CN101553502A

CN101553502A - Hemagglutinin polypeptides, and reagents and methods relating thereto

Info

Publication number: CN101553502A
Application number: CNA2007800300729A
Authority: CN
Inventors: 拉姆·萨西谢卡尔安; 卡西克·维斯瓦那坦; 阿尔特·钱德拉塞卡朗; 拉胡尔·拉曼; 阿拉温德·斯里尼瓦桑; S·拉古拉姆; 维斯瓦那坦·萨西谢卡尔安
Original assignee: Massachusetts Institute of Technology
Current assignee: Massachusetts Institute of Technology
Priority date: 2006-08-14
Filing date: 2007-08-14
Publication date: 2009-10-07
Also published as: CN101501696A

Abstract

The present invention provides a system for analyzing interactions between glycans and interaction partners that bind to them. The present invention also provides HA polypeptides that bind to umbrella-topology glycans, and reagents and methods relating thereto.

Description

Homo agglutinin polypeptide and its related reagent and method

The opinion of right of priority

The application's case is advocated the U.S. Provisional Patent Application case the 60/837th common co-pending of application on August 14th, 2006 according to 35 USC 119 (e), the right of priority that No. the 60/837th, 869, the U.S. Provisional Patent Application case common co-pending of No. 868 and on August 14th, 2006 application.The complete content of each is to be incorporated herein by reference in these existing application cases.

Government supports

The present invention carries out under being supported by the United States Government that the U.S. state-run synthetic medicine institute (National Institute of General MedicalSciences) is authorized according to contract number GM57073 according to contract number U54 GM62116 and NIH (National Institutes of Health).United States Government enjoys some right to the present invention.

Background technology

Influenza has popularity, infectivity, recurrent and fulminant permanent history.Bird flu (comprising the H5N1 bacterial strain) is hyperinfection and potential fatal pathogenic agent, but it only has the limited infection mankind's ability now.Yet, observe in history and find that avian influenza virus gathers its host specificity of change and makes it be easy to infect human sudden change.In fact, thus in the main influenza pandemic disease of eighties of last century both stem to change the avian influenza virus that genetic composition allows the human infection.

Can gather its host specificity of change and make it be easy to infect human significantly concern of sudden change existence for current H5N1, H7N7, H9N2 and H2N2 bird flu bacterial strain.Therefore, in fact whether needs assessment HA albumen can change in these bacterial strains and can be easy to infect human form, and further need to differentiate the HA variant with this ability.Further need to understand the proteic feature of HA that allows usually or forbid different persons under inspection (especially human) infection.Need to be used for to treat effectively or postpone vaccine and therapeutic strategy simultaneously by the caused seizure of disease of influenza virus.

Summary of the invention

The invention provides and have the homo agglutinin polypeptide of specific glycan in conjunction with feature.Specifically, the invention provides in conjunction with homo agglutinin polypeptide with sialylated glycan of umbrella shape topology.In certain embodiments, HA polypeptide of the present invention is in conjunction with having high-affinity and/or specific umbrella shape glycan.In certain embodiments, compare with taper topology glycan, HA polypeptide of the present invention is showed in conjunction with preferred property the umbrella shape glycan.

The present invention provide simultaneously with comprise vaccine the relevant diagnosis of homo agglutinin polypeptide that provides and treatment reagent and method.

Description of drawings

The comparison of the exemplary sequence of Fig. 1: wild-type HA.By NCBI influenza virus sequence library ( Http:// www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) the acquisition sequence.

Fig. 2: the HA glycan is in conjunction with the sequence alignment in territory.Grey: the conserved amino acid that bound sialic acid is related.Red: in conjunction with the related specific amino acids of Neu5Ac α 2-3/6Gal primitive.Yellow: as to influence Q226 (137,138) and the localized amino acid of E190 (186,228).Green: as to combine related amino acid with other monose that is connected to Neu5Ac α 2-3/6Gal primitive (or modification).The sequence of ASI30, APR34, ADU63, ADS97 and Viet04 is to obtain from its crystalline structure separately.Other sequence be from SwissProt ( Http:// us.expasy.org) obtain.Abbreviation: ADA76, A/ duck/A Erbaida (Alberta)/35/76 (H1N1); ASI30, A/ pig/Iowa (Iowa)/30 (H1N1); APR34, A/ Puerto Rico (Puerto Rico)/8/34 (H1N1); ASC18, A/ South Carolina (South Carolina)/1/18 (H1N1); AT91, A/ Texas (Texas)/36/91 (H1N1); ANY18, A/ New York (New York)/1/18 (H1N1); ADU63, A/ duck/Ukraine (Ukraine)/1/63 (H3N8); AAI68, A/ likes to know (Aichi)/2/68 (H3N2); AM99, A/ Moscow (Moscow)/10/99 (H3N2); ADS97, A/ duck/Singapore (Singapore)/3/97 (H5N3); Viet04, A/ Vietnam (Vietnam)/1203/2004 (H5N1).

Fig. 3: the sequence alignment that the conservative subsequence feature of H1 HA is described.

Fig. 4: the sequence alignment that the conservative subsequence feature of H3 HA is described.

Fig. 5: the sequence alignment that the conservative subsequence feature of H5 HA is described.

Fig. 6: the framework that is used to understand the glycan receptor-specific.α 2-3-and/or α 2-6-connect glycan can adopt different topology.

According to the present invention, the HA polypeptide is given it in conjunction with the ability of some described topology and is mediated the ability that different hosts (for example, the mankind) infect.As illustrated among this figure, the present invention specifies two kinds of especially relevant topologys: " taper " topological sum " umbrella shape " topology.α 2-3-and/or α 2-6-connect glycan all can adopt the taper topology, and the taper topology is short chain oligosaccharides that is connected to core or branch oligosaccharides peculiar (but some long-chain oligosaccharides also can adopt this topology).Only α 2-6-connects glycan and can adopt umbrella shape topology (this may increase owing to the conformation diversity that the extra C5-C6 key that exists in the α 2-6-binding is provided), and the umbrella shape topology mainly be by the long-chain oligosaccharides or have long-chain oligosaccharides branch, the branch glycan that especially contains Neu5Ac α 2-6Gal β 1-3/4GlcNAc-primitive adopts.As described herein, the HA polypeptide is given combination and/or the para-infectious ability of Mediated Human to the human receptor in conjunction with the ability of umbrella shape glycan topology.

Fig. 7: HA residue and taper and the comparison of ` mutually of umbrella shape glycan topology.The eutectiferous analysis revealed of HA-glycan, the position of Neu5Ac is with respect to HA binding site no change almost.Relate to such as the conservative residue of F98, S/T136, W153, H183 and L/I194 equal altitudes with contacting of Neu5Ac.Relate to different residues with the contact of other sugar, this depends on that sugared binding is that α 2-3 or α 2-6 and glycan topology are taper or umbrella shape.For instance, in the taper topology, mainly contact Neu5Ac and Gal sugar.E190 and Q226 play the effect of particularly important for this combination.This figure also illustrates other position (for example, 137,145,186,187,193,222) that can participate in conjunction with pyramidal structure.In some cases, different residues can cause the different contacts with different glycan structures.Amino acid whose type in these positions can influence HA polypeptide and the ability that has the receptors bind of different modifying and/or branching pattern in glycan structures.In the umbrella shape topology, come in contact with sugar except that Neu5Ac and Gal.This figure explanation can participate in the residue (for example, 137,145,156,159,186,187,189,190,192,193,196,222,225,226) in conjunction with beveled structure.In some cases, different residues can cause the different contacts with different glycan structures.Amino acid whose type in these positions can influence HA polypeptide and the ability that has the receptors bind of different modifying and/or branching pattern in glycan structures.In certain embodiments, 190 D residue and/or 225 s' D residue causes and the combining of umbrella shape topology.

Fig. 8: exemplary taper topology.This figure illustrates some exemplary (but not exhaustive) glycan structures that adopts the taper topology.

Fig. 9: exemplary umbrella shape topology.This figure illustrates some exemplary (but not exhaustive) glycan structures that adopts the umbrella shape topology.

Figure 10: the epithelial glycan of human bronchial epithelial cell and human colon distributes.For further studying the glycan diversity in the upper respiratory tract tissue, separate N-from HBE (representative upper respiratory tract clone) and connect glycan and use MALDI-MS to analyze.By using sialidase S (α 2-3 specificity) and sialidase A (cracking and SA) pre-treatment sample to confirm the remarkable expression of α 2-6 in HBE.TOF-TOF fragmentation assay by representative mass peak (outstanding with blue-greenish colour) confirms to have the remarkable expression of the glycan of long chain branch topology.For the multifarious reference of glycan in the upper respiratory tract tissue is provided, obtain human colon's epithelial cell (HT29; Representative intestinal tube clone) N-connects glycan and distributes.The reason of selecting described clone is to have showed that current H5N1 virus infects the intestinal tube cell.The remarkable expression of α 2-3 glycan (outstanding with redness) in the HT-29 cell showed in sialidase A and S pre-treatment regulation and control.In addition, long chain branch glycan topology is also not as general as the viewed situation of HBE.Therefore, the human adaptation of H5N1 HA will be referred to be implemented in the high-affinity bonded HA sudden change of the different glycan (for example umbrella shape glycan) of expressing in the human upper respiratory tract tissue.

Figure 11: data mining platform.(A) primary clustering of showing data mining platform in.Feature is to obtain from the data of database object from extracting.With employed data set in the described feature prepared composition class methods with derivation pattern or rule.B shows makes the user data digging method can be applied to the critical software module of glycan array data.

Figure 12: the feature of using in the data mining analysis.This figure is illustrated in the feature of being appointed as representative primitive herein, and it is dissimilar to, triplet and tetrad that the glycan of its explanation from the glycan microarray extracts.Select the fundamental principle of described feature be based on two-(tetrose) with the combining of the glycan binding site of HA.The final data collection comprises the binding signal from the feature of glycan and each HA of screening on described array.In different sorting techniques, service regulations inducing classification method.One of main advantage of described method produces the IF-THEN rule that can be easier to explain for it when comparing with other statistics or the mathematical approach.Two major objectives of classification are: (1) differentiates the enhancing bonded feature that exists on one group of high-affinity glycan part; (2) differentiate to be in and be unfavorable for the bonded feature in the low-affinity glycan part.

Figure 13: the sorter that is used for data mining analysis.This figure provides the table of sorter id and rule.

Figure 14: the conformational map and the solvent accessibility of Neu5Ac α 2-3Gal and Neu5Ac α 2-6Gal primitive.Figure A shows the conformational map of Neu5Ac α 2-3Gal binding.Through enclosing region 2 is observed transoid conformation in APR34_H1_23, ADU63_H3_23 and ADS97_H5_23 eutectic structure.Through enclosing region 1 is observed conformation in AAI68_H3_23 eutectic structure.Figure B shows the conformational map of Neu5Ac α 2-6Gal, and wherein cisoid conformation (through enclosing region 3) all can be observed in the sialylated glycan eutectic of all HA-α 2-6 structure.The solvent-accessible surface that figure C is illustrated in Neu5Ac α 2-3 and α 2-6 sialylated oligosaccharides in indivedual HA-glycan eutectic structures amasss the difference between (SASA).Red and blue-greenish colour post indicate respectively α 2-6 (on the occasion of) or the sialylated glycan of α 2-3 (negative value) in Neu5Ac cause with glycan binding site more and contact.Figure D shows in conjunction with pig and human H1 (H1 _{α 2-3}), bird and human H3 (H3 _{α 2-3}) the sialylated glycan of α 2-3 in NeuAc SASA with combine pig and human H1 (H1 _{α 2-6}) the sialylated glycan of α 2-6 in the SASA of NeuAc between difference.Glaucous H3 _{α 2-3}The indication of negative value post is compared with bird H3, and human H3 HA is less with contacting of Neu5Ac α 2-3Gal.Torsion(al)angle-Φ: C2-C1-O-C3 (for Neu5Ac α 2-3/6 binding); ψ: C1-O-C3-H3 (for Neu5Ac α 2-3Gal) or C1-O-C6-C5 (for Neu5Ac α 2-6Gal); ω: O-C6-C5-H5 (for Neu5Ac α 2-6Gal) binding.Φ, ψ figure be from GlycoMapsDB ( Http:// www.glycosciences.de/modeling/glycomapsdb/) obtain, it is by Martin's Frank doctor (Dr.Martin Frank) and clo Si-Wei Ermufan Der Lieth doctor (Dr.Claus-Wilhelm von der Lieth) (German cancer research institute (German Cancer Research Institute), Germany Hai Mubao (Heidelberg, Germany)) exploitation.Be respectively from the shiny red to the bright green to low-energy color rendering intent from high-energy.

Related residue in the combining of Figure 15: H1, H3 and H5 HA and the sialylated glycan of α 2-3/6.Figure A-D shows the difference (Δ of X-coordinate) of amassing (SASA) in ASI30_H1, APR34_H1, ADU63_H3 and the ADS97_H5 eutectic structure respectively with the solvent-accessible surface of α 2-3 and the interactional residue of the sialylated glycan of α 2-6.Green post corresponding to the residue of glycan direct interaction and light orange post corresponding to the residue that closes on Glu/Asp190 and Gln/Leu226.The Δ of green post more with contacting of the sialylated glycan of α 2-6 on the occasion of the described residue of indication, and that the negative value of Δ is indicated is more with contacting of the sialylated glycan of α 2-3.Figure E summarizes in conjunction with the related residue of the sialylated glycan of α 2-3/6 among H1, H3 and the H5 HA with tabulated form.In conjunction with some related Key residues of the sialylated glycan of α 2-3 be blue and in conjunction with related some Key residues of the sialylated glycan of α 2-6 for red.

Figure 16: Viet04_H5 HA touches combining of the sialylated glycan of α 2-6 (taper topology) with two.The three-dimensional view on surface presents the Viet04_H5 glycan binding site that utilizes being of Neu5Ac α 2-6Gal binding to prolong conformation and (obtains from Toxins, pertussis eutectic structure; PDB ID:1PTO).Lys193 (orange) does not have any contact with the glycan that is this conformation.In conjunction with potential other amino acid that relates to of the glycan that is this conformation is Asn186, Lys222 and Ser227.Yet viewed some contact does not exist in prolonging conformation among the HA in conjunction with the α 2-6 sialylated oligosaccharides that is cisoid conformation.Do not wish to be subjected to the constraint of any particular theory, it should be noted that this shows that prolonging conformation may be not so good as the cisoid conformation ideal with combining of HA.Wherein the Neu5Ac α 2-6Gal β 1-4GlcNAcb branch branch N that is connected to Man α 1-3Man (PDB ID:1LGC) and Man α 1-6Man (PDB ID:1ZAG) structure that is connected glycan is additional to the cis of Neu5Ac α 2-6Gal binding and prolongs on the Neu5Ac α 2-6Gal binding in the Viet04_H5 HA binding site of conformation.Described additional displaying, Neu5Ac α 2-6Gal branched structure of β 1-4GlcNAc and binding site with Man α 1-6Man of the core of being connected to have disadvantageous space overlap (in two kinds of conformations).On the other hand, branched structure with described Man α 1-3Man that is connected to core (as shown in FIG., wherein three seminose cores are purple) with cisoid conformation in Lys193 have space overlap, although but not ideal, still can not with prolong conformation in Lys193 have combination under any situation about contacting.

The generation of Figure 17: WTH1, H3 and H5 HA.Figure A is illustrated in and runs glue and the HA proteic soluble form from H1N1 (A/ South Carolina/1/1918), H3N2 (A/ Moscow/10/1999) and H5N1 (A/ Vietnam/1203/2004) of trace on nitrocellulose filter on the 4-12%SDS-polyacrylamide gel.H1N1 HA is to use goat anti influenza A antibody and anti-goat IgG-HRP to detect.H3N2 is to use ferret anti-H3N2 HA antiserum(antisera) and anti-ferret HRP to detect.H5N1 is to use anti-bird H5N1 HA antibody and anti-rabbit igg-HRP to detect.H1N1 HA and H3N2HA provide with HA0, and H5N1 HA provides with HA0 and HA1.Figure B is illustrated in and runs glue and trace total length H5N1 HA and two kinds of variants (Glu190Asp, Lys193Ser, Gly225Asp, Gln226Leu, " DSDL " on nitrocellulose filter on the SDS-polyacrylamide gel; With GLu190Asp Lys193Ser Gln223LeuGly228Ser, " DSLS ").HA is to use anti-bird H5N1 antibody and anti-rabbit igg-HRP to detect.

Figure 18: the lectin dyeing of upper respiratory tract tissue slice.Utilize the tracheal tissue of jackfruit lectin (Jacalin) (green) and ConA (ConA) (redness) to dye altogether to disclose jackfruit lectin (specificity is connected glycan in conjunction with O) goblet cell preferential and on the tracheae top surface to combine, and ConA (specificity connects glycan in conjunction with N) is preferentially in conjunction with the tracheal cilia epithelial cell.Do not wish to be subjected to the constraint of any particular theory, it should be noted that this discovery shows that goblet cell mainly expresses O and connect glycan, and ciliated epithelial cell is mainly expressed N and connected glycan.Utilize jackfruit lectin and SNA (redness; Specificity is in conjunction with α 2-6) tracheae dye altogether and show combining of SNA and goblet cell and ciliated cell.On the other hand, jackfruit lectin (green) and specificity are showed the very weak not even combination that combines of MAL and false multiple layer tracheal epithelium in conjunction with the common dyeing of the MAL (redness) of the sialylated glycan of α 2-3, but with the regional broad incorporation of the bottom of described tissue.Generally speaking, the indication of lectin dyeing data is connected the remarkable expression and the extensively distribution of the sialylated glycan of α 2-6 of a part that is connected glycan with O respectively with the N in the goblet cell as the cilium on the tracheal epithelium end face.

Figure 19: regroup H1, H3 WT HA combine with the dose response of upper respiratory tract tissue slice and lower respiratory tract tissue slice.At propidium iodide dyeing (redness), show the HA combination with green.The end face of tracheal tissue is mainly expressed the α 2-6 glycan with long chain branch topology.On the other hand, the main express alpha 2-3 glycan of alveolar tissue.H1 HA significantly reduces along with being diluted to 10 μ g/ml from 40 μ g/ml gradually in conjunction with top surface and its combination of tracheae.HI HA only under maximum concentration, also show with some of alveolar tissue a little less than combine.The binding pattern of H3 HA is different from the binding pattern of H1 HA.For instance, H3 HA displaying and tracheae and alveolar tissue under 40 μ g/ml and 20 μ g/ml remarkable combination of cutting into slices.Yet under the concentration of 10 μ g/ml, H3 HA shows main end face in conjunction with tracheal tissue, and combines with alveolar tissue hardly.Generally speaking, the outstanding end face with tracheal tissue of these tissue bond data is with high-affinity bonded importance.In addition, these data also disclose, because H3 HA shows some avidity to the sialylated glycan of α 2-3, therefore not being absolute demand mediates the human infection to the high specific (as confirming by H1 HA) of the sialylated glycan of α 2-6.

