(en)Peptides and polypeptides that elicit immunogenic responses in a mammal; especially neutralizing antibodies, against human and avian influenza strains H1N1, H3N2, H5N1 and H7N7 are disclosed Immunogenic compositions including these peptides, and polypeptides are also provided. Compositions including these peptides and polypeptides with or without adjuvants are disclosed. Nucleic acids and expression cassettes encoding these peptides and polypeptides are also disclosed. Methods of inhibiting infection by influenza, with or without cell entry, are also disclosed using these peptides and polypeptides.

1.ApplicationNumber: US-66405208-A
1.PublishNumber: US-2010285982-A1
2.Date Publish: 20101111
3.Inventor: GOLDING HANA
KHURANA SURENDER

4.Inventor Harmonized: GOLDING HANA(US)
KHURANA SURENDER(US)

5.Country: US
6.Claims:
(en)Peptides and polypeptides that elicit immunogenic responses in a mammal; especially neutralizing antibodies, against human and avian influenza strains H1N1, H3N2, H5N1 and H7N7 are disclosed Immunogenic compositions including these peptides, and polypeptides are also provided. Compositions including these peptides and polypeptides with or without adjuvants are disclosed. Nucleic acids and expression cassettes encoding these peptides and polypeptides are also disclosed. Methods of inhibiting infection by influenza, with or without cell entry, are also disclosed using these peptides and polypeptides.

7.Description:
(en)This application is being filed on 13 Jun. 2008, as a PCT International Patent application in the name of The Government of the United States of America as represented by the Secretary, Department of Health and Human Services, a U.S. national corporation, applicant for the designation of all countries except the US, and Hana Golding, a citizen of the U.S., and Surender Khurana, a citizen of India, applicants for the designation of the US only, and claims priority to U.S. Provisional Patent Application Ser. Nos. 60/929,119, filed Jun. 13, 2007, 61/014,587, filed Dec. 18, 2007, and 61/057,514, filed May 30, 2008.


GOVERNMENT FUNDING
The invention described herein was developed with the support of the Department of Health and Human Services. The United States Government has certain rights in the invention.


FIELD OF THE INVENTION
This invention pertains to the general field of immunology and further to the field of viral immunology and specifically in the field of vaccines, therapeutics and diagnostics for influenza.
BACKGROUND OF THE INVENTION
Influenza is a contagious acute respiratory disease caused by infection of the upper respiratory and gastrointestinal tract by influenza virus. The viral genome is made up of several negative sense single stranded RNA molecules. Several proteins are encoded by the viral genome. NA is a viral surface glycoprotein that cleaves terminal sialic acid residues from carbohydrate moieties on the surfaces of infected cells, promoting the release of progeny viruses. HA is one of the major viral surface glycoproteins and involved in the binding of the virus to sialic acids on the surface of susceptible cells (Uiprasertkul M, et al. Emerg. Infect. Dis. 11, 1036-1041 (2005)). The M2 protein is an ion channel protein. The HA, NA, and M2 protein are present in the viral envelope which is derived from the host cell plasma membrane. A ribonucleoprotein complex comprises an RNA segment associated with nucleoprotein (NP) and three polymerases, PA, PB1, and PB2. The M1 protein is associated with both ribonucleoprotien and the envelope.
Annual epidemics of influenza occur when the antigenic properties of the viral surface protein hemagglutinin (HA) and neuraminidase (NA) are altered. The mechanism of altered antigenicity is twofold: antigenic shift, caused by genetic rearrangement between human and animal viruses after double infection of host cells, which can cause a pandemic; and antigenic drift, caused by small changes in the HA and NA proteins on the virus surface, which can cause influenza epidemics. The emergence of variant virus strains by these two mechanisms is the cause of influenza epidemics.
It is therefore desirable to develop new vaccine candidates likely to generate heterotypic cross-protection and for differential diagnostics for the exposure to avian and seasonal influenza in the face of seasonal vaccinated generated immunity.
SUMMARY OF THE INVENTION
The disclosure provides one or more polypeptides, polynucleotides, and assays useful to generate an immune response to influenza virus, prepare antibodies to influenza virus, and to conduct serodiagnosis or differential diagnosis of influenza virus. The disclosure also provides genome flu phage display libraries useful for epitope mapping and in sero or differential diagnosis. The phage display libraries are utilized to identify portions of influenza polypeptides that bind to antibodies from an infected and/or vaccinated individual. The disclosure provides kits and diagnostic assays
In some embodiments, the disclosure provides one or more isolated and purified influenza polypeptides from an influenza virus that specifically bind to an antibody from a subject infected and/or vaccinated with the influenza virus selected from the group consisting of: a) hemagglutinin (HA), b) neuraminidase (NA), c) basic polymerase 2 (PB2), d) basic polymerase 1(PB1), e) basic polymerase 1 frame 2(PB 1-F2), f) acidic polymerase PA, g) nucleoprotein (NP), h) matrix protein 1 (M1), i) matrix protein 2 (M2), j) Non structural protein NS1, k) Non structural protein NS2, and fragments thereof, wherein the polypeptides exclude the corresponding full length native polypeptides. In some embodiment, the polypeptides are from an influenza virus is selected from the group consisting of H5N1, H3N2, H1N1, and H7N7. In specific embodiments, the polypeptides include one or more of the polypeptides having a sequence of SEQ ID NO:1-123. These polypeptides may form a part of a fusion protein and may be coupled to a solid substrate for use in diagnostic assays. These polypeptides are useful in immunogenic compositions to generate antibodies useful therapeutically, or to provide a protective immune response.
The disclosure also includes polynucleotides encoding the polypeptides and vectors comprising the polynucleotides. In some embodiments, the vector is a phage display vector and a library comprising a plurality of fusion proteins including one or more sequences from an influenza gene segment is formed. In other embodiments, one or more of the polynucleotides are utilized in an immunogenic compositions in order to generate antibodies and/or a protective immune response.
The disclosure also includes methods of using the polypeptides and/or polynucleotides to treat influenza virus infection and to immunize animals. Methods also include assays useful for surveillance of out breaks of infection.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a flow diagram of a process for constructing a gene fragment phage display library from influenza virus genes.
FIG. 2 illustrates a representative gel electrophoresis of genome fragments generated by DNAase digestion of influenza virus genes
FIG. 3 illustrates the sequence diversity of inserts in complete H5N1 gene fragment phage display library.
FIG. 4 illustrates a flow diagram for affinity panning the phage display library.
FIG. 5 illustrates an exemplary B-cell epitope map of hemagglutinin (HA) from H3N2 expressed from a gene fragment phage display library and screened against polyclonal sera collected before vaccination and after the influenza season from convalescent patients in the placebo arm of the study done in 2003-2004 season.
FIG. 6 (a) illustrates epitope sequences recognized by H5N1 neutralizing monoclonal antibody FLA5.10 identified using influenza complete genome-fragment phage display library (GFPDL). Epitope sequences are boxed on the aligned sequence of A/Vietnam/1203/2004 & A/Indonesia/5/05 of the haemagglutinin (HA). Amino acid number 1 corresponds to H3 (A/California/7/2004) amino acid −10. HA sequences recognized by FLA5.10 include amino acids 9-241; (b) illustrates epitope sequences recognized by H5N1 neutralizing monoclonal antibody FLD 21.140 identified using influenza complete genome-fragment phage display library (GFPDL). Epitope sequences are boxed on the aligned sequence of A/Vietnam/1203/2004 & A/Indonesia/5/05 of the haemagglutinin (HA). Amino acid number 1 corresponds to H3 (A/California/7/2004) amino acid −10. HA sequences recognized by FLD 21.140 include amino acids 50-338; and (c) illustrates epitope sequences recognized by H5N1 neutralizing monoclonal antibody FLA 3.14 identified using influenza complete genome-fragment phage display library (GFPDL). Epitope sequences are boxed on the aligned sequence of A/Vietnam/1203/2004 & A/Indonesia/5/05 of the haemagglutinin (HA). Amino acid number 1 corresponds to H3 (A/California/7/2004) amino acid −10. HA sequences recognized by FLA 3.14 include amino acids 47-338.The sequence shown in this figure has accession no. AAW80717. The contact residues shown in each of the figures as encircled or boxed residues were identified using random peptide phage display library (RPL) aligned to HA1 sequence.
FIG. 7 (a) illustrates the binding specificity of 5.10-101(SEQ ID NO:41) with FLA5.10 compared to other Human H5 monoclonal antibodies; and (b) illustrates loss of binding of 5.10-101 peptide to FLA5.10 upon mutation of leucine to Alanine.
FIG. 8 Binding of human monoclonal antibodies to purified HA segment. Serial dilution of MAbs were run on a chip coated with E.coli expressed and purified HA 9-241 (FLA5.10 epitope) to determine the affinity constants using ProtOn system (BioRad) (a) illustrates the binding affinity of H5N1 neutralizing monoclonal antibody FLA 5.10 to recombinant purified protein; (b) illustrates the binding affinity of H5N1 neutralizing monoclonal antibodies FLD 21.140 to recombinant purified protein; and (c) illustrates the binding affinity of H5N1 neutralizing monoclonal antibodies FLD 21.140, FLA 5.10 and FLA 3.14 to recombinant purified protein.
FIG. 9 (a) illustrates the location of the epitope sequences recognized by FLA 5.10 on a crystal structure model of hemagglutinin; (b) illustrates the location of the epitope sequences recognized by FLD 21.140 on a crystal structure model of hemagglutinin; (c) illustrates the location of the epitope sequences recognized by FLA 3.14 on a crystal structure model of hemagglutinin; The residues identified using RPL are shown on the structure of HA (PDB Id-1 JSM) for FLA5.10, FLD21.140 and FLA3.14 respectively. In-vivo challenge studies with suboptimal amounts of FLA5.10 identified two mutated residues that are encircled (in a), and for FLA3.14 (in c) and are represented on the HA structure (in a & c)
FIG. 10 Elucidation of epitope profile recognized by antibodies in individuals that survived H5N1 infections in Vietnam:(a) Reactivity of pooled sera from five H5N1 survivors with recombinant H5 HA (Vietnam 1203) before (closed circles) and after (triangles) adsorption on H5GFPDL. (b) Elucidation of epitope profile in HA and NA recognized by antibodies in individuals that survived H5N1 infections in Vietnam. Alignment of the unique peptide sequences recognized by the pooled sera from H5N1-infected individuals identified using HA and NA GFPDLs. The predicted Influenza encoded proteins are shown and numbered according to the intact complete proteome ( FIG. 14 ). Arrows indicate that inserts are in right orientation with the coding sequence. Each bar represents a unique peptide sequence. The peptide sequences represented in filled bars were either expressed and purified from E.coli or chemically synthesized. These peptides were selected based on the frequency of the phage clones displaying these peptide sequences following affinity selection on H5N1 exposed sera. Their numbers correspond to the peptide IDs in the ELISA assays.
FIG. 11 : Antigenic clusters in structure of HA and NA recognized by antibodies from H5N1/Vietnam-infecetd individuals (A) antigenic clusters in HA identified in FIG. 10 are shown as surface exposed residues on one HA monomer within the HA trimer structure (PDB Id-1JSM). (B) The Neuraminidase conformational epitope (NA-3676-3854) is shown on the tetrameric NA structure (PDB Id-2HTY) with the bound sialic acid.
FIG. 12 : Antibody epitopes in H5N1 internal proteins (HA and NA excluded) recognized by pooled sera from H5N1 (Vietnam) infected individuals (a) Schematic alignment of the unique peptide sequences in various Influenza proteins identified using GFPDL off all internal genes. The predicted influenza encoded proteins are numbered according to the intact complete proteome ( FIG. 14 ). Arrows indicate that inserts are in the right orientation. Each bar represents a unique peptide sequence. The filled rectangles represent the peptide sequences were chemically synthesized and tested with individual H5N1 survivor sera.
FIG. 13 illustrates B cell epitope profile following pre- & post-vaccination with H5N1 subunit vaccine with no adjuvant, alum adjuvant or MF59 adjuvant. Samples were obtained from the clinical trial study described in Bernstein et al, JID; 2008:197; 1-9.
FIG. 14A-C provides the complete proteome of H5N1-A/VIETNAM/1203/2004. Amino acid numbering of H5N1 polypeptides in this patent application is based on the numbering shown in this figure. (SEQ ID NO:124)
FIG. 15A-E provides the complete proteome of H3N2-A/CALIFORNIA/7/2004. Amino acid numbering of H3N2 polypeptides in this patent application is based on the numbering shown in this figure. (SEQ ID NO:136)



