CN114685621A - Norovirus immune composition or kit - Google Patents

Abstract

The invention provides a norovirus immunizing composition or kit, which comprises virus-like particles of norovirus types GII.2, GII.4, GII.6 and GII.17 or active fragments thereof. The invention also provides the use of the composition or the kit. The composition or the kit provided by the invention comprises four antigens, each antigen can generate cross immunity against other valences, and the composition or the kit not only can cover the epidemic valences of various norovirus, but also has the synergy of immunity against the same cross valences among the four antigens.

Description

Norovirus immune composition or kit

Technical Field

The invention belongs to the field of biopharmaceuticals, and relates to a composition or kit for inducing an immune response to norovirus, comprising virus-like particles of norovirus types gii.2, gii.4, gii.6, and gii.17. The invention also provides the use of the composition or the kit.

Background

Norovirus (Norovirus) was first discovered in 1972 by american scholars Kapikian in the feces of diarrhea patients in Norwalk and was named Norwalk virus (Norwalk virus, NoV), which was subsequently named Norovirus on the eighth international committee for virus nomenclature, one of the major pathogens causing outbreaks of human infectious gastroenteritis epidemics and acute diarrhea in infants.

Norovirus is a single-stranded positive-stranded RNA virus belonging to the family caliciviridae, has a diameter of about 26-35nm, is non-enveloped, has a rough, spherical, icosahedral symmetry, and is isolated from the feces of patients with acute gastroenteritis. The norovirus genome is approximately 7.7kb in length and contains 3 Open Reading Frames (ORFs). ORF1 encodes nonstructural proteins, including RNA polymerase (RNA-dependent RNA polymerase, RdRp); ORF2 and ORF3 encode the major (VP1) and minor (VP2) capsid proteins, respectively. Depending on the phylogeny of the gene sequences, norovirus can be divided into 6 gene groups (GI-GVI), each of which is divided into multiple genotypes (genotype). Human infection is mostly caused by the GI and GII gene groups, among which the GII group is the main group; statistically, more than 75% of acute gastroenteritis in humans NoV is caused by GII group infection.

The detection rate and the prevalence of each genotype are greatly different and changed in different periods and different regions, the GII.4 genotype is in the dominant prevalence state in outbreak and sporadic cases worldwide in recent 20 years, other genotypes such as GII.2, GII.3, GII.6, GII.17 and the like have high detection rate in genotypes except the GII.4, and small-scale epidemics are also caused in local regions along with the change of prevalence trends in recent years.

The characteristic of the norovirus that the genotype is various and rapidly varies, and an ideal cell culture system and an animal model do not exist, so that the research and development of anti-norovirus drugs are limited to a certain extent, and effective drugs, vaccines and preventive measures are still lacked. Therefore, the development of effective antiviral drugs and prophylactic vaccines is a focus problem to be solved urgently.

Existing research data show that there is no antibody cross-reactivity and cross-blocking activity between the NoV GI and GII gene groups (korean poise, zhangchong, liumegamine, etc., journal of chinese biologics, 2019). Thus, current research into vaccines against multiple valency norovirus focuses on a variety of artificially synthesized polypeptides.

Based on this, there is currently a need for vaccines against multiple valency norovirus.

Disclosure of Invention

Therefore, the object of the present invention is to provide an immune composition or kit against multiple norovirus types, which overcomes the disadvantages of the prior art. The present inventors have unexpectedly found, through extensive studies on cross-immunity between norovirus valency antigens, a composition or kit for eliciting an immune response against norovirus, which comprises norovirus virus-like particles of types gii.2, gii.4, gii.6 and gii.17, and which can cover both types gi.1 and gii.7, and which also has cross-immunity against type gii.12. The composition or the kit can achieve the aim of covering 7 valences by 4 antigens, and has the superposition of immune action aiming at the same cross valences among four antigens.

The purpose of the invention is realized by the following technical scheme:

in one aspect, the present invention provides a composition for inducing an immune response to norovirus, comprising virus-like particles of gii.2, gii.4, gii.6 and gii.17-type norovirus, or active fragments thereof.

The composition according to the present invention, wherein said norovirus type gii.4 virus-like particle or active fragment thereof comprises or consists of an amino acid sequence selected from one of:

(1) 4, an amino acid sequence shown as SEQ ID NO;

(2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO. 4;

(3) an amino acid sequence having amino acid substitution, deletion or insertion at one or more positions in the amino acid sequence shown as SEQ ID NO. 4; and

(4) 4, wherein X is an integer between 2 and 39; preferably, said X is 27, 37 or 39.

Preferably, said gii.2-type norovirus virus-like particle or active fragment thereof comprises or consists of an amino acid sequence selected from one of:

(1) an amino acid sequence shown as SEQ ID NO. 2;

(2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO 2; and

(3) an amino acid sequence with amino acid substitution, deletion or insertion at one or more positions in the amino acid sequence shown in SEQ ID NO. 2.

Preferably, said gii.6 type norovirus virus-like particle or active fragment thereof comprises or consists of an amino acid sequence selected from one of:

(1) the amino acid sequence shown as SEQ ID NO. 5;

(2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO. 5; and

(3) 5 or an amino acid sequence having amino acid substitution, deletion or insertion at one or more positions in the amino acid sequence shown in SEQ ID NO;

(4) 5, wherein X is an integer between 2 and 27; preferably, said X is 27.

Preferably, said gii.17 type norovirus virus-like particle or active fragment thereof comprises or consists of an amino acid sequence selected from one of:

(1) the amino acid sequence shown as SEQ ID NO. 8;

(2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO. 8; and

(3) an amino acid sequence having amino acid substitution, deletion or insertion at one or more positions in the amino acid sequence shown in SEQ ID NO. 8.

More preferably, said GII.2 type norovirus virus-like particle or active fragment thereof comprises or consists of an amino acid sequence as shown in SEQ ID NO. 2; the GII.4 type norovirus virus-like particle or the active fragment thereof comprises or consists of an amino acid sequence shown as SEQ ID NO. 4; the GII.6 type norovirus virus-like particle or the active fragment thereof comprises or consists of an amino acid sequence shown as SEQ ID NO. 5; the GII.17 type norovirus virus-like particle or the active fragment thereof comprises or consists of an amino acid sequence shown as SEQ ID NO. 8.

The composition according to the present invention, wherein the norovirus may be selected from one or more of norovirus type gi.1, gii.2, gii.4, gii.6, gii.7, gii.12 or gii.17.

Preferably, the composition according to the invention is a pharmaceutical composition, e.g. a vaccine, for the prevention and/or treatment of norovirus infection.