The direct wedding agent quantitative response of Figure 20: H1, H3 and H5 WTHA.Show wild-type H1, H3 and the H5 HA binding signal (regular about 800000 the saturated level that turns to) under various concentration from top to bottom respectively.The legend of glycan is to be shown as illustration, and wherein LN corresponds respectively at the α at LN place 2-3 with 6 ' SLN corresponding to Galbl04GlcNAc and 3 ' SLN and is connected sialic acid with α 2-6.The feature binding pattern that is suitable for infecting human H1 and H3 HA in the scope of 40 μ g/ml dilution dropping to, 5 μ g/ml under saturated level in conjunction with long-chain alpha 2-6 (6 ' SLN-LN) glycan.Although H1 HA is in conjunction with the sialylated glycan high special of long-chain alpha 2-6, H3 HA also with high-affinity in conjunction with the sialylated glycan of short chain α 2-6 (6 ' SLN) and with respect to α 2-6 with than low-affinity in conjunction with long-chain alpha 2-3.The direct wedding agent quantitative response of this of H1 and H3 HA is consistent with the tissue bond pattern.In addition, H1 and H3 HA to the high-affinity of the sialylated glycan of long-chain alpha 2-6 in conjunction with relevant with the broad incorporation of the end face (its expression has the sialylated glycan of α 2-6 of long chain branch topology) of itself and tracheal tissue.This dependency provides to be seen clearly the useful of upper respiratory tract tissue tropism of human adaptability H1 and H3 Has.On the other hand, H5 HA shows that opposite glycan is in conjunction with trend, promptly compare with its relative low-affinity (significant signal is only at 20-40 μ g/ml as seen) to the sialylated glycan of α 2-6, with high-affinity in conjunction with α 2-3 (saturation signal drops to 2.5 μ g/ml from 40 μ g/ml).Therefore, do not wish to be subjected to the constraint of any particular theory, the present inventor proposes: the prerequisite of the human adaptation of HA polypeptide (for example bird H5 HA) is to obtain with high-affinity in conjunction with mainly being expressed in the human ability that goes up the sialylated glycan of long-chain alpha 2-6 (for example umbrella shape topology glycan) in the Gas pipe.

The explanation of HA sequential element

HA sequential element 1

HA sequential element 1 is the sequential element of approximate residue 97-185 (wherein with H3 HA, specifying as a reference the residue position) corresponding to the many HA albumen seen in natural influenza separated strain. Described sequential element has basic structure:

C (Y/F) P X ₁C X ₂W X ₃W X ₄H H P, wherein:

X ₁Approximate 30-45 amino acid long;

X ₂Approximate 5-20 amino acid long;

X ₃Approximate 25-30 amino acid long; And

X ₄Approximate 2 amino acid longs.

In certain embodiments, X ₁Be about 35-45, or about 35-43, or about 35,36,37,38,38,40,41,42 or 43 amino acid longs.In certain embodiments, X ₂Be about 9-15, or about 9-14, or about 9,10,11,12,13 or 14 amino acid longs.In certain embodiments, X ₃Be about 26-28, or about 26,27 or 28 amino acid longs.In certain embodiments, X ₄Has sequence (G/A) (I/V).In certain embodiments, X ₄Has sequence GI; In certain embodiments, X ₄Has sequence GV; In certain embodiments, X ₄Has sequence A I; In certain embodiments, X ₄Has sequence A V.In certain embodiments, HA sequential element 1 comprises disulfide linkage.In certain embodiments, described disulfide linkage bridge residue is corresponding to 97 and 139 (based on standard H3 numbering system used herein).

In certain embodiments, and especially in the H1 polypeptide, X ₁Be about 43 amino acid longs, and/or X ₂Be about 13 amino acid longs, and/or X ₃Be about 26 amino acid longs.In certain embodiments, and especially in the H1 polypeptide, HA sequential element 1 has following structure:

C Y P X _1AT (A/T) is C X (A/S) ₂W X ₃W X ₄H H P, wherein:

X _1AApproximate 27-42, or approximate 32-42, or approximate 32-40, or approximate 26-41, or approximate 31-41, or approximate 31-39, or approximate 31,32,33,34,35,36,37,38,39 or 40 amino acid longs, and X ₂-X ₄As mentioned above.

In certain embodiments, and especially in the H1 polypeptide, HA sequential element 1 has following structure:

C Y P X _1AT (A/T) is C X (A/S) ₂W (I/L) is X (T/V) _3AW X ₄H H P, wherein:

X _1AApproximate 27-42, or approximate 32-42, or approximate 32-40, or approximate 32,33,34,35,36,37,38,39 or 40 amino acid longs;

X _3AApproximate 23-28, or approximate 24-26, or approximate 24,25 or 26 amino acid longs, and X ₂And X ₄As mentioned above.

In certain embodiments, and especially in the H1 polypeptide, HA sequential element 1 comprises following sequence:

Q?L?S?S?I?S?S?F?E?K，

It is usually at X ₁In (be included in X _1AIn) and especially about X ₁Residue 12 places begin (as illustrated in for example Fig. 1-3).

In certain embodiments, and especially in the H3 polypeptide, X ₁Be about 39 amino acid longs, and/or X ₂Be about 13 amino acid longs, and/or X ₃Be about 26 amino acid longs.

In certain embodiments, and especially in the H3 polypeptide, HA sequential element 1 has following structure:

C Y P X _1AS (S/N) is C X (A/S) ₂W X ₃W X ₄H H P, wherein:

X _1AApproximate 27-42, or approximate 32-42, or approximate 32-40, or approximate 23-38, or approximate 28-38, or approximate 28-36, or approximate 28,29,30,31,32,33,34,35,36,37,38,39 or 40 amino acid longs, and X ₂-X ₄As mentioned above.

C Y P X _1AS (S/N) is C X (A/S) ₂W L (T/H) X _3AW X ₄H H P, wherein:

In certain embodiments, and especially in the H3 polypeptide, HA sequential element 1 comprises following sequence:

(L/I)(V/I)A?S?S?G?T?L?E?F，

It is usually at X ₁In (be included in X _1AIn) and especially about X ₁Residue 12 places begin (as illustrated at Fig. 1,2 and 4 for example).

In certain embodiments, and especially in the H5 polypeptide, X ₁Be about 42 amino acid longs, and/or X ₂Be about 13 amino acid longs, and/or X ₃Be about 26 amino acid longs.

In certain embodiments, and especially in the H5 polypeptide, HA sequential element 1 has following structure:

C Y P X _1AS S A C X ₂W X ₃W X ₄H H P, wherein:

C Y P X _1AS S A C X ₂W L I X _3AW X ₄H H P, wherein:

X _1AApproximate 27-42, or approximate 32-42, or approximate 32-40, or approximate 32,33,34,35,36,37,38,39 or 40 amino acid longs; And

In certain embodiments, and especially in the H5 polypeptide, HA sequential element 1 prolongs (promptly in the position corresponding to residue 186-193) through following sequence:

N?D?A?A?E?X?X(K/R)。

In certain embodiments, and especially in the H5 polypeptide, HA sequential element 1 comprises following sequence:

Y?E?E?L?K?H?L?X?S?X?X?N?H?F?E?K，

It is usually at X ₁In and especially about X ₁Residue 6 places begin (as illustrated at Fig. 1,2 and 5 for example).

HA sequential element 2

HA sequential element 2 is approximate sequential element corresponding to the proteic residue 324-340 of many HA (reusing the numbering system based on H3 HA) seen in the natural influenza strain isolated.Described sequential element has basic structure:

G?A?I?A?G?F?I?E。

In certain embodiments, HA sequential element 2 has following sequence:

P X ₁G A I A G F I E, wherein:

X ₁Approximate 4-14 amino acid long, or about 8-12 amino acid long, or about 12,11,10,9 or 8 amino acid longs.In certain embodiments, described sequential element provides HA0 cracking site, thereby allows to produce HA1 and HA2.

In certain embodiments, and especially in the H1 polypeptide, HA sequential element 2 has following structure:

P S (I/V) Q S R X _1AG A I A G F I E, wherein:

X _1AApproximate 3 amino acid longs; In certain embodiments, X _1ABe G (L/I) F.

In certain embodiments, and especially in the H3 polypeptide, HA sequential element 2 has following structure:

P X K X T R X _1AG A I A G F I E, wherein:

X _1AApproximate 3 amino acid longs; In certain embodiments, X _1ABe G (L/I) F.

In certain embodiments, and especially in the H5 polypeptide, HA sequential element 2 has following structure:

P Q R X X X R X X R X _1AG A I A G F I E, wherein:

X _1AApproximate 3 amino acid longs; In certain embodiments, X _1ABe G (L/I) F.

Definition

Avidity: as known in the affiliated field, " avidity " is measuring of specific ligand (for example, HA polypeptide) and its collocation thing (for example, HA acceptor) bonded tightness.Avidity can be measured by different way.

Biological activity: as used herein, phrase " biological activity " is meant in biosystem and especially has the feature of active any reagent in organism.For instance, think when throwing described organism to be had the reagent biologically active of biological action with organism.In a particular embodiment, if albumen or polypeptide biologically active, the so total described albumen or at least a bioactive albumen of polypeptide or the part of polypeptide are commonly referred to " biological activity " part.

Wide spectrum is human in conjunction with (BSHB) H5 HA polypeptide: as used herein, phrase " the wide spectrum mankind are in conjunction with H5 HA " is meant in conjunction with the HA acceptor seen in human epithelium's tissue and especially in conjunction with the pattern of the H5 HA polypeptide of the human HA acceptor with the sialylated glycan of α 2-6.In addition, BSHB H5 HA of the present invention is in conjunction with the multiple different sialylated glycan of α 2-6.In certain embodiments, BSHB H5 HA is in conjunction with the sialylated glycan of different α 2-6 seen in a large amount of human sample, and the virus that contains it has infection population and especially in conjunction with the remarkable ability of upper respiratory tract acceptor in the described colony.In certain embodiments, BSHB H5 HA is in conjunction with umbrella shape glycan as described herein (for example, the sialylated glycan of long-chain alpha 2-6).

Characteristic: as used herein, " characteristic " of phrase albumen or polypeptide is for containing the continuous amino acid fragment or being the segmental part of a large amount of continuous amino acids of the feature of albumen or polypeptide together.Each described continuous fragment will contain at least two amino acid usually.In addition, one of ordinary skill in the art will understand, and proteic feature is needs at least 5,10,15,20 or more a plurality of amino acid usually.In general, characteristic be except that appointed sequence consistence above with the part of total at least one functional character of relevant intact proteins.

Characteristic sequence: " characteristic sequence " is the sequence that sees among polypeptide or all members of nucleic acid family, and therefore one of ordinary skill in the art can use it for the member who defines described family.

The taper topology: phrase " taper topology " is used in reference to some glycan and the three-dimensional arrangement that glycan adopted on the HA acceptor especially in this article.As illustrated in fig. 6, sialylated glycan of α 2-3 or the sialylated glycan of α 2-6 all can adopt the taper topology, and it is peculiar by the short oligonucleotide chain, but some long-chain oligonucleotide also can adopt this conformation.The taper topology is that the glucosides torsion(al)angle by Neu5Ac α 2-3Gal binding characterizes, and it extracts by-60,60 or 180 Φ (C1-C2-O-C3/C6) value and-60 to 60 specified three zones (Figure 14) with least energy conformation of ψ (C2-O-C3/C6-H3/C5) sample approximately.Fig. 8 presents some representativeness (but not exhaustive) example of the glycan that adopts the taper topology.

Corresponding to: as used herein, term " corresponding to " be generally used for representing the position/consistence of amino-acid residue in the HA polypeptide.One of ordinary skill in the art will understand, for the purpose of simplifying, utilize standard numbering system (based on wild-type H3 HA) (as illustrated in for example Fig. 1-5) herein, therefore, for example " corresponding to " in fact the amino acid of 190 residues need not to be the 190th amino acid in the specific amino acids chain, but corresponding to 190 being seen residues in wild-type H3 HA; One of ordinary skill in the art are easy to understand how to differentiate corresponding amino acid.

The separation degree of removing: as used herein, as the amino acid of " separation degree of removal " for to glycan in conjunction with HA amino acid with remote effect.For instance, 1 of removal degree amino acid separation can: (1) with directly combine amino acid interaction; And/or (2) otherwise influence directly in conjunction with amino acid and the interactional ability of the host cell HA acceptor associating glycan of institute; 1 degree amino acid separation of described removal can or can be directly in conjunction with glycan itself.The 2 degree amino acid separations of removing (1) interact with the 1 degree amino acid separation of removing; And/or (2) otherwise influence 1 degree amino acid separation of removal and directly combine the interactional ability of amino acid etc.

Directly in conjunction with amino acid: as used herein, phrase " directly in conjunction with amino acid " is meant the HA polypeptide amino acid with one or more and the associating glycan direct interaction of host cell HA acceptor.

Through through engineering approaches: as used herein, the polypeptide that a kind of aminoacid sequence has been selected by the mankind described in term " through through engineering approaches ".For instance, has the aminoacid sequence that is different from the HA amino acid sequence of polypeptide seen in the natural influenza strain isolated through the HA of through engineering approaches polypeptide.In certain embodiments, has the aminoacid sequence that is different from HA amino acid sequence of polypeptide included in the ncbi database through the HA of through engineering approaches polypeptide.

The H1 polypeptide: when with " H1 polypeptide " when being used for herein, described term is that aminoacid sequence comprises that at least one is as the feature of H1 and make H1 and the HA polypeptide of other other sequential element of HA hypotype phase region.Representative described sequential element can determine such as sequence illustrated among Fig. 1-3 by comparison, and it for example comprises herein the described sequence of H1 specificity embodiment about the HA sequential element.

The H3 polypeptide: when with " H3 polypeptide " when being used for herein, described term is that aminoacid sequence comprises that at least one is as the feature of H3 and make H3 and the HA polypeptide of other other sequential element of HA hypotype phase region.Representative described sequential element can determine by the sequence of comparison described in Fig. 1,2 and 4, and it for example comprises herein the described sequence of H3 specificity embodiment about the HA sequential element.

The H5 polypeptide: when with " H5 polypeptide " when being used for herein, described term is that aminoacid sequence comprises that at least one is as the feature of H5 and make H5 and the HA polypeptide of other other sequential element of HA hypotype phase region.Representative described sequential element can determine by the sequence of comparison described in Fig. 1,2 and 5, and it for example comprises herein the described sequence of H5 specificity embodiment about the HA sequential element.

Homo agglutinin (HA) polypeptide: as used herein, term " homo agglutinin polypeptide " (or " HA polypeptide ") is meant that aminoacid sequence comprises the polypeptide of at least one HA characteristic sequence.Known multiple HA sequence in the affiliated field from the influenza strain isolated; In fact, American National biotechnology information center (National Center for BiotechnologyInformation, comprise till when NCBI) remaining into the application of the application's case 9796 HA sequences database ( Www.ncbi.nlm.nih.gov/genomes/FLU/flu.html).One of ordinary skill in the art can be easy to differentiate with reference to this database and be generally HA polypeptide and/or specific HA polypeptide (for example, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16 polypeptide) or mediate for example sequence of the feature of the HA of specific host infection such as bird, camel, dog, cat, civetta, environment, horse, the mankind, leopard, ermine, mouse, sea dog, stone swallow (stone martin), pig, tiger, whale.For instance, in certain embodiments, the HA polypeptide comprise one or more see seen in the natural influenza virus strain isolated the proteic about residue 97 of HA and 185,324 and 340,96 and 100 and/or 130-230 between the characteristic sequence element.In certain embodiments, the HA polypeptide has in the HA sequential element 1 and 2 that comprises as defined herein the aminoacid sequence of at least one.In certain embodiments, the HA polypeptide has the aminoacid sequence that comprises HA sequential element 1 and 2, in certain embodiments, the separate about 100-200 of described element, or about 125-175, or about 125-160, or about 125-150, or about 129-139, or about 129,130,131,132,133,134,135,136,137,138 or 139 amino acid.In certain embodiments, the HA polypeptide has the aminoacid sequence that comprises the residue that participates in glycan bonded position in regional 96-100 and/or the 130-230.For instance, many HA polypeptide comprise one or more following residue: Tyr98, Ser/Thr136, Trp153, His183 and Leu/Ile194.In certain embodiments, the HA polypeptide comprises at least 2,3,4 or whole 5 described residues.

Through separating: as used herein, term " through separating " is meant that reagent or entity (i) separate from least some its associating components of institute when producing (nature or experimental situation) at first; Or (ii) by artificial generation.Through separation agent or entity can with separate at least about its initial associating other component more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 90%.In certain embodiments, pure through separation agent for surpassing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Long-chain oligosaccharides: for the purpose of this disclosure, comprise that as Nutriflora P at least one has the straight chain of at least 4 saccharide residues, so usually it is considered as " length ".

Alpha-non-natural amino acid: phrase " alpha-non-natural amino acid " is meant to have amino acid whose chemical structure (that is,

Therefore) and can participate at least two peptide bonds but the R group is different from the entity of the being seen group of occurring in nature.In certain embodiments, alpha-non-natural amino acid also can have another non-hydrogen R group, and/or can have one or more other replacements on amino or carboxylic moiety.

Polypeptide: in general, " polypeptide " is for having at least two amino acid whose chains that connect by peptide bond each other.In certain embodiments, polypeptide can comprise 3-5 amino acid at least, and it is connected with other amino acid by means of at least one peptide bond separately.One of ordinary skill in the art will understand, and polypeptide comprises " non-natural " amino acid sometimes or still can choose other entity that is integrated in the polypeptide chain wantonly.

Pure: as used herein, if reagent or entity do not have other component in fact, it is " pure " so.For instance, will contain the preparation that surpasses about 90% particular agent or entity usually and be considered as pure preparation.In certain embodiments, reagent or entity at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%＜or 99% pure.

Short chain oligosaccharides:, as having less than 4 or certainly less than 3 residues in Nutriflora P what straight chain in office, so usually it is considered as " weak point " for the purpose of this disclosure.

Specificity: as known in the affiliated field, " specificity " be specific ligand (for example, the HA polypeptide) distinguishes its measuring in conjunction with the potential ability that combines the thing (for example, bird HA acceptor) of arranging in pairs or groups of collocation thing (for example human HA acceptor, and especially human upper respiratory tract HA acceptor) and other.

Therapeutical agent: as used herein, phrase " therapeutical agent " is meant any reagent that causes required biology or pharmacological action.

Treatment: as used herein, term " treatment " is meant and is used to palliate a disease, one or more symptoms or the aspect of illness or symptom, delays any method that it shows effect, reduces its severity or sickness rate or cause its prevention.For purposes of the present invention, treatment can before the paresthesia epilepsy, during and/or throw afterwards with.

The umbrella shape topology: phrase " umbrella shape topology " is used in reference to some glycan and the three-dimensional arrangement that glycan adopted on the HA acceptor especially in this article.The present invention is contained about in conjunction with the cognition of umbrella shape topology glycan for the proteic feature of HA of the human host infection of mediation.As illustrated in fig. 6, only the sialylated glycan of α 2-6 adopts the umbrella shape topology usually, and the umbrella shape topology is peculiar by long-chain (for example greater than tetrose) oligosaccharides.The example of umbrella shape topology is by providing (referring to for example Figure 14) for the about Φ angle of-60 Neu5Ac α 2-6Gal binding.Fig. 9 presents some representativeness (but not exhaustive) example of the glycan that adopts the umbrella shape topology.

Vaccine inoculation: as used herein, term " vaccine inoculation " is meant to throw with the expection meeting and for example virulence factor is produced immunoreactive composition.For purposes of the present invention, vaccine inoculation can be before being exposed to virulence factor, during and/or throw afterwards with, and in certain embodiments, vaccine inoculation can be before being exposed to the described factor, during and/or throw soon afterwards with.In certain embodiments, the vaccine inoculation repeatedly throwing that comprises the vaccine inoculation composition of appropriate time at interval with.

Variant: as used herein, term " variant " is the relational language of describing the specific HA polypeptide of being paid close attention to and carrying out the relation between sequence " parent " HA polypeptide relatively.If having consistent with parent HA polypeptide, the HA polypeptide of being paid close attention to, so the HA polypeptide of being paid close attention to is considered as " variant " of parent HA polypeptide at the aminoacid sequence that specific position has a small amount of sequence variation.Usually, when comparing with parent, the residue less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% in the described variant is substituted.In certain embodiments, when comparing with parent, variant has 10,9,8,7,6,5,4,3,2 or 1 and is substituted residue.Usually, variant has minute quantity (for example, less than 5,4,3,2 or 1) and is substituted functional residue (that is, participate in particular organisms active residue).In addition, when comparing with parent, variant has usually and is no more than 5,4,3,2 or 1 and adds or disappearance, and does not have usually and add or disappearance.In addition, any interpolation or disappearance are usually less than about 25,20,19,18,17,16,15,14,13,10,9,8,7,6 residues, and usually less than about 5,4,3 or 2 residues.In certain embodiments, parent HA polypeptide is the polypeptide (for example, wild-type HA) that sees in the natural influenza virus strain isolated.

