIATRPTVGGTSGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG AAC32078 F SGGNFIAPEYAYKIVK'IGSSTIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut F SGGNFIAPETAYKIVK GSSTIM5fSALA GTC TTCQTPMGAISSSMPFHSITPLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AAC32078 CPKYVTSG^LVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLRNTPQRERRRKKRGLFGAIAGFIEGGWQGMVGGVYGY SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADQESTQ AIDGVTNKVMSIINKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD AAC32078 GSGAAADQESTQKAIDGVTNKVASI IAK ST&A&AVG&EFSGLERRI&NLN&KMEDGFLD mut GSG¾AADQESTQKAIDGVTNKV¾SIIA MST.¾.¾.¾AVG¾EFSGLARRIA¾.LN.¾.KM¾¾GFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNTELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHKCDNECME AAC32078 V TY TELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CSAACMA mut V TY TELLVLMENERTLDFHDSNVKNLYDKVRLALAG AXTLGNGCFTF H CSA&CKSs mut2

490 500

I I SVK GTYDYPQYSEEARLNREEISG AAC32078

SVANGTYSYPAYSA&ARLA AISG mut

SVAN6TYSAPA&SA&AALAftA-USG mu12 Table 15 - Hong Kong ACJ26330 and mutated sequences

10 20 30 40 50 60

I I I I I I

DHICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGW ACJ26330 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLILTGCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILTGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKD ACJ26330 LLGNPMCSEAISVPAWSYIVE AGPA GLCYPG FGGYAELKHLLSRIG FE I IIP'PG mut LLGNP CSTAISVPAWSYIVETAGPATGLCYPGTFGG&AELAHLLSAIGTFTTITI IPTG mut2

130 140 150 160 170 180

I I I I I I

SWSDHEASLGVSSACPYQGNSSFFRNW LIKKDNAYPTIKKSYNNTNQEDLLVL GIHH ACJ26330 SWSGHEASLGVSSACPTTGGSSFFA W LIKTGGAYPTITKSYTNTNTEDLLVLWGIHH mut SWSGH ASLGVSSACP GGSSFFANWWLIK GGAYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDEAEQTRLYQNPTTYISIGTSTLNQRLVPRIATRSKVNGQSGRIDFFWTILKPNDAIN ACJ26330 PNG&AEQT.&.LYQSPTTYISIGTSTLNQRLVPRIATRS VGGQSGRI FFWTIL PGDAI mut PSG.^AQTALY SPTTAISIGTSTL Q LVPAIATRSTVGGTSGRITFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSEVEYG CNTRCQTPMGAINSSMPFH IHPLTIG ACJ26330 F SGGNFIAPEYAYKIVKiGSSTIMTSAVAYG CxTACQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSTIM S VA G C TACQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNKLVLATGLRNSPQRERRRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ ACJ26330 CPKYVTSG LVLATGLRNSPQRERRRRKRGLFGAIAGFIEGGWQGMVGGWYGY TSM mut TCPK VTSG LVLATGLRNSPQRERRRRKRGLFGAIAGFIEGGWQGMVGGVYGT TSN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD ACJ26330 GSGAAADKESTQKAIDGVTNKVAS 11AKMST&&AAVG&EFSGLERRI&NLNAKMEDGFLD mut GSGAAADKESTQKAIDGVTNKVAS 11ATMSTAAAAVGAEFSGLARRIASLNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHKCDNECME ACJ26330 V TY AELLVLMENERTMFroS VK LYDKVRLQLRG ATTLG GCF FYH CSAACMA mut V TY AELLVLMENERTLDFHDSNVK LYDKVRLALAG ATTLGNGCFTF H CSA&CKA mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG ACJ26330

SVANGTYSYPAYSJU^RLA AISG mut

SVANGTYSAPAJ-SAAA&LAAA&ISG mu12 India

ACZ58110 shares its aa326 epitope with 42 of the other 46 HPAl H5N1 hemagglutinin sequences from India. The sequence of ACZ58110 and its mutated versions are presented in Table 16. The "mut" and "mut2" sequences in Table 23 represent vaccines against the HPAl H5N1 virus in India that has a similar aa326 epitope.

Table 16 - India ACZ58110 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ACZ58110 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFLNVPE SYIVEKINPANDLCYPGTF DYEELKHLLSRINHFEKIQIIPKS ACZ58110 LLGNPMCSEALSVPAWSYIVE IGPA GLCYPGTFGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTALSVPAWSYIVETIGPATGLCYPG FGG&AELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSDHEASSGVSSACPYQGRSSFFRNWWLIKKNDAYP IKISYNNTNQEDLLVL GIHH ACZ58110 S SGH ASSGVSSACPTTGASSFF& WWLIK GGAYPTITI SY NTNTEDLLVLWGIHH mut SWSGH ASSGVSSACP G SSFFANWWLIK GGAYPTITIS NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISVGTSTLNLRLVPKIATRSKVNGQSGRMEFFWTILKPNDAIN ACZ58110 PNGAAEQT.&.LYQSPTTYISVGTSTLNLRLVPKIATRS VGGQSGRMEFFWTIL PGDAI mut PSGAA¾QTALY SPTTAISVGTSTL L LVPiIATRSTVGGTSGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPENAYKIVKKGDSTIMKSELEYG CNTKCQTPIGAINSSMPFH IHPLTIG ACZ58110 F SGGNFIAPENAYKIVKiGSSTIMTSALAYG CxT CQTPIGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSTIM S LA G C T CQTPIGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ ACZ58110 CPKYVTSG^LVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLR SPQGERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD ACZ58110 GSG&AADKESTQKAJDGVT KVAS11AKMST.¾.¾¾AVG.S.EFSGLERRIANLN¾K EAGFLD mut GSGAAADKESTQKAIDGVTNKVAS I IATMST MAVGAEFSGLA&RIASLNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHRCDNECME ACZ58110 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYHACS.¾ACM& mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTF HACSA&CKSS mut2

490 500

I I SVR GTYDYPQYSEESRLKREEISG ACZ58110

SVANGTYSYPAYSA¾.SRLAMAISG mut

SVANGTYSAPAASAASALAS.SA.ISG mu12 Indonesia

Two representative hemagglutinin sequences from Indonesia are listed in Table 5. ABU99029 shares its aa326 epitope with 119 of the other 254 HPAI H5N1 sequences from Indonesia, while AEH59179 shares its epitope with 101 others. The ABU99029 sequence and its mutated versions are presented in Table 17 and those of AEH59179 are in Table 18. The "mut" and "mut2" sequences in these tables represent vaccines against HPAI H5N1 viruses circulating in Indonesia with similar sequences in their aa326 epitopes.

Table 17 - Indonesia ABU99029 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ABU99029 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPANGLCYPGNF DYEELKHLLSRINHFEKIQIIPKS ABU99029 LLGNPMCSEAISVPAWSYIVE AGPA GLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTAISVPAWSYIVETAGPATGLCYPGTFGG&AELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSDHEASLGVSSACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHH ABU99029 S SGHEASLGVSSACP LGASSFFANWWLIKTGSTYPTI RSY NTNTEDLLVLWGIHH mut SWSGH ASLGVSSACP LG SSFFANVVWLI GSTYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISVGTSTLNQRMVPKIATRSKVNGQSGRMEFF TILKP DAIN ABU99029 PNGAAEQT.&LYQSPTTYISVGTSTLNQRMVPKIATRS VGGQSGRM FFWTIL PGDAI mut PGGAA¾QTALY SPTTAISVGTSTL QTIWPTIATRSTVGGTSGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSAIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG ABU99029 F SGGNFIAPEYAYKIVKiGSSAIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSAIM S L G C T CQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDG YGYHHSNEQ ABU99029 CPKYVTSG^LVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFNSLERRIEMLNKKMEDGFLD ABU99029 GSG¾AADKESTQK¾IDGVT KVASI IAKMST.¾.¾¾AVG.S.EFSSLERRI.¾NLN¾K EDGFLD mut GSGAAADKESTQKAIDGVTNKVAS 11ATMSTAAAAVGAEFSSLAARIA.&LNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHKCDNECME ABU99029 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CSGTCMA mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG AXTLGNGCFTF H CSGTCKSs mut2

490 500

I I SIR GTYNYPQYSEEARLKREEISG ABU99029

SIANGTYSYPAYSAAARLAS.SAISG mut

SI.ANGTYSAPAJ^A&A&LAaA&ISG mu12 Table 18 - Indonesia AEH59179 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW AEH59179 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPTNDLCYPGSF DYEELKHLLSRINHFEKIQIIPKS AEH59179 LLGNPMCSEAISVPAWSYIVE AGPTNGLCYPGSFGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTAISVPAWSYIVETAGPTTGLCYPGSFGG&AELAHLLSAIGTFTTITI IPTS mut2