DETAILED DESCRIPTION
Among the eight genes encoded by influenza virus (HA, NA, M1, M2, NP, NS, PA, PB1 and PB2), immunity against hemagglutinin (HA) and neuraminidase (NA) play a central role in protection against influenza. Thus, in developing an effective influenza vaccine for use during a pandemic, HA and NA would be the target antigens along with other antigens, for example, NP.
Highly pathogenic avian H5N1 influenza A virus has caused influenza outbreaks in poultry and migratory birds in Asia, Europe and Africa. A cluster of cases of deadly H5N1 influenza in humans occurred in 2006, in which the WHO deemed that human-to-human transmission was the most probable cause of viral spread. The ability to be transmitted from human to human represents the final barrier to a new pandemic of this deadly avian strain of H5N1 influenza virus, and thus, there remains a need for effective treatments and surveillance assays should such a pandemic arise. Currently licensed human vaccines are strain specific and do not protect against heterotypic influenza viruses. This is problematic, as influenza A (H5N1) continues to evolve into antigenically distinct clades.
The disclosure provides a method of epitope mapping of Influenza virus A proteome using sera from subjects that have been infected and/or vaccinated. The epitope mapping identifies epitopes on influenza virus A proteins that are associated with a protective immune response. The identification of these epitopes is useful in the preparation of immunogenic compositions and antagonists of influenza virus infection. The identification of unique epitopes in infected versus vaccinated, and infected with H5N1 versus H3N2 can be used in assays for surveillance of influenza outbreaks, to distinguish between human and avian influenza strains, to diagnose new infection in previously vaccinated individuals, and rapid analysis of immune response to candidate vaccines in order to predict the likelihood of protection against H5N1 infection.
Definitions
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g., Singleton P and Sainsbury D., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons, Chichester, N.Y. 2001, and Fields Virology 4th ed., Knipe D. M. and Howley P. M. eds, Lippincott Williams & Wilkins, Philadelphia 2001.
The terms “a”, “an” and “the” as used herein are defined to mean “one or more” and include the plural unless the context is inappropriate.
By “'isolated” is meant peptide or protein free from at least some of the components with which it naturally occurs.
“Peptides”, “polypeptides”, and “proteins” are used interchangeably and are defined herein as chains of amino acids (typically L-amino acids) in which the carbonyl group of one amino acid is linked to the amino group of a second amino acid by an amide linkage. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group.
Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminal and increasing in the direction of the carboxy terminal of the peptide. Thus, when one amino acid is said to “follow” another, that amino acid is positioned closer to the carboxy terminal of the peptide than the “preceding” amino acid.
The term “residue” is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a peptide by an amide bond or an amide bond mimetic. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e. amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.
Peptide and protein sequences defined herein are represented by one-letter symbols for amino acid residues as follows:
A alanine L leucine
R arginine K lysine
N asparagine M methionine
D aspartic acid F phenylalanine
C cysteine P proline
Q glutamine S serine
E glutamic acid T threonine
G glycine W tryptophan
H histidine Y tyrosine
I isoleucine V valine
The term “proteome” as used herein refers to all of the proteins expressed by a genome. An exemplary proteome of H5N1 (strain A/VIETNAM/1203/2004) is shown in FIG. 14 . An exemplary proteome of H3N2 (strain A/CALIFORNIA/7/2004) is shown in FIG. 15 .
“Antigen” refers to a molecule which can induce an immune response in an animal, preferably a mammal and most preferably a human. It induces the formation of an antibody. The term includes immunogens.
“Epitope” or “determinant” refers to the antibody binding site on an antigen.
“Antibody” refers to a molecule produced by animals in response to antigen which has the particular property of combining specifically with the antigen which induced its formation.
“Neutralizing antibody” refers to an antibody that blocks viral infection of a cell.
“Neutralizing antigenic epitope” or “neutralizing epitope” refers to an epitope that elicits a neutralizing antibody.
The phrases “specifically binds to a peptide” or “specifically immunoreactive with”, when referring to an antibody, refers to a binding reaction which is determinative of the presence of the peptide, or an antibody to the peptide, in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular peptide and do not bind in a significant amount to other proteins present in the sample. Specific binding to a peptide under such conditions requires an antibody that is selected for its specificity for a particular protein or a particular epitope. In some embodiments, antibodies bind to a protein of one subtype or clade of influenza and not another, for example, antibodies bind to hemagglutinin from H5N1 subtype of influenza and not to H3N2. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
For example, solution or solid phase immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
The term as “clade” as used herein refers to a taxonomic group (such as one of organisms) comprising a single common ancestor and all the descendants of that ancestor.
“Conservative variations” or “conservative modified variations” of a particular sequence refers to amino acids encoded by nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given peptide. Such nucleic acid variations are silent variations, which are one species of conservatively modified variations. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each silent variation of a nucleic acid which encodes a peptide is implicit in any described amino acid sequence. Furthermore, one of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Two polypeptides are said to be “identical” if the sequence of amino acid residues in the two sequences is the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman 1981 Adv Appl Math 2:482-489, by the homology alignment algorithm of Needleman and Wunsch 1970 J Mol Biol 48:443-453, by the search for similarity method of Pearson and Lipman 1988 Proc Natl Acad Sci USA 85:2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
The term “substantial identity” means that a polypeptide comprises a sequence that has at least 55%, 60%, 65%, 70%, 75%, 80%, or 85% sequence identity, preferably 90%, more preferably 95% or more, compared to a reference sequence. Another indication that polypeptide sequences are substantially identical is if one peptide is immunologically reactive with antibodies raised against the disclosed peptide. Thus, the peptides of the invention include peptides immunologically reactive with antibodies raised against the disclosed immunogenic peptides.
The term “phage coat protein” means a protein, at least a portion of which is present on the surface of a phage virus particle. From a functional perspective, a phage coat protein is any protein which associates with a virus particle during the viral assembly process in a host cell, and remains associated with the assembled virus until it infects another host cell. The phage coat protein may be the major coat protein or may be a minor coat protein. A “major” phage coat protein is generally a coat protein which is present in the viral coat at preferably at least about 5, more preferably at least about 7, even more preferably at least about 10 copies of the protein or more. A major phage coat protein may be present in tens, hundreds or even thousands of copies per virion. An example of a major coat protein is the pVIII protein of filamentous phage.
A “fusion protein” and a “fusion polypeptide” refers to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property. The property may be a biological property, such as activity in vitro or in vivo. The property may also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each W other. Preferably, the two portions of the polypeptide are obtained from heterologous or different polypeptides.
“Phage display” is a technique by which variant polypeptides are displayed as fusion proteins to at least a portion of coat protein on the surface of phage, e.g., filamentous phage, particles. A utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently sorted for those sequences that bind to a target antigen with high affinity. Display of peptide and protein libraries on phage has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins through fusions to either gene III or gene VIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol., 3:355-362 (1992), and references cited therein. In monovalent phage display, a protein or peptide library is fused to a gene III or a portion thereof, and expressed at low levels in the presence of wild type gene III protein so that phage particles display 1-3 copies of the fusion proteins. Avidity effects are reduced relative to polyvalent phage so that sorting is on the basis of intrinsic ligand affinity, and phagemid vectors are used, which simplify DNA manipulations. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).
A “phagemid” is a plasmid vector having a bacterial origin of replication, e.g., Co1E1, and a copy of an intergenic region of a bacteriophage. The phagemid may be used on any known bacteriophage, including filamentous bacteriophage and lambdoid bacteriophage. The plasmid will also generally contain a selectable marker for antibiotic resistance. Segments of DNA cloned into these vectors can be propagated as plasmids. When cells harboring these vectors are provided with all genes necessary for the production of phage particles, the mode of replication of the plasmid changes to rolling circle replication to generate copies of one strand of the plasmid DNA and package phage particles. The phagemid may form infectious phage particles. This term includes phagemids which contain a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene as a gene fusion such that the heterologous polypeptide is displayed on the surface of the phage particle.
The term “phage vector” means a double stranded replicative form of a bacteriophage containing a heterologous gene and capable of replication. The phage vector has a phage origin of replication allowing phage replication and phage particle formation. The phage is preferably a filamentous bacteriophage, such as an M13, f1, fd, Pf3 phage or a derivative thereof, or a lambdoid phage, such as lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative thereof.
The term “host cell” means a cell that contains a heterologous nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, avian or mammalian cells, including human cells. Exemplary host cells can include, but are not limited to Vero (African green monkey kidney) cells, BHK (baby hamster kidney) cells, primary chick kidney (PCK) cells, MDCK (Madin-Darby Canine Kidney), 293 cells, and COS cells.
An “immunogenic effective amount” of an influenza polypeptide or polynucleotide refers to an amount of a polypeptide or polynucleotide that is capable of inducing an immune response in an animal. The immune response may be determined by measuring a T or B cell response. Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and/or serum antibodies, by plaque neutralization, complement fixation, enzyme-linked immunosorbent, microneutralization assay, or assays for T cell function. Typically, the induction of an immune response is indicated by the detection of antibodies specific for an influenza polypeptide.
As used herein, the term “immunogenic fragment thereof' refers to a fragment of an influenza polypeptide that is of a sufficient size to elicit an immune response in an animal. Typically, immunogenic fragments are at least 8 amino acids long and may include up to the full-length polypeptide. In some embodiments, an immunogenic fragment is about 9 amino acids, an immunogenic fragment is about 10 amino acids, 15 amino acids, 30 amino acids, or 45 amino acids or longer fragments. The immunogenic fragment is capable of stimulating an antibody or T cell response specific for at least one influenza polypeptide as defined herein. The sequence of immunogenic fragments can be readily predicted using available programs such as Epiredict. The immune response includes both a T and B cell response. In some cases, the immune response is identified by the ability of the fragment to elicit antibodies or to stimulate a T cell response.
A “protective immune response” against influenza virus refers to an immune response exhibited by an animal that is protective against disease when the animal is subsequently exposed to and/or infected with such influenza virus. In some instances, the influenza virus can still cause infection, but the infection is less than serious in non-immune controls. A protective immune response can be characterized by % decrease in morbidity, % increase in survival, a decrease in viral load, an increase in hemagglutinin inhibition titer and/or an increase in neutralization titer. Typically, the protective immune response results in detectable levels of host-engendered serum and secretory antibodies or cytotoxic T-lymphocyte responses that are capable of reacting with antigens from virus of the same strain and/or subgroup and in some cases, also of a different, non-vaccine strain and/or subgroup in vitro and in vivo.
Whole Genome Phage Display Libraries
Vectors
One aspect of the invention includes a replicable expression vector comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a portion of or a segment of influenza viral gene fused to all or a portion of a viral coat protein. Also included is a library of diverse replicable expression vectors comprising a plurality of gene fusions encoding a plurality of different fusion proteins including a plurality of different portions or segments of influenza genes. The vectors can include a variety of components and are preferably constructed to allow for movement of the gene segments between different vectors and/or to provide for display of the fusion proteins in different formats.
Examples of vectors include phage vectors. The phage vector has a phage origin of replication allowing phage replication and phage particle formation. The phage is preferably a filamentous bacteriophage, such as an M13, f1, fd, Pf3 phage or a derivative thereof, or a lambdoid phage, such as lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative thereof.
Examples of viral coat proteins include infectivity protein pIII, major coat protein pVIII, p3, Soc (T4), Hoc (T4), gpD (of bacteriophage lambda), minor bacteriophage coat protein 6 (pVI) (filamentous phage; J Immunol Methods. 1999 Dec. 10; 231(1-2):39-51), variants of the M13 bacteriophage major coat protein (P8) (Protein Sci 2000 April; 9(4):647-54). In some embodiments, influenza gene segments can include from about 30 base pairs up to about 2000 base pairs. When the influenza gene segments or portions thereof include about 300 to 2000 base pairs, the viral coat protein utilized is the pIII protein because this protein can accommodate larger nucleic acid fragments. For gene segments or portions thereof of 300 base pairs or less, pVIII or pVI may also be utilized.
The fusion protein can be displayed on the surface of a phage and suitable phage systems include M13KO7 helper phage, M13R408, M13-VCS, and Phi X 174, pJuFo phage system (J Virol. 2001 August; 75(15):7107-13.v), hyperphage (Nat Biotechnol. 2001 January; 19(1):75-8). The preferred helper phage is M13KO7, and the preferred coat protein is the M13 Phage gene III coat protein. The preferred host is E. coli, and protease deficient strains of E. coli. Vectors, such as the fth1 vector (Nucleic Acids Res. 2001 May 15; 29(10):E50-0) can be useful for the expression of the fusion protein.
Nucleic acid cassettes can be cloned into any suitable vector for expression of a portion of or the entire influenza gene segment. According to methods detailed in the invention, the nucleic acid cassette is cloned into a vector allowing production of a portion of or the entire influenza gene segment fused to all or a portion of a viral coat protein (ie., creating a fusion protein) and displayed on the surface of a particle or cell. While several types of vectors are available, phagemid vectors are the preferred vectors for use herein, as they may be constructed with relative ease, and can be readily amplified. Phagemid vectors generally contain a variety of components including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other necessary components as are known to those of ordinary skill in the art.
The expression vector also can have a secretory signal sequence fused to the DNA encoding each influenza gene segment or portion thereof. This sequence is typically located immediately 5′ to the gene encoding the fusion protein, and will thus be transcribed at the amino terminus of the fusion protein. However, in certain cases, the signal sequence has been demonstrated to be located at positions other than 5′ to the gene encoding the protein to be secreted. This sequence targets the protein to which it is attached across the inner membrane of the bacterial cell. The DNA encoding the signal sequence may be obtained as a restriction endonuclease fragment from any gene encoding a protein that has a signal sequence. Suitable prokaryotic signal sequences may be obtained from genes encoding, for example, gIIIss, pelBss, LamB or OmpF (Wong et al., Gene, 68:1931 (1983), MalE, PhoA and other genes. A preferred prokaryotic signal sequence for practicing this invention is the E. coli heat-stable enterotoxin II (STII) signal sequence and malE.
The vector also typically includes a promoter to drive expression of the fusion protein. Promoters most commonly used in prokaryotic vectors include the gIII promoter, lac Z promoter system, the alkaline phosphatase pho A promoter (Ap), the bacteriophage □ PL promoter (a temperature sensitive promoter), the tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), the tryptophan promoter, and the bacteriophage T7 promoter. While these are the most commonly used promoters, other suitable microbial promoters may be used as well.
The vector can also include other nucleic acid sequences, for example, sequences encoding gD tags, c-Myc epitopes, poly-histidine tags, fluorescence proteins (eg., GFP), or beta-galactosidase protein or glutathione S transferase which can be useful for detection or purification of the fusion protein expressed on the surface of the phage or cell. Nucleic acid sequences encoding, for example, a gD tag, also provide for positive or negative selection of cells or virus expressing the fusion protein. Nucleic acid sequences encoding, for example, a polyhistidine tag, are useful for identifying fusion proteins using immunohistochemistry.
Another useful component of the vectors used to practice this invention is phenotypic selection genes. Typical phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell. By way of illustration, the ampicillin resistance gene (ampr), and the tetracycline resistance gene (tetr) are readily employed for this purpose.
The vector can also include nucleic acid sequences containing unique restriction sites and suppressible stop codons. The unique restriction sites are useful for moving influenza gene segments between different vectors and expression systems. The suppressible stop codons are useful to control the level of expression of the fusion protein and to facilitate purification of soluble influenza gene fragments. For example, an amber stop codon can be read as Gln in a supE host to enable phage display, while in a non-supE host it is read as a stop codon to produce soluble influenza gene fragments without fusion to phage coat proteins.
It is preferable to use vector systems that allow the nucleic acid encoding an influenza gene segment to be easily removed from the vector system and placed into another vector system. For example, appropriate restriction sites can be engineered in a vector system to facilitate the removal of the nucleic acid sequence encoding the influenza gene segment. The restriction sequences are usually chosen to be unique in the vectors to facilitate efficient excision and ligation into new vectors. Influenza gene segments can then be expressed from vectors without extraneous fusion sequences, such as viral coat proteins or other sequence tags.
Introduction of Vectors into Host Cells
Vectors constructed are introduced into a host cell for amplification and/or expression. Vectors can be introduced into host cells using standard transformation methods including electroporation, calcium phosphate precipitation and the like. If the vector is an infectious particle such as a virus, the vector itself provides for entry into the host cell. Transfection of host cells containing a replicable expression vector which encodes the gene fusion and production of phage particles according to standard procedures provides phage particles in which the fusion protein is displayed on the surface of the phage particle.
Selection (Sorting) and Screening for Binders to Targets of Choice
One approach involves constructing a family of variant replicable vectors containing a transcription regulatory element operably linked to a polynucleotide encoding a fusion polypeptide, transforming suitable host cells, culturing the transformed cells to form phage particles which display the fusion polypeptide on the surface of the phage particle, followed by a process that entails selection or sorting by contacting the recombinant phage particles with an antibody that specifically binds an influenza protein so that at least a portion of the population of particles bind to the antibody with the objective to increase and enrich the subsets of the particles which bind from particles relative to particles that do not bind in the process of selection. The selected pool can be amplified by infecting host cells, such as fresh TG1, K91 or XL1-Blue cells, for another round of sorting on the same antibody with different or same stringency.
In some embodiments, the resulting pool of variants are then screened against naïve or synthetic libraries of antibody fragment to identify novel high affinity antibodies. These novel high affinity antibodies can be useful as therapeutic agents as antagonists or agonists, and/or as diagonostic agents. For example, antibodies or antigen binding fragments can be screened for binding to an epitope on a H5N1 clade 1 HA protein and not to H5N1 clade 2 HA. For diagnostic purposes, such antibodies can be used to distinguish whether a subject is infected with clade 1 or clade 2. For therapeutic purposes, an antibody that binds to an epitope on several H5N1 influenza strains or that neutralizes the activity of HA or NA from several different clades would be selected.
Fusion polypeptides can be expressed on the surface of a phage, phagemid particle or a cell and then selected and/or screened for the ability of members of the group of fusion polypeptides to bind a target antibody. The processes of selection for binders to target can also be include sorting on a generic protein such as a tag specific antibody which binds to a tag that is fused to the influenza gene segment.
In some embodiments, a solid support method is employed, the target antibody or polypeptide may be attached to a suitable solid or semi solid matrix which are known in the art such as agarose beads, magnetic beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyalkyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like. Attachment of the target protein to the matrix may be accomplished by methods described in Methods in Enzymology, 44 (1976), or by other means known in the art.
After attachment of the target antibody or polypeptide to the matrix, the immobilized target is contacted with the library expressing the fusion polypeptides under conditions suitable for binding of at least a subset of the phage particle population with the immobilized target antibody or influenza polypeptide frgaments. Normally, the conditions, including pH, ionic strength, temperature and the like will mimic physiological conditions. Bound particles (“binders”) to the immobilized target are separated from those particles that do not bind to the target by washing. Wash conditions can be adjusted to result in removal of all but the high affinity binders. Binders may be dissociated from the immobilized target by a variety of methods. These methods include competitive dissociation using the excess target molecule, altering pH and/or ionic strength, and methods known in the art. Selection of binders typically involves elution from an affinity matrix with a suitable elution material such as acid like 0.1M HCl or excess target molecule. Elution with increasing concentrations of target molecule could elute displayed binding molecules of increasing affinity.
The binders can be isolated and then re-amplified in suitable host cells by infecting the cells with the viral particles that are binders (and helper phage if necessary, e.g. when viral particle is a phagemid particle)and the host cells are cultured under conditions suitable for amplification of the particles that display the desired fusion polypeptide. The phage particles are then collected and the selection process is repeated one or more times until binders of the target antibody or polypeptide are enriched. Any number of rounds of selection or sorting can be utilized. One of the selection or sorting procedures can involve isolating binders that bind to a generic affinity protein such as protein L or an antibody to a polypeptide tag present in a displayed polypeptide such as antibody to the gD protein or polyhistidine.
After binders are identified by binding to the target antibody or polypeptide, the insert sequence is identified using, PCR with suitable primers, and sequenced by typical sequencing method. DNA of the binders can be restriction enzyme digested and then inserted into a vector for protein expression.
Genome of influenza phage display libraries are constructed by isolating cDNAs from each influenza gene segment and partially digesting the cDNAs with DNase. Fragments of 50 to 200 base pairs or 200 to 1000 base pairs are isolated and cloned into a vector containing a nucleic acid encoding a phage coat protein. An exemplary scheme is shown in FIG. 1 . Vectors containing all or portions of influenza gene segments are selected by affinity selection using desired ligand including, without limitation, monoclonal antibodies whether neutralizing or not, vaccinated animal or human sera, post-infection sera. After multiple rounds of affinity selection, clones containing influenza gene fragment inserts are sequenced and may be further selected using post infection and/or post vaccination sera. In some embodiments, the library comprises at least the polynucleotides encoding the sequence of SEQ ID NO:1-31, SEQ ID NO:32-123, or SEQ ID NO:148-176. An exemplary process is shown in FIG. 4 . In embodiments, the libraries contain at least two different sequences from an influenza gene segment and more preferably, at least 3, 4, 5, or any integer up to 100 different sequences from each gene segment.
Epitope Mapping using Whole Genome Phage Display Libraries
Preparation for pandemic influenza threats requires improved cross-reactivity and long-term protection of interpandemic influenza vaccines, the immunogenic peptides provided here can elicit antibody responses based on binding to monoclonal antibodies, sera from vaccinated, infected individuals or convalescent individuals, demonstrating broad cross-protection against interpandemic (seasonal) and pandemic influenza strains. In case of mass vaccination, it would be preferable to rapidly distinguish between exposures to human influenza versus avian influenza, especially in the seasonal influenza vaccinated populations.
A high throughput approach based on the construction of whole viral genome phage display libraries as described herein can identify peptides and polypeptides that specifically bind to antibodies from a subject post infection and/or post vaccination. Separate libraries were created for each influenza strain to express complete sets of protein fragments encoded by human and avian influenza strains H1N1, H3N2, H5N1 and H7N7 or other strains and the peptides are identified by binding of phage clones to the antibodies followed by elution, amplification, and sequencing. The polypeptides encoded by the portions of the influenza gene segments that are selected by binding to the antibodies are identified by sequencing.
Using these libraries, peptides are identified that bind to antibodies that provide broad heterotypic neutralizing activity as well a set of epitopes that are important for serodiagnosis as well as differential diagnosis. The genome flu phage display libraries are incubated with serum samples from a subject vaccinated or infected with an influenza virus of a certain subtype, for example, H3N2 or H5N1 or different clades of H5N1. Using serum samples that are obtained from individuals infected with different strains of influenza with libraries obtained with different subtypes of influenza provides identification of unique as well as shared or overlapping epitopes between different subtypes or clades.
In some embodiments, the epitope mapping includes further mapping using a random dodecamer phage display library and binding to the same antibody or antibodies.
In some embodiments, the peptides listed in the Table 4 and Table 5 below identify highly conserved protective epitopes that are useful in broadly-reactive influenza vaccines. The peptides can be used to generate neutralizing antibodies both in vitro or in vivo that can be used for passive therapy. These peptides would be also useful in studies of viral protein-protein, viral RNA-protein and viral-host protein interactions. The peptides are further applicable to new serological assays for surveillance of pandemic influenza outbreaks. The peptides are further applicable to new serological assays to distinguish between exposure to human and bird influenza strains.
The peptides further provide the means to diagnose true influenza infections in previously vaccinated individuals, rapid analyses of immune sera from pre-clinical and clinical trials of novel influenza vaccines and the ability to map monoclonal and polyclonal antibodies against different influenza gene products.
Polypeptides and Compositions
One aspect of the disclosure provides compositions and methods for priming or enhancing the immune response of an animal to influenza A antigens. The present disclosure provides at least one polypeptide that binds to an antibody from a subject infected with and/or vaccinated with an influenza A virus or subunit thereof and compositions comprising an effective amount of the polypeptide. In some embodiments, an effective amount of the polypeptide is an amount that is effective for treatment of influenza infection. In some embodiments, effectiveness for treatment is determined by a decrease in viral load or a decrease in symptoms. In other embodiments, the effective amount of the polypeptide is effective for inhibition of influenza virus fusion with, or entry into, mammalian cells. In that case, effectiveness of inhibition can be measured by the ability of the peptide to elicit, bind, or stimulate neutralizing antibodies or to elicit or bind hemagglutinin inhibiting antibodies. In yet other embodiments, the effective amount of the polypeptide is effective for eliciting an immune response in a subject.
In embodiments, the disclosure includes one or more isolated and purified influenza polypeptides that specifically bind to an antibody from a subject infected and/or vaccinated with influenza virus selected from the group consisting of: a) hemagglutinin (HA), b) neuraminidase (NA), c) basic polymerase B2 (PB2), d) basic polymerase B1 (PB1), e) basic polymerase frame 2 PB1-F2 (PB1-F2), f) acidic polymerase PA, g) nucleoprotein (NP), h) matrix protein 1 (M1), i) matrix protein 2 (M2), j) non-structural protein 1 (NS1), k) non-structural protein 2 (NS2), and fragments thereof. In some embodiments the polypeptides exclude the corresponding full length native polypeptides. In some embodiments, the influenza virus is selected from the group consisting of H5N1, H3N2, H1N1, and H7N7.