Preferably, the pharmaceutical composition further comprises one or more adjuvants selected from the group consisting of an aluminum adjuvant, a TRL adjuvant, or a saponin adjuvant. More preferably, the aluminium adjuvant is selected from one or more of aluminium hydroxide, aluminium phosphate or aluminium sulphate; further preferably, the aluminum adjuvant is aluminum hydroxide. More preferably, the TRL adjuvant is TRL9 adjuvant; further preferably, the TRL9 adjuvant is a CPG adjuvant; still more preferably, the nucleotide sequence of the CPG adjuvant is selected from one of SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11. More preferably, the saponin adjuvant is QS21 or Iscom adjuvant.

The composition according to the present invention, wherein the norovirus infection is gastroenteritis, preferably viral acute gastroenteritis.

In yet another aspect, the invention also provides an immunoassay kit comprising a composition according to the invention. Preferably, the kit is a kit for detecting norovirus infection.

In a further aspect, the invention also provides the use of a composition according to the invention or a kit according to the invention for the preparation of:

(1) a medicament for the prevention and/or treatment of norovirus infection;

(2) a kit for diagnosing norovirus infection; or

(4) Immunogens for the development of norovirus antibodies.

The use according to the invention, wherein the norovirus infection is gastroenteritis, preferably viral acute gastroenteritis.

The inventors of the present invention found that a composition comprising virus-like particles of norovirus types gii.2, gii.4, gii.6 and gii.17 can cover the gi.1 type, and also has cross-immunity against gii.7 type and gii.12 type, which achieves the purpose of covering 7 valences by 4 antigens, and that there is a superposition of immunity against the same cross-valences among the four antigens, i.e., the composition of the present invention has synergy of immunity against the same cross-valences among the four antigens. For example, gii.4 and gii.6 can simultaneously cover gii.7 type, with a significant increase in antibody titers compared to gii.4 type alone. Therefore, the composition can simultaneously cover various norovirus valence types, reduces the process difficulty of medicine or vaccine preparation, reduces the production cost and has wide market prospect.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the cross-immunization of various valency virus-like particles against various valency norovirus in a composition of the invention;

FIG. 2 shows the cross-immunization of the compositions of the invention against various norovirus types;

FIG. 3 shows the immunological strength of the composition of the present invention against various norovirus types having cross-immunity.

Defining:

unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definition of terms in the field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

It should also be noted that, as used in this specification, the term "or" may be used interchangeably with the term "and/or" unless the context clearly dictates otherwise.

The terms "pharmaceutical composition," "combination drug," and "drug combination" as used herein are used interchangeably to mean at least one drug and any combination thereof that are combined together to achieve a particular purposeSelected pharmaceutically acceptable excipients or combinations of excipients. In certain embodiments, the pharmaceutical compositions include temporally and/or spatially separated combinations, so long as they are capable of acting together to achieve the objectives of the present invention. For example, the ingredients contained in the pharmaceutical composition (e.g., GII.2, GII.4, GII.6, GI.17, or Al (OH))₃) Can be administered to the subject in whole or separately. When the ingredients contained in the pharmaceutical composition are administered separately to a subject, the ingredients may be administered to the subject simultaneously or sequentially.

The term "CpG oligodeoxynucleotide" or "CpG-ODN" as used herein refers to a short single-stranded synthetic DNA molecule containing one or more "CpG" units, wherein C represents cytosine, G represents guanine, and p represents a phosphodiester bond. In particular, the CpG oligodeoxynucleotide is unmethylated. In some embodiments, the CpG-ODN comprises a phosphorothioate linkage or a phosphorothioate backbone. That is, in some embodiments, the CpG-ODN is a phosphorothioate oligodeoxynucleotide (i.e., a thio-oligodeoxynucleotide). Preferably, all internucleotide linkages in said CpG-ODN are phosphorothioate linkages, i.e. said CpG-ODN is a fully thio oligodeoxynucleotide. In other embodiments, the CpG-ODN comprises two or more copies of the 5 '-TTCGTT-3' motif or the 5 '-TCGTCGTCG-3' motif. In particular, the CpG-ODN has a sequence selected from the group consisting of: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO:9), TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO:10) or TCG TCG TCG TCG TCG TCG TCG (SEQ ID NO:11), preferably TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 9).

"saponin" as used herein refers to the active ingredient present in the corresponding plant, in particular QS21, a quillaja saponin, or Iscom adjuvant, an immunostimulating complex adjuvant, in particular Iscom MATRIX (Iscom MATRIX) not comprising an antigen, is a cage-structured adjuvant consisting of phospholipids, saponins, cholesterol.

As used herein, "therapeutically effective amount" or "effective amount" refers to a dosage sufficient to show its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the subject's own condition and severity. Prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and depends on general practitioners and other physicians, often taking into account the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the physician.

The term "mammal" as used herein refers to a human being, but also other animals, such as wild animals (e.g., herou, geranium, crane, etc.), domestic animals (e.g., ducks, geese, etc.) or laboratory animals (e.g., orangutan, monkey, rat, mouse, rabbit, guinea pig, marmot, squirrel, etc.).

In other embodiments, the composition of the invention may further comprise additional additives, such as pharmaceutically acceptable carriers or additives, especially when it is present in the form of a pharmaceutical formulation.

Preferred pharmaceutical carriers are especially water, buffered aqueous solutions, preferably isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides and the like. The type of pharmaceutical carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.

The pharmaceutical composition, vaccine or pharmaceutical preparation according to the invention may be administered by any suitable route, e.g. orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

Detailed Description

The present invention is further described in the following description of the embodiments with reference to the drawings, but the invention is not limited thereto, and those skilled in the art can make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.

Example 1 preparation of Noah antigen

Respectively preparing a plurality of valency norovirus VLP proteins, wherein the amino acid sequences are shown as follows:

the amino acid sequence of the VLP protein of the GII.1 type norovirus is shown as SEQ ID NO. 1.

The amino acid sequence of the VLP protein of the GII.2 type norovirus is shown as SEQ ID NO. 2.

The amino acid sequence of the VLP protein of the GII.3 type norovirus is shown as SEQ ID NO. 3.

The amino acid sequence of the VLP protein of the GII.4 type norovirus is shown as SEQ ID NO. 4.

The amino acid sequence of the VLP protein of the GII.6 type norovirus is shown as SEQ ID NO. 5.

The amino acid sequence of the VLP protein of the GII.7 type norovirus is shown as SEQ ID NO. 6.

The amino acid sequence of the VLP protein of the GII.12 type norovirus is shown as SEQ ID NO. 7.

The amino acid sequence of the VLP protein of the GII.17 type norovirus is shown as SEQ ID NO. 8.