130 140 150 160 170 180

I I I I I I

SWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVL GIHH AEH59179 S SGH ASSGVSSACP LGSPSFFANWWLIKTGSTYPTI KSY NTNTEDLLVLWGIHH mut SWSGH ASSGVSSACP LGSPSFFANWWLI GSTYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISIGTSTLNQRLAPKIATRSKVNGQSGRMEFF TILKP DAIN AEH59179 PNGAAEQT.&LYQSPTTYISIGTSTLNQRLAPKIATRS VGGQSGRM FFWTIL PGDAI mut PSGAA¾QTALY SPTTAISIGTSTL Q LAPiIATRSTVGGTSGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSAIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG AEH59179 F SGGNFIAPEYAYKIVKiGSSAIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSAIM S .L G C T CQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AEH59179 CPKYVTSG¾>LVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLR SPQRESRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD AEH59179 GSG&AADKESTQKAJDGVT KVASI IAK ST&&AAVG&EFSGLERRI&NLN&KMEDGFLD mut GSGAAADKESTQKAIDGVTNKVAS 11ATMSTA¾.¾AVGAEFSGLARRIA.&LNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHKCDNECME AEH59179 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CSGTCMA mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG AXTLGNGCFTF H CSGTCKSs mut2

490 500

I I SIR GTYSYPQYSDEARLKREEISG AEH59179

SIANGTYSYP.&YS.MARLAAA&ISG mut

SI.&NGTYSAPAJ^JUSAALftAAAISG mu12 Thailand

Three representative hemagglutinin sequences from Thailand are listed in Table 5. AAT73266 shares its aa326 epitope with 120 of the 181 other HPAI H5N1 hemagglutinin sequences from Thailand, BAG80800 shares its epitope with 21 others, and AB033748 with 9. The sequence of AAT73266 and of its mutated versions are presented in Table 19, those of BAG80800 are presented in Table 20, and those of AB033748 are in Table 21. The "mut" and "mut2" sequences in these tables represent vaccines against HPAI H5N1 viruses circulating in Thailand with similar sequences in their aa326 epitopes.

Table 19 - Thailand AAT73266 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW AAT73266 D ICIGYHA^STEQVDTII^KWTVT A ILE THSGTLCDLGGVTPLIL&GCSVAG mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPVNDLCYPGDF DYEELKHLLSRINHFEKIQIIPKS AAT73266 LLGNPMCSEAISVPAWSYIVE AGPVNGLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNPMCSmiSVPAWSYIVETAGPV GLCYPGTFGGftftEIAHLLSAISTFXTITIIPTS mut2

130 140 150 160 170 180

I I I I I I

SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHH AAT73266 S SSHEASLGYSSACP TG SSFFANWWLIKTGSTYPTI RSY NTNTEDLLVLWGIHH mut SWSSH ASLGVSSACP G SSFFANVVWLI GSTYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKP DAIN AAT73266 PNGAAEQT.&LYQSPTTYISVGTSTLNQRLVPRIATRS VGGQSGRMEFFWTIL PGDAI mut PSGAA¾QTALY SPTTAISVGTSTL Q LVPAIATRSTVGGTSGRMTFi TILTPSDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG AAT73266 F SNGNFIAPEYAYKIVKiGSSTIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSTIM S L G C T CQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AAT73266 CPKYVTSG^LVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLR SPQRERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD AAT73266 GSGAAADKESTQIAIDGVTNKVASII¾KMST¾¾^VG.¾,EFSGLERRI.¾NLNAK EDGFLD mut GSGAAADKESTQKAIDGVTNKVAS 11ATMSTAASAVGAEFSGLERRIA.&LNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME AAT73266 WTYNAELLVLMENERTMFmS^K LYDKVRLQLRGNATTLG GCFTFYH CSA¾CM& mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG AXTLGNGCFTF H CSA&CKSs mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG AAT73266

SVANGTYSYPAYSA&ARLA&ftAISG mut

SVSN6TYSAPAJ-SAAARLAAAAISG mut2 Table 20 - Thailand BAG80800 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW BAG80800 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPVNDLCYPGDF DYEELKHLLSRINHFEKIQIIPKS BAG80800 LLGNPMCSEAISVPAWSYIVE AGPVNGLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNPMCSmiSVPAWSYIVETAGPV GLCYPGTFGGftftEIAHLLSAISTFXTITIIPTS mut2

130 140 150 160 170 180

I I I I I I

SWSSHEASLGVSSACPYQGKSSFFRNWWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHH BAG80800 S SSHEASLGVSSACP TG SSFFANWWLIKTGSTYPTI RSY NTNTEDLLVLWGIHH mut SWSSH ASLGVSSACP G SSFFANVVWLI GSTYPTITTST NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLIPRIATRSKVNGQSGRMEFFWTILKP DAIN BAG80800 PNGAAEQT.&LYQSPTTYISVGTSTLNQRLIPRIATRS VGGQSGRMEFFWTIL PGDAI mut PSGAA¾QTALY SPTTAISVGTSTL Q LIPAIATRSTVGGTSGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG BAG80800 F SGGNFIAPEYAYKIVKiGSSTIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSTIM S .L G C T CQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQREKRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ BAG80800 CPKYVTSG¾>LVLATGLRNSPQREKRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLR SPQREKRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLMKKMEDGFLD BAG80800 GSG¾AADKESTQK¾IDGVT KVASI IAKMST.¾.¾¾AVG.S.EFSGLERRI.¾NLN¾K EDGFLD mut GSGAAADKESTQKAIDGVTNKVASI IATMST.¾.a.¾AVGAEFSGL¾&RI.MLN.¾.KMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME BAG80800 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CSAACMA mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTF H CSA&CKSS mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG BAG80800

SVANGTYSYPAYSA¾ARLAAA¾ISG mut

SVANGTYSAPAJ-SAAAALASAAISG mu12 Table 21 - Thailand AB033748 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW AB033748 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFSNVPE SYIVEKANPVNDLCYPGDF DYEELKHLLSRINHFEKIQIIPKS AB033748 LLGNPMCSEASSVPAWSYIVE AGPVNGLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLGNP CSTASSVPAWSYIVETAGPVTGLCYPGTFGG¾AELAHLLSAIGTFT ITIIPTS mut2

130 140 150 160 170 180

I I I I I I

SWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL GIHH AB033748 S SSHEASLGYSSACP TG SSFF.S.NWWLIKTGSTYPTI RSY NTNTEDLLVLWGIHH mut SWSSH ASLGVSSACP G SSFFANWWLI GSTYPTITTS NTG LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGR EFFWTILKPNDAIN AB033748 PNGAAEQT.&.LYQSPTTYISVGTSTLNQRLVPRIATRS VGGQSGRMEFFWTIL PGDAI mut PSGAA¾QTALY SPTTAISVGTSTL Q LVPAIATRSTVGG SGRMTFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG AB033748 F SGGNFIAPEYAYKIVKiGSSTIMTSALAYG CxT CQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSTIM S LA G C T CQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQIERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AB033748 CPKYVTSG^LVLATGLRNSPQIERRRKKRGLFGAIAGFIEGGWQG VGGWYGY SM mut TCPK VTSG&LVLATGLR SPQIERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIINKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD AB033748 GSG¾AADKESTQK¾IDGVT KVASI IAKMST.¾.¾¾AVG.S.EFSGLERRI.¾NLN¾K EDGFLD mut GSGAAADKESTQKAIDGVTNKVAS 11ATMSTAAAAVGAEFSGLARRIA.&LNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHKCDNECME AB033748 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CS.¾ACMA mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG AXTLGNGCFTF H CSA&CKSs mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG AB033748

SV&NGTYSYPAYSJU^RLA AISG mut

SVANGTYSAPAJ-SAAAALASAAISG mu12 Viet Nam:

Three representative hemagglutinin sequences from Viet Nam are listed in Table 5. AAT73277 shares its aa326 epitope with 90 of the other 173 HPAI H5N1 sequences from Viet Nam, ACB70548 shares its epitope with 43 others, and AEI26176 with 6. The AAT73277 sequence and its mutated versions are presented in Table 22, those of ACB70548 are in Table 23, and those of AEI26176 are in Table 24. The "mut" and "mut2" sequences in these tables represent vaccines against HPAI H5N1 viruses circulating in Indonesia with similar sequences in their aa326 epitopes.