In embodiments, a polypeptide corresponds to the corresponding H5N1 polypeptide having a sequence as represented by the sequence in the proteome as shown in FIG. 14 . For example, the sequence of the proteome provides a reference sequence for each one of the expressed H5N1 polypeptides. This reference sequence provides for amino acid numbering of H5N1 sequences and to determine amino acid numbering of a corresponding H5N1 sequence. Many examples of H5N1 sequences are known and alignments of these sequences for a polypeptide can readily be obtained as shown in Table 6 (alignments of H5N1 HA sequences) and Table 7 (alignments of H5N1 NA sequences). The sequences as are obtained from publicly available data bases such as the influenza virus database at the NCBI and can be aligned with the reference sequence in order to identify a polypeptide corresponding to that of the reference sequence.
In embodiments, a polypeptide corresponds to the corresponding H3N2 polypeptide having a sequence as represented by the sequence in the proteome as shown in FIG. 15 . For example, the sequence of the proteome provides a reference sequence for each one of the expressed H3N2 polypeptides. This reference sequence provides for amino acid numbering of H3N2 sequences and to determine amino acid numbering of a corresponding H3N2 sequence. Many examples of H3N2 sequences are known and alignments of these sequences for a polypeptide can readily be obtained from publicly available data bases such as the influenza virus database at the NCBI and can be aligned with the reference sequence in order to identify a polypeptide corresponding to that of the reference sequence.
Any of the polypeptides from a strain isolated from nature can be utilized in the compositions described herein, and these polypeptides are referred to as naturally occurring, The amino acid sequences that correspond to the polypeptides that are identified by binding to an antibody from a subject infected or vaccinated with influenza can be determined by aligning the sequence to the reference sequence.
In other embodiments, the corresponding polypeptides may be variants of a reference influenza polypeptide. A variant may have substantial identity to the reference sequence. The term “substantial identity” means that a polypeptide comprises a sequence that has at least 55% to 100% identity or any integer included within the range. In embodiments, the polypeptide has at least, 60%, 65%, 70%, 75%, 80%, or 85% sequence identity, preferably 90%, more preferably 95% or more, compared to the reference sequence of the polypeptides as shown in FIG. 14 or FIG. 15 . Another indication that polypeptide sequences are substantially identical is if one peptide is immunologically reactive with antibodies raised against the disclosed peptide. Thus, the peptides of the disclosure include peptides immunologically reactive with antibodies raised against the disclosed immunogenic peptides. In some embodiments, it may be desirable to align the variant with the reference sequence and retain the amino acid sequence that binds to an antibody from a subject infected and/or vaccinated with an influenza virus. In some embodiments, conservative substitutions may be made without affecting the ability of the polypeptide to bind to such antibodies. In a specific embodiment, a variant polypeptide preferably has 90% sequence identity to the reference polypeptide.
Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. In addition, the crystal structure of many of the influenza virus proteins are known and can be accessed at the Protein Data Bank. Modeling of the effect of any amino acids changes on the structure can be determined by using available computer programs.
Functional domains can also be identified in those polypeptides that have homology to known polypeptides. For example, certain positions in the polypeptide show more variability than others. These positions can be identified using sequence alignments and changes made to those amino acid positions showing high variability (e.g. 3 or more different amino acids in that position when a number of sequences are aligned). For example, when full length HA proteins are aligned from several different isolates or strains, amino acid positions corresponding to positions 55, 102,103, 105, 113, 144, 150 and 162 have variant amino acids as shown in the alignment in Table 6.
The sequences of these functional domains can be compared and aligned to other known sequences that may be provided at the Los Alamos website or GenBank, and locations of amino acid positions for substitutions can be identified as those positions that show a high degree of variability in amino acids, i.e., at least 3 different amino acids are found at that position when different sequences are aligned and compared or have a lower percentage of sequence identity i.e., less than 90% sequence identity. When sequences are aligned, the positions that show variability can either have conservative amino acid substitutions or non-conservative amino acid substitutions. If the position has conservative amino acid substitutions, that would indicate that the amino acid substituted at that position should be of the same type as those observed to be at that position in naturally occurring proteins. In some embodiments, amino acid sequences identified to be associated with virulence can be modified or deleted, such as the HA basic residues PQGERRRKKR/GL (SEQ ID NO: ______)
In some embodiments, it may be desirable to exclude variants of the full length sequence. For example, variants of full length sequences can be made using reverse genetic engineering as described in Wood et al, Nat Rev. Microbiology 2:842(2004). In this case, a naturally occurring sequence can be altered to change the virulence of the encoded protein, such as a modified H5N1 HA lacking or mutated PQGERRRKKR/GL (SEQ ID NO: ______) residues. In embodiments, it is preferred that the variants of full length sequences are changed not more than 10%, that is the variants excluded have at least 90% sequence identity to the corresponding polypeptide.
In some embodiments, the H5N1 HA polypeptide corresponds to a H5N1 HA polypeptide having a sequence of amino acids 2335-2902 of FIG. 14 (SEQ ID NO:124) and comprises the amino acid sequence corresponding to the polypeptide starting at any one of amino acids 2339 to 2365 and ending at any one of amino acids 2581 to 2685 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 HA. In a specific embodiment, the isolated and purified HA polypeptide is selected from the group consisting of a polypeptide comprising amino acids as 2440 to 2484 of SEQ ID NO:124 excluding the corresponding full length sequence of amino acids 2332-2902 SEQ ID NO:124, a polypeptide comprising amino acid 2390 to amino acid 2624 of SEQ ID NO:124 excluding the full length sequence of amino acids 2332-2902 of SEQ ID NO:124, SEQ ID NO:32, SEQ ID NO:54; SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, amino acids 2627 to 2669 of SEQ ID NO:124, amino acids 2642 to 2685 of SEQ ID NO:124,and combinations thereof.
In a specific embodiment, the isolated and purified HA polypeptide is selected from the group consisting of a polypeptide comprising amino acids 2672 to 2902 of SEQ ID NO:124 excluding the sequence of amino acids 2332-2902 SEQ ID NO:124, SEQ ID NO:63, SEQ ID NO:64; SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 NA polypeptide having the sequence of amino acids 3410 to 3855 of SEQ ID NO:124 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 3431 to 3855 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 NA. In specific embodiments, the isolated and purified polypeptide is selected from the group consisting of a polypeptide comprising amino acids 3477 to 3803 of SEQ ID NO:124 excluding the polypeptide having a sequence of 3410 to 3855 of SEQ ID NO:124, a polypeptide comprising amino acid 3807 to amino acid 3832 of SEQ ID NO:124 excluding the polypeptide having the sequence of 3410 to 3855 of SEQ ID NO:124, SEQ ID NO:84, SEQ ID NO:85; SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, amino acids 3638 to 3662 of SEQ ID NO:124, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96 and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 PB2 polypeptide having the sequence of amino acids 1 to 760 of SEQ ID NO:124 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 344 to 516 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 PB2. In specific embodiments, the isolated and purified polypeptide is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO:43 and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 PB 1 polypeptide having the sequence of amino acids 764 to 1612 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 1290 to 1437 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 PB1. In specific embodiments, the isolated and purified polypeptide is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO:46 and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 PB1-F2 polypeptide having the sequence of amino acids 1524 to 1612 of SEQ ID NO:124 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 1524 to 1608 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 PB1-F2. In specific embodiments, the isolated and purified polypeptide is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 PA polypeptide having the sequence of amino acids 1616 to 2331 of SEQ ID NO:124 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 1852 to 2251 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 PA. In a specific embodiments, the isolated and purified polypeptide is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 52, SEQ ID NO:53 and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 NP polypeptide having the sequence of amino acids 2906 to 3403 of SEQ ID NO:124 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 2906 to 3068 or amino acids 3197 to 3399 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 NP. In specific embodiments, the isolated and purified polypeptide is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, amino acids 3347 to 3385 of SEQ ID NO:124 and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 M1 polypeptide having the sequence of amino acids 3859 to 4110 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 3859 to 4109 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 M1. In specific embodiments, the isolated and purified polypeptide is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 97, SEQ ID NO:98,SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105 and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 M2 polypeptide having the sequence of amino acids 4114 to 4211 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 4115 to 4209 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 M2. In a specific embodiment, the isolated and purified M2 polypeptide has the amino acid sequence of SEQ ID NO:107.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 NS1 polypeptide having the sequence of amino acids 4214 to 4428 of SEQ ID NO:124 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 4220 to 4294 or amino acids 4378 to 4428 of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length H5N1 NS1. In specific embodiments, the isolated and purified polypeptide is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112 and combinations thereof.
In some embodiments, the isolated and purified polypeptide corresponds to a H5N1 NS2 polypeptide having the sequence of amino acids 4832 to 4552 and comprises the amino acid sequence corresponding to the polypeptide having the sequence of amino acids 4468 to 4509 (SEQ ID NO:113) of SEQ ID NO:124 or a fragment thereof, wherein the polypeptide does not include the corresponding full length 115N1 NS2.
Other embodiments include any of the H3N2 polypeptides as shown in Table 5, excluding the corresponding full length H3N2 polypeptide. The polypeptides include polypeptides comprising any one of sequences having SEQ ID NO: 1 to 31.
Polypeptides can be Obtained by Recombinant Methods or by Amino Acid Synthesis.
Fusion Polypeptides
As described previously any of these polypeptides can form a fusion polypeptide. A fusion polypeptide can include all or a portion of a viral coat protein. Examples of viral coat proteins include infectivity protein pIII, major coat protein pVIII, p3, Soc (T4), Hoc (T4), gpD (of bacteriophage lambda), minor bacteriophage coat protein 6 (pVI) (filamentous phage; J Immunol Methods. 1999 Dec. 10; 231(1-2):39-51), variants of the M13 bacteriophage major coat protein (P8) (Protein Sci 2000 April; 9(4):647-54). Heterologous sequences encoding gD tags, c-Myc epitopes, poly-histidine tags, fluorescence proteins (eg., GFP), or beta-galactosidase protein or glutathione S transferase which can be useful for detection or purification of the fusion protein expressed on the surface of the phage or cell can be present. In other embodiments, any of the polypeptide identified herein can be combined with a carrier protein such as selected from the group consisting of bovine serum albumin, keyhole limpet hemacyanin, ovalbumin, mouse serum albumin, rabbit serum albumin.
In addition, the polypeptides as described herein can be inserted into the corresponding influenza virus but of a different subtype. For example, a polypeptide comprising the amino acid sequence of amino acids 9-241 of H5N1 HA can be inserted in the place of corresponding residues in H3N2 HA. The insertion can be done by cassette mutagenesis or by mutating one or more of the residues of H3N2 HA to the sequence of the H5N1 HA fragment.
Compositions
One or more of the polypeptides identified by epitope mapping as described herein can be combined in a composition and used to immunize a mammal. Examples of other influenza components include hemagglutinin (HA), neuraminidase (NA), and immunogenic fragments thereof. Examples of conserved influenza components include matrix protein 1 (M1), nucleoprotein (NP), acidic polymerase (PA), basic polymerase 1 (PB1), basic polymerase 2 (PB2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), and immunogenic fragments thereof. In some embodiments, the same polynucleotide encoding one or more HA polypeptides does not encode a nucleoprotein or M1 protein either as individual proteins or as fusions to HA. In other embodiments, the same polynucleotide does not encode matrix protein 1 (M1), nucleoprotein (NP) acidic polymerase (PA), basic polymerase 1 (PB1), basic polymerase 2 (PB2), nonstructural protein 1 (NS 1), or nonstructural protein 2 (NS2). Influenza amino acid and nucleic acid sequences for these variable and conserved influenza components are known in the art and can be found, for example, using GenBank (www-ncbi-nlm-gov) or the Influenza Sequence Database at the Los Alamos website (http://www-flu-lanl-gov).
In some embodiments, the immunogenic compositions of the disclosure can be combined with other influenza vaccines, such as heat killed or subunit vaccines or can be combined with other components of different subtypes using reverse engineering and reassortment.
Compositions can include one or more or a combination of any of the polypeptide described herein, including any of the polypeptides in Tables 4 and Table 5. In some embodiments, the polypeptides are selected that are identified at a frequency in the phage display library of at least 5 clones following affinity selection. In other embodiments, polypeptides are selected that bind to neutralizing antibodies or hemagglutinin inhibiting antibodies. In yet other embodiments, peptides are selected that bind to an antibody in a biological sample with a titer of at least 500 or at least 5 fold increased over control biological sample.
In some embodiments, compositions of the disclosure may consist essentially of one or more of the polypeptides. The compositions that consist essentially include ingredients that do not affect the binding and/or immunogenic properties of the polypeptides such as carriers, excipients, adjuvants, and may exclude the full length corresponding polypeptide.
In some embodiments, the immunogenic compositions of the invention comprise an immunogenic effective amount of the polypeptides as described herein. The immunogenic compositions are useful to provide a protective immune response as well as to provide for monoclonal and/or humanized antibodies for therapeutic purposes.
Compositions may include a carrier, excipient or adjuvant. Adjuvants include,for example, aluminum hydroxide, lipid A, killed bacteria, polysaccharide, mineral oil, Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum phosphate, iron, zinc, a calcium salt, acylated tyrosine, an acylated sugar, a CpG oligonucleotide, a cationically derivatized polysaccharide, an anionically derivatized polysaccharide, a polyphosphazine, a biodegradable microsphere, a monophosphoryl lipid A, MF59, oil in water emulsions AS03 and AS04, ISCOM, and quil A.
Polynucleotides and Vectors
The disclosure also include polynucleotides including one or more of the polypeptides identified herein by epitope mapping. The polynucleotides may be present in a phage vector as a part of a library useful for epitope mapping of immune responses or in a vector useful for immunizing an animal. Phage display vectors have been described previously herein. Polynucleotide sequences encoding any of the polypeptides as described herein can be readily obtained using publicly available databases. Polynucleotide sequence can be prepared using recombinant or synthetic methods.
In some embodiments, one or more polynucleotides is present in a composition in an immunogenic effective amount. In specific embodiments, the polynucleotides encode one or more of the polypeptides as described herein, for example, any one of the polypeptides having a sequence of SEQ ID NO:1-123 or SEQ ID NO:148 to 176. An immunogenic effective amount is an amount of polynucleotide that induces an immune response to the encoded polypeptide when administered to a host, for example an animal. In an embodiment, the polynucleotides are incorporated into host cells in vivo and an immunogenic effective amount of the encoded influenza A polypeptide or fragment thereof is produced in vivo. The actual amount of the immunogenic composition may vary depending on the animal to be immunized, the route of administration and adjuvants. Immunogenic dosages can be determined by those of skill in the art. The immune response can be humoral, cellular, or both. Generally, the immune response inhibits the influenza viral levels in the immunized host compared to influenza viral levels in non-immunized hosts.
An embodiment provides an immunogenic composition according to the present disclosure also including both polypeptides and/ or polynucleotides encoding polypeptides as described herein.
The immunogenic composition optionally includes a pharmaceutically acceptable excipient or carrier. The immunogenic composition may further comprise immunomodulators such as cytokines or chemokines. In some embodiments, a nucleic acid encodes the immunomodulator or adjuvant. Immunomodulators refers to substances that potentiate an immune response including, but not limited to cytokines and chemokines. Examples of cytokines include but are not limited to IL-2, IL-15, IL-12, or GM-CSF.
An embodiment provides an immunogenic composition further comprising an adjuvant. Such adjuvants may include ganglioside receptor-binding toxins (cholera toxin, LT enterotoxin, their B subunits and mutants); surface immunoglobulin binding complex CTA1-DD; TLR4 binding lipopolysaccharide; TLR2-binding muramyl dipeptide; mannose receptor-binding mannan; dectin-1-binding ss 1,3/1,6 glucans; TLR9-binding CpG-oligodeoxynucleotides; cytokines and chemokines; antigen-presenting cell targeting ISCOMATRIX and ISCOM. Adjuvants such as lipids (fatty acids, phospholipids, Freund's incomplete adjuvant in particular), Vaxfectin, M59, polaxomer, anionic copolymers, CpG units, etc. may be added to the composition. In addition, adjuvants able to prime the mucosal immune system following a systemic immunization, include 25(OH)2D3, cholera toxin, CTA1-DD alone or in combination with ISCOM, AS03, and AS04. In some embodiments, the adjuvant may be encoded or expressed by the expression vector used herein.
An embodiment provides an immunogenic composition comprising at least one naked DNA or a naked RNA encoding at least one polypeptide according to the disclosure. Naked DNA or RNA is DNA or RNA that does not have proteins or lipids associated with it.
In certain embodiments, the immunogenic composition comprises at least one recombinant vector or DNA comprising a nucleic acid sequence encoding any of the polypeptides described herein such as those shown in Tables 4 and 5. Examples of vectors include, but are not limited to, recombinant viral vectors, such as poxvirus, vaccinia virus, lenti virus, or adenovirus, and plasmids. Typically a plasmid contains an origin of replication that is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells containing the plasmid. Plasmids of the present invention may include genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in eukaryotic cells. In certain embodiments described herein, a plasmid is a closed circular DNA molecule.
Examples of plasmids that can be used in the present invention include expression vector VR1012 or VR10551 (Vical, San Diego, Calif.). These vectors are built on a modified pUC18 background (see Yanisch et al., 1985, Gene, 33:103-119), and contain a kanamycin resistance gene, the human cytomegalovirus immediate early promoter/enhancer and intron A, and the bovine growth hormone transcription termination signal, and a polylinker for inserting foreign genes (see Hartikka et al., 1996, Hum. Gene Ther., 7:1205-1217). Other commercially available eukaryotic expression vectors can be used in the present invention, including, but not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, pVAX200, and pZeoSV2 (Invitrogen, San Diego, Calif.), plasmid pCI (Promega, Madison, Wis.) and plasmid pDNA-VACC (Nature Tech. Corp., Lincoln, Neb.).
In an embodiment, the immunogenic composition includes a plasmid that comprises a nucleic acid sequence encoding at least one polypeptide or immunogenic fragment thereof from an influenza A virus under the transcriptional control of a promoter region active in a variety of cells. In an embodiment, the promoter region is a human cytomegalovirus (CMV) promoter. In an embodiment, the plasmid is pVR1012. The polypeptides can be naturally occurring, variant, or an immunogenic fragment thereof. To permit selection of plasmid-containing bacteria during the production process, the plasmid may also contain an antibiotic resistance gene with a bacterial origin of replication. DNA is generally less costly to produce than peptide or protein, and is chemically stable under a variety of conditions. DNA is generally administered intramuscularly, using either a needle and syringe or a needle-free injector, or intranasally.
The polypeptide, or fragment thereof, may be expressed in a modified form, such as a fusion protein, and may include secretion signals and/or additional heterologous functional regions. For example, a region of additional amino acids may be added to the N-terminus or C-terminus of the polypeptide to facilitate detection or purification, improve immunogenicity, improve half-life, or improve persistence in the host cell during, for example, purification or subsequent handling and storage. Examples of additional amino acids include peptide tags that may be added to the polypeptide to facilitate detection and/or purification. Such peptide tags include, but are not limited to, His, HA, Avi, biotin, c-Myc, VSV-G, HSV, V5, or FLAG™. Examples of a polypeptide that can enhance immunogenicity include bovine serum albumin, and/or keyhole lymphocyte hemocyanin (KLH). Examples of molecules that improve half-life include polyethylene glycol.
The immunogenic compositions comprising polypeptides and/or polynucleotides of the disclosure can also include a carrier. Carriers include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or animal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ polyethylene glycol (PEG), and PLURONIC™.
The immunogenic compositions comprising polypeptides and/or polynucleotides of the disclosure can be in the form of sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions. For administration as injectable solutions or suspensions, the immunogenic compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
Method of Making Polypeptides and/or Polynucleotides
Polynucleotides encoding influenza polypeptides, recombinant vectors, and host cells containing the recombinant vectors, as well as methods of making such vectors and host cells by recombinant methods are useful to produce the polypeptides as described herein for use in assays or immunogenic compositions.
The polynucleotides of the disclosure may be synthesized or prepared by techniques well known in the art. See, for example, Creighton, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., New York, N.Y. (1983). Nucleotide sequences encoding the influenza polypeptides of the disclosure may be synthesized, and/or cloned, and expressed according to techniques well known to those of ordinary skill in the art. See, for example, Sambrook, et al., Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). In some embodiments, the polynucleotide sequences will be codon optimized for a particular recipient using standard methodologies. For example, the DNA construct encoding a H5N1 HA polypeptide can be codon optimized for expression in humans.
The polynucleotides may be produced by standard recombinant methods known in the art, such as polymerase chain reaction (PCR) or reverse transcriptase PCR (Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), reverse engineering, or the DNA can be synthesized and optimized for expression in bacteria or eukaryotic cells. Primers can be prepared using the polynucleotide sequences that are available in publicly available databases. The polynucleotide constructs may be assembled from polymerase chain reaction cassettes sequentially cloned into a vector containing a selectable marker for propagation in a host. Such markers include but are not limited to dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline, ampicillin, or kanamycin resistance genes for culturing in E. coli and other bacteria.
Representative examples of appropriate hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces and Salmonella typherium, fungal cells such as yeast; insect cells such as Drosophilia S2 and Spodoptera Sf9, animal cells such as CHO, COS, and Bowes melanoma cells, and plant cells. Appropriate culture medium and conditions for the above-described host cells are known in the art.
Introduction of the recombinant vector into the host cell can be effected by injection, by mucosal administration such as by the intranasal route, or by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in standard laboratory manuals such as Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. or Davis et al., 1986, Basic Methods in Molecular Biology. Commercial transfection reagents, such as Lipofectamine (Invitrogen, Carlsbad, Calif.), Effectene (Qiagen, Valencia, Calif.) and FuGENE 6™ (Roche Diagnostics, Indianapolis, Ind.), are also available.
The influenza polypeptide can be recovered and purified from recombinant cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.
Naturally occurring polynucleotides encoding influenza virus polypeptides can be isolated from cloning out viral isolates from infected individuals at various times post infection. Such polynucleotides can be obtained using primers for amplifying polynucleotide encoding a polyeptdie such a showin in Table 5 Such polynucleotides or polypeptides may be utilized in the immunogenic compositions described herein.
The disclosure also includes variants of nucleic acid molecules encoding the polypeptides as described herein. In some embodiments, the disclosure includes polynucleotides having at least about 70% sequence identity, more preferably about 75% sequence identity, more preferably about 80% sequence identity, more preferably about 85% sequence identity, more preferably about 90% sequence identity, more preferably about 95% sequence identity, and even up to 100% sequence identity to a polynucleotide sequence encoding an polypeptide having an amino acid sequence as shown in the reference sequence of FIG. 14 or FIG. 15 . Preferably, the variants generate antibodies that cross react with the corresponding polypeptide from different strains of the same subtype and/or provide protective immunity.
Animal Models
A variety of animal models are available for testing of any of the immunogenic compositions described herein. For example, well-established models include mice, poultry, ferrets, pigs, guinea pigs, or non-human primates. An animal model that provides for an immune response and has a response to challenge with infectious virus is suitable for testing of the immunogenic compositions.
Mouse models systems are available and in some embodiments, include challenge with mouse adapted influenza strains. The mouse model system includes immunizing the mice with an polypeptide or fragment thereof and/or a polynucleotide encoding an polypeptide or fragment thereof. After the mice are immunized, the mice are challenged with an influenza virus strain and evidence of infection can be determined by viral titers in tissues including the respiratory tract or in the case of systemic infection, other tissues as well, and/or by weight loss and/ or death. Suitable mice include BALB/c mice, as well as any of the commercially available mice such as knockout mice and mice that have a human immune system.
Another model system for influenza infection is ferrets. Ferrets are naturally susceptible to infection with human influenza viruses, as well as avian, equine, and swine influenza viruses. Influenza virus infection in ferrets can be detected by detecting viral titers, and/or weight loss, fever, and respiratory symptoms such as nasal discharge. Other symptoms may be detected in ferrets having a systemic infection including neurological symptoms, diarrhea, and lethargy.
Uses and Methods
The present disclosure is also directed to uses and methods for immunizing an animal, including a human, other mammal, or bird, with the immunogenic compositions of the invention to inhibit, control, or prevent influenza infection.
In an embodiment, the method comprises administering to an animal an immunogenic effective amount of an immunogenic composition. An immunogenic effective amount is an amount of polynucleotide and/or polypeptide that induces an immune response to the encoded polypeptide when administered to a host, for example an animal. In an embodiment, the animal is a human, pig, horse, birds including domestic birds, or other animals, especially those used in animal models such as mouse, rat, ferret, or non-human primate. In an embodiment, the polynucleotides are incorporated into host cells in vivo and an immunogenic effective amount of the encoded polypeptide or fragment thereof is produced in vivo. The actual amount of the immunogenic composition may vary depending on the animal to be immunized, the route of administration and adjuvants.
Immunogenic dosages can be determined by those of skill in the art. The immune response may be indicated by T and/or B cell responses. Typically, the immune response is detected by the presence of antibodies that specifically bind to a particular polypeptide. The immune response can also be determined by detecting the presence of neutralizing antibodies or hemagglutinin inhibiting activity. Methods of detecting antibodies to polypeptides are known to those of skill in the art and include such assays as ELISA assays, western blot assays, functional and competition assays. Methods of detecting T cell responses include ELISPOT assays, ICS assays, and in-vitro and in-vivo cytotoxicity assays. The particular region of the polypeptide that is stimulating a T cell or antibody response can be mapped using whole genome phage display libraries as described herein.