Carrier: as used herein, " carrier " is meant the nucleic acid molecule that can transport connected another nucleic acid.In certain embodiments, carrier can be in such as host cells such as eukaryotic cell or prokaryotic cell prokaryocytes extrachromosomal replication and/or express connected nucleic acid.Can guide the carrier of the expression of gene of operability connection to be referred to herein as " expression vector ".

Wild-type: such as in the affiliated field understanding, phrase " wild-type " generally is meant as the standard form at being seen albumen of occurring in nature or nucleic acid.For instance, wild-type HA polypeptide sees in the strain isolated of natural influenza virus.Multiple different wild-type HA sequence is found in NCBI influenza virus sequence library Http:// www.ncbi.nlm.nih.gov/genomes/FLU/FLU.htmlIn.

Embodiment

The invention provides HA polypeptide in conjunction with umbrella shape topology glycan.In certain embodiments, the invention provides the HA polypeptide of being seen umbrella shape topology glycan on the HA acceptor in conjunction with specific target species.For instance, in certain embodiments, the invention provides the HA polypeptide of going up being seen umbrella shape topology glycan in conjunction with human HA acceptor (for example, being seen HA acceptor on the human epithelial cell), and the HA polypeptide in conjunction with being seen umbrella shape topology glycan on the human HA acceptor in the upper respiratory tract especially is provided.

The invention provides HA polypeptide in conjunction with being seen HA acceptor on the human upper respiratory tract cell, and especially provide with through the avidity of indication and/or specificity in conjunction with the HA polypeptide of described acceptor (and/or its glycan, especially its umbrella shape glycan).

Following cognition is contained in the present invention: obtain in conjunction with the ability of umbrella shape topology glycan (for example sialylated glycan of long-chain alpha 2-6) and especially can give the ability that the HA polypeptide variants infects the mankind (wherein its parent HA polypeptide then can not) with high-affinity bonded ability.Do not wish to be subjected to any particular theory to retrain, the present inventor proposes to can be very important and especially propose may not request and other glycan type bonded loss in conjunction with umbrella shape topology glycan.

The present invention further provides all ingredients and the method relevant, comprise the system that for example is used to differentiate it, the strategy that is used to prepare it, in conjunction with its antibody and various relative diagnosis and methods of treatment with HA polypeptide of the present invention.The further describing of some embodiment of these and other aspect of the present invention hereinafter is provided.

Homo agglutinin (HA)

Influenza virus is a ribonucleic acid virus, and its sign is that the adipose membrane coating contains two glycoprotein, homo agglutinin (HA) and the neuraminidase (NA) in the film through being embedded into specific virus.Have 16 kinds of known HA hypotypes and 9 kinds of NA hypotypes, and the number that different influenza bacterial strain is based on bacterial strain HA and NA hypotype is named.Based on the comparison of consensus amino acid sequence and crystalline structure, the HA hypotype is divided into two main groups and four less kinds are branch.Different HA hypotypes needn't have strong aminoacid sequence consistence, but the general three structure of different HA hypotypes is similar each other, have the nuance of several purposes that can be used for classifying.For instance, film far-end subdomain is a kind of usually in order to measure the constitutional features (Russell people such as (Russell), virusology (Virology), 325:287,2004) of HA hypotype with respect to the specific orientation of center alpha-helix.

HA is present in the film with a kind of homotrimer form in 16 kinds of hypotypes that are called H1-H16.Up to now, only three kinds (H1, H2 and H3) have been adapted to the human infection in these hypotypes.Being adapted to of HA infected and human a kind ofly be and preferably compare in conjunction with the bird progenitor cell of the sialylated glycan of α 2-3 through report feature (for example from the HA of popular H1N1 (1918) and H3N2 (1967-68) influenza subtype feature), and it is preferably in conjunction with ability (Si Keer and the Huai Li (Skehel﹠amp of the sialylated glycan of α 2-6; Wiley), biological chemistry annual review (Annu RevBiochem), 69:531,2000; Inferior (the Rogers﹠amp of Roger and bohr; Paulson), virusology, 127:361,1983; People such as Roger, nature (Nature), 304:76,1983; Su Te people such as (Sauter), biological chemistry (Biochemistry), 31:9609,1992; Kang Nuo people such as (Connor), virusology, 205:17,1994; Tell Mu Pei people such as (Tumpey), science (Science), 310:77,2005).Yet following cognition is contained in the present invention: the ability that infects human host is less with the dependency of the glycan that combines specific binding, and bigger with the dependency of the glycan that combines particular topology.Therefore, the present invention confirms the para-infectious HA of Mediated Human in conjunction with umbrella shape topology glycan, shows usually that whereby umbrella shape topology glycan has precedence over the preferred property of taper topology glycan (even taper topology glycan can be the sialylated glycan of α 2-6).

Can be used to several crystalline structure from combination (α 2-3 and the α 2-6 binding) sialylated oligosaccharides of the HA of H1 (mankind and pig), H3 (bird) and H5 (bird) hypotype, and it provides sees clearly (Ai Sen people such as (Eisen) to the molecule of the related specific amino acid of HA and the different interactions of these glycan, virusology, 232:19,1997; Breathe out people such as (Ha), institute of NAS periodical (Proc Natl Acad Sci USA), 98:11181,2001; People such as Kazakhstan, virusology, 309:209,2003; Gai Bolin people such as (Gamblin), science, 303:1838,2004; Stevens people such as (Stevens), science, 303:1866,2004; People such as Russell, sugared connector magazine (Glycoconj J), 23:85,2006; People such as Stevens, science, 312:404,2006).

For instance, differentiate directly and interactional some amino acid of combination glycan separately or in conjunction with the crystalline structure of the H5 (A/ duck/Singapore/3/97) of α 2-3 or α 2-6 sialylated oligosaccharides, and remove once or once above amino acid separation (people such as Stevens, institute of NAS periodical, 98:11181,2001).In some cases, the conformation of described residue is in conjunction with to different aspect the unbound state.For instance, Glu190, Lys193 and Gln226 participate in direct binding interactions all and have not isomorphic map in to unbound states.Near the conformation of the Asn186 of Glu190 in conjunction with also significantly different aspect unbound state.

HA polypeptide of the present invention in conjunction with feature

As mentioned above, following discovery is contained in the present invention: relevant with the ability of the infection of mediation specific host (comprising for example human) in conjunction with umbrella shape topology glycan.Therefore, the invention provides HA polypeptide in conjunction with the umbrella shape glycan.In certain embodiments, HA polypeptide of the present invention with high-affinity in conjunction with the umbrella shape glycan.In certain embodiments, HA polypeptide of the present invention usually with high-affinity and/or specificity in conjunction with multiple different umbrella shapes topology glycan.

In certain embodiments, HA polypeptide of the present invention with high-affinity in conjunction with umbrella shape topology glycan (for example the sialylated glycan of long-chain alpha 2-6, such as Neu5Ac α 2-6Gal β 1-4GlcNAc β 1-3Gal β 1-4GlcNAc-).For instance, in certain embodiments, HA polypeptide of the present invention with combine umbrella shape topology glycan about the para-infectious wild-type HA of Mediated Human (for example H1N1 HA or the H3N2 HA) avidity that viewed avidity is suitable.In certain embodiments, HA polypeptide of the present invention with the avidity that is at least following value in conjunction with the umbrella shape glycan: under the condition suitable 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of viewed value with the para-infectious wild-type HA of Mediated Human.In certain embodiments, HA polypeptide of the present invention combines the umbrella shape glycan with the avidity greater than viewed value under the condition suitable with the para-infectious wild-type HA of Mediated Human.

In certain embodiments, the binding affinity of assessment HA polypeptide of the present invention in a certain concentration range.This strategy especially provides than the remarkable more information of single concentration analysis in examining and determine in multivalence.For instance, in certain embodiments, the binding affinity of assessment HA polypeptide of the present invention in the concentration range that exceeds more than at least 2,3,4,5,6,7,8,9,10 times or 10 times.

In certain embodiments, if HA polypeptide of the present invention is being showed saturation signal such as multivalence glycan array as herein described in examining and determine, it shows high-affinity so.In certain embodiments, be higher than about 400000 or the signal of higher (for example be higher than about 500000,600000,700000,800000 etc.) if HA polypeptide of the present invention is showed in described research, it shows high-affinity so.In certain embodiments, the HA polypeptide is illustrated in 2 times, 3 times, 4 times, the concentration range more than 5 times or 5 times at least, and saturated in conjunction with the umbrella shape glycan in the concentration range that reaches more than 10 times or 10 times in certain embodiments.

In addition, in certain embodiments, HA polypeptide of the present invention than its in conjunction with taper topology glycan more consumingly in conjunction with umbrella shape topology glycan.In certain embodiments, HA polypeptide of the present invention is shown as about relative affinity of 10,9,8,7,6,5,4,3 or 2 for the umbrella shape glycan to the taper glycan.

In certain embodiments, HA polypeptide of the present invention is in conjunction with the sialylated glycan of α 2-6; In certain embodiments, HA polypeptide of the present invention is preferably in conjunction with the sialylated glycan of α 2-6.In certain embodiments, HA polypeptide of the present invention is in conjunction with the multiple different sialylated glycan of α 2-6.In certain embodiments, HA polypeptide of the present invention can not be in conjunction with the sialylated glycan of α 2-3, and in other embodiments, HA polypeptide of the present invention can be in conjunction with the sialylated glycan of α 2-3.

In certain embodiments, HA polypeptide of the present invention is in conjunction with being seen acceptor on the human upper respiratory tract epithelial cell.In certain embodiments, HA polypeptide of the present invention is in conjunction with the HA acceptor in segmental bronchus and/or the tracheae.In certain embodiments, HA polypeptide of the present invention can not be in conjunction with the acceptor in the dark lung, and in other embodiments, HA polypeptide of the present invention can be in conjunction with the acceptor in the dark lung.

In certain embodiments, HA polypeptide combination of the present invention is at least about being seen glycan on the HA acceptor in human upper respiratory tract tissue (for example epithelial cell) more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 95%.

In certain embodiments, HA polypeptide of the present invention is in conjunction with one or more glycan illustrated in fig. 9.In certain embodiments, HA polypeptide of the present invention is in conjunction with multiple glycan illustrated in fig. 9.In certain embodiments, HA polypeptide of the present invention with high-affinity and/or specificity in conjunction with glycan illustrated in fig. 9.In certain embodiments, and compare in conjunction with glycan illustrated in fig. 8, HA polypeptide of the present invention is preferably in conjunction with glycan illustrated in fig. 9.

The invention provides and separatedly have the HA polypeptide of specifying binding specificity, and provide about the umbrella shape glycan have appointment in conjunction with feature through through engineering approaches HA polypeptide.

It is in certain embodiments, of the present invention that to have appointment be the H1 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H2 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H3 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H4 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H5 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H6 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H7 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H8 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H9 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H10 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H11 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H12 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H13 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H14 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H15 polypeptide in conjunction with the HA polypeptide of feature.It is in certain embodiments, of the present invention that to have appointment be the H16 polypeptide in conjunction with the HA polypeptide of feature.

In certain embodiments, of the present invention have appointment in conjunction with the HA polypeptide of feature for the H1 polypeptide, not for the H2 polypeptide and/or be not the H3 polypeptide.

In certain embodiments, HA polypeptide of the present invention does not comprise the H1 albumen from following arbitrary bacterial strain: A/ South Carolina/1/1918; A/ Puerto Rico/8/1934; A/ Taiwan (Taiwan)/1/1986; A/ Texas/36/1991; A/ Beijing (Beijing)/262/1995; A/ Johannesburg (Johannesburg)/92/1996; A/ New Caledonia (New Caledonia)/20/1999; A/ Solomon Islands (Solomon Islands)/3/2006.

In certain embodiments, HA polypeptide of the present invention is not the H2 albumen from arbitrary bacterial strain of the Asian flu transmissible disease of 1957-58.In certain embodiments, HA polypeptide of the present invention does not comprise the H2 albumen from following arbitrary bacterial strain: A/ Japan (Japan)/305+/1957; A/ Singapore/1/1957; A/ Taiwan/1/1964; A/ Taiwan/1/1967.

In certain embodiments, HA polypeptide of the present invention do not comprise the H3 albumen from following arbitrary bacterial strain: A/ like to know/2/1968; A/ Philippines (Phillipines)/2/1982; A/ Mississippi (Mississippi)/1/1985; A/ Leningrad (Leningrad)/360/1986; A/ Sichuan (Sichuan)/2/1987; A/ Shanghai (Shanghai)/11/1987; A/ Beijing/353/1989; A/ Shandong (Shandong)/9/1993; A/ Johannesburg/33/1994; A/ Nanchang (Nanchang)/813/1995; A/ Sydney (Sydney)/5/1997; A/ Moscow/10/1999; A/ Panama (Panama)/2007/1999; A/ Wyoming (Wyoming)/3/2003; A/ Oklahoma (Oklahoma)/323/2003; A/ California (California)/7/2004; A/ Wisconsin (Wisconsin)/65/2005.

Variant HA polypeptide

In certain embodiments, the HA polypeptide is a parent HA variant polypeptides, because except that a small amount of particular sequence changes, the consensus amino acid sequence of its aminoacid sequence and parent HA.In certain embodiments, parent HA is the HA polypeptide (for example wild-type HA polypeptide) seen in the natural strain isolated of influenza virus.

In certain embodiments, HA polypeptide variants of the present invention has the glycan different with its corresponding parent HA polypeptide in conjunction with feature.In certain embodiments, HA variant polypeptide of the present invention has than big avidity and/or the specificity of its homology parent HA polypeptide for umbrella shape glycan (for example comparing with the taper glycan).In certain embodiments, described HA polypeptide variants is the through engineering approaches variant.

In certain embodiments, the glycan that has through changing has sequence alternately or have the sequence that influence the glycan binding site and replace in conjunction with the HA polypeptide variants of feature in residue.In certain embodiments, these are substituted by directly and the interactional amino acid of institute's bonded glycan; In other embodiments, these are substituted by from spending isolating amino acid with 1 of the interactional amino acid removal of institute's bonded glycan, and this is owing to the 1 degree amino acid separation of removing (1) and directly combines the amino acid interaction; (2) otherwise the influence directly in conjunction with amino acid and the interactional ability of glycan, but not with the direct interaction of glycan own; Or (3) otherwise influence directly in conjunction with amino acid and the interactional ability of glycan, and simultaneously and the direct interaction of glycan own.HA polypeptide variants of the present invention contains one or more directly in conjunction with the replacement of any combination of amino acid, one or more first degree amino acid separations, one or more second degree amino acid separations or described situation.In certain embodiments, HA polypeptide variants of the present invention can contain have in addition more high score from one or more amino acid whose replacements of degree.

In certain embodiments, the glycan with change has sequence variation (referring to, Fig. 7 for example) in conjunction with the HA polypeptide variants of feature in the residue of the sugar of contact except that Neu5Ac and Gal.

In certain embodiments, HA compares with the wild-type parent, and the HA polypeptide variants has at least a aminoacid replacement.In certain embodiments, HA compares with homology wild-type parent, and HA polypeptide variants of the present invention has at least two kinds, three kinds, four kinds, aminoacid replacement more than five kinds or five kinds; In certain embodiments, HA polypeptide variants of the present invention has two kinds, three kinds or the replacement of four seed amino acids.In certain embodiments, all these aminoacid replacement all are positioned at the glycan binding site.

In certain embodiments, the HA polypeptide variants has the sequence replacement corresponding to the one or more position in residue 137,145,156,159,186,187,189,190,192,193,196,222,225,226 and 228.In certain embodiments, the HA polypeptide variants has the sequence replacement corresponding to the one or more position in residue 156,159,189,192,193 and 196; And/or has the sequence replacement corresponding to the one or more position in residue 186,187,189 and 190; And/or has the sequence replacement corresponding to the one or more position in residue 190,222,225 and 226; And/or has the sequence replacement corresponding to the one or more position in residue 137,145,190,226 and 228.In certain embodiments, the HA polypeptide variants has the sequence replacement corresponding to the one or more position in residue 190,225,226 and 228.

In certain embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA (for example H5) at the residue place that is selected from the group that forms by following residue: residue 98,136,138,153,155,159,183,186,187,190,193,194,195,222,225,226,227 and 228.In other embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA being selected from the amino acid whose residue place that is arranged in directly in conjunction with the zone of the acceptor of glycan, described residue includes, but is not limited to residue 98,136,153,155,183,190,193,194,222,225,226,227 and 228.In other embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA being selected from the amino acid whose residue place that is positioned at directly in conjunction with the proximity in the zone of the acceptor of glycan, described residue includes, but is not limited to residue 98,138,186,187,195 and 228.

In certain embodiments, HA polypeptide variants of the present invention and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA at the residue place that is selected from the group that forms by following residue: residue 138,186,187,190,193,222,225,226,227 and 228.In other embodiments, HA polypeptide variants of the present invention and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA being selected from the amino acid whose residue place that is arranged in directly in conjunction with the zone of the acceptor of glycan, described residue includes, but is not limited to residue 190,193,222,225,226,227 and 228.In other embodiments, HA polypeptide variants of the present invention and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA being selected from the amino acid whose residue place that is positioned at directly in conjunction with the proximity in the zone of the acceptor of glycan, described residue includes, but is not limited to residue 138,186,187 and 228.

In other embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA at the residue place that is selected from the group that forms by following residue: residue 98,136,153,155,183,194 and 195.In other embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA being selected from the amino acid whose residue place that is arranged in directly in conjunction with the zone of the acceptor of glycan, described residue includes, but is not limited to residue 98,136,153,155,183 and 194.In other embodiments, HA polypeptide variants of the present invention and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA being selected from the amino acid whose residue place that is positioned at directly in conjunction with the proximity in the zone of the acceptor of glycan, described residue includes, but is not limited to residue 98 and 195.

In certain embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA at the isolating amino acid whose residue of 1 degree that is selected from from removing with the interactional amino acid of institute bonded glycan, this be since the 1 degree amino acid separation of removing (1) with directly combine the amino acid interaction; (2) otherwise the influence directly in conjunction with amino acid and the interactional ability of glycan, but not with the direct interaction of glycan own; Or (3) otherwise influence directly in conjunction with amino acid and the interactional ability of glycan, and simultaneously and the direct interaction of glycan own, described residue includes, but is not limited to residue 98,138,186,187,195 and 228.

In other embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA at the isolating amino acid whose residue of 1 degree that is selected from from removing with the interactional amino acid of institute bonded glycan, this be since the 1 degree amino acid separation of removing (1) with directly combine the amino acid interaction; (2) otherwise the influence directly in conjunction with amino acid and the interactional ability of glycan, but not with the direct interaction of glycan own; Or (3) otherwise influence directly in conjunction with amino acid and the interactional ability of glycan, and simultaneously and the direct interaction of glycan own, described residue includes, but is not limited to residue 138,186,187 and 228.

In other embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA at the isolating amino acid whose residue of 1 degree that is selected from from removing with the interactional amino acid of institute bonded glycan, this be since the 1 degree amino acid separation of removing (1) with directly combine the amino acid interaction; (2) otherwise the influence directly in conjunction with amino acid and the interactional ability of glycan, but not with the direct interaction of glycan own; Or (3) otherwise influence directly in conjunction with amino acid and the interactional ability of glycan, and simultaneously and the direct interaction of glycan own, described residue includes, but is not limited to residue 98 and 195.

In certain embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have aminoacid replacement with respect to wild-type parent HA at residue 159 places.

In other embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA at the residue place that is selected from 190,193,225 and 226.In certain embodiments, the HA polypeptide variants and especially the H5 polypeptide variants have one or more aminoacid replacement with respect to wild-type parent HA at the residue place that is selected from 190,193,226 and 228.