Table 22 - Viet Nam AAT73277 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW AAT73277 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLIL&GCSVAGW mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPVNDLCYPGDF DYEELKHLLSRINHFEKIQIIPKS AAT73277 LLGNPMCSEAISVPAWSYIVE AGPVNGLCYPG FGGYAELKHLLSRIG FE I IIP'PS mut LLG PMCSmiSVPAWSYIVETAGPV GLCYPGTFGGftftEIAHLLSAISTFXTITIIPTS mut2

130 140 150 160 170 180

I I I I I I

SWSSHEASLGVSSACPYQGKSSFFRNWWLIKKNSTYPTIKRSYNNTNQEDLLVM GIHH AAT73277 S SSHEASLGYSSACP TG SSFF.S.NWWLIKTGSTYPTI RSY NTNTEDLLVMWGIHH mut SWSSH ASLGVSSACP G SSFFANWWLI GSTYPTITTS NTG LLVMWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFF TILKP DAIN AAT73277 PNGAAEQT.&LYQSPTTYISVGTSTLNQRLVPRIATRS VGGQSGRMEFFWTIL PGDAI mut PSGAA¾QTALY SPTTAISVGTSTL Q LVPAIATRSTVGG SGRMTFi TILTPSDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG AAT73277 F SGGNFIAPEYAYKIVKiGSSTIMTSALAYG CxT CQTPMGAISSSMPFHNIHPLTIG mut FTSGGNFIAPE AYKIVKTGSSTIM S LA G C T CQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ AAT73277 CPKYVTSG^LVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLR SPQRERRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQ AIDGVTNKVMSIIDKMNTQFEAVGREFN LERRIEMLNKKMEDGFLD AAT73277 GSG&AADKESTQKAJDGVT KVASI IAK ST&&&AVG&EFSGLERRI&NLN&KMEDGFLD mut GSGAAADKESTQKAIDGVTNKVAS I IATMST&AAAVG&EFSGLA&RIAALNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME AAT73277 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CS.¾ACMA mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTF H CSA&CKSS mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG AAT73277

SV&NGTYSYPAY SAAARLA AISG mut

SVANGTY SAPAJ-SAAA&LAAa&ISG mu12 Table 23 - Viet Nam ACB70548 and mutated sequences

10 20 30 40 50 60

I I I I I I

DQICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGW ACB70548 D ICIGYHA^STEQVDTII^KWTVT A ILE THSGTLCDLGGVTPLIL&GCSVAG mut DTICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILAGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFINVPE SYIVEKANPVNDLCYPGVF DYEELKHLLSRINHFEKIQIIPKS ACB70548 LLGNPMCSEAISVPAWSYIVE AGPVNGLCYPGVFGGYAELKHLLSRIG FE I IIP'PS mut LLGNPMCS AISVPAWSYIVE AGPV GLCYPGVFGG.¾¾.ELAHLLS¾IG F I IIP S mut2

130 140 150 160 170 180

I I I I I I

SWPSHEASLGVSAACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVI GIHH ACB70548 S PSHEASLGYSAACP TG SSFF.S.NVYWLIK GSTYPTITRSY NTNTEDLLVIWGIHH mut SWPSH ASLGVSAACP G SSFFANWWLI GSTYPTITTST NTG LLVIWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTKLYQNPTTYISVGTSTLNQRLTPRIATRSKVNGQSGRMEFFWTILKP DAIN ACB70548 PNGAAEQTALYQSPTTYISVGTSTLNQRLTPRIATRS VGGQSGRMEFFWTIL PGDAI mut PGGAAAQTALY SPTTAISVGTSTL Q LTPAIATRSTVGGTSGRMEFi TILTPGDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSELEYG CNTKCQTPMGAINSSMPFH IHPLTIG ACB70548 F SGGNFIAPEYAYKIVKiGSSTIMTSALAYG CxT CQTPMGAISSSMPFHNIHPLTIG mut FTSGGNFIAPE AYKIVK GSSTIM S L G C T CQTPMGAISSSMPFHSI PLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQREGRRKKRGLFGAIAGFIEGGWQGMVDG YGYHHSNEQ ACB70548 CPKYVTSG^LVLATGLRNSPQREGRRKKRGLFGAIAGFIEGGWQGMVGGWYGY SM mut TCPK VTSG&LVLATGLR SPQREGRRKKRGLFGAIAGFIEGGWQGMVGGVYG T SN mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQKAIDGVT KVNS 11DKMNTQFEAVGREFN LERRIENLNKKMEDGFLD ACB70548 GSG¾AADKESTQK¾IDGVT KVAS11AKMST.¾.¾¾AVG.S.EFSGLERRI.¾NLN¾K E.S.GFLD mut GSGAAADKESTQKAIDGVTNKVAS I IATMST.¾A.¾AVGAEFSGLA&RI.MLN.¾.KMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLQLRD AKELGNGCFEFYHKCD ECME ACB70548 WTYNAELLVLMENERTMFmS^K LYDKVRLQLRGNATTLG GCFTFYH CSA¾CM& mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTF H CSA&CKSS mut2

490 500

I I SVR GTYDYPQYSEEARLKREEISG ACB70548

SVANGTYSYPAYSA&ARLA&ftAISG mut

SVANGTYSAPA&SA&A&L SG mu12 Table 24 - Viet Nam AEI26176 and mutated sequences

10 20 30 40 50 60

I I I I I I

DHICIGYHANMSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGW AEI26176 DTICIGYHANHSTEQVDTIME NVTVTTATTILE THSGTLCDLGGVTPLILTGCSVAGW mut STICIGYHA^STEQVDTIMEKWTVT A ^ILE T SGTLCDLGGV^PLILTGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFTNVPE SYIVEKANPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKD AEI26176 LLGNPMCSEATSVPAWSYIVE AGPA GLCYPG FGGYAELKHLLSAIG FE I IIP A mut LLGNP CSTATSVPAWSYIVETAGPATGLCYPGTFGG&AELAHLLSAIGTFTTITI IPAA mut2

130 140 150 160 170 180

I I I I I I

SWSDHEASLGVSSACPYQGNSSFFR W LIKKNNAYPTIKKSYNNTNREDLLVL GIHH AEI26176 SWSGHEASLGVSSACPTTGGSSFFANW LIKTGGAYPTI KSYTNTNAEDLLVLWGIHH mut SWSGH ASLGVSSACP GGSSFFANVVWLIK GGAYPTITTS NTG¾ LLVLWGIHH mut2

190 200 210 220 230 240

I I I I I I

PNDEAEQTRLYQNPTTYISIGTSTLNQRLVPRIATRSKVNGQSGRIDFFWTILKPNDAIN AEI26176 PNG&AEQT.&.LYQSPTTYISIGTSTLNQRLVPRIATRS VGGQSGRI FFWTIL PGDAI mut PSGM,¾QTALY SPTTAISIGTSTL Q LVPMATRSTVGGTSGRITFi TILTPSDAI mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSEVEYG CNTRCQTPMGAINSSMPFH IHPLTIG AEI26176 F SGGNFIAPEYAYKIVKiGSSTIM SAVAYG CxTACQTPMGAISSSMPFH IHPLTIG mut FTSGGNFIAPE AYKIVKTGSSTIMTSAVAiGTCTTACQTPMGAISSSMPFHSITPLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNKLVLATGLRNSPQRERRRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNGQ AEI26176 CPKYVTSG LVLATGLRNSPQRERRRRKRGLFGAIAGFIEGGWQGMVGGWYGY TSMG mut TCPK VTSG LVLATGLR SPQRERRRRKRGLFGAIAGFIEGGWQGMVGGVYG T SNGT mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQKAIDGVTNKVNSIIDK NTQFEAVGREFNNLERRIENLNKK EDGFLD AEI26176 GSGAAADKESTQKAIDGVTNKVAS 11AKMST&&AAVGAEFSGLERRIANLN&KMEAGFLD mut GSGAAADKESTQKAIDGVTNKVAS I IATMST MAVGAEFS6LA&RLMLNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

VWTYNAELLVL ENERTLDFHDSNVK LYDKVRLQLRDNAKELGNGCFEFYHKCDDECME AEI26176 V TY AELLVLMENERTMFroS VKraYDKVRLQLRG ATTLG GCF FYH CS.¾ACM& mut V TY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG ATTLGNGCFTF H CSA&CKSS mut2

490 500

I I

SVR GTYDYPQYSEEARLKREEISG AEI26176

SVANGTYSYPAY SJU^RLA AISG mut

SVANGTY SAPAJ-SA&A&LAaA&ISG mu12 [00130] Modified polypeptides for two South Korean H5N1 isolates are shown in Tables 25 and 26.