In some embodiments, the immunogenic composition administered to an animal includes a polynucleotide and/or polypeptides or immunogenic fragments thereof and one or more of variable influenza components, one or more conserved influenza component, or a combination thereof. In an embodiment, the conserved influenza component is M1, NP, PA, PB1, PB2, NS1, NS2, an immunogenic fragment thereof or combination thereof. In some embodiments, the same polynucleotide does not encode an influenza component such as M1 and/ or NP. In other embodiments, the polynucleotide does not encode an influenza component selected from the group consisting of M1, NP, PA, PB1, PB2, NS1, NS2, an immunogenic fragment thereof and combinations thereof.
In an embodiment, an animal is immunized with an immunogenic composition of the invention and then boosted one or more times with the immunogenic composition. In an embodiment, the animal is boosted about 2 to about 4 weeks after the initial administration of the immunogenic composition. If the animal is to be boosted more than once, there is about a 2 to 12 week interval between boosts. In an embodiment, the animal is boosted at about 12 weeks and about 36 weeks after the initial administration of the immunogenic composition. In another embodiment, the animal is a mouse and the mouse is boosted 3 times at 2 week intervals. In yet another embodiment, the animal is a primate and the primate is boosted 1 month and 6 months after the initial administration of the immunogenic composition. The dose used to boost the immune response can include one more cytokines, chemokines, or immunomodulators not present in the priming dose of the immunogenic composition.
The methods of the invention also include prime-boost immunization methods utilizing the immunogenic compositions of the invention. Providing influenza polypeptides in different forms in the prime and boost maximizes the immune response to the polypeptide. In some embodiment, an animal is primed with a polynucleotide encoding a polypeptide, such as shown in Tables 4 or 5, in one vector. The animal may be primed 1 to 8 times. Typically there is a 1, 2, or 3 week interval between administrations. In an embodiment, the animal is primed 3 times at 2 week intervals. The primed animal is then boosted with the same polypeptide or polynucleotide encoding the same polypeptide in a second vector that is different from the first vector. In an embodiment, the animal is boosted with the second vector at least 2 weeks after the last dose of the first viral vector. In an embodiment, the animal is boosted with the second vector at 4 weeks after the last dose of the first viral vector. The dose used to boost the immune response can include one more cytokines, chemokines, immunomodulators, or influenza antigens not present in the priming dose.
Viral delivery vectors are known and commercially available. Examples of viral vectors include, but are not limited to, recombinant poxvirus including vaccinia virus, lentivirus, or adenovirus. In an embodiment, the viral vector is adenovirus type 5. Examples of commercially available viral delivery vectors include, but are not limited to, VIRAPOWER™ lentivirus expression system, VIRAPOWER™ adenovirus expression system (Invitrogen, Carlsbad, Calif.), and ADENO-X adenovirus expression system (Clontech, Mountain View, Calif.).
The methods of the invention also include methods for protecting an animal against a lethal influenza challenge. In some embodiments, the method of the disclosure provides for protective immunity against an infection with virus of the same subtype, and against heterosubtypic virus. In an embodiment, the influenza is a highly pathogenic H5N1.
The methods of the invention can be used to immunize birds to prevent the spread of avian influenza. In an embodiment, the avian influenza is H5N1. In an embodiment, the birds are domesticated poultry.
Any mode of administration can be used in the methods of the inventions so long as the mode results in the expression of the desired peptide or protein, in the desired tissue, in an amount sufficient to generate an immune response to influenza A and/or to generate a prophylactically or therapeutically effective immune response to influenza A in an animal. The immunogenic compositions of the invention can be administered by intramuscular (i.m.), intra-nasally (i.n.), subcutaneous (s.c.), or intrapulmonary route in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, or vehicles. Other suitable routes of administration include, but are not limited to intratracheal, transdermal, intraocular, intranasal, inhalation, intracavity, and intravenous (i.v.) administration. Transdermal delivery includes, but is not limited to intradermal, transdermal, and transmucosal administration. Intracavity administration includes, but is not limited to administration into oral or nasal cavities. The immunogenic compositions can be coated onto particles or nanofibers for delivery or formulated in liposomes.
Administration modes of the present invention include needle injection; catheter infusion; biolistic injectors; particle accelerators such as, for example, “gene guns” or pneumatic “needleless” injectors such as Med-E-Jet (Vahlsing et al., 1994, J. Immunol. Methods, 171:11-22), Pigjet (Schrijver et al., 1997, Vaccine, 15:1908-1916), Biojector (Davis et al., 1994, Vaccine, 12:1503-1509; Gramzinski et al., 1998, Mol. Med., 4: 109-118), AdvantaJet (Linmayer et al., 1986, Diabetes Care, 9:294-297), or Medi-jector (Martins and Roedl, 1979, Occup. Med., 21:821-824); gelfoam sponge depots; other commercially available depot materials such as, for example, hydrogels, osmotic pumps, oral or suppositorial solid (tablet or pill) pharmaceutical formulations, topical skin creams, and decanting, polynucleotide coated suture (Qin, Y., et al., 1999, Life Sci., 65: 2193-2203), or topical applications during surgery. Certain modes of administration are intramuscular needle-based injection and pulmonary application via catheter infusion. Energy-assisted plasmid delivery (EAPD) methods may also be employed to administer the compositions of the invention. One such method involves the application of brief electrical pulses to injected tissues, a procedure commonly known as electroporation. See generally Mir et al., 1999, Proc. Natl. Acad. Sci USA, 96:4262-7; Hartikka et al., 2001, Mol. Ther., 4:407-15; Mathiesen, 1999, Gene Ther., 6:508-14; Rizzuto et al., 2000, Hum. Gen. Ther. 11:1891-900.
The present disclosure is also directed to kits for practicing the methods of the invention. In some embodiments, the kit includes a plasmid expression vector of the invention, a viral vector of the invention, and instructions for priming an animal (including human) with the plasmid expression vector and boosting the animal with the viral vector. In some embodiments, the kit comprises a plasmid expression vector and a viral vector each comprising a polynucleotide encoding a polypeptide from A/H3N2 or A/H5N1. In some embodiments, the kit may further comprise a polypeptide composition. In some cases, the polypeptide of the polypeptide composition and the polypeptide encoded by the plasmid and viral expression vector have the same sequence. In some embodiments, the kit may further comprise at least one adjuvant or immunomodulator. The adjuvant or immunomodulator can be encoded by a polynucleotide. In a specific embodiment, the adjuvant is MF59, CTA1-DD alone or in combination with ISCOM.
Diagnostic Assays
Serodiagnostic or surveillance assays are also provided. Using the epitopes identified by the whole genome libraries, assays and kits can be provided that distinguish between different subtypes of influenza virus infection, for example, H5N1 infection from H3N2 infection; between vaccinated and infected subjects; and between different clades of influenza virus subtypes. These assays can be very important in surveillance of emerging pandemics and especially in countries that do not have the ability to run PCR type assays.
The diagnostic methods of the invention include a method for determining the presence of a
H5N1 infection in a subject comprising analyzing a biological sample to detect the presence of an antibody that specifically binds to one or more polypeptides as described herein, wherein the presence of the antibody is indicative of H5N1 infection. Such polypeptides include anyone of the polypeptides of SEQ ID NO:32 to SEQ ID NO:120 or SEQ ID NO:148 to 176. Preferably a polypeptide is selected that is conserved in H5N1 strains as can be determined using publicly available sequence information such as shown in Tables 6 and 7. All of the peptides in Table 2, 3 and Table 5 from the H5N1 sequence are peptides for differential serodiagnosis development, as evident from absence of ELISA reactivity with the uninfected Vietnam serum samples. These peptides can help differential diagnosis of H5N1 vaccine v/s infected individuals as well as H5N1 infected from other seasonal influenza infected individuals.
In other embodiments, the method further comprises, analyzing the biological sample to detect the presence of an antibody that specifically binds to any one of the polypeptides of SEQ ID NO:1-31, wherein a lack of binding to any one of the polypeptides is indicative of a lack of infection with H3N2 influenza virus and binding to one or more of the polypeptides is indicative of infection with H3N2 strain of influenza.
In yet other embodiments, the method further comprises analyzing the biological sample to detect presence of an antibody that binds to a polypeptide comprising SEQ ID NO:41, wherein binding of the polypeptide is indicative of infection with a strain of H5N1 of clade 1.
Biological samples include serum, tissue, urine samples, and biopsy samples. One or more of the polypeptides may be attached to a solid substrate such as a bead, ELISA plate, dipstick, or microarray.
The presence or absence of the antibody in the biological sample can be determined using methods known to those of skill in the art to detect the antigen antibody complex. Such methods include contacting the antibody antigen complex with a detectably labeled moiety that will bind to the antigen antibody complex and not to antibody or antigen alone.
Antibodies
Polyclonal Antibodies
Polyclonal antibodies to a polypeptide of the disclosure are preferably raised in animals by multiple subcutaneous (sc), intramuscularly (i.m.), intranasally (i.n.) or intraperitoneal (ip) injections of the polynucleotide encoding one or more polypeptides or fragments thereof and/ or the polypeptide, and optionally an adjuvant. Polyclonal antibodies may be useful to treat influenza virus infection via passive immunity or to produce chimeric or humanized antibodies.
It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N═C═NR, where R and R 1 are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with ½ to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
The polyclonal antibodies generated by the immunizations may undergo a screen for antagonist activity or neutralizing activity. Preferably, antibodies to the polypeptide decrease the infectivity of virus produced or inhibit viral levels. In an embodiment, antibodies that specifically bind a HA or NA polypeptide as show in Table 4 or Table 5 reduce or inhibit influenza viral levels. An antagonist antibody would be screened to determine if there was a decrease or inhibition of viral levels in infected cells.
The polyclonal antibodies are also screened by enzyme-linked immunoabsorbent assay (ELISA) to characterize binding. The antigen panel includes all experimental immunogens. Animals with sera samples that test positive for binding to one or more experimental immunogens are candidates for use in monoclonal antibody production. The criteria for selection for monoclonal antibody production is based on a number of factors including, but not limited to, binding patterns against a panel of structured influenza immunogens.
Cross-competition experiments using other mapped Mabs, human sera and peptides can also be performed. Screening methods for identifying antibodies that bind to epitopes shared by all or a number of the influenza viruses of the same subtype, or even of the same clade and not another clade can be selected.
Monoclonal Antibodies
Monoclonal antibodies to a polypeptide of the disclosure may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). Monoclonal antibodies may be useful to treat influenza virus infection via passive immunity or to produce chimeric or humanized antibodies.
In the hybridoma method, a mouse or other appropriate host animal, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the polypeptide used for immunization. Alternatively, cells producing monoclonal antibodies can be obtained from infected or vaccinated individuals. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
The hybridoma cells are than seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or enzyme-linked immunoabsorbent assay (ELISA).
After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies are characterized for specificity of binding using assays as described previously. Antibodies can also be screened for antagonist activity as described previously. Cross-competition experiments using other mapped Mabs, human sera and peptides can also be performed. Screening methods for identifying antibodies that bind to epitopes shared by all or a number of the influenza viruses of the same subtype, or even of the same clade and not another clade can be selected.
Human or Humanized Antibodies
Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. Useful non-human antibodies are monoclonal antibodies that bind specifically to Useful non-human antibodies also include antibodies that inhibit or reduce viral levels. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to improve antibody affinity or functional activity. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech 5:428-433 (1994).
Human antibodies that specifically bind and/or antagonize influenza hemagglutinin or neuraminidase activity and/or inhibit viral infectivity can also be made using the transgenic mice available for this purpose or through use of phage display techniques.
For example, in some embodiments, the polypeptides as described herein are then screened against naïve or synthetic libraries of antibody fragments to identify novel high affinity antibodies. These novel high affmity antibodies can be useful as therapeutic agents as antagonists or agonists, and/or as diagonostic agents. For example, antibodies or antigen binding fragments can be screened for binding to an epitope on a H5N1 clade 1 HA protein and not to H5N1 clade 2 epitope. For diagnostic purposes, such antibodies can be used to distinguish whether a subject is infected with clade 1 or clade 2. For therapeutic purposes, an antibody that binds to an epitope on several H5N1 influenza strains or that neutralizes the activity of HA or NA from several different clades would be selected.
Antibody Conjugates
The antibodies specific for a polypeptide or fragment thereof can be combined with heterologous moieties to provide a detectable label or for targeted delivery of an inhibitory agent or for serodiagnosis.
Detectable labels include radionuclides, biotin, dyes, enzymes, and fluorescent molecules. Inhibitory agents include cytotoxic agents such as toxins.
Compositions
Compositions comprising one or more antibodies may be useful to treat influenza virus infection or to use prophylactically in the event an outbreak of infection arises. Antibodies may be present in the compositions as polyclonal antibodies, chimeric antibodies, human, human antibodies or antigen binding fragments. Compositions may include physiological excipients, or carriers as described previously.
Dosing may be determined by the physician. Guidance for dosing can be found in antibody compositions used to treat rabies, or for any of the antibody compositions currently used to treat cancer.
The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present disclosure.
Example 1
Influenza Whole Genome Flu Phage Display Library (GFPDL)Construction
In order to prepare for the pandemic influenza threat and to improve the cross-reactivity and long-term protection of interpandemic influenza vaccines, it is important to identify all the dominant antibody responses of vaccinated and/or infected individuals. The ability to rapidly distinguish between exposure to human influenza vs. bird influenza will be useful in the surveillance of disease. There is only limited knowledge about the viral proteins/epitopes which are recognized by the immune system of infected individuals. Careful analyses of the immune responses against the new candidate vaccines is required in order to identify the best correlate of protection against seasonal human influenza strains and potential pandemic strains (transmitted from wild birds).
To address these challenges we developed an unbiased high throughout approach based on the construction of Whole Viral Genome Phage Display Libraries, expressing complete sets of protein fragments encoded by several Human and Avian Influenza strains including H1N1, H3N2, H5N1 and H7N7. These libraries are being used for in depth analyses of plasma samples from: a) individuals exposed to human influenza; b) individuals exposed to avian (bird) influenza; c) individuals vaccinated with traditional influenza vaccines; d) individuals vaccinated with new generation vaccines against human and bird influenza viruses.
By using these libraries we have identified a large set of peptides that are shown to provide broad heterotypic neutralizing activity as well a set of epitopes that will be important for serodiagnosis as well as differential diagnosis.
Eight gene segments were cloned from each of the following influenza strains: H1N1-A/New Caledonia/20/99; H3N2-A/California/7/2004; H5N1-A/Vietnam/1203/2004; H5N1 A/Indonesia/5/05; and H7N7-A/Netherlands/2003 were reverse transcribed to form cDNAs of each gene segment and then were digested with DNase in order to form influenza gene segment fragments. These gene fragments were cloned into a phage display vector and phage clones bearing influenza gene segments were selected for binding to antibodies specific for each of the influenza virus proteins. These libraries were utilized to map the antibody response to H3N2 post infection or post vaccination and to map antibody response to H5N1 post infection in human survivors and post vaccination with a H5N1 subunit vaccine.
Cloning of Influenza Gene Segments
A representative scheme for preparing whole genome influenza gene libraries is shown in FIG. 1 . From each strain identified above, cDNAs corresponding to all eight gene segments of the A/H5N1Nietnam/1203/2004 were generated from RNA isolated from egg-grown virus strain and were used for cloning
Phage display libraries were constructed individually for HA & NA genes and the rest of the six gene segments (PB2, PB1, PA, NP, M & NS). Purified DNA containing equimolar ratio of HA & NA (HA-NA) or the rest of the six genes (PB2-NS) were digested with DNase I using DNase shotgun cleavage kit (Novagen) as per manufacturer's instructions to obtain majority of the DNA fragments in the size range of 50-200 and 200-1000 by for each of the two gene segment pools. The digested DNA was isolated from agarose gels, purified, and the overhangs were filled/removed using T4 DNA polymerase. A representative gel electropherogram is shown in FIG. 2 . The end repaired DNA was then dephosphorylated using calf intestinal alkaline phosphatase (OP), and 500 ng of random dephosphorylated blunt ended DNA fragments were ligated with 100 ng of Sma I digested fSKSrf9-3 vector in a 50 μl reaction (25° C.; 1 h) in the presence of 20 U of T4 DNA ligase and 25 U of SrfI. fSKSrf1-9-3 is a gIIIp display based phage vector where the desired polypeptide can be expressed as gIIIp fusion protein constructed in inventor's lab.
Ligated products were used for electroporating E. coli TG1 cells. For producing phages, 10 9 cells from each library were added to 200 ml 2×YT with 20 ug per ml tetracycline and grown for 16 h at 32° C. and 200 rpm. The cell-free phage supernatant was isolated by centrifugation and phage titre determined as Tet r transduction units.
Four libraries were constructed; fSK9-3 HA-NA (50-200 bp), fSK9-3 H5HA-NA (200-1000 bp), fSK9-3 H5t-PB2-NS (50-200 bp) & fSK9-3 H5PB2-NS (200-1000 bp) for each influenza stain.
Affinity Selection of H5N1 MAb or Sera Specific Phages
The influenza genome libraries were then used to characterize the human antibody response to infection and/or vaccination. A representative scheme for affinity panning is shown in FIG. 4 . The phage display library was incubated with antibodies from serum from infected individuals or vaccinated individual bound to a solid substrate or in-solution, unbound phages were washed away, and bound phages were eluted.
For removal of serum components, which could non-specifically interact with phage proteins, sera were incubated with UV-killed M13K07 coated petri dishes. Affinity selection was carried out on antibody coated wells as well as in-solution (with Protein A/G).
Eight wells of Nunc Maxisorp microtitre strips (NUNC Inc, Naperville, Ill., USA) were coated with a mixture of 500 ng each of goat anti-human IgG-Fc specific antibodies and blocked with 350 μl PBST (PBS containing 0.1% Tween-20) containing 2% BSA at RT for 1 h to block the unoccupied reactive sites. After three washes with PBST, 1 μg of MAb or 100-fold dilution (final dilution) of VCSM13-preadsorbed human serum (100 μl in 1% BSA-PBST) was added to wells and incubated for 1 h at RT. The wells were washed thrice with PBST and 10 10 phages (from gene-fragment library) in 100 μl PBST containing 1% BSA were added to all the eight wells and incubated at RT for 1 h. The unbound phages were removed in ten washes with PBST followed by three washes with PBS. The bound phages were eluted by addition of 100 μl of 0.1N HCl (adjusted to pH 2.2 with glycine and BSA added at 1 mg/ml) to each well and incubated for 10 min at 37° C. The eluted phages were collected and neutralized by adding 64 μl of 2 M Tris solution (pH not adjusted).
For in-solution panning, 10 10 phages (from gene-fragment library) in 500 μl PBST containing 1% BSA were added to 200 μl of 50% Ultralink Protein A/G slurry (Pierce) and incubated for 1 h at RT on end-to-end shaker. Following brief centrifugation, 500 μl of supernatant was added to 5 μg of human anti-H5N1 MAb or 100 μl of 10-fold VCSM13-preadsorbed human serum (100 μl in 1% BSA-PBST) and incubated for 1 h at RT on end-to-end shaker, followed by 200 μl of 50% Ultralink Protein A/G slurry (Pierce, Rockford, Ill.) on end-to-end shaker at RT for 1 h. The unbound phages were removed in ten washes with PBST followed by three washes with PBS. The bound phages were eluted by addition of 800 μl of 0.1N HCl (adjusted to pH 2.2 with glycine and BSA), and incubated for 10 min at RT on end-to-end shaker. The eluates was collected and neutralized by adding 64 μl of 2 M Tris solution.
After each round of affinity selection, bound phages were eluted and sequenced. Ninety-six clones were randomly isolated and sequenced to ensure that the library reflected segments of the entire genome. The results for a representative library are shown in FIG. 3 . The results show that several sequences for each gene segment were obtained representing random distribution of size and sequence in the gene-fragment phage display libraries (GFPDL).
Example 2
Epitope Mapping Polyclonal Sera and Monoclonal Antibody Sera using H3N2 Phage Display Library
Epitope mapping using the H3N2 whole genome library with the pool of larger fragments was conducted with polyclonal antisera from individuals pre- & post-vaccinated, or post-infection with H3N2. An exemplary B-cell epitope map of hemagglutinin (HA) from H3N2 expressed from a gene fragment phage display library and screened against polyclonal sera collected before vaccination and after the influenza season from convalescent patients in the placebo arm of the study done in 2003-2004 season is shown in FIG. 5 . The results show that sera taken from individuals pre-vaccination and post infection at end of the seasonal flu season contained antibodies that predominantly reacted with HA, NA, M1 and M2 proteins as compared to sera from individuals before the start of the season.
Some of the clones were isolated and sequenced and are represented as sequences of SEQ ID NO:1-31 of Table 4.
Example 3
Epitope Mapping using H5N1 Library using Monoclonal Antibodies Derived from H5N1 Infected Humans Who Survived Infection
Two H5N1 libraries were constructed as described in Example 1: one library was made from strain H5N1 from Vietnam and another from H5N1 from Indonesia. Examination of strains isolated from different outbreaks has revealed that there are several clades of H5N1 influenza viruses. Clade 1 includes H5N1 strains from Vietnam and Clade 2 includes H5N1 strains from Indonesia.
We characterized the epitopes recognized by the monoclonal antibodies from the infected individuals using the whole genome libraries. These antibodies have been described in Simmons et al, Plos Medicine 4:e178(2007). Three monoclonal antibodies were tested: FLA 5.10, FLD 21.140, and FLA 3.14. Two of these monoclonal antibodies, FLD 21.140, and FLA 3.14, have been shown to protect mice against lethal infection with both the Vietnam and Indonesia strains and thus, are characterized as broadly neutralizing. Monoclonal antibody FLA 5.10 was protective only for the Vietnam strain.
Phage clones were affinity selected as described in Example 1, and were sequenced.
Phage ELISA
Phage ELISA was performed to analyze the reactivity of affinity selected clones using H5N1 positive and normal healthy individual sera. Microtitre plates coated with 1000-fold dilution of anti-phage antibody (GE Healthcare, Piscataway, N.J.) were blocked with EMEM containing 2% BSA and 0.1% Tween-20 (blocking solution). The phages (10 10 /100 μl/well) diluted in blocking solution were then added and incubated for 1 h at room temperature (RT). 100 μl of serially diluted sample was added to the wells in duplicate and incubated at RT for 1 h. All the antibody and phage dilutions were made in blocking solution. Plates were washed with PBST using a microplate washer. The bound serum antibodies were probed with 100 μl of 2000-fold dilution of HRP-conjugated goat anti-human IgG-Fc specific or goat anti-human IgA-α-chain specific antibody. After three washes with PBST followed by three washes with PBS, the reaction was revealed with 100 μl of OPD substrate solution. Cut-off was calculated as mean+3SD (standard deviation) of the phage reactivity in duplicate with three normal random serum samples for each of the phage clone selected for immunoanalysis.
Affinity Selection using Random Peptide Phage Display Library
To further map the epitope regions of the monoclonal antibodies, a phage display library with random dodecamers connected to the p3 protein via a linker was used. This library is referred to as random phage library(RPL). The dodecamers have a sequence of the following formula: X-X-X-X-X-X-X-X-X-X-X-X-X-X, (SEQ ID NO: ______) where X is any naturally occurring amino acid. The library was purchased from New England Biolabs. Each of the monoclonal antibodies were incubated with the dodecamer library and the high affinity binders from the random dodecamer library were isolated and sequenced.
The results for each antibody are shown in FIG. 6 . The HA sequence shown in this figure has accession no. AAW80717.
The results for the H5N1 neutralizing monoclonal antibody FLA5.10 identified using influenza complete genome-fragment phage display library (GFPDL) are shown in FIG. 6A . Epitope sequences are boxed on the aligned sequence of A/Vietnam/1203/2004 and A/Indonesia/5/05 of the haemagglutinin (HA). Amino acid number 1 corresponds to H3 (A/California/7/2004) amino acid -10. HA sequences recognized by FLA5.10 include amino acids 9-241.
The contact residues shown in each of the figures as encircled or boxed residues were identified using random peptide phage display library (RPL) aligned to HA1 sequence. In FIG. 6A , the results for monoclonal antibody FLA5.10 show binding to peptides including amino acids in the receptor binding site: QIIP and EASL. Serine and lysine (K) residues were identified by In-vivo challenge studies with suboptimal amounts of FLA5.10 are shown in italics. When mapped to the crystal structure of HA (as shown in FIG. 9A ), these residues form a contiguous patch on a B sheet in the globular receptor binding domain.
As shown in FIG. 7 (a), monoclonal antibody FLA5.10 binds to a peptide having the amino acid sequence LTAETQIQFFHH (SEQ ID NO:41). This sequence has a partial match to HA sequence as shown in FIG. 6 . Other human antibodies did not bind to this sequence. When the leucine in this sequence is mutated to alanine, the antibody no longer can bind the peptide. See FIG. 7 (b) This may provide the explanation for why monoclonal antibody FLA5.10 does not neutralize or protect mice against infection with the Indonesia strain. The EASL sequence is found in the Vietnam strain but not the Indonesia strain. The sequence in the Indonesia strain is EASS, where the L has been changed to a S.
Monoclonal antibody FLD21.140 was also incubated with the H5N1 Vietnam library and with the random dodecamer library. The results are shown in FIG. 6B . HA sequences from the GFPDL library recognized by FLD 21.140 include amino acids 50-338. The antibody bound to peptides from the RPL library including the amino acid sequence SWS and peptides including YNNT in the 50-338 fragment of the receptor binding domain. Monoclonal antibody 21.140 protects mice against infection with both the Vietnam and Indonesia strains and binds to epitopes containing sequences conserved between the Vietnam and Indonesia strains. These residues also were mapped to the crystal structure of H5N1 HA molecule and are located near the top of the globular receptor binding domain as shown in FIG. 9B .
Monoclonal antibody 3.14 was also incubated with the H5N1 Vietnam library and with the random dodecamer library. The results are shown in FIG. 6C . HA sequences from GFPDL recognized by FLA 3.14 include amino acids 47-338. The antibody bound to peptides from the RPL library including the amino acid sequence GVKP (amino acids 64-67) and peptides including NT (amino acids 231-232). Monoclonal antibody FLA3.14 protects mice against infection with both the Vietnam and Indonesia strains and binds to epitopes containing sequences conserved between the Vietnam and Indonesia strains. These residues also were mapped to the crystal structure of H5N1 HA molecule and even though they are at different ends of the molecule they are brought together by a disulfide bond and appear to flank the globular receptor binding domain as shown in FIG. 9C .
In-vivo challenge studies with suboptimal amounts of FLA5.10 identified two mutated residues that are encircled (in a), and for FLA3.14 (in c) and are represented on the HA structure (in a & c). These mutated residues resulted in virus which is resistant to neutralizing monoclonal antibodies. Asp-61 and Asn-231 were found to be important residues in in-vivo escape studies with FLA3.14. Using two different approaches [epitope mapping using phage display libraries and in vivo escape mutant studies]similar residues were identified as important for the binding of these monoclonal antibodies.
Binding of human monoclonal antibodies to purified HA segments is shown in FIG. 8 . Briefly, serial dilution of MAbs were run on a chip coated with E.coli expressed and purified HA 9-241 (FLA5.10 epitope) to determine the affinity constants using ProtOn system (BioRad). The results show that monoclonal antibody FLD 21.140 bound with the highest affinity to this purified polypeptide exhibiting a Kd of 0.68 nM. Monoclonal antibody FLA 5.10 exhibited a Kd of 34 nM, and antibody FLA3.14 did not bind to this polypeptide at all.
When the recombinant purified polypeptide HA polypeptide 9-241 was incubated with sheep anti-A/Vietnam vaccinated sera, the neutralizing activity was absorbed by the polypeptide. Other peptides including amino acids of HA such as 35-96, 99-121, 120-149, 185-206, and 491-534 did not adsorb neutralizing activity of post infection sera. GST-His has same molecular weight and was purified under similar conditions as HA9-241-His, and was used as negative control. Similar results were obtained with ferret anti-A/Vietnam infected sera. See Table 1.