In certain embodiments, HA polypeptide variants of the present invention and especially the H5 variant have one or more in the following aminoacid replacement: Ser137Ala, Lys156Glu, Asn186Pro, Asp187Ser, Asp187Thr, Ala189Gln, Ala189Lys, Ala189Thr, Glu190Asp, Glu190Thr, Lys193Arg, Lys193Asn, Lys193His, Lys193Ser, Gly225Asp, Gln226Ile, Gln226Leu, Gln226Val, Ser227Ala, Gly228Ser.

In certain embodiments, HA polypeptide variants of the present invention and especially the H5 variant have one or more in the following aminoacid replacement group:

Glu190Asp, Lys193Ser, Gly225Asp and Gln226Leu;

Glu190Asp, Lys193Ser, Gln226Leu and Gly228Ser;

Ala189Gln、Lys193Ser、Gln226Leu、Gly228Ser；

Asp187Ser/Thr、Ala189Gln、Lys193Ser、Gln226Leu、Gly228Ser；

Ala189Lys、Lys193Asn、Gln226Leu、Gly228Ser；

Asp187Ser/Thr、Ala189Lys、Lys193Asn、Gln226Leu、Gly228Ser；

Lys156Glu、Ala189Lys、Lys193Asn、Gln226Leu、Gly228Ser；

Lys193His、Gln226Leu/Ile/Val、Gly228Ser；

Lys193Arg、Gln226Leu/Ile/Val、Gly228Ser；

Ala189Lys、Lys193Asn、Gly225Asp；

Lys156Glu、Ala189Lys、Lys193Asn、Gly225Asp；

Ser137Ala、Lys156Glu、Ala189Lys、Lys193Asn、Gly225Asp；

Glu190Thr、Lys193Ser、Gly225Asp；

Asp187Thr、Ala189Thr、Glu190Asp、Lys193Ser、Gly225Asp；

Asn186Pro、Asp187Thr、Ala189Thr、Glu190Asp、Lys193Ser、Gly225Asp；

Asn186Pro、Asp187Thr、Ala189Thr、Glu190Asp、Lys193Ser、Gly225Asp、Ser227Ala。

In some described embodiment, HA compares with wild-type, and the HA polypeptide has at least a other and replaces, and makes variant increase the avidity and/or the specificity of umbrella shape glycan.

In certain embodiments, HA polypeptide of the present invention (comprising the HA polypeptide variants) has the sequence that comprises D190, D225, L226 and/or S228.In certain embodiments, HA polypeptide of the present invention has the sequence that comprises D190 and D225; In certain embodiments, HA polypeptide of the present invention has the sequence that comprises L226 and S228.

In certain embodiments, with parent HA and especially compare with parent wild-type HA, HA polypeptide variants of the present invention has open binding site.

The part of HA polypeptide or fragment

The present invention further provides characteristic and its coding nucleic acid of HA polypeptide of the present invention.Usually, characteristic is to contain the continuous amino acid fragment or is the segmental part of a large amount of continuous amino acids of the feature of HA polypeptide together.Each described continuous fragment will contain at least two amino acid usually.In addition, one of ordinary skill in the art will understand, and the feature of H5 HA polypeptide is needs at least 5,10,15,20 or more a plurality of amino acid usually.In general, characteristic be except that appointed sequence consistence above also with the part of total at least one functional character of relevant complete H5 polypeptide.In certain embodiments, the characteristic of HA polypeptide of the present invention has glycan in conjunction with feature with relevant total length HA polypeptide.

Produce the HA polypeptide

Can produce HA polypeptide of the present invention and/or its characteristic or its coding nucleic acid by any available means.

For instance, can produce HA polypeptide of the present invention (or characteristic) through through engineering approaches with the host cell systems of expressing HA peptide coding nucleic acid of the present invention by using.

Can use any system to produce HA polypeptide (or characteristic), described system such as ovum, baculovirus, plant, yeast, Madin-Darby canine kidney dirty (Madin Darby Canine Kidney, MDCK) cell or Vero (cercopithecus aethiops kidney) cell.Perhaps or in addition, can use recombinant technology, such as HA polypeptide (or characteristic) being expressed in (Sa Brooker people such as (Sambrook) in the cell by the use expression vector, molecular cloning: laboratory manual (MolecularCloning:A Laboratory Manual), press of cold spring harbor laboratory (CSHL Press), 1989).

Perhaps or in addition, can produce HA polypeptide of the present invention (or its characteristic) by synthesis mode.

Perhaps or in addition, can in the intact virus environment, produce HA polypeptide of the present invention (or its characteristic), and no matter described virus whether in addition for wild-type, through attenuation, through killing etc.Also can in the virus-like particle environment, produce HA polypeptide of the present invention or its characteristic.

In certain embodiments, can separate and/or purifying HA polypeptide (or its characteristic) by influenza virus.For instance, virus can grown such as containing in the ovum such as embryo egg, and material collected under described situation is generally allantoic fluid.Perhaps or in addition, can cultivate by using-system and make any method of viral growth obtain influenza virus.Be used to make the suitable cell substrate of viral growth to comprise that for example the dog kidney cell is (such as MDCK or from MDCK, the clone's of MDCK like cell cell), monkey kidney cell (such as the AGMK cell that comprises the Vero cell), as continuous cell line through cultivating epithelial cell, the 293T cell, the BK-21 cell, the CV-1 cell, or be suitable for producing any other mammalian cell types of the influenza virus that is used for the vaccine purpose, it is easy to obtain (the Rockville's of Maryland, USA the U.S. typical case culture (ATCC of collecting center for example by commercial source, Rockville, Md)).Suitably the cell substrate comprises the human cell again, such as the MRC-5 cell.Suitably the cell substrate is not limited to clone; For example, also comprise such as primary cells such as Embryo Gallus domesticus fibroblasts.

Simultaneously; one of ordinary skill in the art will understand can be by allowing screening rapidly and/or selecting to cultivate under can the condition in conjunction with the HA polypeptide of umbrella shape topology glycan to produce HA (no matter separately or as the part of mixture; comprise part as virion or virus) cell or organism, prepare, differentiate, separate and/or produce HA polypeptide as described herein and variant HA polypeptide especially.For an example only is provided, in certain embodiments, is disclosing and/or helping producing and/or studying many HA variants and may be suitable under the condition in conjunction with the variant of umbrella shape topology glycan (for example with specific specificity and/or avidity).In certain embodiments, produce described many HA variants by evolving at occurring in nature.In certain embodiments, produce described many HA variants by through engineering approaches.In certain embodiments, by through engineering approaches and the described many HA variants of combination results of evolving naturally.

The HA acceptor

HA is by interacting in conjunction with glycoprotein receptor and cell surface.

N-on the HA acceptor connects glycan and mainly mediates combining of HA and HA acceptor.Particularly, lip-deep HA identification of influenza virus particles and the mutually associating sialylated glycan of the lip-deep HA acceptor of cell host.Identification and in conjunction with after, host cell swallows up virocyte and virus and can duplicate and produce more overabsorption to the virion of adjacent cells.

Modify the HA acceptor by near α 2-3 the HA binding site of acceptor or the sialylated glycan of α 2-6, and the binding type of the glycan of receptors bind influences the conformation of the HA binding site of acceptor, therefore influence the specificity of acceptor different HA.

For instance, the glycan binding pocket of bird HA narrows down.According to the present invention, this pocket is in conjunction with the transoid conformation of the sialylated glycan of α 2-3 and/or the taper topology glycan of α 2-3 or α 2-6 connection.

HA acceptor in bird tissue and human dark lung and stomach and intestine (GI) the road tissue is all characterized by the sialylated glycan binding of α 2-3, and in addition (according to the present invention) by the glycan sign of the main employing taper topology that comprises the sialylated and/or sialylated glycan of α 2-6 of α 2-3.

By contrast, the human HA acceptor in segmental bronchus and upper respiratory tract tracheae is to modify through the sialylated glycan of α 2-6.Different with α 2-3 primitive, α 2-6 primitive has extra conformational freedom (Russell people such as (Russell), glycoconjugate magazine (Glycoconj J) 23:85,2006) because of the C6-C5 key.Have the bigger binding pocket of opening in conjunction with the HA of the sialylated glycan of described α 2-6 and hold the free caused structure diversity of conformation thus.In addition, according to the present invention, HA may need in conjunction with the glycan of umbrella shape topology (for example, the sialylated glycan of α 2-6) and especially may need in conjunction with having strong avidity and/or specific described umbrella shape topology glycan, so that mediate the infection of human upper respiratory tract tissue effectively.

Owing to the glycosylation feature of these limited space, the mankind can not infect the virus that contains many wild-type bird HA (for example, bird H5) usually.Particularly, because the human airway part of most possible experience virus (promptly, trachea and bronchus) shortage (for example has the taper glycan, sialylated glycan of α 2-3 and/or short chain glycan) acceptor, and wild-type bird HA usually mainly or only in conjunction with the taper glycan (for example, sialylated glycan of α 2-3 and/or short chain glycan) associating acceptor, so mankind's infected poultry virus seldom.Only enough closely contact with virus so that its can enter in dark lung and/or the gi tract time, the acceptor with umbrella shape glycan (for example, the sialylated glycan of long-chain alpha 2-6) just makes the mankind be infected.

The glycan array

In order to expand rapidly about the interactional current knowledge of known specificity glycan-decorin binding protein (GBP), an international cooperating research is initiated alliance of tissue-function glycogen group student's federation (Consortium for FunctionalGlycomics, CFG; Www.functionalglycomics.org) developed the glycan array, it comprises that several make can be at the glycan structures of novel glycan ligand specificity high throughout screening of G BP.The glycan array comprise unit price and multivalence glycan primitive (that is, being connected to the polyacrylamide main chain) and each array comprise 264 have low (10 μ M) and high (100 μ M) concentration and at the glycan of 6 points of each concentration (referring to Http:// www.functionalglycomics.org/static/consortium/resources/ resourcecoreh5.shtml).

Described array mainly comprises capture N is connected glycan with O the multifarious synthetic glycan of physiology.Except that synthetic glycan, the N that derives from different Mammals glycoprotein connects the glycan mixture and also is presented on the described array.

As used herein, glycan " array " is meant one group of one or more glycan, and it randomly is immobilized onto solid and props up on the Brace thing.In certain embodiments, " array " many glycan of existing as organized arrangement or pattern for two or more positions of physical sepn spatially.Usually, the glycan array will have at least 4,8,16,24,48,96 hundreds of or thousands of discrete locations.Usually, glycan array of the present invention can have any in the various ways.Known various different array formats in the affiliated field applicable to biomolecules.For instance, know maximal number target protein and/or nucleic acid array.One of ordinary skill in the art will understand the standard array form that is suitable for glycan Array row of the present invention rapidly.

In certain embodiments, glycan array of the present invention exists with " microarray " form.Microarray can have the sample position of be separated by 50-200 micron or following distance usually and in millimicro in little not ear concentration range or the immobilization sample of millimicro in the pik scope.Under in the field known array format comprise that each discrete sample position for example for example has the form of 10 microns yardstick.

In certain embodiments, glycan array of the present invention comprises a plurality of glycan that spatially are immobilized onto on the upholder.The present invention provides the glycan molecular array on upholder.As used herein, " upholder " is meant any material that is suitable for array glycan molecule.It will be appreciated by those skilled in the art that and to adopt in the multiple material any.For several examples only are provided, can be used for buttress material of the present invention and comprise hydrophobic film, for example soluble cotton, PVDF or nylon membrane.Described film be know in the affiliated field and can available from the Bole company of for example Britain He Moer Hempstead (Bio-Rad, Hemel Hempstead, UK).

In other embodiments, the upholder of carrying glycan array can comprise metal oxide.Suitably metal oxide includes, but is not limited to titanium oxide, tantalum oxide and aluminum oxide.The example of described material can be matched BH12 4QH imagination road Sigma aldrich company limited (Sigma-Aldrich Company Ltd, Fancy Road, Poole, Dorset.BH12 4QH UK) available from Britain more.In other embodiments, above support is a metal oxide gel, or comprises metal oxide gel.Think that metal oxide gel provides the immobilization with the molecule of auxiliary carbohydrate containing of big surface-area in given naked eyes visibility region.

Other that can be used according to the invention or alternative buttress material comprise gel, for example silica gel or alumina gel.The example of described material can available from the Merck group of for example Darmstadt, Germany (Merck KGaA, Darmstadt, Germany).

In certain embodiments of the present invention, the glycan array being immobilized in can be on the upholder of opposing size or change of shape between the normal usage period.For instance, upholder can be the slide glass that is coated with the constituent materials that is applicable to the array glycan.Simultaneously, some matrix material can desirably provide stability to upholder.

Confirm that as this paper array of the present invention is applicable to differentiates and/or characterize different HA polypeptide and it is in conjunction with feature.In certain embodiments, on described array, H5 polypeptide of the present invention is tested, to assess its ability in conjunction with umbrella shape topology glycan (for example, the sialylated glycan of α 2-6 and especially with the sialylated glycan of the long-chain alpha 2-6 of umbrella shape topological arrangement).

In fact, the invention provides and can be used for characterizing HA polypeptide binding ability and/or as the sialylated glycan of α 2-6 of the diagnosis thing that for example detects human bonded HA polypeptide and the array of the sialylated glycan of optional α 2-3.In certain embodiments, array of the present invention contains the glycan (for example sialylated glycan of α 2-6 and the especially sialylated glycan of long-chain alpha 2-6) that is umbrella shape topology form.One of ordinary skill in the art will be apparent, and described array is applicable to and characterizes or detect any HA polypeptide, comprise the HA polypeptide seen in for example natural influenza strain isolated and the researchist is designed and/or the HA polypeptide of preparation.

In certain embodiments, described array comprises that being seen glycan (for example on human HA acceptor of expression and the especially human upper respiratory tract HA acceptor, the umbrella shape glycan, it will be the sialylated glycan of α 2-6, the especially sialylated glycan of long-chain alpha 2-6 usually) about glycan more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 95%.In certain embodiments, array of the present invention comprises some or all glycan structures described in Figure 10.In certain embodiments, array comprises at least about these the described glycan more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 95%.

The invention provides and use the glycan array to differentiate or characterize the proteic method of HA.In certain embodiments, in fact, described method comprises following steps: (1) provides the sample that contains the HA polypeptide for example; (2) sample is contacted with the glycan array; (3) detect combining of one or more glycan on HA polypeptide and the array.

The appropriate sources that contains the sample of the HA polypeptide that will contact with glycan array according to the present invention includes, but is not limited to the pathology sample, such as blood, blood serum, peripheral blood monocyte/peripheral blood lymphocyte (PBMC/PBL), saliva, urine, ight soil, throat swab, dermatosis swab, celiolymph, cervical smear, purulence sample, food matrix with from the tissue of the various parts (such as brain, spleen and liver) of health.Perhaps or in addition, other appropriate sources that contains the sample of HA polypeptide includes, but is not limited to such as soil, water and floral environmental samples.Other sample comprises the laboratory sample of the through engineering approaches HA polypeptide that is for example designed by the researchist and/or prepare.Also can use other sample of not enumerating as yet.The known multiple detection system that is suitable for examining and determine in conjunction with the HA polypeptide of glycan array of the present invention in the affiliated field.For instance, the HA polypeptide can with before or after array contacts through detectability mark (directly or indirectly); Then, can detect combination by detecting the localization mark.In certain embodiments, can use scanning device to check specific position on the array.

Perhaps or in addition, the method that can use for example calorimetric, fluorescence or radioassay system or other marking method or other and not require mark is measured and the combining of the glycan that is arranged in array.Usually, fluoroscopic examination is usually directed to directly detect array and monitor fluorescent signal with fluorescence molecule.Perhaps or in addition, can be used for the molecule that indirect fluorescent detects through mark (for example with biotin labeling) and detect array (under described situation) by testing the combination of fluorescently-labeled streptavidin.Perhaps or in addition, can use the fluorescent quenching method, the glycan that wherein is arranged in array is detected through fluorescent mark and with test molecule (its can through or can be without different fluorophore marks).In described embodiment, with the fluorescence that combining of array worked and sent by the glycan that is arranged in array to eliminate, therefore the loss by fluorescent emission detects combination.Perhaps or in addition, can be in the presence of radioactive substance the living tissue sample of growth (thereby obtaining) through radiolabeled probe detect the glycan that is arranged in array.Can detect combination among the described embodiment by measuring radioactive emission.Described method is applicable to measures combining and/or combination degree of HA polypeptide and glycan array of the present invention.In certain embodiments of the present invention, can further use described method to differentiate and/or characterize disturbs or change glycan-interactional reagent of HA polypeptide.For example, method hereinafter described can be used for especially differentiating whether be believed in fact can be like this really with the interactional molecule of carbohydrate, or differentiates whether molecule has and the interactional ability of carbohydrate unexpectedly.

The present invention also provides the method for using array of the present invention for example to detect the particular agent in the test sample book.For instance, described method can comprise following steps: (1) makes glycan array and test sample book (for example, being considered to contain the sample of HA polypeptide) contact; (2) detect combining of any reagent and described array in the test sample book.

In addition, can use with combining of array of the present invention and for example measure interactional kinetics between wedding agent and the glycan.For instance, the method that is used to measure interaction dynamics of the present invention can may further comprise the steps: (1) makes the glycan array contact with the institute test molecule; (2) measure wedding agent and be arranged in interactional kinetics between the glycan of array.

Can measure the interactional kinetics of the arbitrary glycan in wedding agent and the array of the present invention by the real-time change of the colorimetric that for example described in detail as mentioned or fluorescent signal.Whether described method especially can be used for for example measuring the particular combination agent can be with than interacting with high combination degree and the same carbohydrate of the interactional different wedding agents of particular carbon hydrate.

Certainly, should be appreciated that and to assess the glycan combination of HA polypeptide of the present invention at non-existent glycan sample in the array format itself or source.For instance, can make HA polypeptide conjunctive tissue sample of the present invention and/or clone to assess its glycan in conjunction with feature.Suitably clone comprises any in for example multiple mammal cell line, and especially performance contains the clone of the HA acceptor of umbrella shape topology glycan (for example, wherein at least some can be sialylated glycan of α 2-6 and the sialylated glycan of long-chain alpha 2-6 especially).In certain embodiments, employed clone shows the indivedual glycan with umbrella shape topology.In certain embodiments, employed clone shows multiple glycan.In certain embodiments, clone is available from clinical separation strain; In some cases, it distributes and/or ubiquity through keeping or handle having required glycan.In certain embodiments, the epithelial glycan feature of tissue samples and/or the clone performance Mammals upper respiratory tract.

Data mining platform

As discussed here, according to the present invention, can be by differentiating and/or characterize the HA polypeptide in conjunction with mining data, structural information (for example HA crystalline structure) and/or the protein structure predictor of research from glycan.

The explanation present inventor uses the key step that relates in the particular data method for digging of (and illustration) herein among Figure 11.These steps relate to the operation at three elements: data object, feature and sorter." data object " is for being stored in the raw data in the database.Under the situation of glycan array data, the chemical descriptor of glycan structures aspect monose and binding with and with the binding signal composition data object of the different GBP that screened.The characteristic of data object is " feature ".Selection comes the data of description object based on rule or pattern that feature obtained." sorter " is for being used for the rule or the pattern of the relation between particular category or the definite feature that data object is clustered to.Sorter provides with the special characteristic that glycan satisfied of high-affinity in conjunction with GBP.These rules are divided into two classes: (1) is present in one group of feature on the high-affinity glycan part, thinks that it can strengthen combination; (2) should not be present in feature in the high-affinity glycan part, think that it is in conjunction with unfavorable.

Data mining platform used herein comprises software module interact with each other (Figure 11) and carries out operation mentioned above.Feature extractor will be as the interface of CFG database so that extract feature, and the employed Relational database based on described object of CFG helps determining flexibly of feature.

Feature extraction and data are prepared:

The characteristic features that glycan from the glycan array extracts is listed in the table 1.

The feature that table 1. extracts from the glycan on the glycan array.