Table 25 - ABW73807 (South Korea HPAI H5N1 HA) and mutated variants*

10 20 30 40 50 60

I I I I I I

DQICIGYHAlSnaSTEQVDTIMEKNVTVTHAQDILEKTHMGKLCDLDGMKPLILKDCSVAGW ABW73807 D ICIGYIIA^STEQVDTIMEKNVTVTTAT ILETTHSG LCDLGGM PLIL GCSVAGW mut DTIClGYHj-mSTEQVDTII^ia^TVTTA TILETTTSGTLCDLGGMTPLILTGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKS ABW73807 LLGNPMC3EALSVPAWSYIVETIGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTS mut LLGNPMCSTALSVPAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTS mut2

130 140 150 160 170 180

I I I I I I

S SDHEASSGYSSACPYQGRSSFFRNVVWLIKKNDAYPTIKRSYNNTNQEDLLVL GIHH ABW73807 SWSGH?ASSGVSSACP GASSFFANWWLIKTGGAYPTI?RSYTNTN EDLLVLWGIHH mut SWSGHTASSGVSSACPTTGASSFFANVV LIKTGGAYPTITTSTTNTGTTTLLVL GIHH mut2

190 200 210 220 230 240

I I I I I I

PNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFF TILKP DAIN ABW73807 PNGAAEQTALYQSPTTYISVGTSTLNQRLVPKIATRSTVGGQSGRMTFFWTILTPGDAIT mut PGGAAAQTALY SPTTAISVGTSTLTQTLVPTIATRS VGGTSGRM F TIL PGDAIT mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFH IHPLTIG ABW73807 FTSGGNFIAPENAYKIVK GSSTIM SALAYGTC T CQTPIGAISSSMPFH IHPLTIG mut FTSGG FIAPETAYKIVKTGSSTIMTSALATGTCxTTCQTPIGAISSSMPFHSITPLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQ ABW73807 CPKYVTSGALVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGWYGYTTSNTT mut TCPK V SGALVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGVYG SNT mut2

370 380 390 400 410 420

I I I I I I

GSGYAADKESTQKAIDGVTNKVNS 11DKMNTQFEAVGREFN LERRIENLNKKMEDGFLD ABW73807 GSGAAADKESTQKAIDGVTNKVASIIAKMST AAAVGAEFSGLERRIANLNAKMEAGFLD mut GSGAAADKESTQIAIDGVTNKVASI IA MSTAAAAVGAEFSGLAARI ALNAKMAAGFLD mut2

430 440 450 460 470 480

I I I I I I

V TY AELLVLMENERTMFHDS^K LYDKVRLQLRD AKELGNGCFEFYHRCD ECME ABW73807 V TYNAELLVLMENERTMFmS VKraYDKVRLQLRG A LGNGCFTFYHACSAACRA mut VWTY AELLVLMENERTLDFHDSNVKNLYDKVRLALAG A TLGNGCFTFTHAC}AACMA mut2

490 500

I I SVR GTYDYPQYSEESRLKREEISG AB 73807

SVANGTY8YPAYSAASRLAAA¾.ISG mut

SVANGTYSAPAASAASALAAA ISG mu12 Table 26 - AEJ90156 (South Korea HPAI H5N1 HA) and mutated

variants

10 20 30 40 50 60

I I I I I I

DHICIGYIIANMSTEQVDTIMEKNVTVTHAQDILEKTHMGKLCDLNGVKPLILKDCSVAGW AEJ90156

D ICIGYIIA^STEQVDTIMEKNVTVTTAT ILETTTSG LCDLGGV PLIL GCSVAGW mut

DTICIGYHA-TOSTEQVDTI-^IQJVT TAT ILE TTSGTLCDLGGV PLILTGCSVAGW mut2

70 80 90 100 110 120

I I I I I I

LLGNPLCDEFINVPEWSYIVEKAKPANDLCYPGNF DYEELKHLLSRINHFEKIQIIPKD AEJ90156

LLGNPLC3EAISVPAWSYIVETATPANGLCYPGTFGGYAELKHLLSAIGTFETITIIPTG mut

LLGNPLCSTAISVPA SYIVETATPATGLCYPGTFGGA ELAHLLSAIGTFTTITIIPTG mut2

130 140 150 160 170 180

I I I I I I

S SEHEASLGYSAACSYQGNSSFFRNVVWLIKKDNAYPTIKKGYNNTNQEDLLVL GIHH AEJ90156

SWSTHEASLGVSAACS GGSSFFANWWLIK GGAYPTI'TKGYTNTN EDLLVLWGIHH mut

S STHTASLGVSAACSTTGGSSFFANVV LIKTGGAYPTITTGTTNTGTTTLLVL GIHH mut2

190 200 210 220 230 240

I I I I I I

PNDEAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKINGQSGRIDFFWTILKP DAIH AEJ90156

PNGAAEQTALYQSPTTYISIGTSTLNQRLVPKIATRSTIGGQSGRITFFWTILTPGDAIT mut

PGGAAAQTALY SPTTAISIGTSTLTQ LVPTIATRS IGGTSGRI F TIL PGDAIT mut2

250 260 270 280 290 300

I I I I I I

FESNGNFIAPEYAYKIVKKGDSTIMKSEVEYGNCNTRCQTPIGAINSSMPFH IHPLTIG AEJ90156

FESGGNFIAPEYAYKIVK GSSTIM SAVAYGTC TACQTPIGAISSSMPFH IHPLTIG mut

FTSGGNFIAPETAYKIVKTGSSTIMTSAVATGTCTTACQTPIGAISSSMPFHSITPLTIG mut2

310 320 330 340 350 360

I I I I I I

ECPKYVKSNKLVLATGLRNSPQRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQG AEJ90156

CPKYVTSGTLVLATGLRNSPQRERRRKRGLFGAIAGFIEGGWQGMVGGWYGYTTSNTTG mut

TCPK V SG LVLATGLRNSPQRERRRKRGLFGAIAGFIEGGWQGMVGGVYGT SNT G mut2

370 380 390 400 410 420

I I I I I I

SGYAADKESTQKAIDGVTNKV SIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDV AEJ90156

SGAAADKESTQKAIDGVTNKVASII KMSTAAAAVGAEFSGLERRIANLNAKMEDGFLDV mut

SGAAADKESTQKAIDGVTNKVASI IATMSTAAAA.VGAEFSGLARRIAALNAKMAAGFLDV mut2

430 440 450 460 470 480

I I I I I I TYNAELLVLMENERTI^FHDSNVKNLYDKVRLQLKDNAKELG GCFEFYHKCNNECMES AEJ90156

TYNAELLVLMENERT^F^S K^YDKVRLQLKGNA LG GCFTFYHTCSAACMAS mut

WTYMAELLVLMENERTLDFHDS- KNLYDKVRLAL GMAT LGNGCF FTHTCSAACMAS mut2

490 500

I I VR GTYDYPQYSEEARLKREEISG AEJ90156

VA GTY8YPAYSA ARLAA I SG mut

VANGTYSAPAASAAAA.LAAA ISG mut2

[00131] Tables 6 through 26 present sequences representing vaccines that are useful against HPAI H5N1 in the nine countries where the virus is known to be present in high incidence. These candidate vaccines are meant to focus the antibody response to a part of hemagglutinin that most probably will not change in view of its vital importance in the ability of the virus to invade target cells. This means that the vaccines could be effective for use every year, even if a new strain of the virus emerges.

[00132] Examination of the aa326 epitopes listed in Table 5 reveals the existence of epitopes that are common to viruses found in several countries. The most common is the epitope of AAT73266 from Thailand which is shared by AAX53505 from China, by ABC66568 from Hong Kong, by ABU99029 from Indonesia, and by AAT73277 from Viet Nam, i.e., by 405 of the 1271 HPAI H5N1 sequences available from the CBl Influenza Virus Database as of December 31, 2011. Another aa326 epitope that is common is that of ACU24777 from Bangladesh, which is shared by ABE68921 from China, and by ABY76247 from Egypt, i.e., by 225 of the sequences. The AAT73266 vaccine alone might be able to protect against one third of possible cases, while the simultaneous use of the AAT73266 and ABY76247 vaccines might be able to protect against half. Further, most of the more reactive residues are common among the various aa326 epitopes and several of the differences are conservative substitutions, e.g., lysine vs. arginine, so that the AAT73266/ABY76247 vaccine combination may already be protective against most, if not all, HPAI H5N1 strains.

[00133] Two vaccines are shown for each of the hemagglutinins analyzed here. One is the result of the replacement of residues that are at least 40% exposed (the "mut" sequence); the other by the replacement of residues that are at least 25% exposed (the "mut2" sequence). For AAT73266, the "mut" and "mut2" replacements result in sequences that are 81.8% and 73.7%) identical to the unmutated sequence, respectively. Similar numbers are obtained for the other hemagglutinins (not shown). This degree of sequence similarity is generally accepted as indicative of close similarity in tertiary structure, so that the proper presentation of the aa326 epitope to the immune system can be expected for all the proposed vaccines. The "mut" sequence should result in a more faithful preservation of the hemagglutinin structure, in view of the fewer replacements. On the other hand, as the antigenicity plots in Figure 1 show, the "mut2" replacements would result in a greater reduction in the overall antigenicity of the molecule, while emphasizing that of the aa326 epitope.