The preparation method comprises the following steps: optimizing the nucleic acid sequence of the target protein sequence according to the target protein sequence to ensure that the codon of the target protein sequence accords with a yeast expression system, and synthesizing a target gene; connecting the synthesized target gene with pMAUR (S.C) KARS1 plasmid by enzyme digestion connection, transforming Top 10 competence, selecting positive monoclonal, and sequencing and verifying the positive monoclonal; amplifying a large amount of monoclonal thallus, and extracting a large amount of plasmids conforming to electrotransformation by using a plasmid extraction kit; the plasmid was transformed into uracil auxotrophic hansenula competent cells by electroporation transformation, and positive transformed cells were obtained using G418 resistance selection.

Inoculating the recombinant strain to a yeast extract peptone glucose culture medium for activation, transferring the activated strain to the yeast extract peptone glucose culture medium for amplification culture, and supplementing methanol to the culture medium for induction culture once every 24 hours and twice after the strain is cultured to a plateau period. And collecting thalli after induction, and breaking and purifying the thalli to obtain the protein.

Example 2 animal immunization experiment

2.1 Experimental animals

Balb/c mice, female, 6 weeks old, 36 animals, Shanghai Ling laboratory animals technology Limited.

2.2 materials of the experiment

Various valency norovirus VLP proteins, were prepared from example 1.

Aluminum hydroxide: purchased from beijing baiolai bock technologies ltd.

2.3 Experimental groups

TABLE 1 groups of animal immunization experiments

2.4 animal immunization

1) The administration route is as follows: and (4) carrying out intramuscular injection.

2) The administration method comprises the following steps: mice were selected for left posterior thigh injection at 100. mu.l/mouse.

3) Frequency and duration of administration: the administration is carried out 1 time every 3 weeks for 3 times, namely, the administration is injected in 0 week, 3 week and 6 week respectively, blood is collected in two weeks after the first immunization, three weeks after the second immunization and endpoint blood collection in two weeks after the third immunization.

4) The serum collection method comprises the following steps: standing whole blood in a constant temperature incubator at 37 deg.C for 40-50min, standing at 4 deg.C for 1h, centrifuging at 12000rpm at 4 deg.C for 10min, sucking supernatant, and freezing in a medical low-temperature storage box for use.

EXAMPLE 3 Cross-Block testing of pharmaceutical compositions

3.1 reagent Material

1) BSA (bovine serum albumin, Beyotime Co.), TMB color former (Thermo Co.), HBGA (Glycotech Co.), rabbit polyclonal antiserum (Kyoto biosome), goat anti-rabbit IgG-HRP (horse anti-rabbit IgG labeled with horseradish peroxidase, SIGMA Co.), avidin plate (Thermo Co.).

2) Washing liquid: 0.1M PB buffer

Diluting liquid: 0.1M PB buffer + 0.25% BSA

Stopping liquid: 2M H₂SO₄

Prepared by the Nanjing Changtai Weixin biological Co Ltd;

3) norovirus VLP protein: forms GI.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.12 and GII.17, respectively, from example 1, 8 μ g/ml

3.2 detection step

1) The avidin plate was washed 1 time with 200. mu.l/well wash;

2) HBGA was diluted to 4. mu.g/ml, added to the avidin plate of step 1) at 100. mu.l/well, and incubated at 25 ℃ for 1 h;

3) respectively diluting the serum from 20 times to 1280 times in a 2-fold ratio in a 96-well plate, wherein the final volume is 60 mu l, 60 mu l of monovalent norovirus VLP protein is added into each well and mixed to form a VLP-serum mixture, and a positive control (serum-free) and a blank control (diluent) are established at the same time and incubated for 1h at 4 ℃;

4) washing the HBGA-coated avidin plate of step 2) with 200. mu.l/well of washing solution 2 times, pipetting 100. mu.l of VLP-serum mixture from the 96-well plate of step 3) and adding to the avidin plate, and incubating at 4 ℃ for 2 h;

5) washing avidin plate with 200 μ l/well washing solution for 3 times, adding rabbit antiserum (1:10000), and incubating at 4 deg.C for 1 h;

6) washing avidin plate with 200 μ l/well washing solution for 3 times, adding goat anti-rabbit IgG-HRP (1:10000), and incubating at 37 deg.C for 40 min;

7) washing avidin plate with 200 μ l/hole washing solution for 3 times, adding TMB developing solution with 100 μ l/hole, and developing at 25 deg.C for 5-15 min;

8) and (4) terminating: adding stop solution into 100 mul/hole to stop reaction;

9) reading: OD450nm values (corrected for OD630 nm) were determined.

Detection standard: the blocking index% ((1-serogroup a value/positive control a value) × 100%) and BT50, the highest dilution of serum that was able to block 50% of VLP binding to HBGA, was calculated.

Setting a critical value (Cut-off): with Al (OH)₃The 2-fold of group BT50 was used as a cut-off value to determine whether it had a cross-immunity effect.

3.3 results of the experiment

The experimental results are shown in fig. 1 and 2: figure 1 shows the cross-immunization of individual single antigens. It can be seen that gii.2 has cross-immunity on gii.12, gii.4 has cross-immunity on gii.1, gii.2, gii.7 and gii.12, gii.6 has cross-immunity on gii.7, and gii.17 has cross-immunity on gii.6, gii.7 and gii.12.

FIG. 2 shows that the composition provided by the present invention, which contains four valency type norovirus VLP proteins of GII.2, GII.4, GII.6 and GII.17, can cover seven norovirus, wherein GI.1, GII.7 and GII.12 are of the coverage increase type due to cross immunity, wherein the cross immunity against GI.1 is strongest, and BT50 can reach more than 150.

Example 4 IgG antibody level detection of pharmaceutical compositions

4.1 reagent Material

1) Skimmed milk powder (BD Co., Ltd.), PBS (China fir gold bridge Co., Ltd.), carbonate (SIGMA Co., Ltd.), Tween 20(BIO-LINK Co., Ltd.), TMB color developing solution (Thermo Co., Ltd.), horseradish peroxidase-labeled goat anti-rabbit IgG (hereinafter referred to as goat anti-rabbit IgG-HRP, SIGMA Co., Ltd.)

2) Coating liquid: 0.5% (g/100ml) carbonate solution

Wash buffer PBST: 10g/L PBS +0.5ml/L Tween 20

Diluting liquid: 2% skim milk PBST

Stopping liquid: 2M H₂SO₄

All the above are prepared by Nanjing Tokyo Weixin biomedicine Co., Ltd;

4.2 detection step

1) Coating: respectively diluting the norovirus type GI.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.12 and GII.17 VLP proteins to 1 mu g/ml by using a coating solution, coating an ELISA enzyme linked plate, 50 mu l/hole and standing overnight at 4 ℃;

2) washing: washing the plate with buffer solution PBST for 3 times, 2-3 min/time;

3) and (3) sealing: adding 5% of sealing liquid milk into the mixture, sealing the mixture at the temperature of 37 ℃ for 1h at a hole of 200 mu l;

4) washing: washing the plate with buffer solution PBST for 3 times, 2-3 min/time;

5) a first antibody: adding diluted serum in multiple proportion, diluting from 90 times to 196830 times in 3 times, setting two blank control holes at 50 μ l/hole, and incubating at 37 deg.C for 1 h;

6) washing: washing the plate with buffer solution PBST for 3 times, 2-3 min/time;

7) secondary antibody: HRP-goat anti-mouse IgG antibody (1:10000), incubating at 37 ℃ for 40 min;

8) washing: washing the plate with buffer solution PBST for 3 times, 2-3 min/time;

9) color development: adding TMB color development solution, 50 μ l/hole, developing for 10 min;

10) and (4) terminating: stopping the reaction by 50 mul/hole of stop solution;

11) reading: OD450nm values (corrected for OD630 nm) were determined on an enzyme-linked analyzer.