Rule-based sorting algorithm is used the feature described in this table to differentiate and is characterized and specificity GBP bonded pattern.

The ultimate principle of these special characteristics that selection is showed is that the glycan binding site on the GBP holds two-tetrose usually.Use captures information (tree root is at reducing end) about monose in the glycan structures and binding based on the representation of tree.This representation helps the extraction of various features, comprises advanced features, such as (Figure 12) such as monose triplet sets through connecting.The data preparation relates to the branch bar tabulation of the feature (table 1) of each glycan that produces all glycan in the glycan array and extracted.At rule-based classification (vide infra), from this master meter of glycan and its feature, select a subclass to determine control and specific GBP or GBP group bonded AD HOC.

Sorter:

Developed dissimilar sorters and used it in many application.It mainly is divided into three primary categories: the mathematical approach, distance method and logical approach.These different methods with and merits and demerits be discussed in Wei Si (Weiss) and Ying Dukeya (Indrukhya) (predictive data excavation-practice guideline (Predictive data mining-A practicalguide.) the root Kaufman that rubs (Morgan Kaufmann) in detail, San Francisco (San Francisco), 1998) in.For this application-specific, we select a kind of method that is called rule induction, and it belongs to logical approach.If producing, the rule induction sorter is-pattern of rule (IF-THEN rule) form so.

If logical approach and especially generation-major advantage such as sorters such as rule induction methods of rule is so, when comparing with other statistics or the mathematical approach, the result of sorter can more easily obtain explaining.This makes can explore the rule found or the structure and the biological significance of pattern.Use the exemplary rule (table 1) that feature produced of description in the early time to be- IfGlycan Contain" Galb4GlcNAcb3Gal[B] " and Do not contain" Fuca3GlcNAc[B] ", ThatGlycan will be with higher affinity in conjunction with galactose agglutinin 3.The algorithm that the ad hoc rules inductive algorithm that uses is is in the case researched and developed as Wei Si and Ying Dukeya (predictive data excavation-practice guideline rub root Kaufman, San Francisco, 1998).

Combination degree

Determine the differentiation low-affinity and the high-affinity bonded threshold value of each glycan array screening data set.

Nucleic acid

In certain embodiments, the invention provides the nucleic acid of the feature or the biologic activity part of coding HA polypeptide or HA polypeptide.In other embodiments, the invention provides and the encode nucleic acid complementary nucleic acid of the feature of HA polypeptide or HA polypeptide or biologic activity part.

In other embodiments, the invention provides and the encode nucleic acid molecule of nucleic acid hybridization of the feature of HA polypeptide or HA polypeptide or biologic activity part.Can be with described nucleic acid for example as introduction or probe.Only lift several examples, described nucleic acid can be used as the introduction in the polymerase chain reaction (PCR), the introduction that is used for hybridizing the probe of (comprising in situ hybridization) and/or is used for reverse transcription PCR (RT-PCR).

In certain embodiments, nucleic acid can be DNA or RNA and can be strand or double-stranded.In certain embodiments, nucleic acid of the present invention can comprise one or more non-natural nucleotide; In other embodiments, nucleic acid of the present invention only comprises natural nucleotide.

Antibody

The invention provides the antibody of HA polypeptide of the present invention.These antibody can be mono-clonal or polyclonal antibody, and can prepare by in the known multiple technologies of one of ordinary skill in the art any (referring to for example, Ha Lu (Harlow) and blue Buddhist nun (Lane), antibody: laboratory manual (Antibodies:A Laboratory Manual), Cold Spring Harbor Laboratory press, 1988).For instance, can be by comprising the cell culture technology that produces monoclonal antibody or via the antibody gene transfection is prepared antibody to allow producing recombinant antibodies in suitable bacterium or mammalian cell host.

Medical composition

In certain embodiments, the invention provides medical composition, described composition comprises the nucleic acid of the feature of HA polypeptide, coding said polypeptide, described polypeptide or biological active fragment or nucleic acid, in conjunction with described polypeptide or segmental antibody, with described polypeptide or combine its interactional small molecules of glycan etc.

The present invention forgives by throwing with described medical composition of the present invention and treats influenza infection.In certain embodiments, realize treatment by throwing with vaccine.Up to now, although obtaining prominent achievement aspect the research and development influenza vaccines, but still there is the space of further improving.The invention provides and comprise HA polypeptide of the present invention and especially comprise vaccine in conjunction with the HA polypeptide of umbrella shape glycan (for example the umbrella shape glycan that connects of α 2-6, such as the sialylated glycan of long-chain alpha 2-6).

Only cite an actual example, to the reduced immunogenicity of small part owing to H5 HA, generation can't be succeedd usually to the trial that the H5N1 bacterial strain has specific vaccine in the mankind.In a research, through showing the antibody titer that produced 1: 40, this is not that sufficient to guarantee prevents that the height that is infected from tiring at the vaccine of H5N1 bacterial strain.In addition, produce even dosage that 1: 40 antibody titer of appropriateness is required (90 μ g of 2 dosage purified kill virus or antigen) is 12 times of (Qu Anuo people such as (Treanor) of common used dosage under the situation of common seasonal current influenza virus vaccine, New England Journal of Medicine (N Eng J Med), 354:1343,2006).Other research shows that similarly current H5 vaccine does not have hyperimmunization originality (Bu Ruisen people such as (Bresson), lancet (Lancet), 367:1657,2006).In certain embodiments, use one or more be intended to allow to use than the H5 HA albumen of low dosage and/or reach high immunogenicity strategy (referring to for example, Ace Rake (Enserink), science, 309:996,2005) allocate vaccine of the present invention.For instance, in certain embodiments, (for example via using tree-shaped polymkeric substance) improves multivalence; In certain embodiments, use one or more adjuvants, or the like.In certain embodiments, the invention provides vaccine and these vaccines to human person under inspection's throwing with.In certain embodiments, vaccine is the one or more composition that comprises in following each thing: (1) inactivation of viruses; (2) live attenuated influenza virus, for example replication defective virus; (3) HA polypeptide of the present invention or its feature or biologic activity part; (4) nucleic acid of coding HA polypeptide or its feature or biologic activity part; (5) dna vector of coding HA polypeptide or its feature or biologic activity part; And/or (6) expression system, for example express the cell of one or more desires as antigenic influenza proteins.

Therefore, in certain embodiments, the invention provides inactivated influenza vaccine.In certain embodiments, inactivated influenza vaccine comprises a kind of in the following three types antigen preparation: inactivated whole virus; Subviral particle wherein destroys purified virion with lipin dissolving coating (" cracking " vaccine) by sanitising agent or other reagent; Or purified HA polypeptide (" subunit " vaccine).In certain embodiments, can handle inactivation of virus by using formaldehyde, beta-propiolactone, ether, ether and sanitising agent (such as tween-80), cetyl trimethylammonium bromide (CTAB) and triton (Triton) N101, Sodium desoxycholate and tricresyl phosphate (positive butyl ester).Deactivation can occur in (from the virus for preparing) and purify after the allantoic fluid or before ovum; Separate and purified virus particle (Nicholson people such as (Nicholson) volume by centrifugal, influenza textbook (Textbook of Influenza), U.S. Backwill scientific publication company (Blackwell Science), plum steps on (Maiden), the Maryland State (MA), 1998).Be the usefulness of assessment vaccine, can use unidirectional radioimmunodiffusion (SRD) test (Si Qieerde people such as (Schild), World Health Organization's bulletin (Bull.World Health Organ.), 52:43-50﹠amp; 223-31,1975; Mo Situo people such as (Mostow), clinical microbiology magazine (J.Clin.Microbiol), 2:531,1975).

The present invention also provides Gripovax and method of attenuating alive to be known by affiliated field.In certain embodiments, realize attenuation via using such as reverse geneticses such as site-directed mutagenesises.

In certain embodiments, the influenza virus that is used in vaccine grows in ovum, for example grows in containing the embryo egg, and under described situation, collected material is an allantoic fluid.Perhaps or in addition, can cultivate by using-system and make any method of viral growth obtain influenza virus.Be used to make the suitable cell substrate of viral growth to comprise that for example the dog kidney cell is (such as MDCK or from MDCK, the clone's of MDCK like cell cell), monkey kidney cell (such as the AGMK cell that comprises the Vero cell), as continuous cell line through cultivating epithelial cell, the 293T cell, the BK-21 cell, the CV-1 cell, or be suitable for producing any other mammalian cell types (comprising the airway epithelia cell) of the influenza virus that is used for the vaccine purpose, it is easy to obtain (the Rockville's of Maryland, USA the U.S. typical case culture (ATCC of collecting center for example by commercial source, Rockville, Md)).Suitably the cell substrate comprises the human cell again, such as the MRC-5 cell.Suitably the cell substrate is not limited to clone; For example, also comprise such as primary cells such as Embryo Gallus domesticus fibroblasts.

In certain embodiments, vaccine of the present invention further comprises one or more adjuvants.For instance, can use aluminium salt (Bai Le people such as (Baylor), vaccine (Vaccine), 20:S18,2002) and monophosphoryl lipid A (MPL; Lapie people such as (Ribi), (1986), bacterial endotoxin immunology and immunopharmacology (Immunology andImmunopharmacology of bacterial endotoxins), Pu Lainan publishing company (Plenum Publ.Corp.), New York (NY), the 407th page, 1986) as the adjuvant in the human vaccine.Perhaps or in addition, testing the new compound that in human vaccine, is used as adjuvant at present, such as MF59 (U.S. Kai Long group (Chiron Corp.), Http:// www chiron com/investors/pressreleases/2005/051028.html), CPG 7909 (Cusparia people such as (Cooper), vaccine, 22:3136,2004) and Saponin/TSM, such as QS21 (Fu Qikean (Ghochikyan) etc. goes into vaccine, 24:2275,2006).

In addition, the immunogenic adjuvant of known some enhanced flow influenza vaccine in the affiliated field is such as poly-[two (carboxylic foundation phenoxy group) phosphorus nitrence] (PCCP; Send grace people such as (Payne), vaccine 16:92,1998), interferon-(people such as (Cao) Cao, vaccine, 10:238,1992), segmented copolymer P1205 (CRL1005; Card thatch (Katz) people of etc.ing, vaccine, 18:2177,2000), white element-2 (IL-2 are situated between; Mu Wuke people such as (Mbwuike), vaccine, 8:347,1990) and polymethylmethacrylate (PMMA; Gram as special people such as (Kreuter), medical science magazine (J.Pharm.Sci.), 70:367,1981).

Except that vaccine, the present invention also provides other to be applicable to the therapeutic composition of treatment viral infection.For instance, in certain embodiments, realize treatment by throwing with disturbing HA polypeptide expression of the present invention or active medicament.For instance, can use comprise antibody (antibody that contains the virion of the specific HA polypeptide HA polypeptide of umbrella shape glycan (for example in conjunction with) such as identification), nucleic acid (such as with HA sequence complementary nucleotide sequence, it can be used for RNAi), competition realizes treatment in conjunction with the composition of glycan, competition glycan-interactional small molecules of HA polypeptide or sugared simulant (glycomometics) or its any combination of HA acceptor.In certain embodiments, use the different medicaments that have different structure in a large number.In certain embodiments, therapeutic composition comprises one or more multivalence medicaments.In certain embodiments, treatment be included in expose or doubtful exposure after urgent soon throw with.

Usually, except that one or more such as the sterile biocompatible supporting agent inertia reagent such as (including, but is not limited to sterilized water, physiological saline, buffer saline or dextrose solution), medical composition also will comprise therapeutical agent.Perhaps or in addition, described composition can contain any in the multiple additives, such as stablizer, damping fluid, vehicle or sanitas.In certain embodiments, medical composition will comprise through capsule envelope, catch or be combined in the therapeutical agent in lipid vesicle, biological available and/or biocompatibility and/or biodegradable substrate or other particulate.

Can separately or make up one or more other treatment agent (including, but is not limited to vaccine and/or antibody) throws and medical composition of the present invention." combination " do not plan to hint medicament must throw simultaneously with or must be used to send through allotment, though these delivering methods are also within the scope of the invention.Usually, will be to throw and described medicament at determined dosage of each medicament and time-histories.In addition, the present invention forgives combination and can improve biological usability, reduction or improve metabolism, suppress to drain or improve and send medical composition of the present invention at the medicament of the intravital distribution of body.Though can use medical composition of the present invention to treat any person under inspection (for example any animal) who needs that has, most preferably use its treatment human.

Can throw and medical composition of the present invention by number of ways, comprise in per os, intravenously, intramuscular, intra-arterial, subcutaneous, the ventricle, through skin, intracutaneous, per rectum, intravaginal, intraperitoneal, part (by pulvis, ointment, emulsifiable paste or drops), per mucous membrane, through the oral cavity, with per os or through nasal spray or aerosol form throw with.Usually, optimal throwing and approach will rely on multiple factor, comprise the character (for example its stability in gastrointestinal tract environment) of medicament, patient's situation (for example patient whether can tolerate oral administration with) etc.At present, per os or be most commonly used to therapeutical agent directly is delivered to lung and respiratory system through nasal spray or aerosol approach.Yet, considering that medicine sends may improving of science, the present invention forgives by any suitable pathways and sends medical composition of the present invention.

The appropriate device that is used to send intracutaneous medical composition as herein described comprises the hour hand device, such as being described in United States Patent (USP) the 4th, 886, No. 499, No. the 5th, 190,521, United States Patent (USP), United States Patent (USP) the 5th, 328, No. 483, United States Patent (USP) the 5th, 527, No. 288, No. the 4th, 270,537, United States Patent (USP), United States Patent (USP) the 5th, 015, No. 235, United States Patent (USP) the 5th, 141, device in No. the 5th, 417,662, No. 496, United States Patent (USP).Also can throw and the intracutaneous composition by the device of the effective penetration length of restriction pin in skin, described device is such as the device and its functional equivalent that are described among the WO99/34850 (being incorporated herein by reference).Effusive hydrofluidic syringe or pin that stratum corneum and generation reach corium are suitable equally with the jet jet apparatus that aqueous vaccine is delivered to corium via thrusting.The jet jet apparatus is to be described in for example following patent: United States Patent (USP) the 5th, 480, No. 381, United States Patent (USP) the 5th, 599, No. 302, United States Patent (USP) the 5th, 334, No. 144, United States Patent (USP) the 5th, 993, No. 412, United States Patent (USP) the 5th, 649, No. 912, United States Patent (USP) the 5th, 569, No. 189, United States Patent (USP) the 5th, 704, No. 911, United States Patent (USP) the 5th, 383, No. 851, United States Patent (USP) the 5th, 893, No. 397, United States Patent (USP) the 5th, 466, No. 220, United States Patent (USP) the 5th, 339, No. 163, United States Patent (USP) the 5th, 312, No. 335, United States Patent (USP) the 5th, 503, No. 627, United States Patent (USP) the 5th, 064, No. 413, United States Patent (USP) 5,520,639 No., United States Patent (USP) the 4th, 596, No. 556, United States Patent (USP) the 4th, 790, No. 824, United States Patent (USP) the 4th, 941, No. 880, United States Patent (USP) the 4th, 940, No. 460, WO 97/37705 and WO 97/13537.It is suitable equally to use pressurized gas to come the vaccine of powder quick form to pass the trajectory powder/granule delivery apparatus that skin outer layer arrives corium.In addition, in typical Man Tuo (mantoux) method of intracutaneous dispensing, can use conventional syringe.

Common consideration item in the allotment of medical agent and the manufacturing is found in for example Lei Mingdun medical science (Remington ' sPharmaceutical Sciences), the 19th edition, Mike publishing company (Mack Publishing Co.), Easton (Easton), Pennsylvania (PA) is in 1975.

Diagnosis/test kit

The invention provides to be used for detecting the HA polypeptide of pathology sample and especially to detect and (for example have specific glycan in conjunction with feature, in conjunction with the umbrella shape glycan, the sialylated glycan of α 2-6, the test kit of the HA polypeptide sialylated glycan of long-chain alpha 2-6 etc.), described pathology sample comprises (but being not limited to) blood, blood serum, peripheral blood monocyte/peripheral blood lymphocyte (PBMC/PBL), saliva, urine, ight soil, throat swab, the dermatosis swab, celiolymph, cervical smear, the purulence sample, food matrix and from the health each several part (such as brain, spleen and liver) tissue.The present invention also is provided for the test kit of the HA polypeptide of paying close attention in the testing environment sample (including, but is not limited to soil, water and flora).Also can use other sample of not enumerating as yet.

In certain embodiments, test kit of the present invention can comprise that one or more specific detection have the medicament of specific glycan in conjunction with the HA polypeptide of feature.Described medicament can comprise that for example some HA polypeptide of specific recognition (for example, HA polypeptide in conjunction with umbrella shape glycan and/or sialylated glycan of α 2-6 and/or the sialylated glycan of long-chain alpha 2-6) antibody, it can be used for by the described HA polypeptide of ELISA, immunofluorescence and/or immunoblotting specific detection.These antibody also can be used for virus neutralization test, wherein handle sample and test it infects institute's cultured cells with respect to unprocessed sample ability with the specific antibody of the HA polypeptide paid close attention to.If the virus in the described sample contains described HA polypeptide, antibody will neutralize viral and prevent that it from infecting institute's cultured cells so.Perhaps or in addition, also described antibody can be used for HA and suppress test,, handle and test it and condense erythrocytic ability with respect to unprocessed sample with the specific antibody of specific HA polypeptide or HA polypeptide group wherein from given sample separation HA albumen.If the virus in the sample contains described HA polypeptide, so antibody will in and HA polypeptide active and prevent its cohesion red blood corpuscle (Ha Lu and blue Buddhist nun, antibody: laboratory manual, Cold Spring Harbor Laboratory press, 1988; Http:// www.who.int/csr/resources/publications/influenza/WHO_CDS _ CSR_NCS_2002_5/en/Index.html; Http:// www.who.int/csr/disease/avian influenza/guidelines/labtests/en/index.html).In other embodiments, described medicament can comprise specificity in conjunction with the coding specific HA polypeptide and can be used for by the Nucleotide of RT-PCR or the described HA polypeptide of in situ hybridization specific detection nucleic acid ( Http:// www.who.int/csr/resources/publications/influenza/WHO_CDS _ CSR_NCS_2002_5/en/ index.html Http:// www.who.int/csr/disease/avian influenza/guidelines/labtests/en/index.html).In certain embodiments, before detecting amplification from the nucleic acid of sample separation.In certain embodiments, diagnostic reagent can be through the detectability mark.

The present invention provides simultaneously and contains the test kit that is used to produce the reagent of influenza virus and vaccine of the present invention.The inclusion of test kit includes, but is not limited to contain the expression plasmid of the HA Nucleotide (or feature or biologic activity part) of the HA polypeptide (or feature or biologic activity part) that coding pays close attention to.Perhaps or in addition, test kit can contain the expression plasmid of the HA polypeptide (or feature or biologic activity part) that expression pays close attention to.The user also can comprise the expression plasmid that does not contain virogene, so that can incorporate the HA Nucleotide from any influenza virus of paying close attention to into.Test kit also can comprise mammal cell line, includes, but is not limited to Vero and mdck cell system.In certain embodiments, diagnostic reagent can be through the detectability mark.

In certain embodiments, test kit used according to the invention can comprise reference sample, is used to handle sample, the specification sheets of testing, the specification sheets that is used for explanation results, damping fluid and/or tests necessary other reagent.In certain embodiments, test kit can comprise the antibody group.

In certain embodiments of the present invention, can use glycan array as discussed above as diagnostic reagent and/or test kit.

In certain embodiments, use glycan array of the present invention and/or test kit to carry out dose response research, with assessment HA polypeptide and the combination of umbrella shape glycan under a plurality of dosage (for example, as described herein).Described research provides the seeing clearly in conjunction with the especially useful of feature of the HA polypeptide of being tested, and is particularly useful for assessing the specificity combination.The dose response of this type is in conjunction with discovering many suitable application.Only cite an actual example, it can help to follow the trail of in the HA polypeptide variants of relevant series the evolution in conjunction with feature, and no matter described series is via natural evolution, has a mind to through engineering approaches or both combinations produce.