TABLE 1



ADSORPTION OF NEUTRALIZATION ACTIVITY

USING HA PEPTIDE SEQUENCES






Peptides added
TITER






Sheep anti-A/Vietnam/1203/04-HA-sera






No peptide
640


HA 9-241 − FLOW-THROUGH
<40


HA 9-241 − ELUATE
640


GST-His − FLOW-THROUGH
640


GST-His − ELUATE
<40


HA 35-96 + HA 99-121 FLOW-THROUGH
640


HA 120-149 + HA 185-206 FLOW-THROUGH
640


HA 491-534 FLOW-THROUGH
640



Ferret anti-A/Vietnam/1203/04-infected sera (200






No peptide
640


HA 9-241 FLOW-THROUGH
<40


HA 35-96 + HA 99-121 FLOW-THROUGH
640


HA 120-149 + HA 185-206 FLOW-THROUGH
640


HA 491-534 FLOW-THROUGH
640





indicates data missing or illegible when filed















































This data suggests that most of the neutralizing antibodies in either the vaccinated sheep sera or the infected ferret sera can be adsorbed by a H5-HA 9-241 protein. Moreover most neutralizing antibodies are conformation dependent as most smaller peptides within HA9-241 cannot adsorb any neutralizing reactivity from either the vaccinated or infected sera. Therefore, HA9-241 will be a good vaccine candidate to generate high-affinity neutralizing antibodies as well as to be used a bait for production of neutralizing monoclonal antibodies by various approaches.
Example 4
Elucidation of Epitope Profile of H5N1 Survivors
Individual serum samples were obtained from 5 survivors of H5N1/ Vietnam infection was evaluated for epitope specificity. Epitope specificity was determined both by affinity selection using GFPDL followed by peptide ELISA using chemically synthesized peptides or recombinantly expressed peptides.
Peptide ELISA
Biotinylated peptides (1 μg/well) were captured onto wells coated with 500 ng of streptavidin. After blocking with PBST containing 2% Milk, serial dilutions of human serum in blocking solution were added to each well, incubated for 1 hr at RT, followed by addition of 2000-fold dilution of HRP-conjugated goat anti-human IgG-Fc specific or goat anti-human IgA-α-chain specific antibody and developed by 100 μl of OPD substrate solution. Absorbance was measured at 490 nm. Cut-off was calculated as mean+3SD of the peptide reactivity (in duplicate) with three normal random serum samples for each of the phage clone selected for immunoanalysis.
The results are shown in FIG. 10 . The pooled serum was incubated with H5-HA-GFPDL and antibodies binding to H5-HA that remained after incubation were determined in ELISA. The H5-HA-GFPDL adsorbed all of the antibodies that specifically bind to HA. See FIG. 10A .
The B cell epitope profile of the phage that bound to the pooled sera was determined by isolating and sequencing clones that bound to the pooled sera. The results For HA and NA are shown in FIG. 10 (b). The unique peptide sequences recognized by the pooled sera from H5N1-infected individuals identified using HA and NA GFPDLs were aligned to the reference sequence for HA and NA. Their numbers correspond to the peptide IDs in the ELISA assays. The predicted influenza encoded proteins are shown and numbered according to the intact complete proteome reference sequence ( FIG. 14 ). Arrows indicate that inserts are in right orientation with the coding sequence. Each bar represents a unique peptide sequence.
The peptide sequences represented in filled bars were either expressed and purified from E.coli or chemically synthesized. These peptides were selected based on the frequency of the phage clones displaying these peptide sequences following affinity selection on H5N1 exposed sera. (See Table 5) The peptides were analyzed for binding to antibodies in the sera. ELISA reactivities of sera from individual H5N1-infected patients (Viet1-5) and pooled sera from 20 uninfected age-matched women from Vietnam with HA1 peptides 1-8, HA2 peptide 9-14, and NA peptides 15-21 (as indicated in FIG. 10 b) The end-point titers are shown. Days post admission represent the time of serum collection for each patient. See Table 2.








TABLE 2





IgG RESPONSES












Viet-1
Viet-2
Viet-3
Viet-4
Viet-5

Uninfected







Days Post-Admission
ANTIGENIC
Vietnam











ALIGNED ID
PEPTIDE
54
69
73
113
182
CLUSTER
Sera













1
HA-2376-2659
12500
>12500
>12500
>12500
2500

500

2
HA-2339-2581
>12500
12500
12500
12500
500

100

3
HA-2365-2427
500
2500
2500
500
100
I
<100

4
HA-2431-2453
2500
12500
2500
500
100

100

5
HA-2452-2481
500
500
500
500
100
II
<100

6
HA-2517-2538
500
500
500
500
100

<100

7
HA-2627-2668
12500
2500
500
500
100
III
<100

8
HA-2641-2684
2500
2500
2500
2500
500

<100

9
HA-2682-2703
500
500
100
100
100
IV
<100

10
HA-2695-2756
500
2500
500
500
100

100

11
HA-2703-2731
500
500
500
500
500
V
<100

12
HA-2722-2762
500
500
500
500
500

100

13
HA-2759-2814
500
500
500
500
100

<100

14
HA-2838-2866
2500
2500
>12500
>12500
>12500
VI
<100

15
NA-3676-3854
2500
12500
12500
12500
2500

100

16
NA-3431-3481
500
2500
500
500
100

<100

17
NA-3489-3530
500
500
500
500
100

<100

18
NA-3541-3576
500
100
500
500
100

<100

19
NA-3638-3662
500
100
100
100
100

<100

20
NA-3659-3689
500
500
2500
500
100

<100

21
NA-3834-3854
500
100
100
100
100

<100




















































The results show that for HA, 3 antigenic clusters are found on HA1 and three on HA2. The antigenic clusters on HA1 include amino acids 2365-2427 (SEQ ID NO:54) and 2431-2453, (SEQ ID NO:57) in antigenic cluster I, amino acids 2452-2481 (SEQ ID NO:59) and amino acids 2517 to 2538 (SEQ ID NO:60) in antigenic cluster II, and amino acids 2627-2668 of SEQ ID NO:124 and amino acids 2642-2685 of SEQ ID NO:124 in antigenic cluster III.
At least, a peptide of amino acids HA 2339-2581 (SEQ ID NO:56) shows good agreement with epitope mapping using monoclonal antibodies FLA 5.10 and FLD 21.140. Peptides of amino acids HA 2376-2659 (SEQ ID NO:55) showed good correlation with antibody binding sites of monoclonal antibody FLA3.14. At least two of the peptides also map to sites that sialic acid of the receptor are thought to bind: peptide HA-2517-2538 (SEQ ID NO:60) and peptide HA-2431-2453 (SEQ ID NO:57). These antigenic clusters are shown on the crystal structure of HA as shown in FIG. 11A .
Antigenic clusters on HA2 include: amino acids HA-2682-2703 (SEQ ID NO:63); amino acids 2695-2756 (SEQ ID NO:65); amino acids HA-2703-2731 (SEQ ID NO:66); amino acids HA-2722-2762 (SEQ ID NO:70); amino acids HA-2759-2814 (SEQ ID NO:71); and amino acids 2838-2866 (SEQ ID NO:74). These antigenic clusters are shown on the crystal structure of HA as shown in FIG. 11A .
Antigenic clusters on neuraminidase include: amino acids 3676-3854,(SEQID NO:85); amino acids 3431-3481(SEQID NO:84); 3489-3530 (SEQID NO:86); 3541-3576 (SEQID NO:88); 3639-3662 of SEQID NO:124; amino acids 3659-3689 (SEQID NO:91); and amino acids NA-3834-3854 (SEQID NO:89).
The results for other proteins encoded by the viral genome are shown in FIG. 12 a. End-point titers of individual patients and pooled sera from 20 uninfected age-matched women from Vietnam against each epitopic site were determined by ELISA against peptides derived from: PB2 (#1), PB1 (#2-3), PB1-F2 (#4-7), PA (#8), NP (#9-12), M1 (#13-16), M2e (17), M2 (#18), NS1 (#19-20), and NS2 (#21) When the peptides were analyzed by binding to individual serum samples, as shown in Table 3, other major epitopes were detected on NP, M1, and the M2 ectodomain. See Table 3
All the peptide sequences described above were obtained by affinity selection of H5N1 survivors, so any of these peptides can serve as a protective epitope. Also absence of ELISA reactivity with the uninfected Vietnam serum samples showed that these peptides can also be used for development of serodiagnosis of H5N1 infection as well as differential diagnosis of vaccine v/s infected individuals.





TABLE 3



(B) END-POINT TITER OF INDIVIDUAL POST-H5N1 EXPOSURE

SERA WITH GFPDL SELECTED PEPTIDES






IgG RESPONSES











Viet-1
Viet-2
Viet-3
Viet-4
Viet-5
Uninfected






Days Post-Admission
Vietnam










ALIGNED ID
PEPTIDE
54
69
73
113
182
Sera












1
PB2-344-375
500
500
500
100
100
<100

2
PB1-1348-1361
500
500
500
500
100
<100

3
PB1-1420-1437
500
500
500
500
100
<100

4
PB1-F2-1524-1598
500
500
500
500
100
<100

5
PB1-F2-1525-1572
500
500
500
100
100
<100

6
PB1-F2-1560-1592
500
100
500
500
100
<100

7
PB1-F2-1570-1605
500
500
500
100
100
<100

8
PA-2202-2251
500
100
500
100
100
<100

9
NP-2906-2929
500
500
100
100
100
<100

10
NP-2955-3011
500
2500
100
12500
100
100

11
NP-3263-3305
500
500
500
2500
500
<100

12
NP-3346-3384
2500
500
500
500
500
<100

13
M1-3866-3894
12500
12500
2500
500
2500
<100

14
M1-3859-3909
2500
>12500
12500
500
2500
100

15
M1-4040-4104
500
12500
100
2500
2500
100

16
M1-4080-4109
500
2500
500
2500
2500
<100

17
M2e-4114-4137
>12500
2500
>12500
2500
100
<100

18
M2-4180-4206
500
500
100
100
<100
<100

19
NS1-4235-4254
2500
2500
500
500
100
<100

20
NS1-4393-4417
500
500
500
500
100
<100

21
NS2-4467-4508
500
500
100
100
100
<100

























































Example 5
Elucidation of Epitope Profile of Individuals Vaccinated with H5N1 Subunit Vaccine
Pooled and individual serum samples were obtained from individuals vaccinated with H5N1 (A/Vietnam/1203/2004) subunit vaccine with or without adjuvant. The study has been described in Bernstein et al, JID; 2008:1977; 1-9. These serum samples were evaluated for epitope specificity. The adjuvants tested included alum and MF59 adjuvant.
The pooled serum was incubated with H5-HA-GFPDL. The B cell epitope profile of the phage that bound to the pooled sera was determined by isolating and sequencing clones that bound to the pooled sera. The results for HA and NA are shown in FIG. 13 . Those peptide sequences delineated with unfilled squares represent peptides that adsorbed neutralizing activity of the sera. In the pre-vaccine sera, a single peptide in the HA2 ectodomain was identified after 3 rounds of affmity selection. This was unexpected as these individuals have never been vaccinated or exposed to H5N1 before in their lifetime. On homology blast search, it was found that there is only one residue change in the 73 amino acid long peptide between the HA of A/Vietnam/1203/2004 and the H1N1 (A/New Caledonia/20/99) showing 98% homology. So this peptide selection is probably due to antibodies generated following vaccination or infection due to the A/New Caledonia/20/99 strain that also cross-react with the corresponding peptide sequence in HA of A/Vietnam/1203/2004.
The results show that the sera obtained from individuals vaccinated with H5N1 subunit vaccine in MF59 adjuvant had a broader antibody response to HA1 than those vaccinated with the vaccine in alum or no adjuvant. In MF59, the neutralizing antibodies were predominantly associated with antibodies to HA1. Also a shift of antibody response was observed in individuals vaccinated with MF59 adjuvant vaccine compared to the no adjuvant or Alum adjuvant vaccine. An increase in antibody response to NA was also seen in those patients vaccinated with H5N1.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.
The disclosure has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the disclosure.








TABLE 4



Influenza Peptide

Sequence ID


Name
PEPTIDE SEQUENCE
Number



PB2 571-596
YNKMEFEPFQSLVPKAIKGQYSGFV
SEQ. ID.




No. 1



PB2 696-756
LGKEDRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQT
SEQ. ID.


ATKRIRM
No. 2



PB1 918-948
GLTANESGRLIDFLKDVMESMDKEEMEITT
SEQ. ID.



No. 3



PA 1720-1748
EKPKFLPDLYDYKENRFIEIGVTRREVH
SEQ. ID.



No. 4



PA 2211-2231
IESMIEAESSIKEKD
SEQ. ID.



No. 5



HA 2369-2424
LVFAQKLPGNDNSAATLCLGHHAVPNRTMVKTITNDQIEVTNATELVQRGKT
SEQ. ID.


VESC
No. 6



HA 2388-2408
HAVPNGTIVKTITNDQIEV
SEQ. ID.



No. 7



HA 2391-2465
AGPNGTIVKTITNDQIEVTNATELVLSSSTGGICDSPHQILDGENCTLINALLGD
SEQ. ID.


PQCDGFQNKKWDLFVERSK
No. 8



HA 2634-2711
SGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKL
SEQ. ID.


ATGMRNVPEKQTRGIFGAIAGFI
No. 9



HA 2681-2717
VKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWE
SEQ. ID.



No. 10



HA 2772-2824
SEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERT
SEQ. ID.



No. 11



NP 2923-2941
SGSRVDNHSLSDIKVMASQGTKRSYEQM
SEQ. ID.



No. 12



NP 2984-2997
IQNSLTIEKMVLS
SEQ. ID.



No. 13



NP 3033-3064
RELVLYDKKKIRRIWRQANNGDDATAGLTHIM
SEQ. ID.



No. 14



NP 3322-3363
NTNQQRASAGQISTQPTFSVQRNLPFDKTTIMAAFTGNTEGRTSD
SEQ. ID.



No. 15



NP 3368-3392
RAEIIRMMEGAKPEEVSFRGRGVFE
SEQ. ID.



No. 16



NP-NA 3398-3432
PPKSDGSTSAAAAEAGVKMNPNQKIITIGSVFLTIS
SEQ. ID.



No. 17



NA 3415-3434
PPKSDGSTRAAAAEAGVKMNPNQKIITIGSVSLTISTI
SEQ. ID.



No. 18



NA 3478-3498
IVYLTNTTIEKEICPKLAEY
SEQ. ID.



No. 19



NA 3582-3626
LGTKQVCIAWSSSSCHDGKAWLHVCVTGDDKNATASFIYNGRLVD
SEQ. ID.



No. 20



NA 3631-3669
WSKEILRTQESECVCINGTCTVVMTDGSASGKADTKIL
SEQ. ID.



No. 21



NA 3754-3797
NNEEGGHGVKGWAFDDGNDVWMGRTISEKLRSGYETFKVIEG
SEQ. ID.



No. 22



NA 3868-3881
TYGTGSWPDGADINLMP
SEQ. ID.



No. 23



M1 3907-3943
VETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLE
SEQ. ID.



No. 24



M1 4007-4119
REITFHGAKEIALSYSAGALASCMGLIYNRMGAVTTESAFGLICATCEQIADSQ
SEQ. ID.


HKSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQA
No. 25


RQMVQAM



M1 4033-4084
IYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQMVATTNPLIKHENRMVLA
SEQ. ID.



No. 26



M1 4101-4161
AAEAMEVASQARQMVQAMRAIGTHPSSSTGLKNDLLENLRAYQKRMGVQM
SEQ. ID.


QRAKI
No. 27



NS1 4291-4312
KQVVDQELSDAPFLDRLRRDQ
SEQ. ID.



No. 28



NS1 4323-4360
LHIKAATHVGKQIVEKILKEESDEALKMTMVSTPASRY
SEQ. ID.



No. 29



NS1 4456-4472
PRGLEWNDNTVRVSKNLQ
SEQ. ID.



No. 30



H3-RPL-LIAV
LNPSRLEVNSGINPGPRHHGLHHARNSNRS
SEQ. ID.



No. 31



H5-HA-9-241
LFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCD
SEQ. ID.


LDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDF
No. 32


NDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWL


IKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTST


LNQRLVPRIATRSKVNGQSGRMEFFWTILK



H5-HA-RPL-112
LTAEKKGGFSS
SEQ. ID.



No. 33



H5-HA-RPL-408
VIEPPYSWLHAP
SEQ. ID.