3.6 results of the experiment

According to the figure 3, the pharmaceutical composition has immune response to GI.1, GII.7 and GII.12, and the antibody titer can reach more than 10000 levels. Compared with the IgG antibody level of the GII.4 type monoclonal antibody aiming at each valence type, both GII.7 and GII.12 are improved by more than 10 times, and the synergy of antigen cross action is realized.

In conclusion, the composition provided by the invention can achieve the purpose of preventing seven-valence norovirus infection through the cross immunization of four antigens, has the synergy of immunization against the same cross valence among the four antigens, enhances the antibody level of the coverage valence, reduces the antigen types, reduces the process difficulty, and has high clinical application value and wide market prospect.

Although the present invention has been described in detail above, those skilled in the art will appreciate that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. The scope of the invention is not limited to the above detailed description, and the modifications and variations are intended to fall within the scope of the claims. Although only examples of specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principle and spirit of the invention, and these changes or modifications are all within the scope of the invention.

Sequence listing

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Ala Pro Leu Gly Val Pro Asp Phe Gln Gly Arg Val Phe Gly Ile Ile

325 330 335

Ser Gln Arg Asp Lys His Asn Ser Pro Gly His Asn Glu Pro Ala Asn

340 345 350

Arg Gly His Asp Ala Val Val Pro Thr Tyr Thr Ala Gln Tyr Thr Pro

355 360 365

Lys Leu Gly Gln Ile Gln Ile Gly Thr Trp Gln Thr Asp Asp Leu Thr

370 375 380

Val Asn Gln Pro Val Lys Phe Thr Pro Val Gly Leu Asn Asp Thr Glu

385 390 395 400

His Phe Asn Gln Trp Val Val Pro Arg Tyr Ala Gly Ala Leu Asn Leu

405 410 415

Asn Thr Asn Leu Ala Pro Ser Val Ala Pro Val Phe Pro Gly Glu Arg

420 425 430

Leu Leu Phe Phe Arg Ser Tyr Ile Pro Leu Lys Gly Gly Tyr Gly Asn

435 440 445

Pro Ala Ile Asp Cys Leu Leu Pro Gln Glu Trp Val Gln His Phe Tyr

450 455 460

Gln Glu Ala Ala Pro Ser Met Ser Glu Val Ala Leu Val Arg Tyr Ile

465 470 475 480

Asn Pro Asp Thr Gly Arg Ala Leu Phe Glu Ala Lys Leu His Arg Ala

485 490 495

Gly Phe Met Thr Val Ser Ser Asn Thr Ser Ala Pro Val Val Val Pro

500 505 510

Ala Asn Gly Tyr Phe Arg Phe Asp Ser Trp Val Asn Gln Phe Tyr Ser

515 520 525

Leu Ala Pro Met Gly Thr Gly Asn Gly Arg Arg Arg Val Gln

530 535 540

<210> 3

<211> 548

<212> PRT

<213> Norovirus (Norovirus)