In certain embodiments, use glycan array of the present invention and/or test kit to induce, differentiate and/or select the HA polypeptide, and/or have required HA polypeptide variants in conjunction with feature.For instance, in certain embodiments, use glycan array of the present invention and/or test kit that the colony of HA polypeptide is applied evolution (for example, screening and/or selection) pressure.

Illustration

Example 1: about the framework of the binding specificity of H1, H3 and H5 HA and α 2-3 and the sialylated glycan of α 2-6

From the crystalline structure of the HA of H1 (PDB ID:1RD8,1RU7,1RUY, 1RV0,1RVT, 1RVX, 1RVZ), H3 (PDBID:1MQL, 1MQM, 1MQN) and H5 (1JSN, 1JSO, 2FK0) with and provide the molecule of residue related in interacting about specificity HA-glycan to see clearly with the mixture of α 2-3 and/or α 2-6 sialylated oligosaccharides.Recently, comprise the glycan receptor-specific that wild-type on the glycan array of multiple α 2-3 and the sialylated glycan of α 2-6 and mutant are described bird and human H1 and H3 hypotype in detail by screening.

Asp 190Glu among the HA of human epidemic virus in 1918 sudden change makes its specificity be reversed the sialylated glycan of α 2-3 (people such as Stevens, molecular biology magazine (J.Mol.Biol), 355:1143,2006 from α 2-6; Karl-Heinz Grasser people such as (Glaser), Journal of Virology (J.Virol), 79:11533,2005).On the other hand, dual sudden change Glu190Asp on the bird H1 (A/ duck/A Erbaida/35/1976) and Gly225Asp make its specificity be reversed the sialylated glycan of α 2-6 from α 2-3.Under the situation of H3 hypotype, Gln226 becomes Leu and Gly228 becomes the amino acid change of Ser and relevant (the Rogers people such as (Rogers) of change of its preferred property from α 2-3 to the sialylated glycan of α 2-6 between bird H3N8 virus strain in 1963 and the popular human H3N2 virus strain of 1967-68, nature, 304:76,1983).In the ferret model, use 1918 H1N1 virus of highly pathogenic and virulence to confirm relation (Ta Mupei T.M. (Tumpey T.M.) waits people, science, 315:655,2007) between HA glycan binding specificity and the propagation efficiency.

Become the bird α preferred property of 2,3 sialylated acceptors (AV18) can produce the virus that to propagate from the preferred sex reversal of

human α

2,6 sialylated glycan (SC18) acceptors of parent the receptors bind specificity.On the other hand, a kind of

blend alpha

2,3/

α

2,6 sialylated glycan specificity virus (A/ New York/1/18 (NY18)) do not represent propagated, be to be similarly the specific A/ of

blend alpha

2,3/ α, 2,6 sialylated glycan Texas/36/91 (Tx91) virus effect spread can be arranged unexpectedly.In addition, as indicated above, various highly pathogenic H5N1 virus bacterial strains are also showed

blend alpha

2,3/

α

2,6 sialylated glycan specificitys (hillside plot S. people such as (Yamada S.), natural 444:378,2006), and still can be in interpersonal propagation.Cause following problem about the sialylated glycan specificity of HA and the mixing resultant of propagation.First, whether the sialylated glycan seen in the human upper respiratory tract exists diversity, can and this explain the specificity and the tissue tropism of virus? second, how may existence combine the slight change of the glycan conformation that works for α 2-3 and/or the sialylated glycan of α 2-6 and HA glycan binding pocket? generally speaking, what be glycan in conjunction with the requirement of A type influenza virus HA about human adaptability?

Replace the structural limitations that is applied in conjunction with the sialylated glycan of α 2-3 for H1, H3 and H5 HA by the glycan topological sum

The analysis indication of all HA-glycan eutectic structures: the orientation of Neu5Ac sugar (SA) is to fix with respect to HA glycan binding site.Grappling SA relates to different HA hypotype camber conservative amino acid Phe95, Ser/Thr136, Trp153, His183, Leu/Ile194 group.Therefore, HA is controlled by the interaction of HA glycan binding site and glucosides Sauerstoffatom and the sugar except that SA to the specificity of α 2-3 or α 2-6.

The conformation of Neu5Ac α 2-3Gal binding makes Gal among the α 2-3 and the carbohydrate mapping except that Gal in the tapered zone of the glucosides torsion(al)angle control that is subjected to this binding place (Fig. 6).The representative region of least energy conformation is by-60 or 60 or 180 Φ value appointment approximately, wherein ψ from-60 to 60 sampling (Figure 14).In these least energy zones, the sugar among the α 2-3 except that Gal stretches out outside the HA glycan binding site.This also can be apparent from the eutectic structure of HA and α 2-3 primitive (Neu5Ac α 2-3Gal β 1-3/4GlcNAc-), and wherein the Φ value is generally about 180 (being called transoid conformation).Transoid conformation makes α 2-3 primitive stretch out outside the pocket.This hint is that the center will be to HA in conjunction with having maximum effect (Fig. 7) at Gal and/or GlcNAc (or GalNAc) the sugar branched structural changes in place (sulfation and fucosylation) with three sugar (or trisaccharide) α 2-3 primitive.This structure implication with obtain from data mining analysis about the different sorters of sialylated glycan bonded three classes of HA and α 2-3 conform to (table 3).The common characteristic of all these three classifications is not for existing Neu5Ac α 2-3Gal and GalNAc α/β 1-4Gal.Crystal structure analysis shows that the GalNAc that is connected to the Gal of Neu5Ac α 2-3Gal causes with proteic unfavorable space and contacts, and this conforms to sorter.

Outside the conservative anchor point of desalivation acid bonded, relate to two Key residues Gln226 and Glu190 in conjunction with Neu5Ac α 2-3Gal primitive.Be positioned at the glucosides Sauerstoffatom interaction (Figure 15, figure C, D) of the Gln226 and the Neu5Ac α 2-3Gal binding of binding site bottom.Be positioned at Glu190 on the opposite side of Gln226 and Neu5Ac and Gal monose interact (Figure 15, figure C, D).In addition, the conservative residue A la138 of bird HA camber (contiguous Gln226) and Gly228 (contiguous Glu190) can relate to promoting for the correct conformation of sialylated glycan interactional Gln226 of the best of α 2-3 and Glu190 in (Figure 15).As viewed in crystalline structure, human H1 hypotype APR34 contains all four amino acid Ala138, Glu190, Gln226 and Gly228 and in conjunction with the sialylated glycan of α 2-3 (Figure 14, figure B).

Additional glycan binding site will provide about Glu190 side chain location and its additionally seeing clearly HA and the sialylated glycan bonded influence of α 2-3 in the crystalline structure of AAI68_H3_23, ADU67_H3_23 and APR34_H1_23.The side chain of Glu190 among the H1 HA stretches into the side chain far away (about 1 dust) of binding site than the Glu190 among the H3 HA.This is attributable to the amino acid difference of the Ser186 among Pro 186 and the H3 HA among the H1 HA of contiguous Glu190 residue.Shown in the data mining analysis of glycan microarray data, this change of Glu190 side chain conformation can with bird H1 (and non-bird H3) with appropriate avidity in conjunction with the sialylated glycan of some α 2-6 relevant (table 3).In addition, replace the interaction that Ser (the significant change between bird and the human H3 hypotype) will change the conformation of Glu190 and disturb the Neu5Ac α 2-3Gal of human H3HA and transoid conformation with Gly228.This will be described in further detail by the not isomorphic map (non-transoid conformation) of viewed Neu5Ac α 2-3Gal primitive in human AAI68_H3_23 eutectic structure.The best with bird H3 HA that described primitive provided that the best contact gear ratio with human H3 HA binding site that Neu5Ac α 2-3Gal primitive in this conformation is provided is transoid conformation contacts few (Figure 14).Owing to this contact loss, Gly228Ser among old friend's class H3 HA sudden change makes its glycan binding site not too help interaction with the sialylated glycan of α 2-3.This structure observation conforms to the result's (table 3) who is obtained by data mining analysis, and it shows that human H3 HA only has the avidity of appropriateness for the sialylated glycan of some α 2-3.

How does structural changes about Neu5Ac α 2-3Gal influence the interaction of HA-glycan? be positioned at the conservative Lys193 (Fig. 5) of bird H5 camber so that its with Neu5 Ac α 2-3Gal β 1-4GlcNAc in 6-O sulfation Gal and/or 6-O sulfation GlcNAc interaction.Can verify this observations by data mining analysis, wherein only bird H5 is combined in Gal or the Sulfated sialylated glycan of α 2-3 in GlcNAc place (table 3) with high-affinity.222 primary amino acid can be in a similar manner with Neu5Ac α 2-3Gal β 1-3GlcNAc primitive in 4-O sulfation GlcNAc or the 6-O sulfation GlcNAc in the Neu5Ac α 2-3Gal β 1-4GlcNAc primitive interact.On the other hand, disturb fucosylation GlcNAc in Neu5Ac α 2-3Gal β 1-4 (Fuc α 1-3) the GlcNAc primitive potentially such as the Lys222 among H1 and the H5 and the huge side chains such as Trp222 among the H3.This structure observation conclusive evidence is about bird H3 and H5 bacterial strain viewed classifier rules α 2-3 C type (table 3), and this shows at GlcNAc place fucosylation in conjunction with unfavorable.Because the amino acid in Viet04_H5 HA and the ADS97_H5 HA glycan binding site separately much at one, so the association class of the two and the sialylated glycan of α 2-3 seemingly (table 3).

Therefore, in conjunction with the sialylated glycan of α 2-3, except that the residue of grappling Neu5Ac, at the conservative Glu190 of all bird H1, H3 and H5 hypotype camber and Gln226 for most important in conjunction with Neu5Ac α 2-3Gal primitive.With GlcNAc or GalNAc contact and α 2-3 primitive in relate to 137,186,187,193 and 222 amino acid such as replacements such as sulfation and fucosylations.Show different binding specificities from the HA of H1, H3 and H5 for the sialylated glycan of different α 2-3 that exists in the glycan microarray.Amino-acid residue in these positions is not conservative in different HA, and this explains different binding specificities.

Replace the structural limitations that is applied in conjunction with the sialylated glycan of α 2-6 for H1 and H3 HA by the glycan topological sum

Under the situation of Neu5Ac α 2-6Gal binding, the existence of extra C6-C5 key provides the conformation handiness of increase.The position of Gal and sugar subsequently will be crossed over than the much bigger umbrella shape zone (Fig. 6) of conical region in α 2-3 situation among the α 2-6.Length on oligosaccharides is decided, and will be referred to contact with the Neu5Ac at place, described umbrella shape topology bottom and the best of Gal sugar and sugar subsequently in conjunction with α 2-6.To adopt the taper topology potentially such as short chain α 2-6 oligosaccharides such as Neu5Ac α 2-6Gal β 1-3/4Glc.On the other hand, GlcNAc among the α 2-6 primitive Neu5Ac α 2-6Gal β 1-4GlcNAc-but not the existence of Glc will help the umbrella shape topology potentially, this can contact and be stablized by the best Van der Waals (van derWaals) between the acetyl carbon of GlcNAc and Neu5Ac.Yet α 2-6 primitive also can adopt the taper topology, so that can compensate the stability that is provided by the umbrella shape topology such as extra factors such as branch and HA combinations.Can be by interacting the stable taper topology that connects the α 2-6 primitive that the branched part of a plurality of short oligosaccharides exists in the glycan as N in the sugar.On the other hand, the α 2-6 primitive in long oligosaccharides branch (tetrose at least) helps the umbrella shape topology.The above-mentioned viewpoint of eutectic structural support of H1 and H3 HA and α 2-6 primitive (Neu5Ac α 2-6Gal β 1-4GlcNAc-), wherein be about-60 Φ (being called cisoid conformation) and cause sugar except that Neu5Ac α 2-6Gal towards the bending of HA albumen, thus with best contact (Fig. 7) of binding site.

In H1 HA, adding will provide about amino acid whose see clearly related to the specificity of the sialylated glycan of α 2-6 is provided from the HA of human H1N1 (A/ South Carolina/1/1918) hypotype and the glycan binding domains of ASI30_H1_26 and APR34_H1_26.Location Lys222 and Asp225 with Neu5Ac α 2-6Gal primitive in the Sauerstoffatom of Gal interact.Location Asp190 and Ser/Asn193 are with the extra monose GlcNAc α 1-3Gal with Neu5Ac α 2-6Gal α 1-4GlcNAc α 1-3Gal primitive interact (Figure 15, figure A, B).

In the H1 HA of human epidemic virus strain in 1918, Asp190, Lys222 and Asp225 high conservative.Although amino acid Gln226 is conservative all birds and human H1 hypotype camber, but as if comparing in conjunction with the effect in the sialylated glycan of α 2-3 (in bird H1 hypotype) with it, it does not relate in conjunction with the sialylated glycan of α 2-6 (in human H1 hypotype).Data mining analysis about the glycan array result of the wild-type of bird and human H1 HA and mutant form confirms that further above-mentioned amino acid is in the effect (table 3) aspect the sialylated glycan of α 2-6.The Glu190Asp/Gly225Asp double mutant of bird H1 HA makes itself and combine oppositely (table 3) of the sialylated glycan of α 2-6.In addition, the Lys222Leu mutant of human ANY18_H1 remove its with described array on the combining of all sialylated glycan, this with Lys222 glycan in conjunction with in vital role conform to.

For discriminating provides H3N2 HA specific amino acid in conjunction with the sialylated glycan of α 2-6, the glycan of the HA of additional human H3N2 (AAI68_H3), ADU63_H3_26 and ASI30_H1_26 is in conjunction with the territory.Show for the analysis of these additional structures, location Leu226 with provide contact with the best Van der Waals of the C6 atom of Neu5 α 2-6Gal primitive and locate Ser228 with sialic O9 interaction.Ser228 among the human H3 also interacts (different with the Gly228 that can't carry out among the described interactional bird ADU63_H3) with Glu190, thereby influences its side chain conformation.Compare the side chain of Glu190 about 0.7 dust that in binding site, is shifted slightly among the human H3 HA with the Glu190 among the bird H3 HA.These difference limit human H3 HA in conjunction with the ability of the sialylated glycan of α 2-3 and preferably relevant in conjunction with the sialylated glycan of α 2-6 with it.Therefore, Gln226Leu and Gly228Ser sudden change causes glycan receptor-specific reverse of bird H3 and human H3 hypotype during prevailing disease in 1967.

From the HA of the popular H3N2 of 1967-68 with from the comparison reveals of the HA of (after nineteen ninety) H3 hypotype in the recent period, in hypotype in the recent period, Glu190 is mutated into Asp.Because the Asp190 of location among the human H3 to be advantageously interacting with these glycan, so this sudden change further strengthens combining of human H3 and the sialylated glycan of α 2-6.This structure implication will further be proved conclusively by the data mining analysis about the glycan array data of human H3 hypotype (A/ Moscow/10/1999).This HA comprises Asp190, Leu226 and Ser228 (Fig. 2) and the sialylated glycan of α 2-6 is showed extremely strong preferred property (table 3).

Above-mentioned observations highlights H1 and combines similarity and difference between the sialylated glycan of α 2-6 with H3 HA.In H1 and H3 HA, location Asp190 with Ser/Asn193 so that can advantageously contact (Figure 15 schemes A, B) with the monose except that Neu5Ac α 2-6Gal primitive.Amino acid between H1 and the H3 HA with and the difference that contacts with the sialylated glycan of α 2-6 be provided for different surfaces and ion complementarity in conjunction with these glycan.Neu5Ac α 2-6Gal binding has the conformational freedom of Duoing than Neu5Ac α 2-3Gal.Therefore, have bigger binding pocket opening in conjunction with the HA of the sialylated glycan of α 2-6 and hold this conformation freedom.Providing with the best Van der Waals of Neu5Ac α 2-6Gal and contact although locate Leu226 among the human H3 HA, is not the best by the ion contact of the primitive therewith that Gln226 provided among the H1 HA.On the other hand, in H1, amino acid Lys222 provides than the Trp222 among the H3 with Asp225 and more contacts with the best ion of the sialylated glycan of α 2-6 with Gly225.

About wild-type and mutant H5 HA and the sialylated glycan bonded of α 2-6 structural limitations

Show that by the interaction that different aminoacids provided among H1 and the H3 HA current bird H5N1 HA can be mutated into H1 sample or H3 sample glycan binding site so that make its glycan receptor-specific reverse with the sialylated glycan of α 2-6.According to said frame, further prove conclusively the H1 of supposition of H5 HA and the sudden change of H3 sample and such as hereinafter argumentation tested.

Provide about the H1 sample bonded of H5 HA and the sialylated glycan of α 2-6 for the analysis of additional ASI30_H1_26, APR34_H1_26, ADS97_H5_26 and Viet04_H5 structure and to see clearly.Because H1 and H5 HA belong to the same structure clade, so the total similar topological sum amino acid distribution of its glycan binding site people such as (, virusology, 325:287,2004) Russells.To locate to provide the best with Gal Neu5Ac α 2-6Gal primitive similar Lys that is similar among the H1 HA to contact at the conservative Lys222 of bird H5 HA camber.Glu190 among the Viet04_H5 does not provide the necessity with Neu5Ac α 2-6Gal β 1-4GlcNAc primitive that is similar to H1 to contact with Gly225 (substituting Asp190 and Asp225 among the H1).Therefore sudden change can improve and the contacting of the sialylated glycan of α 2-6 the Glu190Asp among the H5 HA potentially with Gly225Asp.

Analysis about the glycan binding pocket of the interaction except that GlcNAc and H1 and H5 HA in the Neu5Ac α 2-6Gal β 1-4GlcNAc β 1-3Gal β 1-4Glc oligosaccharides is showed, although the Ser/Asn193 among the H1 HA provides with the favourable of penult Gal and contacts, similar Lys193 among the H5 and GlcNAc β 1-3Gal primitive have disadvantageous space overlap.Therefore, the Lys193Ser sudden change can provide the extra favourable contact with the sialylated glycan of α 2-6 (together with Glu190Asp and Gly225As sudden change).

The conservative Gln226 of H1 HA camber also guards in bird H5 HA.Because Gln226 plays not too positive effect (discussing as mentioned) at H1 HA aspect the sialylated glycan of α 2-6,, amino acid mutation one-tenth such as hydrophobic amino acids such as Leu contact with the Van der Waals of C6 atom of Gal in the Neu5Ac α 2-6Gal primitive so can strengthening it potentially.

Additional ADU63_H3_26, AAI68_H3, ADS97_H5_26 and Viet04_H5 will provide about the H3 sample bonded of H5 HA and the sialylated glycan of α 2-6 and see clearly.Although this additional glycan binding site of structurally aiming at H5 and H3 HA, it is good like that not as H5 aims at the structure between the H1.The favourable Van der Waals with Neu5 α 2-6Gal primitive that is provided with Ser228 by the Leu226 among the H3 HA respectively contacts to contact in H5 HA (having Gln226 and Gly228) with ion and does not exist.Because Leu226 and Ser228 for most important in conjunction with the sialylated glycan of α 2-6 among the human H3 HA, contact so the Gln226Leu among the H5 HA suddenlys change can provide potentially with the best of the sialylated glycan of α 2-6 with Gly228Ser.In addition, even when relatively H3 is with H5, with the Lys193 location so that with through locating to provide the Ser193 among the favourable human H3 HA that contacts to compare, Lys193 will have disadvantageous space with the monose except that Neu5Ac α 2-6Gal primitive and contact.Although the HA from the popular H3N2 of 1967-68 comprises Glu190, thereby the Asp 190 among the H5 HA will provide through the location with the preferable ion of Neu5Ac α 2-6Gal primitive in the longer oligosaccharides and contact.