No. 34



H5-HA-RPL-161
YTHMQDSRFSRL
SEQ. ID.



No. 35



H5-HA-RPL-486
SLSPNPLIIIR
SEQ. ID.



No. 36



H5-HA-RPL-411
SLSPKLIGSSLD
SEQ. ID.



No. 37



H5-HA-RPL-102
LTAEKKKKKFFI
SEQ. ID.



No. 38



H5-HA-RPL-462
SLSPKTMHHHQT
SEQ. ID.



No. 39



H5-HA-RPL-457
FQTHMHHPFNQI
SEQ. ID.



No. 40



H5-HA-RPL-101
LTAETQIQFFHH
SEQ. ID.



No. 41



H5-PB2-344-375
EVLTGNLQTLKIRVHEGYEEFTMVGRRATAILR
SEQ. ID.



No. 42



H5-PB2-447-516
QNWGIEPIDNVMGMIGILPDMTPSTEMSLRGVRVSKMGVDEYSSTERVVVSID
SEQ. ID.


RFLRVRDQRGNVLLSPE
No. 43



H5-PB1-1290-1451
TVIKNNMINNDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFELKKL
SEQ. ID.


WEQTRSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCNPLNPF
No. 44


VSHKEIEVNNAVVMPAHGPAKSMEYDAVATTHSWIPKRNRSILNTSQRGILED


EQMY



H5-PB1-1348-1361
KAGLLVSDGGPNLY
SEQ. ID.



No. 45



H5-PB1-1420-1437
DAVATTHSWIPKRNRSIL
SEQ. ID.



No. 46



H5-PB1-F2-1524-1598
EQGQDTPWTQSTEHTNIQKRGSGQQTQRLEHPNSTRLMDHYLRIMSPVGTHK
SEQ. ID.


QIVYWKQWLSLKNPTQGSLKTR
No. 47



H5-PB1-F2-1525-1572
QGQDTPWTQSTEHTNIQKRGSGQQTQRLEHPNSTRLMDHYLRIMSPVG
SEQ. ID.



No. 48



H5-PB1-F2-1548-1608
QTQRLEHPNSTRLMDHYLRIMSPVGTHKQIVYWKQWLSLKNPTQGSLKTRVL
SEQ. ID.


KRWKLFNKQ
No. 49



H5-PB1-F2-1560-1592
LMDHYLRIMSPVGTHKQIVYWKQWLSLKNPTQG
SEQ. ID.



No. 50



H5-PB1-F2-1570-1605
PVGTHKQIVYWKQWLSLKNPTQGSLKTRVLKRWKLF
SEQ. ID.



No. 51



H5-PA-1852-1966
EPNGCIEGKLSQMSKEVNARIEPFLKTTPRPLRLPDGPPCSQRSKFLLMDALKL
SEQ. ID.


SIEDPSHEGEGIPLYDAIKCMKTFFGWKEPNIVKPHEKGINPNYLLAWKQVLAE
No. 52


LQDIENE



H5-PA-2202-2251
QSLQQIESMIEAESSVKEKDMTKEFFENKSETWPIGESPKGVEEGSIGKV
SEQ. ID.



No. 53



H5-HA-2365-2427
VDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMC
SEQ. ID.


DEFINVPEWS
No. 54



H5-HA-2376-2659
VTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWS
SEQ. ID.


YIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVS
No. 55


SACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAA


EQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAI


NFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFH


NIHPLTIGECPKYVKSN



H5-HA-2339-2581
VLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKL
SEQ. ID.


CDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPG
No. 56


DFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVV


WLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVG


TSTLNQRLVPRIATRSKVNGQSGRMEFFWT



H5-HA-2431-2453
EKANPVNDLCYPGDFNDYEELKH
SEQ. ID.



No. 57



H5-HA-2436-2543
NDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWL
SEQ. ID.


IKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQN
No. 58



H5-HA-2452-2481
KHLLSRINHFEKIQIIPKSSWSSHEASLGV
SEQ. ID.



No. 59



H5-HA-2517-2538
TNQEDLLVLWGIHHPNDAAEQT
SEQ. ID.



No. 60



H5-HA-2520-2566
EDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATR
SEQ. ID.



No. 61



H5-HA-2568-2649
KVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEY
SEQ. ID.


GNCNTKCQTPMGAINSSMPFHNIHPLTI
No. 62



H5-HA-2682-2703
LFGAIAGFIEGGWQGMVDGWYG
SEQ. ID.



No. 63



H5-HA-2686-2748
IAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIID
SEQ. ID.


KMNTQFEAVGR
No. 64



H5-HA-2695-2756
QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEA
SEQ. ID.


VGREFNNLERR
No. 65



H5-HA-2703-2731
GYHHSNEQGSGYAADKESTQKAIDGVTNK
SEQ. ID.



No. 66



H5-HA-2706-2808
HSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIE
SEQ. ID.


NLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRD
No. 67



H5-HA-2706-2843
HSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIE
SEQ. ID.


NLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRD
No. 68


NAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYS



H5-HA-2707-2753
SNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNL
SEQ. ID.



No. 69



H5-HA-2722-2762
QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNK
SEQ. ID.



No. 70



H5-HA-2759-2814
NLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRD
SEQ. ID.


NAKELG
No. 71



H5-HA-2823-2866
KCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQI
SEQ. ID.



No. 72



H5-HA-2829-2864
MESVRNGTYDYPQYSEEARLKREEISGVKLESIGIY
SEQ. ID.



No. 73



H5-HA-2838-2866
DYPQYSEEARLKREEISGVKLESIGIYQI
SEQ. ID.



No. 74



H5-NP-2906-2929
MASQGTKRSYEQMETGGERQNATE
SEQ. ID.



No. 75



H5-NP-2915-3011
YEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDYEGRLIQNSITI
SEQ. ID.


ERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVR
No. 76



H5-NP-2915-3068
YEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDYEGRLIQNSITI
SEQ. ID.


ERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVRELILYDKEEI
No. 77


RRIWRQANNGEDATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRM



H5-NP-2939-3055
SGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVLSAFDERRNRYLEEHPSAGKD
SEQ. ID.


PKKTGGPIYRRRDGKWVRELILYDKEEIRRIWRQANNGEDATAGLTHLMIWH
No. 78


SNLNDATYQR



H5-NP-2955-3011
SDYEGRLIQNSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGK
SEQ. ID.


WVR
No. 79



H5-NP-3197-3229
EREGYSLVGIDPFRLLQNSQVFSLIRPNENPAH
SEQ. ID.



No. 80



H5-NP-3214-3261
NSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTRVVPRG
SEQ. ID.



No. 81



H5-NP-3263-3305
LSTRGVQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQR
SEQ. ID.



No. 82



H5-NP-3329-3399
TIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKATNPIVP
SEQ. ID.


SFDMNNEGSYFFGDNAE
No. 83



H5-NA-3431-3481
QIGNMISIWVSHSIHTGNQHQSEPISNTNFLTEKAVASVKLAGNSSLCPIN
SEQ. ID.



No. 84



H5-NA-3469-3504
VKLAGNSSLCPINGWAVYSKDNSIRIGSKGDVFVIR
SEQ. ID.



No. 85



H5-NA-3489-3530
DNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKH
SEQ. ID.



No. 86



H5-NA-3522-3541
QGALLNDKHSNGTVKDRSPH
SEQ. ID.



No. 87



H5-NA-3541-3576
HRTLMSCPVGEAPSPYNSRFESVAWSASACHDGTSW
SEQ. ID.



No. 88



H5-NA-3548-3613
PVGEAPSPYNSRFESVAWSASACHDGTSWLTIGISGPDNGAVAVLKYNGIITDT
SEQ. ID.


IKSWRNNILRTQ
No. 89



H5-NA-3618-3662
ACVNGSCFTVMTDGPSNGQASHKIFKMEKGKVVKSVELDAPNYHY
SEQ. ID.



No. 90



H5-NA-3659-3689
NYHYEECSCYPNAGEITCVCRDNWHGSNRPW
SEQ. ID.



No. 91



H5-NA-3676-3854
CVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSSN
SEQ. ID.


GAYGVKGFSFKYGNGVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDI
No. 92


VAITDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGV


NSDTVGWSWPDGAELPFTID



H5-NA-3704-3840
CSGVFGDNPRPNDGTGSCGPVSSNGAYGVKGFSFKYGNGVWIGRTKSTNSRS
SEQ. ID.


GFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPELTGLDCIRPCF
No. 93


WVELIRGRPKESTIWTSGSSISFCGVNSDTVG



H5-NA-3758-3809
EMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPELTGLDCIRPCFW
SEQ. ID



No. 94



H5-NA-3821-3854
TIWTSGSSISFCGVNSDTVGWSWPDGAELPFTID
SEQ. ID.



No. 95



H5-NA-3834-3854
VNSDTVGWSWPDGAELPFTID
SEQ. ID.



No. 96



H5-M1-3859-3889
MSLLTEVETYVLSIIPSGPLKAEIAQKLEDV
SEQ. ID.



No. 97



H5-M1-3859-4013
MSLLTEVETYVLSIIPSGPLKAEIAQKLEDVFAGKNTDLEALMEWLKTRPILSPL
SEQ. ID.


TKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDRAVKLYKKLKREI
No. 98


TFHGAKEVALSYSTGALASCMGLIYNRMGTVTTEVAFGLVCATCEQIA



H5-M1-3863-3909
TEVETYVLSIIPSGPLKAEIAQKLEDVFAGKNTDLEALMEWLKTRPI
SEQ. ID.



No. 99



H5-M1-3866-3894
ETYVLSIIPSGPLKAEIAQKLEDVFAGKN
SEQ. ID.



No. 100



H5-M1-3989-4048
IYNRMGTVTTEVAFGLVCATCEQIADSQHRSHRQMATITNPLIRHENRMVLAS
SEQ. ID.


TTAKAME
No. 101



H5-M1-4002-4110
FGLVCATCEQIADSQHRSHRQMATITNPLIRHENRMVLASTTAKAMEQMAGS
SEQ. ID.


SEQAAEAMEIANQARQMVQAMRTIGTHPNSSAGLRDNLLENLQAYQKRMGV
No. 102


QMQRFK



H5-M1-4040-4104
ASTTAKAMEQMAGSSEQAAEAMEIANQARQMVQAMRTIGTHPNSSAGLRDN
SEQ. ID.


LLENLQAYQKRMGV
No. 103



H5-M1-4054-4110
SEQAAEAMEIANQARQMVQAMRTIGTHPNSSAGLRDNLLENLQAYQKRMGV
SEQ. ID.


QMQRFK
No. 104



H5-M1-4080-4109
HPNSSAGLRDNLLENLQAYQKRMGVQMQRF
SEQ. ID.



No. 105



H5-M2-4115-4137
SLLTEVETPTRNEWECRCSDSSD
SEQ. ID.



No. 106



H5-M2-4181-4209
VPESMREEYRQEQQSAVDVDDGHFVNIEL
SEQ. ID.



No. 107



H5-NS1-4220-4284
SSFQVDCFLWHVRKRFADQELGDAPFLDRLRRDQKSLRGRGNTLGLDIETATR
SEQ. ID.


AGKQIVERILEG
No. 108



H5-NS1-4236-4255
ADQELGDAPFLDRLRRDQKS
SEQ. ID.



No. 109



H5-NS1-4283-4294
EGESDKALKMPA
SEQ. ID.



No. 110



H5-NS1-4378-4397
TGEDVKNAIGVLIGGLEWND
SEQ. ID.



No. 111



H5-NS1-4393-4428
LEWNDNTVRVTETIQRFAWRNSDEDGRLPLPPNQKR
SEQ. ID.



No. 112



H5-NS2-4468-4509
SLKLYRDSLGETVMRMGDFHSLQIRNGKWREQLSQKFEEIRW
SEQ. ID.



No. 113



348-H5-HA-RPL
WTPIHLTTKVTL
SEQ. ID.



No. 114



368-H5-HA-RPL
WSYSWFYNTSYE
SEQ. ID.



No. 115



A2.19-RPL-227
VWNPYIWSAPFS
SEQ. ID.



No. 116



A2.19-RPL-121
GVWPNATHFPSS
SEQ. ID.



No. 117



21E12-RPL-315
WWDTPHSWWTMR
SEQ. ID.



No. 118



3F3-RPL-108
WGLFGVSPHVQS
SEQ. ID.



No. 119



3F3-RPL-101
QARWPVTSPYWP
SEQ. ID.



No. 120



H5-HA-47-338
AQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVE
SEQ. ID.


KANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACP
No. 121


YQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQT


KLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFE


SNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHP


LTIGECPKYVKSNRLVLATGLRNS



H5-HA-2627-2669
KCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRN
SEQ. ID.



No. 122



H5-HA-2642-2685
HNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGA
SEQ. ID.



No. 123

































































































































































































































































































































































































































TABLE 5



FREQUENCY OF SELECTED PHAGE CLONES WITH H5N1 EXPOSED SURVIVORS USING H5N1 GENE-


FRAGMENT PHAGE DISPLAY LIBARIES






Influenza Peptide

FRE-


Name
PEPTIDE SEQUENCE
QUENCY









H5-PB2-344-375


EVLTGNLQTLKIRVHEGYEEFTMVGRRATAILR


9





H5-PB2-447-516
QNWGIEPIDNVMGMIGILPDMTPSTEMSLRGVRVSKMGVDEYSSTERVVVSIDRFLRVRDQRGNVLLSPE
2



H5-PB1-1290-1451
TVIKNNMINNDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGPNLYN
2


IRNLHIPEVCLKWELMDEDYQGRLCNPLNPFVSHKEIEVNNAVVMPAHGPAKSMEYDAVATTHSWIPKRNRSI


LNTSQRGILEDEQMY




H5-PB1-1348-1361


KAGLLVSDGGPNLY


7





H5-PB1-1420-1437


DAVATTHSWIPKRNRSIL


6





H5-PB1-F2-1524-1598


EQGQDTPWTQSTEHTNIQKRGSGQQTQRLEHPNSTRLMDHYLRIMSPVGTHKQIVYWKQWLSLKNPTQGSLKTR


13





H5-PB1-F2-1525-1572


QGQDTPWTQSTEHTNIQKRGSGQQTQRLEHPNSTRLMDHYLRIMSPVG


5




H5-PB1-F2-1548-1608
QTQRLEHPNSTRLMDHYLRIMSPVGTHKQIVYWKQWLSLKNPTQGSLKTRVLKRWKLFNKQ
4




H5-PB1-F2-1560-1592


LMDHYLRIMSPVGTHKQIVYWKQWLSLKNPTQG


6





H5-PB1-F2-1570-1605


PVGTHKQIVYWKQWLSLKNPTQGSLKTRVLKRWKLF


7




H5-PA-1852-1966
EPNGCIEGKLSQMSKEVNARIEPFLKTTPRPLRLPDGPPCSQRSKFLLMDALKLSIEDPSHEGEGIPLYDAIK
3


CMKTFFGWKEPNIVKPHEKGINPNYLLAWKQVLAELQDIENE



H5-PA-1904-1927
KLSIEDPSHEGEGIPLYDAIKCMK (SEQ ID NO: 148)
1



H5-PA-1945-1973
INPNYLLAWKQVLAELQDIENEEKIPKTK (SEQ ID NO: 149)
1




H5-PA-2202-2251


QSLQQIESMIEAESSVKEKDMTKEFFENKSETWPIGESPKGVEEGSIGKV


8




H5-HA-2349-2389
KSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNG(SEQ ID NO: 150)
1




H5-HA-2365-2427


VDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWS


9





H5-HA-2376-2659


VTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDY


17




EELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQED




LLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESN




GNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSN





H5-HA-2339-2581


VLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWL


13




LGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSA




CPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQ




RLVPRIATRSKVNGQSGRMEFFWT





H5-HA-2431-2453

EKANPVNDLCYPGDFNDYEELKH (SEQ ID NO: 151)

36




H5-HA-2436-2543
NDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTN
3


QEDLLVLWGIHHPNDAAEQTKLYQN (SEQ ID NO: 152)




H5-HA-2452-2481


KHLLSRINHFEKIQIIPKSSWSSHEASLGV


26




H5-HA-2484-2514
ACPYQGKSSFFRNVVWLIKKNSTYPTIKRSY
1




H5-HA-2517-2538


TNQEDLLVLWGIHHPNDAAEQT


16




H5-HA-2520-2566
EDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATR
9



H5-HA-2568-2649
KVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMP
4


FHNIHPLTI



H5-HA-2603-2637
AYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINS (SEQ ID NO: 153)
3



H5-HA-2614-2639
IMKSELEYGNCNTKCQTPMGAINSSM (SEQ ID NO: 154)
1




H5-HA-2627-2669

KCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRN (SEQ ID NO: 155)

17




H5-HA-2632-2651
MGAINSSMPFHNIHPLTIGE (SEQ ID NO: 156)
2




H5-HA-2642-2685

HNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGA (SEQ ID NO: 157)

9




H5-HA-2648-2670
TIGECPKYVKSNRLVLATGLRNS (SEQ ID NO: 158)
2




H5-HA-2682-2703


LFGAIAGFIEGGWQGMVDGWYG


13




H5-HA-2686-2748
IAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGR
4




H5-HA-2695-2756


QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERR


34





H5-HA-2703-2731


GYHHSNEQGSGYAADKESTQKAIDGVTNK


26




H5-HA-2703-2753
GYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNL
3



H5-HA-2706-2808
HSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAEL
36


LVLMENERTLDFHDSNVKNLYDKVRLQLRD



H5-HA-2706-2843
HSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAEL
19


LVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYS



H5-HA-2707-2753
SNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNL
17



H5-HA-2707-2786
SNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELL
4


VLMENER (SEQ ID NO: 159)



H5-HA-2709-2727
EQGSGYAADKESTQKAIDG (SEQ ID NO: 160)
1



H5-HA-2711-2730
GSGYAADKESTQKAIDGVTN (SEQ ID NO: 161)
6



H5-HA-2716-2791
ADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTL
3


DFH (SEQ ID NO: 162)



H5-HA-2722-2749
QKAIDGVTNKVNSIIDKMNTQFEAVGRE (SEQ ID NO: 163)
8



H5-HA-2724-2756
AIDGVTNKVNSIIDKMNTQFEAVGREFNNLERR (SEQ ID NO: 164)
12




H5-HA-2722-2762


QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNK


9




H5-HA-2729-2750
TNKVNSIIDKMNTQFEAVGREF (SEQ ID NO: 165)
5




H5-HA-2759-2814


NLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELG


54




H5-HA-2805-2860
QLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLES (SEQ ID NO: 166)
2



H5-HA-2805-2828
QLRDNAKELGNGCFEFYHKCDNEC (SEQ ID NO: 167)
1



H5-HA-2823-2866
KCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQI
172



H5-HA-2829-2864
MESVRNGTYDYPQYSEEARLKREEISGVKLESIGIY
32




H5-HA-2838-2866


DYPQYSEEARLKREEISGVKLESIGIYQI


154





H5-NP-2906-2929


MASQGTKRSYEQMETGGERQNATE


8




H5-NP-2915-3011
YEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVLSAFDERRNRYLEEH
13


PSAGKDPKKTGGPIYRRRDGKWVR



H5-NP-2915-3068
YEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVLSAFDERRNRYLEEH
3


PSAGKDPKKTGGPIYRRRDGKWVRELILYDKEEIRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQRTRALV


RTGMDPRM



H5-NP-2939-3055
SGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVR
6


ELILYDKEEIRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQR




H5-NP-2955-3011


SDYEGRLIQNSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGRWVR


26




H5-NP-2964-3022
NSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVRELILYDKEEIR(SEQID NO: 168)
11



H5-NP-3197-3229
EREGYSLVGIDPFRLLQNSQVFSLIRPNENPAH
2



H5-NP-3214-3261
NSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTRVVPRG
3




H5-NP-3263-3305


LSTRGVQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNOQR


8




H5-NP-3329-3399
TIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKATNPIVPSFDMNNEGSYFFGDNAE
2




H5-NP-3347-3385

TEIIRMMESARPEDVSFQGRGVFELSDEKATNPIVPSFD (SEQ ID NO: 169)

9





H5-NA-3431-3481


QIGNMISIWVSHSIHTGNQHQSEPISNTNFLTEKAVASVKLAGNSSLCPIN


7




H5-NA-3453-3480
EPISNTNFLTEKAVASVKLAGNSSLCPI (SEQ ID NO: 170)
3



H5-NA-3469-3504
VKLAGNSSLCPINGWAVYSKDNSIRIGSKGDVFVIR
2




H5-NA-3489-3530


DNSIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKH


11




H5-NA-3522-3541
QGALLNDKHSNGTVKDRSPH
1




H5-NA-3541-3576


HRTLMSCPVGEAPSPYNSRFESVAWSASACHDGTSW


9




H5-NA-3548-3613
PVGEAPSPYNSRFESVAWSASACHDGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQ
1



H5-NA-3578-3606
TIGISGPDNGAVAVLKYNGIITDTIKSWR (SEQ ID NO: 171)
2



H5-NA-3618-3650
ACVNGSCFTVMTDGPSNGQASHKIFKMEKGKVV (SEQ ID NO: 172)
1



H5-NA-3618-3662
ACVNGSCFTVMTDGPSNGQASHKIFKMEKGKVVKSVELDAPNYHY
4




H5-NA-3638-3662

HKIFKMEKGKVVKSVELDAPNYHY (SEQ ID NO: 173)