<400> 3

Met Lys Met Ala Ser Asn Asp Ala Thr Pro Ser Asn Asp Gly Ala Ala

1 5 10 15

Gly Leu Val Pro Glu Ile Asn Asn Glu Ala Met Ala Leu Asp Pro Val

20 25 30

Ala Gly Ala Ala Ile Ala Ala Pro Leu Thr Gly Gln Gln Asn Ile Ile

35 40 45

Asp Pro Trp Ile Met Asn Asn Phe Val Gln Ala Pro Gly Gly Glu Phe

50 55 60

Thr Val Ser Pro Arg Asn Ser Pro Gly Glu Val Leu Leu Asn Leu Glu

65 70 75 80

Leu Gly Pro Glu Ile Asn Pro Tyr Leu Ala His Leu Ala Arg Met Tyr

85 90 95

Asn Gly Tyr Ala Gly Gly Phe Glu Val Gln Val Val Leu Ala Gly Asn

100 105 110

Ala Phe Thr Ala Gly Lys Ile Ile Phe Ala Ala Ile Pro Pro Asn Phe

115 120 125

Pro Ile Asp Asn Leu Ser Ala Ala Gln Ile Thr Met Cys Pro His Val

130 135 140

Ile Val Asp Val Arg Gln Leu Glu Pro Val Asn Leu Pro Met Pro Asp

145 150 155 160

Val Arg Asn Asn Phe Phe His Tyr Asn Gln Gly Ser Asp Ser Arg Leu

165 170 175

Arg Leu Val Ala Met Leu Tyr Thr Pro Leu Arg Ala Asn Asn Ser Gly

180 185 190

Asp Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Ser Pro

195 200 205

Glu Phe Ser Phe Asn Phe Leu Val Pro Pro Thr Val Glu Ser Lys Thr

210 215 220

Lys Pro Phe Thr Leu Pro Ile Leu Thr Ile Ser Glu Met Ser Asn Ser

225 230 235 240

Arg Phe Pro Val Pro Ile Asp Ser Leu His Thr Ser Pro Thr Glu Asn

245 250 255

Ile Val Val Gln Cys Gln Asn Gly Arg Val Thr Leu Asp Gly Glu Leu

260 265 270

Met Gly Thr Thr Gln Leu Leu Pro Ser Gln Ile Cys Ala Phe Arg Gly

275 280 285

Val Leu Thr Arg Ser Thr Ser Arg Ala Ser Asp Gln Ala Asp Thr Ala

290 295 300

Thr Pro Arg Leu Phe Asn Tyr Tyr Trp His Ile Gln Leu Asp Asn Leu

305 310 315 320

Asn Gly Thr Pro Tyr Asp Pro Ala Glu Asp Ile Pro Gly Pro Leu Gly

325 330 335

Thr Pro Asp Phe Arg Gly Lys Val Phe Gly Val Ala Ser Gln Arg Asn

340 345 350

Pro Asp Ser Thr Thr Arg Ala His Glu Ala Lys Val Asp Thr Thr Ala

355 360 365

Gly Arg Phe Thr Pro Lys Leu Gly Ser Leu Glu Ile Ser Thr Glu Ser

370 375 380

Gly Asp Phe Asp Gln Asn Gln Pro Thr Arg Phe Thr Pro Val Gly Ile

385 390 395 400

Gly Val Asp His Glu Ser Asp Phe Gln Gln Trp Ser Leu Pro Asp Tyr

405 410 415

Ser Gly Gln Phe Thr His Asn Met Asn Leu Ala Pro Ala Val Ala Pro

420 425 430

Asn Phe Pro Gly Glu Gln Leu Leu Phe Phe Arg Ser Gln Leu Pro Ser

435 440 445

Ser Gly Gly Arg Ser Asn Gly Ile Leu Asp Cys Leu Val Pro Gln Glu

450 455 460

Trp Val Gln His Phe Tyr Gln Glu Ser Ala Pro Ala Gln Thr Gln Val

465 470 475 480

Ala Leu Val Arg Tyr Val Asn Pro Asp Thr Gly Arg Val Leu Phe Glu

485 490 495

Ala Lys Leu His Lys Leu Gly Phe Met Thr Ile Ala Lys Asn Gly Asp

500 505 510

Ser Pro Ile Thr Val Pro Pro Asn Gly Tyr Phe Arg Phe Glu Ser Trp

515 520 525

Val Asn Pro Phe Tyr Thr Leu Ala Pro Met Gly Thr Gly Asn Gly Arg

530 535 540

Arg Arg Val Gln

545

<210> 4

<211> 540

<212> PRT

<213> Norovirus (Norovirus)

<400> 4

Met Lys Met Ala Ser Ser Asp Ala Asn Pro Ser Asp Gly Ser Ala Ala

1 5 10 15

Asn Leu Val Pro Glu Val Asn Asn Glu Val Met Ala Leu Glu Pro Val

20 25 30

Val Gly Ala Ala Ile Ala Ala Pro Val Ala Gly Gln Gln Asn Val Ile

35 40 45

Asp Pro Trp Ile Arg Asn Asn Phe Val Gln Ala Pro Gly Gly Glu Phe

50 55 60

Thr Val Ser Pro Arg Asn Ala Pro Gly Glu Ile Leu Trp Ser Ala Pro

65 70 75 80

Leu Gly Pro Asp Leu Asn Pro Tyr Leu Ser His Leu Ala Arg Met Tyr

85 90 95

Asn Gly Tyr Ala Gly Gly Phe Glu Val Gln Val Ile Leu Ala Gly Asn

100 105 110

Ala Phe Thr Ala Gly Lys Val Ile Phe Ala Ala Val Pro Pro Asn Phe

115 120 125

Pro Thr Glu Gly Leu Ser Pro Ser Gln Val Thr Met Phe Pro His Ile

130 135 140

Val Val Asp Val Arg Gln Leu Glu Pro Val Leu Ile Pro Leu Pro Asp

145 150 155 160

Val Arg Asn Asn Phe Tyr His Tyr Asn Gln Ser Asn Asp Pro Thr Ile

165 170 175

Lys Leu Ile Ala Met Leu Tyr Thr Pro Leu Arg Ala Asn Asn Ala Gly

180 185 190

Asp Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Ser Pro

195 200 205

Asp Phe Asp Phe Ile Phe Leu Val Pro Pro Thr Val Glu Ser Arg Thr

210 215 220

Lys Pro Phe Ser Val Pro Val Leu Thr Val Glu Glu Met Thr Asn Ser

225 230 235 240

Arg Phe Pro Ile Pro Leu Glu Lys Leu Phe Thr Gly Pro Ser Ser Ala

245 250 255

Phe Val Val Gln Pro Gln Asn Gly Arg Cys Thr Thr Asp Gly Val Leu

260 265 270

Leu Gly Thr Thr Gln Leu Ser Pro Val Asn Ile Cys Thr Phe Arg Gly

275 280 285

Asp Val Thr His Ile Thr Gly Ser Arg Asn Tyr Thr Met Asn Leu Ala

290 295 300

Ser Gln Asn Trp Asn Asn Tyr Asp Pro Thr Glu Glu Ile Pro Ala Pro

305 310 315 320

Leu Gly Thr Pro Asp Phe Val Gly Lys Ile Gln Gly Val Leu Thr Gln

325 330 335

Thr Thr Arg Thr Asp Gly Ser Thr Arg Gly His Lys Ala Thr Val Tyr

340 345 350

Thr Gly Ser Ala Asp Phe Ala Pro Lys Leu Gly Arg Val Gln Phe Glu

355 360 365

Thr Asp Thr Asp His Asp Phe Glu Ala Asn Gln Asn Thr Lys Phe Thr

370 375 380

Pro Val Gly Val Ile Gln Asp Gly Ser Thr Thr His Arg Asn Glu Pro

385 390 395 400

Gln Gln Trp Val Leu Pro Ser Tyr Ser Gly Arg Asn Thr His Asn Val

405 410 415

His Leu Ala Pro Ala Val Ala Pro Thr Phe Pro Gly Glu Gln Leu Leu

420 425 430

Phe Phe Arg Ser Thr Met Pro Gly Cys Ser Gly Tyr Pro Asn Met Asp

435 440 445

Leu Asp Cys Leu Leu Pro Gln Glu Trp Val Gln Tyr Phe Tyr Gln Glu

450 455 460

Ala Ala Pro Ala Gln Ser Asp Val Ala Leu Leu Arg Phe Val Asn Pro

465 470 475 480

Asp Thr Gly Arg Val Leu Phe Glu Cys Lys Leu His Lys Ser Gly Tyr

485 490 495

Val Thr Val Ala His Thr Gly Gln His Asp Leu Val Ile Pro Pro Asn

500 505 510

Gly Tyr Phe Arg Phe Asp Ser Trp Val Asn Gln Phe Tyr Thr Leu Ala

515 520 525

Pro Met Gly Asn Gly Thr Gly Arg Arg Arg Ala Val

530 535 540

<210> 5

<211> 547

<212> PRT

<213> Norovirus (Norovirus)