The effect of above-mentioned residue will further be proved conclusively (table 3) by the data mining analysis at the glycan array data of the wild-type of Viet04_H5 and mutant form.Any glycan structures of double mutant Glu190Asp/Gly225Asp debond, this is because it loses in conjunction with the amino acid Glu190 of the sialylated glycan of α 2-3 and Lys193 for having spatial interference in conjunction with the sialylated glycan of α 2-6.Similar with double mutant, Gln226Leu/Gly228Ser is in conjunction with the sialylated glycan of some α 2-3 (α 2-3 Type B sorter), but also in conjunction with single two sialylated glycan of α 2-6 (α 2-6 A type sorter) that touch.

About the described and two sialylated glycan bonded of α 2-6 analysis revealeds that touch, the Neu5Ac α 2-6Gal binding in this glycan can be potentially prolonging conformation in conjunction with double mutant, but only have less contact (Figure 16).In addition, the Neu5Ac α 2-6Gal on the Mal α 1-3Man branch is than the more advantageously combination of identical primitive on the Man α 1-6Man branch, and the identical primitive on the described Man α 1-6Man branch has disadvantageous space with the glycan binding site of H5 HA and contacts (Figure 16).The Gln226Leu/Gly228Ser double mutant is consistent with interference bonded Lys193 for the narrower specificity of the sialylated glycan of α 2-6.

Do not wish to be subjected to the constraint of any particular theory, the inventor proposes the prerequisite of the human A of adaptation type influenza virus HA for to obtain with the ability of high-affinity in conjunction with long α 2-6 (mainly expressing) in people's upper respiratory tract.For instance, the multifarious aspect of glycan is through the branched length of the end capped lactose amine of sialic acid.This captures (table 3) by two different characteristicss by the sialylated glycan of the resulting α 2-6 of data mining analysis.Feature characterizes by the Neu5Ac α 2-6Gal β 1-4GlcNAc that is connected to N and connects the Man of core and thereby another feature is to characterize by being connected to the described primitive that another lactose amine unit forms longer branch (adopting the umbrella shape topology usually).Therefore, the broad incorporation of mutant H5 HA and the upper respiratory tract only these mutant with high-affinity combine adopt the umbrella shape topology have the glycan of long α 2-6 the time just possible.For instance, according to the present invention, the desired combination pattern comprises in conjunction with the umbrella shape glycan described in Fig. 9.

By contrast, we notice first about the modified H5 HA albumen recent report of (containing Gly228Ser and Gln226Leu/Gly228Ser replaces), it is only showed in conjunction with single two on the glycan array and touches α 2-6 saliva acidic group-lactosaminoglycan structure (people such as Stevens, science 312:404,2006).Therefore, as described herein, described modified H5 HA albumen is not BSHB H5 HA.

The clone of example 2:HA, baculovirus are synthetic, expression and purifying

Homo agglutinin in the virus exists with trimeric form and is fixed on the film.The total length of HA constructs that body has the N-terminal signal peptide and C-terminal is striden the film sequence.Recombinant expressed for HA uses the weak point of the HA that allows secretory protein to construct body usually.Produce this short solubility by N-terminal signal peptide and construct body with Gp67 signal peptide sequence displacement HA, and C-terminal stride diaphragm area through after meet trypsinase cracking site and 6X-His mark ' foldon ' sequence displacement (people such as Stevens, the molecular biology magazine, 355:1143,2006).Total length and the soluble form of HA all are expressed in the insect cell.The suspension culture of baculovirus infection Sf-9 cell in Sf900 IISFM substratum (U.S. hero Life Technologies, Inc. (Invitrogen)) with the HA that contains total length or soluble form.Infect the 72-96 hour collecting cell in back.

Homo agglutinin (HA) from A/ Vietnam/1203/2004 is by breathing out the friendly present of Dao Erfujia-Sha (AdolfoGarcia-Sastre).Use speed to become (QuikChange) II XL site-directed mutagenesis test kit (this special smart company (Stratagene) of the U.S.) and speed becomes many site-directed mutagenesises test kit (this special smart company of the U.S.), with this " wild-type " (WT) HA as two kinds of templates that body DSLS and DSDL are constructed in different sudden changes of generation.Use based on the program PrimerX of website ( Http:// bioinformatics.org/primerx/) be designed for the introduction of mutagenesis, and synthetic by U.S. hero Life Technologies, Inc..Use TOPO ligation is with WT and suddenly change HA gene time cloning in entry vector pENTR-DTOPO (U.S. hero Life Technologies, Inc.).Use path (Gateway) clone technology will contain the entry vector and baculovirus expression system (BaculoDirect) linear DNA (U.S. hero Life Technologies, Inc.) reorganization of WT and mutator gene.In each time cloning step, carry out the DNA sequencing to confirm the accuracy of sequence.Use prepared recombinant baculovirus DNA to come transfection meadow mythimna separata (Spodoptera frugiperda) Sf-9 cell (U.S. hero Life Technologies, Inc.) to produce the protovirus reserve.

By according to king people such as (Wang), (2006) vaccine, the method that 24:2176 revises is from the membrane portions purifying total length HA of infected cell.In fact concise and to the point, collect from the cell of 150ml culture by centrifugal, and under 4 ℃, extract cell pellet and reach 30min with the 1% superfine holder NP-9 of 30ml in buffer A (20mM sodium phosphate, 1.0mM EDTA, 0.01% superfine holder (Tergitol)-NP9,5% glycerine, pH 5.93).Then, make extract, stand the centrifugal 15min of reaching under the 000g 6.Using 0.45 micron filter filtering supernatant and it is loaded into has before used buffer A to reach on the equilibrated Q/SP tubing string (General Electric medical treatment group (GE healthcare), Piscataway (Piscataway), New Jersey (NJ)).After the loading, with 20ml buffer A washing tubing string.Then, disconnect anionresin tubing string Q and use buffer B (20mM sodium phosphate, 0.03% superfine holder, 5% glycerine of 5 5ml parts, pH 8.2) and the damping fluid C of two 5ml parts (20mM sodium phosphate, 150mM NaCl, 0.03% superfine holder, 5% glycerine, pH 8.2) use the SP tubing string to come eluted protein.To contain and pay close attention to proteic elution fraction to some extent and pool together and use the Amy to agree ultrafiltration (Amicon Ultra) 100 K NMWL film filters (Millipore Corp. (Millipore)) to make it stand ultrafiltration.Albumen is concentrated and reconstruct in PBS.

Use the scheme described in the people (2004) such as Stevens, by the HA of the supernatant liquor purification of soluble form of infected cell.Concise and to the point in fact, concentrated supernatant and by using Ni-NTA bead (complete smart company (Qiagen)) to carry out the affinity chromatography and from spissated cell conditioned medium liquid recovery solubility HA.Compile the elution fraction that contains HA and at 10mMTris-HCl, 50mM NaCl; PH 8.0 dialysis.Use list-Q HR10/10 tubing string (Pharmacia (Pharmacia)), the dialysis sample is carried out ion exchange chromatography.The elution fraction that will contain HA pools together and uses the willing ultrafiltration 100K NMWL film filter (Millipore Corp.) of Amy to make it stand ultrafiltration.Albumen is concentrated and reconstruct in PBS.

Verify proteic existence the in the sample by carrying out the western-blot analysis with anti-bird H5N1 HA antibody.Via spot immune calibrating (using as a reference), measure the protein concentration of WT and mutant available from the WT H5 HA of albumen scientific company (Protein Sciences Inc).In the various experiments of being carried out, the protein concentration of finding H5 HA (WT and mutant) is usually in 20-50 mcg/ml scope.Based on given batch protein concentration, use 1: 10-1: the suitable serial dilution (referring to Figure 17) in 100 scopes.

Example 3: application data is excavated the glycan binding specificity that platform is studied HA

Research and development H5N1 hypotype and the sialylated glycan of α 2-3/6 combine framework (Fig. 7).This framework comprises two complementation analysises.First analysis relate to use H1, H3 and H5 HA-glycan eutectic structural system analysis HA glycan binding site with and with the interaction (table 2) of α 2-3 and the sialylated glycan of α 2-6.

This analysis provides interactional important the seeing clearly to HA glycan binding site and the sialylated glycan of multiple α 2-3/6 (glycan that comprises umbrella shape or taper topology).Second analysis relates to the data digging method of analysis about the glycan array data of different H1, H3 and H5 HA.This data mining analysis makes the constitutional features relevant (table 3) of the glycan in the strong, weak of different wild-types and mutant HA and non-binding dose and the microarray.

Importantly, the micro-structure variation of the sialylated binding of these dependencys (sorter) capture α 2-3/6 and/or different topology are to the influence in conjunction with different HA.Such as hereinafter argumentation, the dependency of the glycan feature that will obtain from data mining analysis is positioned on the HA glycan binding site, thereby the bonded framework of systematically studying H1, H3 and H5 HA and α 2-3 and the sialylated glycan of α 2-6 (glycan that comprises different topology) is provided.

Only cite an actual example, this frame application is illustrated the chain length of α 2-6 oligonucleotide chain, especially how to become more more important than the nuance about the structural changes of glycan under the situation of branch degree in H5 HA according to the present invention.For instance, and at comparing, have three of single α 2-6 primitive and touch structure and will influence the HA-glycan and combine with the two structures of touching with longer α 2-6 primitive about the structural changes of indivedual α 2-6 primitives.This can determine (table 3) by the different lengths dependency sorter of the α 2-6 primitive that is used for herein obtaining from data mining.

Example 4: the wide spectrum mankind are in conjunction with H5 HA polypeptide

In specific embodiments more of the present invention, the HA polypeptide is the H5 polypeptide.In some described embodiment, H5 polypeptide of the present invention is showed in conjunction with (for example high-affinity and/or specificity combination) umbrella shape glycan.In certain embodiments, H5 polypeptide of the present invention is called " the human combination of wide spectrum " (BSHB) H5 polypeptide.

Initial design phrase " wide spectrum human in conjunction with " (BSHB) refers to that the H5 polypeptide is in conjunction with the HA acceptor seen in human epithelium's tissue and especially in conjunction with the human HA acceptor that is characterized by the sialylated glycan of α 2-6.Usually discuss about the HA polypeptide as above, in certain embodiments, BSHB H5 HA polypeptide of the present invention is in conjunction with being seen acceptor on the human upper respiratory tract epithelial cell.In addition, BSHB H5 HA polypeptide of the present invention is in conjunction with the multiple different sialylated glycan of α 2-6.In certain embodiments, BSHB H5 HA polypeptide is in conjunction with the umbrella shape glycan.

In certain embodiments, BSHB HA HA polypeptide of the present invention is in conjunction with the HA acceptor in segmental bronchus and/or the tracheae.In certain embodiments, BSHB H5 HA polypeptide can not be in conjunction with the acceptor in the dark lung, and in other embodiments, BSHBH5 HA polypeptide can be in conjunction with the acceptor in the dark lung.In other embodiments, BSHB H5 HA polypeptide can not be in conjunction with the sialylated glycan of α 2-3, and BSHB H5 HA polypeptide can be in conjunction with the sialylated glycan of α 2-3 in other embodiments.

In certain embodiments, BSHB H5 HA polypeptide of the present invention is the variant of parent H5 HA (for example, the H5 HA seen in the natural influenza isolate).For instance, in certain embodiments, compare with wild-type H5 HA, BSHB H5 HA polypeptide of the present invention has at least one aminoacid replacement or has the aminoacid replacement that at least one influences the glycan binding site in the glycan binding site.In certain embodiments, described being substituted by directly and the interactional amino acid of institute's bonded glycan; In other embodiments, described being substituted by from spending isolating amino acid with 1 of the interactional amino acid removal of institute's bonded glycan, this is owing to the 1 degree amino acid separation of removing (1) and directly combines the amino acid interaction; (2) otherwise the influence directly in conjunction with amino acid and the interactional ability of glycan, but not with the direct interaction of glycan own; Or (3) otherwise influence directly in conjunction with amino acid and the interactional ability of glycan, and simultaneously and the direct interaction of glycan own.BSHB H5 HA polypeptide of the present invention contains one or more directly in conjunction with the replacement of any combination of amino acid, one or more first degree amino acid separations, one or more second degree amino acid separations or described situation.In certain embodiments, BSHB H5 HA polypeptide of the present invention can contain and has even one or more amino acid whose replacements of higher separation degree.

In certain embodiments, compare with wild-type H5 HA, BSHB H5 HA polypeptide of the present invention has at least two kinds, three kinds, four kinds, aminoacid replacement more than five kinds or five kinds; In certain embodiments, BSHB H5HA polypeptide of the present invention has two kinds, three kinds or the replacement of four seed amino acids.In certain embodiments, all these aminoacid replacement all are positioned at the glycan binding site.

In certain embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA at the residue place that is selected from the group that is made up of following residue: residue 98,136,138,153,155,159,183,186,187,190,193,194 195,222,225,226,227 and 228.In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA being selected from the amino acid whose residue place that is arranged in directly in conjunction with the zone of the acceptor of glycan, and described residue includes, but is not limited to residue 98,136,153,155,183,190,193,194,222,225,226,227 and 228.In other embodiments, BSHBH5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA being selected from the amino acid whose residue place that is positioned at directly in conjunction with the contiguous place in the zone of the acceptor of glycan, and described residue includes, but is not limited to residue 98,138,186,187,195 and 228.

In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA at the residue place that is selected from the group that is made up of following residue: residue 138,186,187,190,193,222,225,226,227 and 228.In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA being selected from the amino acid whose residue place that is arranged in directly in conjunction with the zone of the acceptor of glycan, and described residue includes, but is not limited to residue 190,193,222,225,226,227 and 228.In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA being selected from the amino acid whose residue place that is positioned at directly in conjunction with the contiguous place in the zone of the acceptor of glycan, and described residue includes, but is not limited to residue 138,186,187 and 228.

In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA at the residue place that is selected from the group that is made up of following residue: residue 98,136,153,155,183,194 and 195.In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA being selected from the amino acid whose residue place that is arranged in directly in conjunction with the zone of the acceptor of glycan, and described residue includes, but is not limited to residue 98,136,153,155,183 and 194.In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA being selected from the amino acid whose residue place that is positioned at directly in conjunction with the contiguous place in the zone of the acceptor of glycan, and described residue includes, but is not limited to residue 98 and 195.

In certain embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA at the isolating amino acid whose residue of 1 degree that is selected from from the interactional amino acid removal of institute's bonded glycan, and this is owing to the 1 degree amino acid separation of removing (1) and directly combines the amino acid interaction; (2) otherwise the influence directly in conjunction with amino acid and the interactional ability of glycan, but not with the direct interaction of glycan own; Or (3) otherwise influence directly in conjunction with amino acid and the interactional ability of glycan, and simultaneously and the direct interaction of glycan own, described residue includes, but is not limited to residue 98,138,186,187,195 and 228.

In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA at the isolating amino acid whose residue of 1 degree that is selected from from the interactional amino acid removal of institute's bonded glycan, and this is owing to the 1 degree amino acid separation of removing (1) and directly combines the amino acid interaction; (2) otherwise the influence directly in conjunction with amino acid and the interactional ability of glycan, but not with the direct interaction of glycan own; Or (3) otherwise influence directly in conjunction with amino acid and the interactional ability of glycan, and simultaneously and the direct interaction of glycan own, described residue includes, but is not limited to residue 138,186,187 and 228.

In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA at the isolating amino acid whose residue of 1 degree that is selected from from the interactional amino acid removal of institute's bonded glycan, and this is owing to the 1 degree amino acid separation of removing (1) and directly combines the amino acid interaction; (2) otherwise the influence directly in conjunction with amino acid and the interactional ability of glycan, but not with the direct interaction of glycan own; Or (3) otherwise influence directly in conjunction with amino acid and the interactional ability of glycan, and simultaneously and the direct interaction of glycan own, described residue includes, but is not limited to residue 98 and 195.

In certain embodiments, BSHB H5 HA polypeptide has aminoacid replacement with respect to wild-type H5 HA at residue 159 places.

In other embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA at the residue place that is selected from 190,193,225 and 226.In certain embodiments, BSHB H5 HA polypeptide has one or more aminoacid replacement with respect to wild-type H5 HA at the residue place that is selected from 190,193,226 and 228.In certain embodiments, HA polypeptide variants of the present invention and especially the H5 variant have one or more in the following aminoacid replacement: Ser137Ala, Lys156Glu, Asn186Pro, Asp187Ser, Asp187Thr, Ala189Gln, Ala189Lys, Ala189Thr, Glu190Asp, Glu190Thr, Lys193Arg, Lys193Asn, Lys193His, Lys193Ser, Gly225Asp, Gln226Ile, Gln226Leu, Gln226Val, Ser227Ala, Gly228Ser.

Glu190Asp, Lys193Ser, Gly225Asp and Gln226Leu;

Glu190Asp, Lys193Ser, Gln226Leu and Gly228Ser;

Ala189Gln、Lys193Ser、Gln226Leu、Gly228Ser；

Asp187Ser/Thr、Ala189Gln、Lys193Ser、Gln226Leu、Gly228Ser；

Ala189Lys、Lys193Asn、Gln226Leu、Gly228Ser；

Asp187Ser/Thr、Ala189Lys、Lys193Asn、Gln226Leu、Gly228Ser；

Lys156Glu、Ala189Lys、Lys193Asn、Gln226Leu、Gly228Ser；

Lys193His、Gln226Leu/Ile/Val、Gly228Ser；

Lys193Arg、Gln226Leu/Ile/Val、Gly228Ser；

Ala189Lys、Lys193Asn、Gly225Asp；

Lys156Glu、Ala189Lys、Lys193Asn、Gly225Asp；

Ser137Ala、Lys156Glu、Ala189Lys、Lys193Asn、Gly225Asp；

Glu190Thr、Lys193Ser、Gly225Asp；

Asp187Thr、Ala189Thr、Glu190Asp、Lys193Ser、Gly225Asp；

Asn186Pro、Asp187Thr、Ala189Thr、Glu190Asp、Lys193Ser、Gly225Asp；

In certain embodiments, BSHB H5 HA polypeptide of the present invention has the Amino acid sequence signature of H1 HA.For instance, in certain embodiments, described H1 sample BSHB H5 HA polypeptide has Glu190Asp, Lys193Ser, Gly225Asp and the Gln226Leu of replacement.

In certain embodiments, BSHB H5 HA polypeptide of the present invention has the Amino acid sequence signature of H1 HA.For instance, in certain embodiments, described H3 sample BSHB H5 HA contains and replaces Glu190Asp, Lys193Ser, Gln226Leu and Gly228Ser.

In certain embodiments, compare with wild-type H5 HA, BSHB H5 HA polypeptide of the present invention has open binding site.In certain embodiments, BSHB H5 HA polypeptide of the present invention is in conjunction with the sialylated glycan of following α 2-6:

With its combination.In certain embodiments, BSHB H5 HA polypeptide of the present invention is in conjunction with the glycan of following structure:

With its combination; And/or

And/or

And/or

With its combination.In certain embodiments, BSHB H5 HA polypeptide of the present invention combination

And/or

In certain embodiments, in conjunction with

In certain embodiments, in conjunction with

And in certain embodiments, in conjunction with And/or

In certain embodiments, BSHB H5 HA polypeptide of the present invention is in conjunction with umbrella shape topology glycan.In certain embodiments, BSHB H5 HA polypeptide of the present invention is in conjunction with at least some glycan described in Fig. 9 (for example sialylated glycan of α 2-6).In certain embodiments, BSHB H5 HA polypeptide of the present invention is in conjunction with the multiple glycan described in Fig. 9.

In certain embodiments, BSHB H5 HA polypeptide combination of the present invention is at least about being seen glycan on the HA acceptor in human upper respiratory tract tissue (for example epithelial cell) more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 95%.

Example 5: the glycan diversity in the human upper respiratory tract tissue

Lectin is showed the diversity of α 2-3 and the distribution of α 2-6 in upper respiratory tract tissue in conjunction with research.The sialylated glycan of Study on dyeing indication α 2-6 connects (ciliated cell) is connected glycan (in goblet cell) with O the remarkable distribution (Figure 18) of a part on the end face of tracheal epithelium as N-.On the other hand, the interior region of tracheal tissue mainly comprises the α 2-3 that is distributed on the N-connection glycan.Probing into the sialylated glycan acceptor of which kind of α 2-6 for a long time is present in the human lung.