[00134] The residues that constitute the aa326 epitope come from two adjacent molecules.

Therefore, the vaccine should be presented as a hemagglutinin trimer - the natural biological assembly. In a particular embodiment, a weakened virus, generated by reverse genetics (see, e.g., Neumann et al. 1999) and expressing the gene for the mutated hemagglutinin, can be used to present the the antigen. Target Selection

[00135] Two constructs were generated for ABW73807 mut2 wrt Arg326 (SEQ ID NO:64) and ABW73807 target-less mut2 wrt Arg326 (SEQ ID NO:65). Each sequence was codon optimizated to boost protein expression levels. The constructs were cloned into bacterial expression vectors. Sequences were amplified and subcloned into a proper bacterial expression vector and subcloned gene authenticity was confirmed by restriction enzyme digest and sequencing.

[00136] Two liters of bacterial culture were induced with IPTG and cell pellets harvested for protein purification. Cell pellets were disrupted by sonication. Protein purification was performed by affinity column chromatography, gel filtration, ion exchange, and hydrophobic column chromatography. SDS-PAGE and Western-blot analysis was performed and shown in Figures 2 and 3.

Immune Response Induction

[00137] Immunogenicity was assessed of the ABW73807 mut 2 wrt Arg326 and the ABW73807 target-less mut 2 wrt Arg326 antigens in a mouse model. Immune responses were tested using an ELISA assay for the detection of mouse IgG specific for these antigens.

[00138] The B57/BL6 mice were immunized subcutaneously with 100 ug of ABW73807 mut 2 wrt Arg326 or the ABW73807 target-less mut 2 wrt Arg326 mixed in complete Freund's adjuvant (CFA). The mice were injected with phosphate buffered saline (PBS) served as a negative control. The blood was collected from the tail vein and the serum was separated, aliquoted and stored at -20°C.

[00139] The basic ELISA procedure can be summarized as follows: The plates were coated at 100 μΐ/well with the test antigens and incubated overnight at 4°C in a humidity chamber. After washing the plate 3 times with PBS/0.05% Tween 20, the plates were blocked for 1 hour with 0.1% gelatin/PBS/0.05% Tween 20. The wash step was repeated and serum samples were added at 100 μΐ/well for a 2 hour incubation at room temperature. The plates were washed once again and the secondary antibody was diluted in blocking buffer, added at 100 μΐ/well, and incubated for 2 hours at room temperature. The plates were washed again, with PBS/0.05% Tween 20 as before and 4 times with Final Wash (0.4 M Tris/0.15 M NaCl, pH 7.5) and then plated with 100 μΐ well substrate solution (pNPP tablets dissolved in substrate buffer). The plates were read at 405 and 650 nm at timepoints listed in the table below. Control wells consisted of blocking controls (BC, which do not receive antigen), conjugate controls (CC, which do not receive serum), and substrate blanks (SB, which do not receive sample or substrate).

] Summary of results

OD values ranging from 1.660 (20 μg/ml) to 1.038 (5 μg/ml) in response to the antigen, ABW73807 target-less mut 2 wrt Arg326. The conjugate controls produced mean OD values ranging from 0.107 (5 μg/ml) to 0.091 (10 &'ιηΐ) and blocking controls produced a mean OD of 0.108 (Gl, PBS) and 0.119 (G3, target-less mut 2). The substrate blank mean OD values ranged from 0.046 (20 μg/ml and 5 μ^πιΐ) to 0.044 (10 μ^πιΐ).

The positive control produced mean OD values of 3.833 and 3.764 for plates 1 and 2, respectively.

The values below are the mean OD values at 16 hours for serum diluted 1 :100 at the 405nm wavelength. On Plate I, the control group (Gl, PBS) produced mean OD values ranging from 0.789 (10 μg/ml) to 0.546 (5 μg/ml) in response to the antigen, ABW73807 mut 2 wrt Arg326. Also on Plate 1, antigen immunized group (G2, mut 2) produced mean OD values ranging from 3.136(10 μg/ml) to 1.873(5 μg/ml) in response to the antigen, ABW73807 mut 2 wrt Arg326. The conjugate controls produced mean OD values ranging from 0.203 (10 μg/ml) to 0.131 (5 μg/ml) and blocking controls produced a mean OD of 0.389 (Gl, PBS) and 0.371 (G2, mut 2). The substrate blank mean OD values ranged from 0.072 (10 μ^πιΐ) to 0.057 (5 μ^πιΐ).

On Plate 2, the control group (Gl, PBS) produced mean OD values ranging from 0.840 (20 μ^πιΐ) to 0.602 (5 μ^ηιΐ) in response to the antigen, ABW73807 target-less mut 2 wrt Arg326. Also on Plate 2, the antigen immunized group (G3, target-less mut 2) produced OD values at the maximum OD of 4.000 for all concentrations of the antigen, ABW73807 target-less mut 2 wrt Arg326. The conjugate controls produced mean OD values ranging from 0.129 (20 μg/ml) to 0.112 (10 μg/ml) and blocking controls produced a mean OD of 0.443 (Gl, PBS) and 0.575 (G3, target-less mut 2). The substrate blank mean OD ranged from 0.047 (20 μ^πιΐ) to 0.045 (10 μg/ml).

The positive control produced OD values at the maximum OD of 4.000 for both plates.

Conclusions: Immunization produced antigen-specific responses in the mouse groups. The antigen-specific response produced a direct correlation with the concentration of the antigen coated on the ELISA plate.

Scope of the experiment: Two (2) plates were coated ABW73807 mut 2 wrt Arg326 (Lot# MB1343-1) and ABW73807 target-less mut 2 wrt Arg326 (Lot# MB1343-2) diluted in PBS at 20 μ^πιΐ, 10 μ^πιΐ, and 5 μg/ml. Also, on each plate the positive control, C57BL6 Mouse Serum (Lot#14516) was coated at 1 :20000 in PBS. One (1) plate was coated for each antigen. The three serum samples were from pooled mouse serum (except Group 3 due to the death of two mice in the group) and diluted 1 : 100 and 1 : 1000 in blocking buffer. Plate 1 received the control PBS serum and the mouse serum from

ABW73807 mut 2 wrt Arg326 immunized mice. Plate 2 received the control PBS serum and the mouse serum from the ABW73807 target- less mut 2 wrt Arg326 immunized mouse. The secondary antibody, Goat Anti-Mouse IgG AP (Southern Biotech, cat#l 030-04, lot#D5012-RG63B) was diluted 1 :1000 in blocking buffer. pNPP

N2913-01-04 tablets were dissolved in substrate buffer to 1 mg/ml.

Results: The values below are the mean OD values at 20 minutes for serum diluted 1 : 100 at the 405nm wavelength. On Plate 1, the control group (Gl, PBS) produced mean OD values ranging from 0.104 (5 μ^πιΐ) to 0.100 (10 μ&'ιηΐ) in response to the antigen, ABW73807 mut 2 wrt Arg326. Also on Plate 1, antigen immunized group (G2, mut 2) produced mean OD values ranging from 3.929(20 μ^ΐηΐ) to 3.239(5 g ml) in response to the antigen, ABW73807 mut 2 wrt Arg326. The conjugate controls produced mean OD values ranging from 0.093 (20 μ^πιΐ) to 0.092 (5 μ^πιΐ and 10 μ^πιΐ) and blocking controls produced a mean OD of 0.098 (Gl, PBS) and 0.111 (G2, mut 2). The substrate blank mean OD values ranged from 0.052 (10 μg/ml) to 0.048 (20 μ^πιΐ).

On Plate 2, the control group (Gl, PBS) produced mean OD values ranging from 0.113 (20 μg/ml) to 0.108 (10 &^ηιΐ) in response to the antigen, ABW73807 target-less mut 2 wrt Arg326. Also on Plate 2, the antigen immunized group (G3, target-less mut 2) produced mean OD values ranging from 2.842(20 μ^ηιΐ) to 2.319(5 μ^ηιΐ) in response to the antigen, ABW73807 target-less mut 2 wrt Arg326. The conjugate controls produced mean OD values ranging from 0.091 (20 μg/ml) to 0.090 (10 μ^'πά and 5 μ^πιΐ) and blocking controls produced a mean OD of 0.108 (Gl, PBS) and 0.106 (G3, target-less mut 2). The substrate blank mean OD ranged from 0.050 (20 ^ηιΐ) to

0.047 (ΙΟ μ πύ).