<400> 5

Met Lys Met Ala Ser Asn Asp Ala Ala Pro Ser Asn Asp Gly Ala Ala

1 5 10 15

Asn Leu Val Pro Glu Ala Thr Asn Glu Val Met Ala Leu Glu Pro Val

20 25 30

Val Gly Ala Ser Ile Ala Ala Pro Val Val Gly Gln Gln Asn Ile Ile

35 40 45

Asp Pro Trp Ile Arg Glu Asn Phe Val Gln Ala Pro Gln Gly Glu Phe

50 55 60

Thr Val Ser Pro Arg Asn Ser Pro Gly Glu Met Leu Leu Asn Leu Glu

65 70 75 80

Leu Gly Pro Glu Leu Asn Pro Tyr Leu Ser His Leu Ser Arg Met Tyr

85 90 95

Asn Gly Tyr Ala Gly Gly Met Gln Val Gln Val Val Leu Ala Gly Asn

100 105 110

Ala Phe Thr Ala Gly Lys Ile Ile Phe Ala Ala Val Pro Pro His Phe

115 120 125

Pro Val Glu Asn Ile Ser Ala Ala Gln Ile Thr Met Cys Pro His Val

130 135 140

Ile Val Asp Val Arg Gln Leu Glu Pro Val Leu Leu Pro Leu Pro Asp

145 150 155 160

Ile Arg Asn Arg Phe Phe His Tyr Asn Gln Glu Asn Thr Pro Arg Met

165 170 175

Arg Leu Val Ala Met Leu Tyr Thr Pro Leu Arg Ala Asn Ser Gly Glu

180 185 190

Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Ala Pro Asp

195 200 205

Phe Glu Phe Thr Phe Leu Val Pro Pro Thr Val Glu Ser Lys Thr Lys

210 215 220

Pro Phe Thr Leu Pro Ile Leu Thr Leu Gly Glu Leu Ser Asn Ser Arg

225 230 235 240

Phe Pro Ala Pro Ile Asp Met Leu Tyr Thr Asp Pro Asn Glu Ala Ile

245 250 255

Val Val Gln Pro Gln Asn Gly Arg Cys Thr Leu Asp Gly Thr Leu Gln

260 265 270

Gly Thr Thr Gln Leu Val Pro Thr Gln Ile Cys Ser Phe Arg Gly Thr

275 280 285

Leu Val Ser Gln Thr Ser Arg Ser Ala Asp Ser Thr Asp Ser Ala Pro

290 295 300

Arg Val Arg Ser His Pro Leu His Val Gln Leu Lys Asn Leu Asp Gly

305 310 315 320

Thr Pro Tyr Asp Pro Thr Asp Glu Val Pro Ala Val Leu Gly Ala Ile

325 330 335

Asp Phe Lys Gly Thr Val Phe Gly Val Ala Ser Gln Arg Asn Thr Thr

340 345 350

Gly Asn Ser Ile Gly Ala Thr Arg Ala His Glu Val His Ile Asp Thr

355 360 365

Thr Asn Pro Arg Tyr Thr Pro Lys Leu Gly Ser Val Leu Met Tyr Ser

370 375 380

Glu Ser Asn Asp Phe Asp Asp Gly Gln Pro Thr Arg Phe Thr Pro Ile

385 390 395 400

Gly Met Gly Ala Asp Asp Trp His Gln Trp Glu Leu Pro Glu Tyr Ser

405 410 415

Gly His Leu Thr Leu Asn Met Asn Leu Ala Pro Ala Leu Ala Pro Ala

420 425 430

Phe Pro Gly Glu Arg Ile Leu Phe Phe Arg Ser Val Val Pro Ser Ala

435 440 445

Gly Gly Tyr Gly Ser Gly His Ile Asp Cys Leu Ile Pro Gln Glu Trp

450 455 460

Val Gln His Phe Tyr Gln Glu Ala Ala Pro Ser Gln Ser Ala Val Ala

465 470 475 480

Leu Ile Arg Tyr Val Asn Pro Asp Thr Gly Arg Asn Ile Phe Glu Ala

485 490 495

Lys Leu His Arg Glu Gly Phe Ile Thr Val Ala Asn Ser Gly Asn Asn

500 505 510

Pro Ile Val Val Pro Pro Asn Gly Tyr Phe Arg Phe Glu Ala Trp Val

515 520 525

Asn Gln Phe Tyr Thr Leu Thr Pro Met Gly Thr Gly Gln Gly Arg Arg

530 535 540

Arg Val Gln

545

<210> 6

<211> 540

<212> PRT

<213> Norovirus (Norovirus)