Essence diversity (Figure 10) and the significantly expression of the sialylated glycan of α 2-6 on air flue on the mankind showed in the distributional analysis of MALDI-MS glycan.Significantly, use the fragmentation of the representative mass peak of MALDITOF-TOF to confirm that the longer oligosaccharides branch with a plurality of lactose amine (lactosamine) tumor-necrosis factor glycoproteins compares the glycan topology (Figure 10) that distributes widely with short oligosaccharides branch.Glycan distributes and topological multifarious reference in the air flue in order to provide, and the N-from human colon's epithelial cell (HT29) is connected glycan carry out the MALDI-MS analysis.Known current H5N1 virus mainly infects intestinal tube, and therefore selects these cells to be used as representative intestinal tube cell.The glycan of HT29 cell distributes and significantly is different from the distribution of HBE, and wherein α 2-3 distributes significantly and long-chain oligosaccharides branch glycan topology (Figure 10) not as observed.

Produce the data of Figure 18 by the following method.Buy fixing and by the section of paraffin-embedded human tracheal tissue by formalin (Formalin) from America Biological Science Co., Ltd (US Biological). With tissue slice deparaffnize and rehydrated after, use streptavidin / vitamin H blocking-up test kit (carrier laboratory (Vector Labs)) to block the endogenous vitamin H.Then, use jackfruit lectin (O is connected glycan have specificity), biotinylation concanavalin A (Concanavalin A) (the Con A of FITC mark, α is connected mannose residue have specificity, described residue is for constituting the part that N connects the core oligosaccharide structure of glycan), biotinylation bosom Chinese scholartree (Maackia amurensis) lectin (MAL, to SA α 2,3-gal has specificity) and biotinylation golden elder (Sambuccus nigra) lectin (SNA, to SA α 2,6-gal has specificity) (carrier laboratory, 10 μ g / ml in PBS) will cut into slices and cultivate 3 hours with 0.5% tween 20.After TBST washing (Tris buffer saline), with not Lip river (Alexa fluor) 546 streptavidins (2 μ g / ml in the PBS) cultivation 1 hour of will cut into slices of Arax with 0.5% tween 20 with 1% tween 20.Use the TBST washed and inspecting under the poly-little Mirror of burnt significant (the poly-altogether little Mirror Intraoperative of burnt were of Cai Si (Zeiss) LSM510 laser scanning) altogether.At room temperature (RT) carries out all cultivations .

Use following method to produce the data of Figure 10.When cell reach＞during 90% Long Full, utilize 100mM Citrate trianion normal saline buffer solution collecting cell (about 70 * 10 ⁶Individual), and at the back isolated cell film of handling and homogenize with proteinase inhibitor (card encyclopaedia nurse biochemical corp (Calbiochem)).Handling cytolemma with PNGaseF (Niu Yinglun biotech company (NewEngland Biolabs)) partly and with reaction mixture cultivation under 37 ℃ spends the night.Make reaction mixture boiling 10min so that enzyme deactivation, and use Sai Pu-Parker (Sep-Pak) C18 SPE barrel (water generation company (Waters)) to remove peptide and albumen through de-glycosylation.Further desalination of glycan and use graphitized carbon Solid-Phase Extraction tubing string (Su Puke company (Supelco)) purifying are become neutral (25% acetonitrile part) and acid (50% acetonitrile that contains 0.05% trifluoroacetic acid) part.Use soft ionization condition (the extraction time of lag of acceleration voltage 22kV, grid voltage 93%, lead 0.3% and 150ns), analyze acidic moiety with positive and negative mode respectively by MALDI-TOF MS.Peak value proofreaied and correct be sodiated material not.Confirm the remarkable expression of the sialylated glycan of α 2-6 by the sample pre-treatment of using sialidase A and S to carry out.Then, under 37 ℃, use 0.1U urine Arthrobacter (Arthrobacter ureafaciens) sialidase (sialidase A, non-specific) or pneumonia in the 50mM of 100mL final volume sodium phosphate (pH 6.0)

Coccus (Streptococcuspneumoniae) sialidase (sialidase S has specificity to the sialylated glycan of α 2-3) will be cultivated 24 hours through separating glycan.Analyze neutrality and acidic moiety with positive and negative mode respectively by MALDI-TOF MS.

Example 6:H1 and H3 HA combine with the dose response of human lung tissue

The end face of tracheal tissue is mainly expressed the α 2-6 glycan with long branch topology.On the other hand, the main express alpha 2-3 glycan of alveolar tissue.H1 HA significantly reduces (Figure 19) gradually in conjunction with top surface and its combination of tracheae along with being diluted to 10 μ g/ml from 40 μ g/ml.H1 HA only under maximum concentration, also show with some of alveolar tissue a little less than combine.The binding pattern of H3 HA is different from H1 HA, and wherein H3 HA is illustrated in 40 and 20 μ g/ml and significantly combines (Figure 19) with tracheae and alveolar tissue section down.Yet, HA under the concentration of 10 μ g/ml, show mainly in conjunction with the end face of tracheal tissue and with the very weak not even combination of combining of alveolar tissue.Generally speaking, the tissue bond data point out 1) H1 and H3 HA combine with the high-affinity of the end face of tracheal tissue; With 2) when H3 HA showed avidity (more relatively low than α 2-6) to α 2-3, H1 HA was to α 2-6 high special.

Use the data among following method generation Figure 19.Respectively from US biological substrate company (US Biomax, Inc) and America Biological Science Co., Ltd buy fixing and by paraffin-embedded human tissue lung and tracheae section by formalin (Formalin). With tissue slice deparaffnize, rehydrated and use the 1% BSA among the PBS to cultivate 30 minutes to prevent non-specific binding.Make H1N1 and H3N2 HA respectively with one-level antibody (mouse anti 6X His mark, the large company of moral (Abeam )) and secondary antibody (Arax is Lip river 488 goat anti-mouse not, US hero Life Technologies, Inc.) with 4:2:1 ratios at pre-compound 20 minutes on ice.Formed mixture is diluted among the 1% BSA -PBS, reaches the final HA concentration of 40,20 or 10 μ g / ml.Then, tissue slice was at room temperature cultivated 3 hours with the HA antibody complex.To cut into slices with propidium iodide (US hero Life Technologies, Inc ., In TBST 1:100) counterstaining, thorough washing and inspecting under the poly-little Mirror of burnt significant (the poly-altogether little Mirror Intraoperative of burnt were of Cai Si LSM510 laser scanning) altogether then.

Example 7: wild-type HA polypeptide directly combines with the dose response of the glycan of different topology

As described herein, the present invention forgives following cognition: the HA polypeptide is relevant with the ability of the infection of HA polypeptide mediation human host with the combination of the glycan that has herein the particular topology that is called " umbrella shape " topology.This case description mediates direct result and the explanation human infection and the dependency between the umbrella shape glycan combines in conjunction with research of the different HA polypeptide of different host infections.

Directly use the glycan array usually, wherein use main polymer chain that specified glycan structures (for example unit price or multivalence) is presented on the upholder (for example glass slide or orifice plate) usually in conjunction with calibrating.In so-called " sequence " calibrating, trimerization HA polypeptide is combined with array and then, for example use firsts and seconds antibody to detect through mark (for example through FITC or horseradish peroxidase-labeled).In " multivalence " calibrating, at first make trimerization HA and firsts and seconds antibody (usually with 4: 2: 1 HA: one-level: the secondary ratio) compound, make 12 glycan binding sites of each pre-compound HA existence, and trimerization HA contacted with array.Usually in a series of HA concentration, carry out combination calibrating, so that obtain about information to the relative affinity of the different glycan in the array.

For instance, the array that use has different glycan carries out directly in conjunction with research, described glycan such as 3 ' SLN, 6 ' SLN, 3 ' SLN-LN, 6 ' SLN-LN and 3 ' SLN-LN-LN, wherein LN represents Gal β 1-4GlcNAc, 3 ' expression Neu5Ac α 2-3 and 6 ' expression Neu5Ac α 2-6.Specifically, use before and to clean three times high-bond 384 orifice plates with PBS biotinylation glycan (50 μ l 120pmol/ml) is cultivated spend the night (in PBS, under 4 ℃) through the streptavidin coating.Then, use PBS with plate washing three times removing excessive glycan, and do not have and further handle promptly directly use.

With 4: 2: 1 HA: one-level: the secondary ratio, HA albumen, one-level antibody (mouse anti 6X His mark) and the secondary antibody (HRP bonded goat anti-mouse IgG) of an amount of His mark were cultivated 15 minutes on ice.Then, the 1%BSA among the use PBS supplies mixture (that is pre-compound HA) to the final volume of 250 μ l.Then, the pre-compound HA of 50 μ l is added in 384 orifice plates in the hole of glycan coating and at room temperature cultivated 2 hours.Then, use the PBS that contains 0.05% tween 20 with hole washing 3 times, and use PBS washing 3 times then.According to the specification sheets of manufacturers, use the red superoxide enzyme reagent kit of dust Price (Amplex) (U.S. hero Life Technologies, Inc., California (CA)) to assess the HRP activity.The serial dilution of research HA pre-composite.Comprise suitable feminine gender (non-sialylated glycan) and background (no glycan or do not have HA) contrast, and all calibratings are repeated three times.The result is provided among Figure 20.

One of the binding pattern of known human adaptability H1 and H3 HA be characterized as its in the scope of 40 μ g/ml dilution dropping to, 5 μ g/ml under saturated level in conjunction with long-chain alpha 2-6 (6 ' SLN-LN) (Figure 20).Although H1 HA is in conjunction with long-chain alpha 2-6 high special, H3 HA also with high-affinity in conjunction with short chain α 2-6 (6 ' SLN) and with respect to α 2-6 with than low-affinity in conjunction with long-chain alpha 2-3 (Figure 20).The direct wedding agent quantitative response of H1 and H3 HA is consistent with the tissue bond pattern.In addition, H1 is relevant with its broad incorporation with the end face of the tracheal tissue of expressing the α 2-6 glycan with long branch topology with the high-affinity combination of long-chain alpha 2-6 with H3 HA.This dependency provides the useful of upper respiratory tract tissue tropism that the mankind are adapted to H1 and H3 HA to see clearly.On the other hand, the H5 HA that tests show that opposite glycan is in conjunction with trend, wherein with to the relative low-affinity of α 2-6 (significant signal only under 20-40 μ g/ml as seen) compare, it with high-affinity in conjunction with α 2-3 (saturation signal drops to 2.5 μ g/ml from 40) (Figure 20).

Equivalent

One of ordinary skill in the art only use normal experiment will recognize the many equivalents that maybe can determine specific embodiment of the present invention as herein described.Scope of the present invention does not plan to be limited to above-mentioned embodiment, but as encloses described in the claim.

Claims

1. through engineering approaches HA polypeptide, it is in conjunction with umbrella shape topology glycan.

2. through engineering approaches HA polypeptide according to claim 1, wherein said umbrella shape topology glycan comprises the sialylated glycan of α 2-6.

3. through engineering approaches HA polypeptide according to claim 1 and 2, wherein said HA polypeptide with high-affinity in conjunction with described umbrella shape topology glycan.

4. through engineering approaches HA polypeptide according to claim 3, wherein said HA polypeptide combines described umbrella shape topology glycan with the avidity suitable with the avidity of the human adaptability HA of the para-infectious wild-type of Mediated Human.

5. through engineering approaches HA polypeptide according to claim 3, wherein said HA polypeptide think that at least 50% the avidity of avidity of the para-infectious wild-type HA of Mediated Human is in conjunction with described umbrella shape topology glycan.

6. through engineering approaches HA polypeptide according to claim 3, wherein said HA polypeptide think that at least 70% the avidity of avidity of the para-infectious wild-type HA of Mediated Human is in conjunction with described umbrella shape topology glycan.

7. through engineering approaches HA polypeptide according to claim 3, wherein said HA polypeptide think that at least 80% the avidity of avidity of the para-infectious wild-type HA of Mediated Human is in conjunction with described umbrella shape topology glycan.

8. through engineering approaches HA polypeptide according to claim 3, wherein said HA polypeptide think that at least 90% the avidity of avidity of the para-infectious wild-type HA of Mediated Human is in conjunction with described umbrella shape topology glycan.

9. through engineering approaches HA polypeptide according to claim 3, wherein said HA polypeptide think that at least 100% the avidity of avidity of the para-infectious wild-type HA of Mediated Human is in conjunction with described umbrella shape topology glycan.

10. through engineering approaches HA polypeptide according to claim 1 and 2 is wherein compared with taper topology glycan, and described HA polypeptide is preferably in conjunction with described umbrella shape topology glycan.

11. through engineering approaches HA polypeptide according to claim 10, wherein said HA polypeptide be at least 2 relative affinity with respect to the topological glycan of taper in conjunction with umbrella shape topology glycan.

12. through engineering approaches HA polypeptide according to claim 10, wherein said HA polypeptide be at least 3 relative affinity with respect to the topological glycan of taper in conjunction with umbrella shape topology glycan.

13. through engineering approaches HA polypeptide according to claim 10, wherein said HA polypeptide be at least 4 relative affinity with respect to the topological glycan of taper in conjunction with umbrella shape topology glycan.

14. through engineering approaches HA polypeptide according to claim 10, wherein said HA polypeptide be at least 5 relative affinity with respect to the topological glycan of taper in conjunction with umbrella shape topology glycan.

15. through engineering approaches HA polypeptide according to claim 10, wherein said HA polypeptide be at least 10 relative affinity with respect to the topological glycan of taper in conjunction with umbrella shape topology glycan.

16. one kind in conjunction with umbrella shape topology glycan through separating the HA polypeptide, described HA polypeptide is not the H1 albumen from following arbitrary bacterial strain: A/ South Carolina (South Carolina)/1/1918, A/ Puerto Rico (Puerto Rico)/8/1934, A/ Taiwan (Taiwan)/1/1986, A/ Texas (Texas)/36/1991, A/ Beijing (Beijing)/262/1995, A/ Johannesburg (Johannesburg)/92/1996, A/ New Caledonia (New Caledonia)/20/1999, A/ Solomon Islands (Solomon Islands)/3/2006; Or from the H2 albumen of following arbitrary bacterial strain: A/ Japan (Japan)/305+/1957, A/ Singapore (Singapore)/1/1957, A/ Taiwan/1/1964, A/ Taiwan/1/1967; Or like to know (Aichi)/2/1968 from the H3 albumen of following arbitrary bacterial strain: A/, A/ Philippines (Phillipines)/2/1982, A/ Mississippi (Mississippi)/1/1985, A/ Leningrad (Leningrad)/360/1986, A/ Sichuan (Sichuan)/2/1987, A/ Shanghai (Shanghai)/11/1987, A/ Beijing/353/1989, A/ Shandong (Shandong)/9/1993, A/ Johannesburg/33/1994, A/ Nanchang (Nanchang)/813/1995, A/ Sydney (Sydney)/5/1997, A/ Moscow/10/1999, A/ Panama (Panama)/2007/1999, A/ Wyoming (Wyoming)/3/2003, A/ Oklahoma (Oklahoma)/323/2003, A/ California (California)/7/2004, A/ Wisconsin (Wisconsin)/65/2005.

17. the characteristic of a through engineering approaches HA polypeptide, it is in conjunction with umbrella shape topology glycan.

18. the characteristic of a HA polypeptide, described HA polypeptide are not the H1 albumen from following arbitrary bacterial strain: A/ South Carolina/1/1918, A/ Puerto Rico/8/1934, A/ Taiwan/1/1986, A/ Texas/36/1991, A/ Beijing/262/1995, A/ Johannesburg/92/1996, A/ New Caledonia/20/1999, A/ Solomon Islands/3/2006; Or from the H2 albumen of following arbitrary bacterial strain: A/ Japan/305+/1957, A/ Singapore/1/1957, A/ Taiwan/1/1964, A/ Taiwan/1/1967; Or like to know from the H3 albumen of following arbitrary bacterial strain: A//2/1968, A/ Philippines/2/1982, A/ Mississippi/1/1985, A/ Leningrad/360/1986, A/ Sichuan/2/1987, A/ Shanghai/11/1987, A/ Beijing/353/1989, A/ Shandong/9/1993, A/ Johannesburg/33/1994, A/ Nanchang/813/1995, A/ Sydney/5/1997, A/ Moscow/10/1999, A/ Panama/2007/1999, A/ Fujian (Fujian)/411/2002, A/ Wyoming/3/2003, A/ Oklahoma/323/2003, A/ California/7/2004, A/ Wisconsin/65/2005, wherein said characteristic is in conjunction with umbrella shape topology glycan.

19. a peptide species, it comprises according to claim 17 or 18 described characteristics.

20. a nucleic acid, it is encoded according to claim 17 or 18 described characteristics.

21. a nucleic acid, its polypeptide according to claim 19 of encoding.

22. a carrier, it contains nucleic acid according to claim 20.

23. a carrier, it contains nucleic acid according to claim 21.

24. a host cell, it contains nucleic acid according to claim 20.

25. a host cell, it contains nucleic acid according to claim 21.

26. a host cell, it contains carrier according to claim 22.

27. a host cell, it contains carrier according to claim 23.

28. an antibody, itself and the through engineering approaches HA polypeptide combination that combines umbrella shape topology glycan.

29. antibody, it is in conjunction with the HA polypeptide, and described HA polypeptide is not the H1 albumen from following arbitrary bacterial strain: A/ South Carolina/1/1918, A/ Puerto Rico/8/1934, A/ Taiwan/1/1986, A/ Texas/36/1991, A/ Beijing/262/1995, A/ Johannesburg/92/1996, A/ New Caledonia/20/1999, A/ Solomon Islands/3/2006; Or from the H2 albumen of following arbitrary bacterial strain: A/ Japan/305+/1957, A/ Singapore/1/1957, A/ Taiwan/1/1964, A/ Taiwan/1/1967; Or like to know from the H3 albumen of following arbitrary bacterial strain: A//2/1968, A/ Philippines/2/1982, A/ Mississippi/1/1985, A/ Leningrad/360/1986, A/ Sichuan/2/1987, A/ Shanghai/11/1987, A/ Beijing/353/1989, A/ Shandong/9/1993, A/ Johannesburg/33/1994, A/ Nanchang/813/1995, A/ Sydney/5/1997, A/ Moscow/10/1999, A/ Panama/2007/1999, A/ Fujian/411/2002, A/ Wyoming/3/2003, A/ Oklahoma/323/2003, A/ California/7/2004, A/ Wisconsin/65/2005, wherein said HA polypeptide is in conjunction with umbrella shape topology glycan.

30. according to claim 28 or 29 described antibody, described antibody is polyclonal.

31. according to claim 28 or 29 described antibody, described antibody is monoclonal.

32. a virion, it comprises the through engineering approaches HA polypeptide in conjunction with umbrella shape topology glycan.

33. one kind by throwing the method for the treatment of influenza infection with composition, described composition comprises following each thing: in conjunction with the through engineering approaches HA polypeptide of umbrella shape topology glycan, comprise polypeptide in conjunction with the characteristic fragment of the through engineering approaches HA polypeptide of umbrella shape topology glycan, with the through engineering approaches HA polypeptide that combines umbrella shape topology glycan or its characteristic bonded antibody, coding in conjunction with the through engineering approaches HA polypeptide of the topological glycan of umbrella shape or the nucleic acid of its characteristic, or its combination.

34. a glycan array, it comprises at least about the being seen glycan structures on the in-house HA acceptor of the human upper respiratory tract more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 95%.

35. a method that is used to differentiate or characterize the HA polypeptide, described method comprises following steps:

Provide and contain the proteic sample of HA;

Described sample is contacted with glycan Array row according to claim 26; With

Detect combining of one or more glycan on HA and the described array.