The positive control produced mean OD values of 2.132 and 1.715 for plates 1 and 2, respectively.

The values below are the mean OD values after 60 minutes of development for serum diluted 1 : 100 at the 405nm wavelength) On Plate 1, the control group (Gl, PBS) produced mean OD values ranging from 0.136 (5 μg/ml) to 0.127 (10 μ^ιηΐ and 20 μ§/'ν \) in response to the antigen, ABW73807 mut 2 wrt Arg326. Also on Plate

1, antigen immunized group (G2, mut 2) produced mean OD values ranging from 4.000 (10 μg/ml and 20

to 3.935 (5 μ /πιΓ) in response to the antigen, ABW73807 mut 2 wrt Arg326. The conjugate controls produced mean OD values ranging from 0.100 (5 μ^ητΐ) to 0.098 (10 μg/ml and 20 μ&'ηύ) and blocking controls produced a mean OD of 0.123 (Gl, PBS) and 0.140 (G2, mut 2). The substrate blank mean OD values ranged from 0.052 (10 μ^ηιΐ) to 0.048 (20 μg/ml).

On Plate 2, the control group (Gl, PBS) produced mean OD values ranging from 0.151 (20 μg/ml) to 0.149 (10 μξ/ναϊ) in response to the antigen, ABW73807 target-less mut 2 wrt Arg326. Also on Plate 2, the antigen immunized group (G3, target-less mut 2) produced mean OD values ranging from 4.000 (20 μg/ml and 10 μg/ml) to 3.841 (5 μg/ml) in response to the antigen, ABW73807 target-less mut 2 wrt Arg326. The conjugate controls produced mean OD values ranging from 0.092 (20 μ^πιΐ) to 0.091 (10 μ^πιΐ and 5 μ^πιΐ) and blocking controls produced a mean OD of 0.134 (Gl, PBS) and 0.144 (G3, target-less mut 2). The substrate blank mean OD values ranged from 0.049 (20 μ^πιΐ and 5 μ^πιΐ) to 0.048 (10 μ^).

The positive control produced mean OD values of 3.786 and 3.660 for plates 1 and 2, respectively.

Conclusion: Immunization produced significantly high antigen-specific responses in all immunized groups. This response was visible in both the ABW73807 target-less mut 2 wrt Arg326 immunized mice and the ABW73807 mut 2 wrt Arg326 immunized mice.

SEQUENCES

SEQ ID NO:l - ACU24777 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVS SACPYQGRSSFFRNVVWLIKKNDAYPTIKISYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SVGTSTLNLRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHRCDNECMESVRNGTYDYPQYSEESRLKREEISG

SEQ ID NO: 2 - ACU24777 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEALSVPAWSYIVETIGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTTGASSFFANVVWLIKTGGAYPTITISYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNLRLVPKIATRSTVGGQSGRMTFFWTILTPGDAITFTSGGNFIAPENAYKIVKTGSSTI MTSALAYGTCTTTCQTPIGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHACSAACMASVANGTYSYPAYSAASRLAAAAISG

SEQ ID NO: 3 - ACU24777 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTALSVPAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTTGASSFFANWWLIKTGGAYPTITISTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTLTLVPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPIGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHACSAACMASVANGTYSAPAASAASALAAAAISG

SEQ ID NO: 4 - AAX53505 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSNHEASSGVS SACPYLGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 5 - AAX53505 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETASPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTLGTSSFFANVVWLIKTGSTYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPKIATRSTVGGQSGRMTFFWTILTPGDAITFTSNGNFIAPEYAYKIVKTGSSAI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 6 - AAX53505 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETASPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTLGTSSFFANWWLITTGSTYPTI STTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSAI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLARRIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 7 - AAY21163 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSNHEASSGVS SACPYNGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLILWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDTINFESNGNFIAPEYAYKIVKKGDSAI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVKNGTYDYPRYSEEARLNREEISG

SEQ ID NO: 8 - AAY21163 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTGGTSSFFANVVWLIKTGSTYPTITRSYTNTNTEDLLILWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPKIATRSTVGGQSGRMTFFWTILTPGDTITFTSGGNFIAPEYAYKIVKTGSSAI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 9 - AAY21163 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTGGTSSFFANVVWLITTGSTYPTITTSTTNTGTTTLLILWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTPGDTITFTSGGNFIAPETAYKIVKTGSSAI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVGGVYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 10 - ABE68921 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVS SACPYQGRSSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 11 - ABE68921 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEALSVPAWSYIVETIGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTTGASSFFANWWLIKTGGAYPTI RSYTNTNTEDLLVLWGIHHFTSGGNFIAPENAYKIVK TGSSTIMTSALAYGTCTTTCQTPIGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQGE RRRKKRGLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMS TAAAAVGAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQL RGNATTLGNGCFTFYHACSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 12 - ABE68921 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTALSVPAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTTGASSFFANVVWLIKTGGAYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPIGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHACSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 13 - ABY76247 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVS SACPYQGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPENAYKIVKKGDSTI MKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 14 - ABY76247 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEALSVPAWSYIVETIGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTTGASSFFANVVWLIKTGGAYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPKIATRSTVGGQSGRMTFFWTILTSGDAITFTSGGNFIAPENAYKIVKTGSSTI MTSALAYGTCTTTCQTPIGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHACSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 15 - ABY76247 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTALSVPAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTTGASSFFANVVWLIKTGGAYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTSGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPIGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHACSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 16 - ADD21363 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFPNVSEWSYIVEKINPANDLCYPGNFNNYEELKHLLSRINRFEKIQIIPKSSWPDHEASLGVS SACPYQGGPSFYRNWWLIKKNNTYPTIKESYHNTNQEDLLVLWGIHHPNDEEEQTRIYKNPTTYI SVGTSTLNQRLVPKIATRSKVNGQSGRVEFFWTILKSNDTINFESNGNFIAPENAYKIVKKGDSTI MKSELEYGNCSTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGEGRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSI IDKMNTQFEAV GREFNNLEKRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 17 - ADD21363 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAPSVSAWSYIVETIGPATGLCYPGTFGGYAELKHLLSRIGTFTTITIIPTSSWPGHEASLGVS SACPTTGGPSFYANVVWLIKTGGTYPTITESYTNTNTEDLLVLWGIHHPNGAAAQTAIYTSPTTYI SVGTSTLNQRLVPKIATRSTVGGQSGRVTFFWTILTSGDTITFTSGGNFIAPENAYKIVKTGSSTI MTSALAYGTCSTTCQTPIGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQGEGRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLEKRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHACSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 18 - ADD21363 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAPSVSAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWPGHTASLGVS SACPTTGGPSFYANVVWLIKTGGTYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTAIYTSPTTAI SVGTSTLTQTLVPTIATRSTVGGTSGRVTFTWTILTSGDTITFTSGGNFIAPETAYKIVTTGSSTI MTSALATGTCSTTCQTPIGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQGEGRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAKRIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHACSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 19 - AAL31381 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKASPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSNHEASSGVS SACPYHGKSSFFRNVVWLIKKNSAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSI IDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVKNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 20 - AAL31381 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETASPVNGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTTGTSSFFANVVWLIKTGSAYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPKIATRSTVGGQSGRMTFFWTILTPGDAITFTSNGNFIAPEYAYKIVKTGSSAI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 21 - AAL31381 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETASPVTGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTTGTSSFFANWWLITTGSAYPTI STTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSAI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVGGVYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLARRIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 22 - ABC66568 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKNSWSSHEASLGVS SACPYQGKSSFFRNVVWLIKKNSAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 23 - ABC66568 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPAASWSSHEASLGVS SACPTTGTSSFFANVVWLIKTGSAYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPKIATRSTVGGQSGRMTFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSAI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 24 - ABC66568 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPAASWSSHTASLGVS SACPTTGTSSFFANVVWLITTGSAYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSAI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 25 - AAC32078 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILERTHNGKLCDLNGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKASPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSNHDASSGVS SACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPEIATRPKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADQESTQKAIDGVTNKVNSIINKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNTELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVKNGTYDYPQYSEEARLNREEISG

SEQ ID NO: 26 - AAC32078 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILEATHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETASPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHDASSGVS SACPTLGASSFFANWWLIKTGSTYPTI RSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPEIATRPTVGGQSGRMTFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSTI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADQESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEDGFLDVWTYNTELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 27 - AAC32078 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILEATTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETASPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTLGASSFFANVVWLITTGSTYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPAIATRPTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNTPQRERRRKKR GLFGAIAGFIEGGWQGMVGGVYGYTTSNTTGSGAAADQESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLARRIAALNAKMAAGFLDVWTYNTELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 28 - ACJ26330 - HAwt

DHICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWSDHEASLGVS SACPYQGNSSFFRNVVWLIKKDNAYPTIKKSYNNTNQEDLLVLWGIHHPNDEAEQTRLYQNPTTYI SIGTSTLNQRLVPRIATRSKVNGQSGRIDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEVEYGNCNTRCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRERRRRKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 29 - ACJ26330 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILTGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTGSWSGHEASLGVS SACPTTGGSSFFANVVWLIKTGGAYPTITKSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SIGTSTLNQRLVPRIATRSTVGGQSGRITFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSTI MTSAVAYGTCTTACQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGTLVLATGLRNSPQRERRRRKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 30 - ACJ26330 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILTGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTGSWSGHTASLGVS SACPTTGGSSFFANVVWLIKTGGAYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SIGTSTLTQTLVPAIATRSTVGGTSGRITFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSAVATGTCTTACQTPMGAISSSMPFHSITPLTIGTCPKTVTSGTLVLATGLRNSPQRERRRRKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLARRIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 31 - ACZ58110 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFLNVPEWSYIVEKINPANDLCYPGTFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVS SACPYQGRSSFFRNVVWLIKKNDAYPTIKISYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SVGTSTLNLRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHRCDNECMESVRNGTYDYPQYSEESRLKREEISG

SEQ ID NO: 32 - ACZ58110 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEALSVPAWSYIVETIGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTTGASSFFANVVWLIKTGGAYPTITISYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNLRLVPKIATRSTVGGQSGRMEFFWTILTPGDAITFTSGGNFIAPENAYKIVKTGSSTI MTSALAYGTCTTTCQTPIGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHACSAACMASVANGTYSYPAYSAASRLAAAAISG

SEQ ID NO: 33 - ACZ58110 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTALSVPAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTTGASSFFANVVWLIKTGGAYPTITISTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTLTLVPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPIGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHACSAACMASVANGTYSAPAASAASALAAAAISG

SEQ ID NO: 34 - ABU99029 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANGLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASLGVS SACPYLGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SVGTSTLNQRMVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSI IDKMNTQFEAV GREFNSLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISG

SEQ ID NO: 35 - ABU99029 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHEASLGVS SACPTLGASSFFANVVWLIKTGSTYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRMVPKIATRSTVGGQSGRMTFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSAI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSSLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSGTCMASIANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 36 - ABU99029 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASLGVS SACPTLGASSFFANVVWLITTGSTYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTMVPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSAI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSSLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSGTCMASIANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 37 - AEH59179 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVS SACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SIGTSTLNQRLAPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESIRNGTYSYPQYSDEARLKREEISG

SEQ ID NO: 38 - AEH59179 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPTNGLCYPGSFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTLGSPSFFANVVWLIKTGSTYPTITKSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SIGTSTLNQRLAPKIATRSTVGGQSGRMTFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSAI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQRESRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSGTCMASIANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 39 - AEH59179 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPTTGLCYPGSFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTLGSPSFFANVVWLITTGSTYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SIGTSTLTQTLAPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSAI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQRESRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLARRIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSGTCMASIANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 40 - AAT73266 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVS SACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 41 - AAT73266 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPVNGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSSHEASLGVS SACPTTGTSSFFANVVWLIKTGSTYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPRIATRSTVGGQSGRMEFFWTILTPGDAITFTSNGNFIAPEYAYKIVKTGSSTI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 42 - AAT73266 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPVTGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSSHTASLGVS SACPTTGTSSFFANVVWLITTGSTYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPAIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLERRIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAARLAAAAISG

SEQ ID NO: 43 - BAG80800 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVS SACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLIPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQREKRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 44 - BAG80800 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPVNGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSSHEASLGVS SACPTTGTSSFFANVVWLIKTGSTYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLIPRIATRSTVGGQSGRMEFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSTI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQREKRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 45 - BAG80800 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPVTGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSSHTASLGVS SACPTTGTSSFFANVVWLITTGSTYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLIPAIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQREKRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASI IATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO:46 - AB033748 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFSNVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVS SACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQIERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIINKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 47 - AB033748 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEASSVPAWSYIVETAGPVNGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSSHEASLGVS SACPTTGTSSFFANVVWLIKTGSTYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPRIATRSTVGGQSGRMEFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSTI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQIERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 48 - AB033748 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTASSVPAWSYIVETAGPVTGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSSHTASLGVS SACPTTGTSSFFANVVWLITTGSTYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPAIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQIERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASI IATMS AAAAV GAEFSGLARRIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 49 - AAT73277 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVS SACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVMWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 50 - AAT73277 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPVNGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSSHEASLGVS SACPTTGTSSFFANVVWLIKTGSTYPTITRSYTNTNTEDLLVMWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPRIATRSTVGGQSGRMEFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSTI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 51 - AAT73277 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPVTGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSSHTASLGVS SACPTTGTSSFFANVVWLITTGSTYPTITTSTTNTGTTTLLVMWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPAIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQRERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 52 - ACB70548 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPVNDLCYPGVFNDYEELKHLLSRINHFEKIQIIPKSSWPSHEASLGVS AACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVIWGIHHPNDAAEQTKLYQNPTTYI SVGTSTLNQRLTPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQREGRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 53 - ACB70548 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSEAISVPAWSYIVETAGPVNGLCYPGVFGGYAELKHLLSRIGTFETITIIPTSSWPSHEASLGVS AACPTTGTSSFFANVVWLIKTGSTYPTITRSYTNTNTEDLLVIWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLTPRIATRSTVGGQSGRMEFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSTI MTSALAYGTCTTTCQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQREGRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 54 - ACB70548 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILAGCSVAGWLLGNPM CSTAISVPAWSYIVETAGPVTGLCYPGVFGGAAELAHLLSAIGTFTTITIIPTSSWPSHTASLGVS AACPTTGTSSFFANVVWLITTGSTYPTITTSTTNTGTTTLLVIWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLTPAIATRSTVGGTSGRMEFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPMGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQREGRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 55 - AEI26176 - HAwt

DHICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWLLGNPM CDEFTNVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWSDHEASLGVS SACPYQGNSSFFRNVVWLIKKNNAYPTIKKSYNNTNREDLLVLWGIHHPNDEAEQTRLYQNPTTYI SIGTSTLNQRLVPRIATRSKVNGQSGRIDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSEVEYGNCNTRCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRERRRRKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNGQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHKCDDECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 56 - AEI26176 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGVTPLILTGCSVAGWLLGNPM CSEATSVPAWSYIVETAGPATGLCYPGTFGGYAELKHLLSAIGTFETITIIPAASWSGHEASLGVS SACPTTGGSSFFANVVWLIKTGGAYPTITKSYTNTNAEDLLVLWGIHHPNGAAEQTALYQSPTTYI SIGTSTLNQRLVPRIATRSTVGGQSGRITFFWTILTPGDAITFTSGGNFIAPEYAYKIVKTGSSTI MTSAVAYGTCTTACQTPMGAISSSMPFHNIHPLTIGTCPKYVTSGTLVLATGLRNSPQRERRRRKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNGTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 57 - AEI26176 - HAmut2

STICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILTGCSVAGWLLGNPM CSTATSVPAWSYIVETAGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPAASWSGHTASLGVS SACPTTGGSSFFANVVWLIKTGGAYPTITTSTTNTGATTLLVLWGIHHPGGAAAQTALYTSPTTAI SIGTSTLTQTLVPAIATRSTVGGTSGRITFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSAVATGTCTTACQTPMGAISSSMPFHSITPLTIGTCPKTVTSGTLVLATGLRNSPQRERRRRKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNGTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 58 - ABW73807 - HAwt

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGMKPLILKDCSVAGWLLGNPM CDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVS SACPYQGRSSFFRNVVWLIKKNDAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYI SVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTI MKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKE LGNGCFEFYHRCDNECMESVRNGTYDYPQYSEESRLKREEISG

SEQ ID NO: 59 - ABW73807 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTHSGTLCDLGGMTPLILTGCSVAGWLLGNPM CSEALSVPAWSYIVETIGPATGLCYPGTFGGYAELKHLLSRIGTFETITIIPTSSWSGHTASSGVS SACPTTGASSFFANVVWLIKTGGAYPTITRSYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SVGTSTLNQRLVPKIATRSTVGGQSGRMTFFWTILTPGDAITFTSGGNFIAPENAYKIVKTGSSTI MTSALAYGTCTTTCQTPIGAISSSMPFHNIHPLTIGTCPKYVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAV GAEFSGLERRIANLNAKMEAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRGNATT LGNGCFTFYHACSAACMASVANGTYSYPAYSAASRLAAAAISG