8





H5-NA-3659-3689


NYHYEECSCYPNAGEITCVCRDNWHGSNRPW


9





H5-NA-3676-3854


CVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGAYGVKGFSFKYGNGVWIGRT


39




KSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKEST




IWTSGSSISFCGVNSDTVGWSWPDGAELPFTID




H5-NA-3703-3723
ICSGVFGDNPRPNDGTGSCGP (SEQ ID NO: 174)
2



H5-NA-3704-3840
CSGVFGDNPRPNDGTGSCGPVSSNGAYGVKGFSFKYGNGVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVK
23


QDIVAITDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVG



H5-NA-3758-3809
EMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPELTGLDCIRPCFW
2



H5-NA-3821-3854
TIWTSGSSISFCGVNSDTVGWSWPDGAELPFTID
4




H5-NA-3834-3854


VNSDTVGWSWPDGAELPFTID


19





H5-M1-3859-3889


MSLLTEVETYVLSIIPSGPLKAEIAQKLEDV


65




H5-M1-3859-4013
MSLLTEVETYVLSIIPSGPLKAEIAQKLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVPSERG
2


LQRRRFVQNALNGNGDPNNMDRAVKLYKKLKREITFHGAKEVALSYSTGALASCMGLIYNRMGTVTTEVAFGL


VCATCEQIA



H5-M1-3863-3909
TEVETYVLSIIPSGPLKAEIAQKLEDVFAGKNTDLEALMEWLKTRPI
35




H5-M1-3866-3894


ETYVLSIIPSGPLKAEIAQKLEDVFAGKN


102




H5-M1-3989-4048
IYNRMGTVTTEVAFGLVCATCEQIADSQHRSHRQMATITNPLIRHENRMVLASTTAKAME
3



H5-M1-4002-4110
FGLVCATCEQIADSQHRSHRQMATITNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEIANQARQMVQAM
10


RTIGTHPNSSAGLRDNLLENLQAYQKRMGVQMQRFK



H5-M1-4012-4055
IADSQHRSHRQMATITNPLIRHENRMVLASTTAKAMEQMAGSSE (SEQ ID NO: 175)
5




H5-M1-4040-4104


ASTTAKAMEQMAGSSEQAAEAMEIANQARQMVQAMRTIGTHPNSSAGLRDNLLENLQAYQRMGV


174




H5-M1-4050-4078
MAGSSEQAAEAMEIANQARQMVQAMRTIG (SEQ ID NO: 176)
2



H5-M1-4054-4110
SEQAAEAMEIANQARQMVQAMRTIGTHPNSSAGLRDNLLENLQAYQKRMGVQMQRFK
88




H5-M1-4080-4109


HPNSSAGLRDNLLENLQAYQKRMGVQMQRF


45





H5-M2-4115-4123


SLLTEVETP


37





H5-M2-4124-4138


TRNEWECRCSDSSDP


29





H5-M2-4181-4209


VPESMREEYRQEQQSAVDVDDGHFVNIEL


17




H5-NS1-4220-4284
SSFQVDCFLWHVRKRFADQELGDAPFLDRLRRDQKSLRGRGNTLGLDIETATRAGKQIVERILEG
10




H5-NS1-4236-4255


ADQELGDAPFLDRLRRDQKS


6




H5-NS1-4283-4294
EGESDKALKMPA
1



H5-NS1-4378-4397
TGEDVKNAIGVLIGGLEWND
1




H5-NS1-4393-4428


LEWNDNTVRVTETIQRFAWRNSDEDGRLPLPPNQKR


5





H5-NS2-4468-4509


SLKLYRDSLGETVMRMGDFHSLQIRNGKWREQLSQKFEEIRW


5






































































































































































































































































TABLE 6





Positions from 1 till 60



Consensus sequence
ICKMEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKL



ABD16284
A/Thailand/NK165/2005 (H5N1)
-----R....F.................................................



ABE01046
A/Egypt/2782-NAMRU3/2006 (H5N1)
---------------------.......................................



ABI36040
A/Indonesia/CDC184/2005 (H5N1)
-------------------.........................................



ABI36041
A/Indonesia/CDC194P/2005 (H5N1)
-------------------.........................................



ABI36044
A/Indonesia/CDC292N/2005 (H5N1)
-------------------.........................................



ABI36045
A/Indonesia/CDC292T/2005 (H5N1)
-------------------.........................................



ABI36046
A/Indonesia/CDC326N/2006 (H5N1)
-------------------.........................................



ABI36047
A/Indonesia/CDC326N2/2006 (H5N1)
-------------------.........................................



ABI36049
A/Indonesia/CDC326T/2006 (H5N1)
-------------------.........................................



ABI36056
A/Indonesia/CDC370T/2006 (H5N1)
-------------------.........................................



ABI36057
A/Indonesia/CDC390/2006 (H5N1)
-------------------.........................................



ABM90544
A/Indonesia/CDC1047S/2007 (H5N1)
---........S................................................



ABU53968
A/Egypt/2629-NAMRU3/2007 (H5N1)
-------------------.........................................



ABU53969
A/Egypt/2630-NAMRU3/2007 (H5N1)
-------------------.........................................



ABU53970
A/Egypt/2631-NAMRU3/2007 (H5N1)
-------------------.........................................



ABU53971
A/Egypt/2750-NAMRU3/2007 (H5N1)
-------------------.........................................



ABW74701
A/Indonesia/TLL001/2006 (H5N1)
---.........................................................



ABW74704
A/Indonesia/TLL004/2006 (H5N1)
---.........................................................



ABW74706
A/Indonesia/TLL006/2006 (H5N1)
---.........................................................



ABW74707
A/Indonesia/TLL007/2006 (H5N1)
---.........................................................



AAD52043
A/Hong Kong/485/97 (H5N1)
---.........T........................................R......



ABI36198
A/Indonesia/CDC523/2006 (H5N1)
---.........................................................



AAF74330
A/Hong Kong/483/97 (H5N1)
---.........T........................................R......



AAF74331
A/Hong Kong/486/97 (H5N1)
---.........T........................................R......



ABC72655
A/Thailand/676/2005 (H5N1)
---.......F.................................................



ABE97626
A/Vietnam/CL17/2004 (H5N1)
-----------------------------------.........................



ABE97633
A/Vietnam/CL119/2005 (H5N1)
---.......F.................................................





Positions from 61 till 120


Consensus sequence
C DLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEEL



ABD16284
A/Thailand/NK165/2005 (H5N1)
. ........................................P..........D.......



ABE01046
A/Egypt/2782-NAMRU3/2006 (H5N1)
. ............................L...........I..................



A3I36040
A/Indonesia/CDC184/2005 (H5N1)
. ...........................................T.......S.......



ABI36041
A/Indonesia/CDC194P/2005 (H5N1)
. ...........................................T.G.....S.......



ABI36044
A/Indonesia/CDC292N/2005 (H5N1)
. ...........................................T.......S.......



ABI36045
A/Indonesia/CDC292T/2005 (H5N1)
. ...........................................T.......S.......



ABI36046
A/Indonesia/CDC326N/2006 (H5N1)
. ...........................................T.......S.......



A5I36047
A/Indonesia/CDC326N2/2006 (H5N1)
. ...........................................T.......S.......



ABI36049
A/Indonesia/CDC326T/2006 (H5N1)
. ...........................................T.......S.......



ABI36056
A/Indonesia/CDC370T/2006 (H5N1)
. ..........K................................T.......S.......



ABI36057
A/Indonesia/CDC390/2006 (H5N1)
. ..........K................................T.......S.......



ABM90544
A/Indonesia/CDC1047S/2007 (H5N1)
. ..........K................................T.......S.......



ABU53968
A/Egypt/2629-NAMRU3/2007 (H5N1)
. N...........................L...........I..................



ABU53969
A/Egypt/2630-NAMRU3/2007 (H5N1)
. ............................L...........I..................



ABU53970
A/Egypt/2631-NAMRU3/2007 (H5N1)
. N...........................L...........I..................



ABU53971
A/Egypt/2750-NAMRU3/2007 (H5N1)
. ............................L...........I..........D.......



ABW74701
A/Indonesia/TLL001/2006 (H5N1)
. ...........................................T.......S.......



ABW74704
A/Indonesia/TLL004/2006 (H5N1)
. ............................T..............T.......S.......



ABW74706
A/Indonesia/TLL006/2006 (H5N1)
. ...........................................T.......S.......



ABW74707
A/Indonesia/TLL007/2006 (H5N1)
. ...........................................T.......S.......



AAD52043
A/Hong Kong/485/97 (H5N1)
. ..N......................................S.................



ABI36198
A/Indonesia/CDC523/2006 (H5N1)
. ...........................................T.......S.......



AAC32099
A/Hong Kong/483/97 (H5N1)
. ..N......................................S.................



AAC40508
A/Hong Kong/156/97 (H5N1)
. ..N......................................S.................



AAD21153
A/Hong Kong/486/97 (H5N1)
. ..N......................................S.................



ABE97624
A/Vietnam/CL01/2004 (H5N1)
. ...........................................V.......D.......



ABE97628
A/Vietnam/CL26/2004 (H5N1)
. ...........................................V.......D.......



ABE97629
A/Vietnam/CL36/2004 (H5N1)
. ...........................................V.......D.......



ABE97630
A/Vietnam/CL100/2004 (H5N1)
. ..........K..............................D.V...............



ABE97631
A/Vietnam/CL105/2005 (H5N1)
. ........................................V..V.......V.......





Positions from 121 till 180


Consensus sequence
KHLLSRINHFEKIAQIIPKSSWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKNSTYPTI



ABD16284
A/Thailand/NK165/2005 (H5N1)
.............-.........S....V..........K....................



ABE01046
A/Egypt/2782-NAMRU3/2006 (H5N1)
.............-.......................................DNA....



ABI36040
A/Indonesia/CDC184/2005 (H5N1)
.............-.......................L.SP...................



ABI36041
A/Indonesia/CDC194P/2005 (H5N1)
.............-.......................L.SP...................



ABI36044
A/Indonesia/CDC292N/2005 (H5N1)
.............-.......................L.SP...................



ABI36045
A/Indonesia/CDC292T/2005 (H5N1)
.............-.......................L.SP...................



ABI36046
A/Indonesia/CDC326N/2006 (H5N1)
.............-.......................L.SP...................



ABI36047
A/Indonesia/CDC326N2/2006 (H5N1)
.............-.......................L.SP...................



ABI36049
A/Indonesia/CDC326T/2006 (H5N1)
.............-.......................L.SP...................



ABI36056
A/Indonesia/CDC370T/2006 (H5N1)
.............-.......................L.SP...................



ABI36057
A/Indonesia/CDC390/2006 (H5N1)
.............-.......................L.SP...................



ABM90544
A/Indonesia/CDC1047S/2007 (H5N1)
.............-.......................L.SP...................



ABU53968
A/Egypt/2629-NAMRU3/2007 (H5N1)
.............-.....N........-.....................T..DNA....



ABU53969
A/Egypt/2630-NAMRU3/2007 (H5N1)
.............-.......................................DNA....



ABU53970
A/Egypt/2631-NAMRU3/2007 (H5N1)
.............-.....N........-.....................T..DNA....



ABU53971
A/Egypt/2750-NAMRU3/2007 (H5N1)
.............-........................................NA....



ABW74701
A/Indonesia/TLL001/2006 (H5N1)
.............-.......................L.S....................



ABW74704
A/Indonesia/TLL004/2006 (H5N1)
.............-.......................L.SP.....A.............



ABW74706
A/Indonesia/TLL006/2006(H5N1)
.............-.......................L.SP...................



ABW74707
A/Indonesia/TLL007/2006(H5N1)
.............-.......................L.SP...................



AAD52043
A/Hong Kong/485/97(H5N1)
.............-.........N.D...........L.................S....



ABI36198
A/Indonesia/CDC523/2006(H5N1)
.............-.......................L.SP...................



AAC32099
A/Hong Kong/483/97(H5N1)
.......S.....-.........N.D...........L.K....................



AAC40508
A/Hong Kong/156/97(H5N1)
.............-.........N.D...........L.................A....



AAD21153
A/Hong Kong/486/97(H5N1)
.............-.........N.D...........L.................A....



ABE97625
A/Vietnam/CL02/2004(H5N1)
.............-.........S....L..........E....................



ABE97627
A/Vietnam/CL20/2004(H5N1)
.............-.........S....L..........K....................



ABE97632
A/Vietnam/CL115/2005(H5N1)
.............-.........S....L...A......K....................





Positions from 181 till 240


Consensus sequence
KRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVN



ABD16284
A/Thailand/NK165/2005(H5N1)
............................K......................R........



ABE01046
A/Egypt/2782-NAMRU3/2006(H5N1)
............................................................



ABI36040
A/Indonesia/CDC184/2005(H5N1)
.K.....................G...............I....................



ABI36041
A/Indonesia/CDC194P/2005(H5N1)
.K.....................................I....................



ABI36044
A/Indonesia/CDC292N/2005(H5N1)
.K.....................................I....................



ABI36045
A/Indonesia/CDC292T/2005(H5N1)
.K.....................................I....................



ABI36046
A/Indonesia/CDC326N/2006(H5N1)
.K.....................................I....................



ABI36047
A/Indonesia/CDC326N2/2006(H5N1)
.K.....................................I....................



ABI36049
A/Indonesia/CDC326T/2006(H5N1)
.K.....................................I....................



ABI36056
A/Indonesia/CDC370T/2006(H5N1)
.K.....................................I....................



ABI36057
A/Indonesia/CDC390/2006(H5N1)
.E.....................................I....................



ABM90544
A/Indonesia/CDC1047S/2007(H5N1)
.K....................NEE..............I....................



ABU53968
A/Egypt/2629-NAMRU3/2007(H5N1)
............................................................



ABU53969
A/Egypt/2630-NAMRU3/2007(H5N1)
............................................................



ABU53970
A/Egypt/2631-NAMRU3/2007(H5N1)
............................................................



ABU53971
A/Egypt/2750-NAMRU3/2007(H5N1)
............................................................



ABW74701
A/Indonesia/TLL001/2006(H5N1)
.K.....................................I....................



ABW74704
A/Indonesia/TLL004/2006(H5N1)
.K.....................................I....................



ABW74706
A/Indonesia/TLL006/2006(H5N1)
.K.....................................I....................



ABW74707
A/Indonesia/TLL007/2006(H5N1)
.K.....................................I............M.......



AAD52043
A/Hong Kong/485/97(H5N1)
...................................................E....P...



ABI36198
A/Indonesia/CDC523/2006(H5N1)
.K....................N................I....................



ABE97626
A/Vietnam/CL17/2004(H5N1)
..............M.............K......................R........



ABE97633
A/Vietnam/CL119/2005(H5N1)
..............M....................................R........



AAC32099
A/Hong Kong/483/97(H5N1)
............................K......................E....P...



AAC40508
A/Hong Kong/156/97(H5N1)
.................V..........K......................E....P...



AAD21153
A/Hong Kong/486/97(H5N1)
............................K......................E....P...



ABE97627
A/Vietnam/CL20/2004(H5N1)
..............M.............K......................R........



ABE97632
A/Vietnam/CL115/2005(H5N1)
..............M............AK......................R........



ABE97634
A/Vietnam/CL2009/2005(H5N1)
..............M............AK......................R........





Positions from 241 till 300


Consensus sequence
GQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTPKCQ



ABD16284
A/Thailand/NK165/2005(H5N1)
........................................................-...



ABE01046
A/Egypt/2782-NAMRU3/2006(H5N1)
..............S................N........................-...



ABI36040
A/Indonesia/CDC184/2005(H5N1)
..........................................A.............-...



ABI36041
A/Indonesia/CDC194P/2005(H5N1)
..........................................A.............-...



ABI36044
A/Indonesia/CDC292N/2005(H5N1)
.............N............................A.............-...



ABI36045
A/Indonesia/CDC292T/2005(H5N1)
.............N............................A.............-...



ABI36046
A/Indonesia/CDC326N/2006(H5N1)
.............N............................A.............-...



ABI36047
A/Indonesia/CDC326N2/2006(H5N1)
.............N............................A.............-...



ABI36049
A/Indonesia/CDC326T/2006(H5N1)
.............N............................A.............-...



ABI36056
A/Indonesia/CDC370T/2006(H5N1)
..........................................A.............-...



ABI36057
A/Indonesia/CDC390/2006(H5N1)
..........................................A.............-...



ABM90544
A/Indonesia/CDC1047S/2007(H5N1)
..........................................A........S....-...



ABU53968
A/Egypt/2629-NAMRU3/2007(H5N1)
..............S................N........................-...



ABU53969
A/Egypt/2630-NAMRU3/2007(H5N1)
..............S................N........................-...



ABU53970
A/Egypt/2631-NAMRU3/2007(H5N1)
..............S................N........................-...



ABU53971
A/Egypt/2750-NAMRU3/2007(H5N1)
..............S.........................................-...



ABW74701
A/Indonesia/TLL001/2006(H5N1)
..........................................A.............-...



ABW74704
A/Indonesia/TLL004/2006(H5N1)
..........................................A.............-...



ABW74706
A/Indonesia/TLL006/2006(H5N1)
..........................................A.............-...



ABW74707
A/Indonesia/TLL007/2006(H5N1)
..........................................A.............-...



AAD52043
A/Hong Kong/485/97(H5N1)
........................................................-...



ABI36198
A/Indonesia/CDC523/2006(H5N1)
..........................................A.............-...



ABE97626
A/Vietnam/CL17/2004(H5N1)
........................................................-...



ABE97633
A/Vietnam/CL119/2005(H5N1)
........................................................-...



AAC32099
A/Hong Kong/483/97(H5N1)
.....I..................................................-...



AAC40508
A/Hong Kong/156/97(H5N1)
........................................................-...



AAD21153
A/Hong Kong/486/97(H5N1)
........................................................-...



ABE97625
A/Vietnam/CL02/2004(H5N1)
........................................................-...



ABE97627
A/Vietnam/CL20/2004(H5N1)
........................................................-...



ABE97632
A/Vietnam/CL115/2005(H5N1)
........................................................-...





Positions from 301 till 360


Consensus sequence
TPMGAIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAI



ABD16284
A/Thailand/NK165/2005(H5N1)
...---..........................................K...........



ABE01046
A/Egypt/2782-NAMRU3/2006(H5N1)
..I---............................I..........G..............



ABI36040
A/Indonesia/CDC184/2005(H5N1)
...---..........................................S...........



ABI36041
A/Indonesia/CDC194P/2005(H5N1)
...---..........................................S...........



ABI36044
A/Indonesia/CDC292N/2005(H5N1)
...---..........................................S...........



ABI36045
A/Indonesia/CDC292T/2005(H5N1)
...---..........................................S...........



ABI36046
A/Indonesia/CDC326N/2006(H5N1)
...---..........................................S...........



ABI36047
A/Indonesia/CDC326N2/2006(H5N1)
...---..........................................S...........



ABI36049
A/Indonesia/CDC326T/2006(H5N1)
...---..........................................S...........



ABI36056
A/Indonesia/CDC370T/2006(H5N1)
...---..........................................S...........



ABI36057
A/Indonesia/CDC390/2006(H5N1)
...---...........................K..............S...........



ABM90544
A/Indonesia/CDC1047S/2007(H5N1)
...---.........................S...............S...........



ABU53968
A/Egypt/2629-NAMRU3/2007(H5N1)
..I---........................................G.............



ABU53969
A/Egypt/2630-NAMRU3/2007(H5N1)
..I---........................................G.............



ABU53970
A/Egypt/2631-NAMRU3/2007(H5N1)
..I---........................................G.............



ABU53971
A/Egypt/2750-NAMRU3/2007(H5N1)
..I---........................................G.............



ABW74701
A/Indonesia/TLL001/2006(H5N1)
...---................T.........................S...........



ABW74704
A/Indonesia/TLL004/2006(H5N1)
...---..........................................S...........



ABW74706
A/Indonesia/TLL006/2006(H5N1)
...---..........................................S...........



ABW74707
A/Indonesia/TLL007/2006(H5N1)
...---..........................................S...........



AAD52043
A/Hong Kong/485/97(H5N1)
...---.....................................T................



ABI36198
A/Indonesia/CDC523/2006(H5N1)
...---..........................................S...........



AAF74330
A/Hong Kong/483/97(H5N1)
...---.....................................A................



AAF74331
A/Hong Kong/486/97(H5N1)
...---.....................................T................



ABC72655
A/Thailand/676/2005(H5N1)
...---..........................................K...........



ABE97626
A/Vietnam/CL17/2004(H5N1)
...---......................................................



ABE97633
A/Vietnam/CL119/2005(H5N1)
...---......................................................





Positions from 361 till 420


Consensus sequence
AGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV



ABD16284
A/Thailand/NK165/2005(H5N1)
............................................................



ABE01046
A/Egypt/2782-NAMRU3/2006(H5N1)
............................................................



ABI36040
A/Indonesia/CDC184/2005(H5N1)
....................................R.......................



ABI36041
A/Indonesia/CDC194P/2005(H5N1)
............................................................



ABI36044
A/Indonesia/CDC292N/2005(H5N1)
............................................................



ABI36045
A/Indonesia/CDC292T/2005(H5N1)
............................................................



ABI36046
A/Indonesia/CDC326N/2006(H5N1)
............................................................



ABI36047
A/Indonesia/CDC326N2/2006(H5N1)
............................................................



ABI36049
A/Indonesia/CDC326T/2006(H5N1)
............................................................



ABI36056
A/Indonesia/CDC370T/2006(H5N1)
............................................................



ABI36057
A/Indonesia/CDC390/2006(H5N1)
............................................................



ABM90544
A/Indonesia/CDC1047S/2007(H5N1)
............................................................