<400> 6

Met Lys Met Ala Ser Asn Asp Ala Ala Pro Ser Asn Asp Gly Ala Ala

1 5 10 15

Gly Leu Val Pro Glu Ile Asn Asn Glu Val Met Pro Leu Glu Pro Val

20 25 30

Ala Gly Ala Ser Leu Ala Thr Pro Val Ala Gly Gln His Asn Ile Ile

35 40 45

Asp Pro Trp Ile Arg Asn Asn Phe Val Gln Ala Pro Ala Gly Glu Phe

50 55 60

Thr Val Ser Pro Arg Asn Ser Pro Gly Glu Ile Leu Leu Asp Leu Glu

65 70 75 80

Leu Gly Pro Glu Leu Asn Pro Tyr Leu Ala His Leu Ala Arg Met Tyr

85 90 95

Asn Gly His Ala Gly Gly Met Glu Val Gln Ile Val Leu Ala Gly Asn

100 105 110

Ala Phe Thr Ala Gly Lys Ile Ile Phe Ala Ala Ile Pro Pro Gly Phe

115 120 125

Pro Tyr Glu Asn Leu Ser Pro Ser Gln Ile Thr Met Cys Pro His Val

130 135 140

Ile Ile Asp Val Arg Gln Leu Glu Pro Val Leu Leu Pro Met Pro Asp

145 150 155 160

Ile Arg Asn Asn Phe Phe His Tyr Asn Gln Ser Asn Asp Pro Lys Leu

165 170 175

Arg Leu Leu Ala Met Leu Tyr Thr Pro Leu Arg Ala Asn Asn Ser Gly

180 185 190

Asp Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Lys Pro Ser Pro

195 200 205

Asp Phe Glu Phe Thr Phe Leu Val Pro Pro Thr Val Glu Ser Lys Thr

210 215 220

Lys Gln Phe Thr Leu Pro Ile Leu Lys Ile Ser Glu Met Thr Asn Ser

225 230 235 240

Arg Phe Pro Val Pro Val Glu Met Met Tyr Thr Ala Arg Asn Glu Asn

245 250 255

Gln Val Val Gln Pro Gln Asn Gly Arg Val Thr Leu Asp Gly Glu Leu

260 265 270

Leu Gly Thr Thr Pro Leu Leu Ala Val Asn Ile Cys Lys Phe Lys Gly

275 280 285

Glu Val Ile Ala Lys Asn Gly Asp Val Arg Ser Tyr Arg Met Asp Met

290 295 300

Glu Ile Thr Asn Thr Asp Gly Thr Pro Ile Asp Pro Thr Glu Asp Thr

305 310 315 320

Pro Gly Pro Ile Gly Ser Pro Asp Phe Gln Gly Ile Leu Phe Gly Val

325 330 335

Ala Ser Gln Arg Asn Lys Asn Glu Gln Asn Pro Ala Thr Arg Ala His

340 345 350

Glu Ala Asn Ile Asn Thr Gly Gly Asp Gln Tyr Ala Pro Lys Leu Ala

355 360 365

Gln Val Lys Phe Phe Ser Glu Ser Gln Asp Phe Glu Val His Gln Pro

370 375 380

Thr Val Phe Thr Pro Val Gly Val Val Gly Asp Ser Ser His Pro Phe

385 390 395 400

Arg Gln Trp Val Leu Pro Arg Tyr Gly Gly His Leu Thr Asn Asn Thr

405 410 415

His Leu Ala Pro Ala Val Ala Pro Leu Phe Pro Gly Glu Gln Ile Leu

420 425 430

Phe Phe Arg Ser Gln Ile Pro Ser Ser Gly Gly His Glu Ser Gly Tyr

435 440 445

Val Asp Cys Leu Val Pro Gln Glu Trp Val Gln His Phe Tyr Gln Glu

450 455 460

Ala Ala Thr Ala Gln Ser Glu Val Ala Leu Ile Arg Phe Ile Asn Pro

465 470 475 480

Asp Thr Gly Arg Val Leu Phe Glu Ala Lys Leu His Lys Gln Gly Phe

485 490 495

Ile Thr Val Ala His Thr Gly Asp Asn Pro Ile Val Met Pro Pro Asn

500 505 510

Gly Tyr Phe Arg Phe Glu Ala Trp Val Asn Gln Phe Tyr Ser Leu Ala

515 520 525

Pro Val Gly Thr Gly Asn Gly Arg Arg Arg Ile Gln

530 535 540

<210> 7

<211> 535

<212> PRT

<213> Norovirus (Norovirus)

<400> 7

Met Lys Met Ala Ser Asn Asp Ala Ala Pro Ser Asn Asp Gly Ala Ala

1 5 10 15

Gly Leu Val Pro Glu Val Asn Asn Glu Thr Met Ala Leu Glu Pro Val

20 25 30

Ala Gly Ala Ser Ile Ala Ala Pro Leu Thr Gly Gln Asn Asn Val Ile

35 40 45

Asp Pro Trp Ile Arg Leu Asn Phe Val Gln Ala Pro Asn Gly Glu Phe

50 55 60

Thr Val Ser Pro Arg Asn Ser Pro Gly Glu Val Leu Leu Asn Leu Glu

65 70 75 80

Leu Gly Pro Glu Leu Asn Pro Tyr Leu Ala His Leu Ser Arg Met Tyr

85 90 95

Asn Gly Tyr Ala Gly Gly Val Glu Val Gln Val Leu Leu Ala Gly Asn

100 105 110

Ala Phe Thr Ala Gly Lys Leu Val Phe Ala Ala Val Pro Pro His Phe

115 120 125

Pro Leu Glu Asn Ile Ser Pro Gly Gln Ile Thr Met Phe Pro His Val

130 135 140

Ile Ile Asp Val Arg Thr Leu Glu Pro Val Leu Leu Pro Leu Pro Asp

145 150 155 160

Val Arg Asn Asn Phe Phe His Tyr Asn Gln Gln Asn Glu Pro Arg Met

165 170 175

Arg Leu Val Ala Met Leu Tyr Thr Pro Leu Arg Ser Asn Gly Ser Gly

180 185 190

Asp Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Ser Pro

195 200 205

Asp Phe Asp Phe Asn Tyr Leu Val Pro Pro Thr Val Glu Ser Lys Thr

210 215 220

Lys Pro Phe Thr Leu Pro Ile Leu Thr Ile Gly Glu Leu Thr Asn Ser

225 230 235 240

Arg Phe Pro Val Pro Ile Asp Glu Leu Tyr Thr Ser Pro Asn Glu Ser

245 250 255

Leu Val Val Gln Pro Gln Asn Gly Arg Cys Ala Leu Asp Gly Glu Leu

260 265 270

Gln Gly Thr Thr Gln Leu Leu Pro Thr Ala Ile Cys Ser Phe Arg Gly

275 280 285

Arg Ile Asn Gln Lys Val Ser Gly Glu Asn His Val Trp Asn Met Gln

290 295 300

Ile Thr Asn Ile Asn Gly Thr Pro Phe Asp Pro Thr Glu Asp Val Pro

305 310 315 320

Ala Pro Leu Gly Thr Pro Asp Phe Ser Gly Lys Leu Phe Gly Val Leu

325 330 335

Ser Gln Arg Asp His Asp Asn Ala Cys Arg Ser His Asp Ala Val Ile

340 345 350

Ala Thr Asn Ser Ala Lys Phe Thr Pro Lys Leu Gly Ala Ile Gln Ile

355 360 365

Gly Thr Trp Glu Gln Asp Asp Val His Ile Asn Gln Pro Thr Lys Phe

370 375 380

Thr Pro Val Gly Leu Phe Glu Ser Glu Gly Phe Asn Gln Trp Thr Leu

385 390 395 400

Pro Asn Tyr Ser Gly Ala Leu Thr Leu Asn Met Gly Leu Ala Pro Pro

405 410 415

Val Ala Pro Thr Phe Pro Gly Glu Gln Ile Leu Phe Phe Arg Ser His

420 425 430

Ile Pro Leu Lys Gly Gly Val Ala Asp Pro Val Ile Asp Cys Leu Leu

435 440 445

Pro Gln Glu Trp Ile Gln His Leu Tyr Gln Glu Ser Ala Pro Ser Gln

450 455 460

Thr Asp Val Ala Leu Ile Arg Phe Thr Asn Pro Asp Thr Gly Arg Val

465 470 475 480

Leu Phe Glu Ala Lys Leu His Arg Ser Gly Tyr Ile Thr Val Ala Asn

485 490 495

Thr Gly Ser Arg Pro Ile Val Val Pro Ala Asn Gly Tyr Phe Arg Phe

500 505 510

Asp Ser Trp Val Asn Gln Phe Tyr Ser Leu Ala Pro Met Gly Thr Gly

515 520 525

Asn Gly Arg Arg Arg Val Gln

530 535

<210> 8

<211> 540

<212> PRT

<213> Norovirus (Norovirus)