SEQ ID NO: 60 - ABW73807 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGMTPLILTGCSVAGWLLGNPM CSTALSVPAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTSSWSGHTASSGVS SACPTTGASSFFANVVWLIKTGGAYPTITTSTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSALATGTCTTTCQTPIGAISSSMPFHSITPLTIGTCPKTVTSGALVLATGLRNSPQGERRRKKR GLFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAV GAEFSGLAARIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATT LGNGCFTFTHACSAACMASVANGTYSAPAASAASALAAAAISG

SEQ ID NO: 61 - AEJ90156 - HAwt

DHICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWLLGNPL CDEFINVPEWSYIVEKAKPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWSEHEASLGVS AACSYQGNSSFFRNVVWLIKKDNAYPTIKKGYNNTNQEDLLVLWGIHHPNDEAEQTRLYQNPTTYI SIGTSTLNQRLVPKIATRSKINGQSGRIDFFWTILKPNDAIHFESNGNFIAPEYAYKIVKKGDSTI MKSEVEYGNCNTRCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRERRRKRG LFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVG REFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLKDNAKEL GNGCFEFYHKCNNECMESVRNGTYDYPQYSEEARLKREEISG

SEQ ID NO: 62 - AEJ90156 - HAmut

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILTGCSVAGWLLGNPL CSEAISVPAWSYIVETATPANGLCYPGTFGGYAELKHLLSAIGTFETITIIPTGSWSTHEASLGVS AACSTTGGSSFFANVVWLIKTGGAYPTITKGYTNTNTEDLLVLWGIHHPNGAAEQTALYQSPTTYI SIGTSTLNQRLVPKIATRSTIGGQSGRITFFWTILTPGDAITFESGGNFIAPEYAYKIVKTGSSTI MTSAVAYGTCTTACQTPIGAISSSMPFHNIHPLTIGTCPKYVTSGTLVLATGLRNSPQRERRRKRG LFGAIAGFIEGGWQGMVGGWYGYTTSNTTGSGAAADKESTQKAIDGVTNKVASIIAKMSTAAAAVG AEFSGLERRIANLNAKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLKGNATTL GNGCFTFYHTCSAACMASVANGTYSYPAYSAAARLAAAAISG

SEQ ID NO: 63 - AEJ90156 - HAmut2

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGVTPLILTGCSVAGWLLGNPL CSTAISVPAWSYIVETATPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTGSWSTHTASLGVS AACSTTGGSSFFANVVWLIKTGGAYPTITTGTTNTGTTTLLVLWGIHHPGGAAAQTALYTSPTTAI SIGTSTLTQTLVPTIATRSTIGGTSGRITFTWTILTPGDAITFTSGGNFIAPETAYKIVKTGSSTI MTSAVATGTCTTACQTPIGAISSSMPFHSITPLTIGTCPKTVTSGTLVLATGLRNSPQRERRRKRG LFGAIAGFIEGGWQGMVGGVYGTTTSNTTGSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAVG AEFSGLARRIAALNAKMAAGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALTGNATTL GNGCFTFTHTCSAACMASVANGTYSAPAASAAAALAAAAISG

SEQ ID NO: 64 - ABW73807 mut2 wrt Arg326

DTICIGYHANNSTEQVDTIMEKNVTVTTATTILETTTSGTLCDLGGMTPLILTGCSVAGW LLGNPMCSTALSVPAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTS SWSGHTASSGVSSACPTTGASSFFANVVWLIKTGGAYPTITTSTTNTGTTTLLVLWGIHH PGGAAAQTALYTSPTTAISVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTPGDAIT FTSGGNFIAPETAYKIVKTGSSTIMTSALATGTCTTTCQTPIGAISSSMPFHSITPLTIG TCPKTVTSGALVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVGGVYGTTTSNTT GSGAAADKESTQKAIDGVTNKVASIIATMSTAAAAVGAEFSGLAARIAALNAKMAAGFLD VWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLALAGNATTLGNGCFTFTHACSAACMA SVANGTYSAPAASAASALAAAAISG

SEQ ID NO: 65 - ABW73807 target-less mut2 wrt Arg326

DTICIGYTASGSTATVSTIMETGVTVTTATTILETTTSGTLCDLGGMTPLILTGCSVAGW LLGNPMCSTALSVPAWSYIVETIGPATGLCYPGTFGGAAELAHLLSAIGTFTTITIIPTS SWSGHTASSGVSSACPTTGASSFFANVVWLIKTGGAYPTITTSTTNTGTTTLLVLWGIHH PGGAAAQTALYTSPTTAISVGTSTLTQTLVPTIATRSTVGGTSGRMTFTWTILTPGDAIT FTSGGNFIAPETAYKIVKTGSSTIMTSALATGTCTTTCQTPIGAISSSMPFHSITPLTIG TCPKTVTSGALVLATGLRNSPTGTAAATTAGLFGAIAGFIAGGATGMVGGVYGTTTSNTT GSGAAASTASTAAAIAGVTAKVASIIATMSTAAAAVGAEFSGLAARIAALNAKMAAGFLD VWTYNAELLVLMENERTLDFHDSNVKALYDAVRLALAGNATTLGGGCFTFTHACSAACMA SVAAGTYSAPAASAASALAAAAISG

Claims

WHAT IS CLAIMED IS:

1. An isolated avian influenza virus hemaglutinin (HA) polypeptide which comprises a native cleavage site corresponding to about amino acids 316 to about 345 of HA and further comprises at least one mutation as specified in Table 1.

2. The polypeptide of claim 1, wherein the polypeptide comprises a native influenza HA amino acid sequence at amino acids corresponding to about 6 to about 18.

3. The polypeptide of claims 1 or 2, wherein the polypeptide comprises a native influenza HA amino acid sequence at amino acids corresponding to about 20 to about about 28.

4. The polypeptide of any of claims 1-3, wherein the polypeptide comprises a native influenza HA amino acid sequence at amino acids corresponding to about 367 to about 377.

5. An isolated avian influenza virus HA polypeptide which comprises a native cleavage site corresponding to about amino acids 6 to about 18, about 20 to about 28, about 316 to about 345, and about 367 to about 377, wherein the polypeptide further comprises at least one mutation as specified in Table 1.

6. An isolated avian influenza HA polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to a mutated polypeptide shown in any of Tables 6-26.

7. An isolated avian influenza HA polypeptide, wherein the polyeptide comprises an amino acid sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 64, and 65.

8. The polypeptide of claim 7, comprising an amino acid sequence at least 90% identical to a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 64, and 65.

9. The polypeptide of claim 8, comprising an amino acid sequence at least 95% identical to a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 64, and 65.

10. The polypeptide of claim 9, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 64, and 65.

11. An isolated avian HA polypeptide fragment encompassing at least 100, at least 200, at least 300, at least 400, or at least 500 amino acids of the polypeptide of any of claims 1-10.

12. An isolated polynucleotide encoding the polypeptide of any of claims 1-11.

13. A vector comprising the isolated polynucleotide of claim 12.

14. The vector of claim 13, which is a recombinant influenza virus.

1 . A host cell comprising the vector of claim 13.

16. A method of producing an avian influenza HA polypeptide comprising culturing the host cell of claim 15 under conditions that result in expression of the polypeptide.

17. An avian influenza vaccine composition comprising the polypeptide, polynucleotide, or vector of any of claims 1-14.

18. The vaccine composition of claim 17, further comprising an adjuvant.

19. A method of vaccinating a subject susceptible to avian influenza infection comprising administering an effective amount of the polypeptide, polynucleotide, vector, or vaccine composition of any of claims 1-14, 17 or 18, and wherein said subject is an avian species.

20. The method of claim 19, wherein said avian is a chicken, turkey, ostrich, pigeon, game hen, squab, guinea fowl, pheasant, quail, duck, goose, or emu.

21. The method of claim 20, wherein said avian is a chicken.

22. The method of any of claims 19-21, wherein the polypeptide, polynucleotide, vector, or vaccine composition is administered via drinking water or spraying.

23. The method of any of claims 19-22, wherein the dose is within the range of about 0.25 mL to 2.0 mL per avian member.

24. The method of any of claims 19-23 wherein said vaccine is administered in no more than one dose.

25. The method of any of claims 19-24, wherein the method comprises a prime -boost administration regime.

26. The method of any of claims 19-25, wherein the method is therapeutic.

27. The method of any of claims 19-25, wherein the method is prophylactic.

28. A method of treating avian influenza infection comprising administering an effective amount of the polypeptide, polynucleotide, vector, or vaccine composition of any of claims 1-14, 17 or 18 to a subject in need thereof.