ABU53968
A/Egypt/2629-NAMRU3/2007(H5N1)
............................................................



ABU53969
A/Egypt/2630-NAMRU3/2007(H5N1)
............................................................



ABU53970
A/Egypt/2631-NAMRU3/2007(H5N1)
............................................................



ABU53971
A/Egypt/2750-NAMRU3/2007(H5N1)
..................................................N.........



ABW74701
A/Indonesia/TLL001/2006(H5N1)
............................................................



ABW74704
A/Indonesia/TLL004/2006(H5N1)
............................................................



ABW74706
A/Indonesia/TLL006/2006(H5N1)
............................................................



ABW74707
A/Indonesia/TLL007/2006(H5N1)
............................................................



AAD52043
A/Hong Kong/485/97(H5N1)
...............................Q..................N.........



ABI36198
A/Indonesia/CDC523/2006(H5N1)
............................................................



AAF74330
A/Hong Kong/483/97(H5N1)
...............................Q..................N.........



AAF74331
A/Hong Kong/486/97(H5N1)
..................................................N.........



ABC72655
A/Thailand/676/2005(H5N1)
............................................................



ABE97626
A/Vietnam/CL17/2004(H5N1)
............................................................



ABE97633
A/Vietnam/CL119/2005(H5N1)
............................................................





Positions from 421 till 480


Consensus sequence
GREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQL



ABD16284
A/Thailand/NK165/2005(H5N1)
............................................................



ABE01046
A/Egypt/2782-NAMRU3/2006(H5N1)
............................................................



ABI36040
A/Indonesia/CDC184/2005(H5N1)
............................................................



ABI36041
A/Indonesia/CDC194P/2005(H5N1)
............................................................



ABI36044
A/Indonesia/CDC292N/2005(H5N1)
............................................................



ABI36045
A/Indonesia/CDC292T/2005(H5N1)
............................................................



ABI36046
A/Indonesia/CDC326N/2006(H5N1)
............................................................



ABI36047
A/Indonesia/CDC326N2/2006(H5N1)
............................................................



ABI36049
A/Indonesia/CDC326T/2006(H5N1)
............................................................



ABI36056
A/Indonesia/CDC370T/2006(H5N1)
.....S......................................................



ABI36057
A/Indonesia/CDC390/2006(H5N1)
............................................................



ABM90544
A/Indonesia/CDC1047S/2007(H5N1)
............................................................



ABU53968
A/Egypt/2629-NAMRU3/2007(H5N1)
............................................................



ABU53969
A/Egypt/2630-NAMRU3/2007(H5N1)
............................................................



ABU53970
A/Egypt/2631-NAMRU3/2007(H5N1)
............................................................



ABU53971
A/Egypt/2750-NAMRU3/2007(H5N1)
............................................................



ABW74701
A/Indonesia/TLL001/2006(H5N1)
............................................................



ABW74704
A/Indonesia/TLL004/2006(H5N1)
............................................................



ABW74706
A/Indonesia/TLL006/2006(H5N1)
............................................................



ABW74707
A/Indonesia/TLL007/2006(H5N1)
............................................................



AAD52043
A/Hong Kong/485/97(H5N1)
............................................................



ABI36198
A/Indonesia/CDC523/2006(H5N1)
............................................................



AAF74330
A/Hong Kong/483/97(H5N1)
............................................................



AAF74331
A/Hong Kong/486/97(H5N1)
............................................................



ABC72655
A/Thailand/676/2005(H5N1)
............................................................



ABE97626
A/Vietnam/CL17/2004(H5N1)
............................................................



ABE97633
A/Vietnam/CL119/2005(H5N1)
............................................................





Positions from 481 till 540


Consensus sequence
RDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQI



ABD16284
A/Thailand/NK165/2005(H5N1)
........................................................I...



ABE01046
A/Egypt/2782-NAMRU3/2006(H5N1)
................R..........................................



ABI36040
A/Indonesia/CDC184/2005(H5N1)
.........................I.....N............................



ABI36041
A/Indonesia/CDC194P/2005(H5N1)
.........................I.....N............................



ABI36044
A/Indonesia/CDC292N/2005(H5N1)
.........................I.....N............................



ABI36045
A/Indonesia/CDC292T/2005(H5N1)
.........................I.....N............................



ABI36046
A/Indonesia/CDC326N/2006(H5N1)
.........................I.....N............................



ABI36047
A/Indonesia/CDC326N2/2006(H5N1)
.........................I.....N............................



ABI36049
A/Indonesia/CDC326T/2006(H5N1)
.........................I.....N............................



ABI36056
A/Indonesia/CDC370T/2006(H5N1)
.........................I.....N............................



ABI36057
A/Indonesia/CDC390/2006(H5N1)
.........................I.....N............................



ABM90544
A/Indonesia/CDC1047S/2007(H5N1)
.........................I.....N............................



ABU53968
A/Egypt/2629-NAMRU3/2007(H5N1)
................R...........................................



ABU53969
A/Egypt/2630-NAMRU3/2007(H5N1)
................R...........................................



ABU53970
A/Egypt/2631-NAMRU3/2007(H5N1)
................R...........................................



ABU53971
A/Egypt/2750-NAMRU3/2007(H5N1)
................R.......................................I...



ABW74701
A/Indonesia/TLL001/2006(H5N1)
......................I..I.....N............................



ABW74704
A/Indonesia/TLL004/2006(H5N1)
.........................I.....N............................



ABW74706
A/Indonesia/TLL006/2006(H5N1)
.........................I.....N............................



ABW74707
A/Indonesia/TLL007/2006(H5N1)
.........................I.....N............................



AAD52043
A/Hong Kong/485/97(H5N1)
..........................K...............N...........M.....



ABI36198
A/Indonesia/CDC523/2006(H5N1)
.........................I.....N............................



AAF74330
A/Hong Kong/483/97(H5N1)
..........................K...............N...........M.....



AAF74331
A/Hong Kong/486/97(H5N1)
..........................K...............N...........M.....



ABC72655
A/Thailand/676/2005(H5N1)
........................................................I...



ABE97626
A/Vietnam/CL17/2004(H5N1)
........................................................I...



ABE97633
A/Vietnam/CL119/2005(H5N1)
........................................................I...





Positions from 541 till 583


Consensus sequence
LSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIKFCEDRL



ABD16284
A/Thailand/NK165/2005(H5N1)
.....................................LES.--



ABE01046
A/Egypt/2782-NAMRU3/2006(H5N1)
....................F...........-----------



ABI36040
A/Indonesia/CDC184/2005(H5N1)
................M...................-------



ABI36041
A/Indonesia/CDC194P/2005(H5N1)
................M...................-------



ABI36044
A/Indonesia/CDC292N/2005(H5N1)
................M...................-------



ABI36045
A/Indonesia/CDC292T/2005(H5N1)
................M...................-------



ABI36046
A/Indonesia/CDC326N/2006(H5N1)
................M...................-------



ABI36047
A/Indonesia/CDC326N2/2006(H5N1)
................M...................-------



ABI36049
A/Indonesia/CDC326T/2006(H5N1)
................M...................-------



ABI36056
A/Indonesia/CDC370T/2006(H5N1)
................I...................-------



ABI36057
A/Indonesia/CDC390/2006(H5N1)
................I...................-------



ABM90544
A/Indonesia/CDC1047S/2007(H5N1)
................I...................-------



ABU53968
A/Egypt/2629-NAMRU3/2007(H5N1)
....................F............----------



ABU53969
A/Egypt/2630-NAMRU3/2007(H5N1)
....................F............----------



ABU53970
A/Egypt/2631-NAMRU3/2007(H5N1)
....................F............----------



ABU53971
A/Egypt/2750-NAMRU3/2007(H5N1)
....................F............----------



ABW74701
A/Indonesia/TLL001/2006(H5N1)
................M...................-------



ABW74704
A/Indonesia/TLL004/2006(H5N1)
................I...................-------



ABW74706
A/Indonesia/TLL006/2006(H5N1)
................M...................-------



ABW74707
A/Indonesia/TLL007/2006(H5N1)
................I...................-------



AAD52043
A/Hong Kong/485/97(H5N1)
.......................--------------------



ABI36198
A/Indonesia/CDC523/2006(H5N1)
................I...................-------



AAF74330
A/Hong Kong/483/97(H5N1)
..L....................--------------------



AAF74331
A/Hong Kong/486/97(H5N1)
.......................--------------------



ABC72655
A/Thailand/676/2005(H5N1)
....................................-------



ABE97626
A/Vietnam/CL17/2004(H5N1)
..........---------------------------------



ABE97633
A/Vietnam/CL119/2005(H5N1)
................................-----------
































































































































































































































































































































































































































































































































































































































TABLE 7



Positions from 1 till 60




Consensus sequence

IFLREQKQEFKMNPNQKIITIGSICMVIGIVSLML

IGNMDISIWGVSHSIQTGNQHQAE








ABC72646
A/Thailand/676/2005(H5N1)
-----------....K...........T.M.........L-....-.....H....QK..



ABI36200
A/Indonesia/CDC523/2006(H5N1)
-----------.............................-....-......K.......



ABI36347
A/Indonesia/CDC624/2006(H5N1)
-----------.............................-....-....V.........



ABI36380
A/Indonesia/CDC634/2006(H5N1)
-----------.............................-....-....T.K.......



ABI49409
A/Indonesia/CDC742/2006(H5N1)
-----------....R.............M..........-....-..............



ABJ98530
A/Thailand/RPNP/2005(H5N1)
------.........K...........T.M.........L-....-L.R..H....QK..



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
-----------.............................-....-..............



ABU80632
A/Anhui/T2/2006(H5N1)
-----------.............................-....-..........R...



AAC32089
A/Hong Kong/156/97(H5N1)
-----------................V..I........I-..V.-...I...WHPN.P.



AAD16788
A/Hong Kong/486/97(H5N1)
-----------................V..IN.......T-..V.-...I.K.WHPN.P.



AAD16799
A/Hong Kong/514/97(H5N1)
-----------................V..I........I-..V.-...I...WHPN.P.



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
-----------....K...........T.M.........L-....-.....H.....K..



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
------------------------------------------------------------



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
-----------................T........V...-....-.....H........



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
-----------................T........V...-....-.....N........



ABI34143
A/Guangzhou/1/2006(H5N1)
--------------------....................-....-............VG



ABI36014
A/Indonesia/CDC7/2005(H5N1)
-----------.............................-....-..............



AB136084
A/Indonesia/CDC292N/2005(H5N1)
-----------.............................-....-..............





Positions from 61 till 120




Consensus sequence

PCNQSIITYENNTWVNQTYVNISNTNFLTEKAVAS

TLAGNSSLCPI

GWAVYSKDNSIR








ABC72646
A/Thailand/676/2005(H5N1)
.--------------------...............K..........N............



ABI36200
A/Indonesia/CDC523/2006(H5N1)
S--------------------.....P.........................H....N..



ABI36347
A/Indonesia/CDC624/2006(H5N1)
S--------------------.....P.........................H....N..



ABI36380
A/Indonesia/CDC634/2006(H5N1)
S--------------------.....P.........................H....N..



ABI49409
A/Indonesia/CDC742/2006(H5N1)
S--------------------.....P.........................H....N..



ABJ98530
A/Thailand/RPNP/2005(H5N1)
.--------------------...............K..........N............



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
S--------------------.....P......V..................H....N..



ABU80632
A/Anhui/T2/2006(H5N1)
.--------------------V...K.....................S....H.......



AAC32089
A/Hong Kong/156/97(H5N1)
..-------------------NQSI..Y..Q.A..............S...I........



AAD16788
A/Hong Kong/486/97(H5N1)
..-------------------NQSI..Y..Q.A..............S...I........



AAD16799
A/Hong Kong/514/97(H5N1)
..-------------------NQSI..Y..Q.A..............S...I....K...



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
.--------------------...............K..........N............



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
------------------------------------------------------------



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
.--------------------...............K..........N............



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
.--------------------...A...........K..........N............



ABI34143
A/Guangzhou/1/2006(H5N1)
.--------------------...............................H.......



ABI36014
A/Indonesia/CDC7/2005(H5N1)
S--------------------.....P.........................H....N..



ABI36084
A/Indonesia/CDC292N/2005(H5N1)
S--------------------.....P.........................H....N..





Positions from 121 till 180




Consensus sequence

IGSKGDVFVI

EPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPHRTLMSCPVGEAP








ABC72646
A/Thailand/676/2005(H5N1)
..............................S.............................



ABI36200
A/Indonesia/CDC523/2006(H5N1)
............................................................



ABI36347
A/Indonesia/CDC624/2006(H5N1)
............................................................



ABI36380
A/Indonesia/CDC634/2006(H5N1)
............................................................



ABI49409
A/Indonesia/CDC742/2006(H5N1)
............................................................



ABJ98530
A/Thailand/RPNP/2005(H5N1)
..............................S.............................



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
............................................................



ABU80632
A/Anhui/T2/2006(H5N1)
............................................................



AAC32089
A/Hong Kong/156/97(H5N1)
...............................................Y............



AAD16788
A/Hong Kong/486/97(H5N1)
..........K....................................Y............



AAD16799
A/Hong Kong/514/97(H5N1)
...............................................YG.........T.



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
............................................................



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
------------------------------------------------------------



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
............................................................



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
............................................................



ABI34143
A/Guangzhou/1/2006(H5N1)
............................................................



ABI36014
A/Indonesia/CDC7/2005(H5N1)
............................................................



ABI36084
A/Indonesia/CDC292N/2005(H5N1)
............................................................





Positions from 181 till 240




Consensus sequence

SPYNSRFESVAWSASACHDGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQ








ABC72646
A/Thailand/676/2005(H5N1)
................................S...........................



ABI36200
A/Indonesia/CDC523/2006(H5N1)
.................................E..........................



ABI36347
A/Indonesia/CDC624/2006(H5N1)
.................................E..........................



ABI36380
A/Indonesia/CDC634/2006(H5N1)
.................................E..........................



ABI49409
A/Indonesia/CDC742/2006(H5N1)
.................................E..........................



ABJ98530
A/Thailand/RPNP/2005(H5N1)
................................S......................T....



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
.................................E..........................



ABU80632
A/Anhui/T2/2006(H5N1)
............................................................



AAC32089
A/Hong Kong/156/97(H5N1)
....................I.......................................



AAD16788
A/Hong Kong/486/97(H5N1)
....................I..................................T....



AAD16799
A/Hong Kong/514/97(H5N1)
...................SI......................M.........K......



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
............................................................



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
-----------L................................................



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
............................................................



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
............................................................



ABI34143
A/Guangzhou/1/2006(H5N1)
...........................................M................



ABI36014
A/Indonesia/CDC7/2005(H5N1)
.................................E..........................



ABI36084
A/Indonesia/CDC292N/2005(H5N1)
.................................E..........................





Positions from 241 till 300




Consensus sequence

ESECACVNGSCFTVMTDGPSNGQASYKIFKMGEKGKVVKSVELDAPNYHYEECSCYPDAG








ABC72646
A/Thailand/676/2005(H5N1)
.........................H.....-D...........................



ABI36200
A/Indonesia/CDC523/2006(H5N1)
...............................-............................



ABI36347
A/Indonesia/CDC624/2006(H5N1)
...............................-............................



ABI36380
A/Indonesia/CDC634/2006(H5N1)
...............................-............................



ABI49409
A/Indonesia/CDC742/2006(H5N1)
...............................-...........V................



ABJ98530
A/Thailand/RPNP/2005(H5N1)
.........................H....W-K...WLNQSQ..................



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
...............................-............................



ABU80632
A/Anhui/T2/2006(H5N1)
...............................-..........................D.



AAC32089
A/Hong Kong/156/97(H5N1)
.....................E........I-...R.......N................



AAD16788
A/Hong Kong/486/97(H5N1)
.....................E........I-...R.......N................



AAD16799
A/Hong Kong/514/97(H5N1)
.....................E........I-...R.......N................



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
.........................H.....-............................



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
.........................H.....-............................



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
.........................H.....-............................



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
.........................H.....-............................



ABI34143
A/Guangzhou/1/2006(H5N1)
....V..........................-............................



ABI36014
A/Indonesia/CDC7/2005(H5N1)
...............................-............................



ABI36084
A/Indonesia/CDC292N/2005(H5N1)
...............................-............................





Positions from 301 till 360




Consensus sequence

EITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSPNGAYGV








ABC72646
A/Thailand/676/2005(H5N1)
......................................T..............S..T...



ABI36200
A/Indonesia/CDC523/2006(H5N1)
...................................................M........



ABI36347
A/Indonesia/CDC624/2006(H5N1)
...................................................M........



ABI36380
A/Indonesia/CDC634/2006(H5N1)
..................................I................MF.......



ABI49409
A/Indonesia/CDC742/2006(H5N1)
...................................................M........



ABJ98530
A/Thailand/RPNP/2005(H5N1)
........................G.............T..............S..T...



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
...................................................M........



ABU80632
A/Anhui/T2/2006(H5N1)
............................................................



AAC32089
A/Hong Kong/156/97(H5N1)
......................................S..............L......



AAD16788
A/Hong Kong/486/97(H5N1)
......................................S..............L......



AAD16799
A/Hong Kong/514/97(H5N1)
......................................S..............L......



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
.....................................................S......



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
.....................................................S......



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
.....................................................S......



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
......................................T..............S......



ABI34143
A/Guangzhou/1/2006(H5N1)
............................................................



ABI36014
A/Indonesia/CDC7/2005(H5N1)
...................................................M........



ABI36084
A/Indonesia/CDC292N/2005(H5N1)
...................................................M........





Positions from 361 till 420




Consensus sequence

KGFSFKYGNGVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSF








ABC72646
A/Thailand/676/2005(H5N1)
............................................................



ABI36200
A/Indonesia/CDC523/2006(H5N1)
...................................G........................



ABI36347
A/Indonesia/CDC624/2006(H5N1)
...................................G........................



ABI36380
A/Indonesia/CDC634/2006(H5N1)
...................................G........................



ABI49409
A/Indonesia/CDC742/2006(H5N1)
...................................G........................



ABJ98530
A/Thailand/RPNP/2005(H5N1)
............................................................



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
...................................G........................



ABU80632
A/Anhui/T2/2006(H5N1)
...................................G........................



AAC32089
A/Hong Kong/156/97(H5N1)
...................S......................L....I............



AAD16788
A/Hong Kong/486/97(H5N1)
...................S......................L....I............



AAD16799
A/Hong Kong/514/97(H5N1)
...................S......................L....I............



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
............................................................



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
.......................-------------------------------------



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
............................................................



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
............................................................



ABI34143
A/Guangzhou/1/2006(H5N1)
..................PS........................................



ABI36014
A/Indonesia/CDC7/2005(H5N1)
...................................G........................



ABI36084
A/Indonesia/CDC292N/2005(H5N1)
...................................G...............N........





Positions from 421 till 480




Consensus sequence

VQHPELTGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVGWSWPDGAELPFT








ABC72646
A/Thailand/676/2005(H5N1)
............................................................



ABI36200
A/Indonesia/CDC523/2006(H5N1)
...............................................S............



ABI36347
A/Indonesia/CDC624/2006(H5N1)
...............................................S............



ABI36380
A/Indonesia/CDC634/2006(H5N1)
...............................................S............



ABI49409
A/Indonesia/CDC742/2006(H5N1)
..............................................AS............



ABJ98530
A/Thailand/RPNP/2005(H5N1)
.........N......................................G...--------



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
...............................................S............



ABU80632
A/Anhui/T2/2006(H5N1)
..............----------------------------------------------



AAC32089
A/Hong Kong/156/97(H5N1)
I........N.M...............K................................



AAD16788
A/Hong Kong/486/97(H5N1)
I........N.M...............K.........................D......



AAD16799
A/Hong Kong/514/97(H5N1)
I........N.M...............K..............D..........D......



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
............................................................



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
------------------------------------------------------------



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
...........................................T................



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
............................................................



ABI34143
A/Guangzhou/1/2006(H5N1)
...............................................S.......-----



ABI36014
A/Indonesia/CDC7/2005(H5N1)
...............................................S.....D....--



ABI36084
A/Indonesia/CDC292N/2005(H5N1)
...............................................S.....D....--





Positions from 481 till 484




Consensus sequence

IDKY








ABC72646
A/Thailand/676/2005(H5N1)
...-



ABI36200
A/Indonesia/CDC523/2006(H5N1)
...-



ABI36347
A/Indonesia/CDC524/2006(H5N1)
...-



ABI36380
A/Indonesia/CDC634/2006(H5N1)
...-



ABI49409
A/Indonesia/CDC742/2006(H5N1)
...-



ABJ98530
A/Thailand/RPNP/2005(H5N1)
----



ABM90513
A/Indonesia/CDC1046/2007(H5N1)
...-



ABU80632
A/Anhui/T2/2006(H5N1)
----



AAC32089
A/Hong Kong/156/97(H5N1)
...-



AAD16788
A/Hong Kong/486/97(H5N1)
...-



AAD16799
A/Hong Kong/514/97(H5N1)
...-



AAS89006
A/Thailand/4(SP-528)/2004(H5N1)
...-



AAV73978
A/Viet Nam/DN-33/2004(H5N1)
----



AAZ72720
A/Viet Nam/BL-014/2005(H5N1)
...-



AAZ72721
A/Viet Nam/DT-036/2005(H5N1)
...-



ABI34143
A/Guangzhou/1/2006(H5N1)
----



ABI36014
A/Indonesia/CDC7/2005(H5N1)
----



ABI36084
A/Indonesia/CDC292N/2005(H5N1)
----