<400> 8

Met Lys Met Ala Ser Asn Asp Ala Ala Pro Ser Asn Asp Gly Ala Ala

1 5 10 15

Gly Leu Val Pro Glu Gly Asn Asn Glu Thr Leu Pro Leu Glu Pro Val

20 25 30

Ala Gly Ala Ala Ile Ala Ala Pro Val Thr Gly Gln Asn Asn Ile Ile

35 40 45

Asp Pro Trp Ile Arg Thr Asn Phe Val Gln Ala Pro Asn Gly Glu Phe

50 55 60

Thr Val Ser Pro Arg Asn Ser Pro Gly Glu Ile Leu Leu Asn Leu Glu

65 70 75 80

Leu Gly Pro Asp Leu Asn Pro Tyr Leu Ala His Leu Ser Arg Met Tyr

85 90 95

Asn Gly Tyr Ala Gly Gly Val Glu Val Gln Val Leu Leu Ala Gly Asn

100 105 110

Ala Phe Thr Ala Gly Lys Ile Leu Phe Ala Ala Val Pro Pro Asn Phe

115 120 125

Pro Val Glu Phe Leu Ser Pro Ala Gln Ile Thr Met Leu Pro His Leu

130 135 140

Ile Val Asp Val Arg Thr Leu Glu Pro Ile Met Ile Pro Leu Pro Asp

145 150 155 160

Val Arg Asn Thr Phe Phe His Tyr Ser Asn Gln Pro Asn Ser Arg Met

165 170 175

Arg Leu Val Ala Met Leu Tyr Thr Pro Leu Arg Ser Asn Gly Ser Gly

180 185 190

Asp Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Thr Pro

195 200 205

Asp Phe Glu Phe Thr Tyr Leu Val Pro Pro Ser Val Glu Ser Lys Thr

210 215 220

Lys Pro Phe Ser Leu Pro Ile Leu Thr Leu Ser Glu Leu Thr Asn Ser

225 230 235 240

Arg Phe Pro Val Pro Ile Asp Ser Leu Phe Thr Ala Gln Asn Asn Val

245 250 255

Leu Gln Val Gln Cys Gln Asn Gly Arg Cys Thr Leu Asp Gly Glu Leu

260 265 270

Gln Gly Thr Thr Gln Leu Leu Pro Ser Gly Ile Cys Ala Phe Arg Gly

275 280 285

Arg Val Thr Ala Gln Ile Asn Gln Arg Asp Arg Trp His Met Gln Leu

290 295 300

Gln Asn Leu Asn Gly Thr Thr Tyr Asp Pro Thr Asp Asp Val Pro Ala

305 310 315 320

Pro Leu Gly Thr Pro Asp Phe Lys Gly Val Val Phe Gly Met Val Ser

325 330 335

Gln Arg Asn Val Gly Asn Asp Ala Pro Gly Ser Thr Arg Ala Gln Gln

340 345 350

Ala Trp Val Ser Thr Tyr Ser Pro Gln Phe Val Pro Lys Leu Gly Ser

355 360 365

Val Asn Leu Arg Ile Ser Asp Asn Asp Asp Phe Gln Phe Gln Pro Thr

370 375 380

Lys Phe Thr Pro Val Gly Val Asn Asp Asp Asp Asp Gly His Pro Phe

385 390 395 400

Arg Gln Trp Glu Leu Pro Asn Tyr Ser Gly Glu Leu Thr Leu Asn Met

405 410 415

Asn Leu Ala Pro Pro Val Ala Pro Asn Phe Pro Gly Glu Gln Leu Leu

420 425 430

Phe Phe Arg Ser Phe Val Pro Cys Ser Gly Gly Tyr Asn Gln Gly Ile

435 440 445

Ile Asp Cys Leu Ile Pro Gln Glu Trp Ile Gln His Phe Tyr Gln Glu

450 455 460

Ser Ala Pro Ser Gln Ser Asp Val Ala Leu Ile Arg Tyr Val Asn Pro

465 470 475 480

Asp Thr Gly Arg Thr Leu Phe Glu Ala Lys Leu His Arg Ser Gly Tyr

485 490 495

Ile Thr Val Ala His Ser Gly Asp Tyr Pro Leu Val Val Pro Ala Asn

500 505 510

Gly His Phe Arg Phe Asp Ser Trp Val Asn Gln Phe Tyr Ser Leu Ala

515 520 525

Pro Met Gly Thr Gly Asn Gly Arg Arg Arg Ala Gln

530 535 540

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcgttcgttc gttcgttcgt t 21

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcgttcgttc gttcgttcgt tcgtt 25

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcgtcgtcgt cgtcgtcgtc g 21

Claims (10)

1. A composition for eliciting an immune response to norovirus, comprising virus-like particles of gii.2, gii.4, gii.6, and gii.17-type norovirus, or active fragments thereof.

2. The composition of claim 1, wherein the norovirus type GII.4-like particle, or active fragment thereof, comprises or consists of an amino acid sequence selected from one of:

(1) 4, an amino acid sequence shown as SEQ ID NO;

(2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO. 4;

(3) an amino acid sequence having amino acid substitution, deletion or insertion at one or more positions in the amino acid sequence shown as SEQ ID NO. 4; and

(4) 4, amino acids from the X position to the 480 position of the amino acid sequence shown in SEQ ID NO, wherein the integer between X2 and 39; preferably, said X is 27, 37 or 39;

preferably, said gii.2-type norovirus virus-like particle or active fragment thereof comprises or consists of an amino acid sequence selected from one of:

(1) an amino acid sequence shown as SEQ ID NO. 2;

(2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO 2; and

(3) an amino acid sequence having amino acid substitution, deletion or insertion at one or more positions in the amino acid sequence shown as SEQ ID NO. 2;

preferably, said gii.6 type norovirus virus-like particle or active fragment thereof comprises or consists of an amino acid sequence selected from one of:

(1) the amino acid sequence shown as SEQ ID NO. 5;

(2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO. 5; and

(3) 5 or an amino acid sequence having amino acid substitution, deletion or insertion at one or more positions in the amino acid sequence shown in SEQ ID NO;

(4) 5, wherein X is an integer between 2 and 27; preferably, said X is 27;

preferably, said gii.17 norovirus virus-like particle or active fragment thereof comprises or consists of an amino acid sequence selected from one of:

(1) the amino acid sequence shown as SEQ ID NO. 8;

(2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence set forth in SEQ ID NO. 8; and

(3) an amino acid sequence having amino acid substitutions, deletions or insertions at one or more positions in the amino acid sequence shown in SEQ ID NO. 8.

3. The composition of claim 1 or 2, wherein the GII.2 type norovirus virus-like particle, or active fragment thereof, comprises or consists of the amino acid sequence set forth as SEQ ID NO 2; the GII.4 type norovirus virus-like particle or the active fragment thereof comprises or consists of an amino acid sequence shown as SEQ ID NO. 4; the GII.6 type norovirus virus-like particle or the active fragment thereof comprises or consists of an amino acid sequence shown as SEQ ID NO. 5; the GII.17 type norovirus virus-like particle or the active fragment thereof comprises or consists of an amino acid sequence shown as SEQ ID NO. 8.

4. The composition of any one of claims 1 to 3, wherein the norovirus is selected from one or more of the noroviruses type GI.1, GII.2, GII.4, GII.6, GII.7, GII.12, or GII.17.

5. The composition of any one of claims 1 to 4, wherein the composition is a pharmaceutical composition, such as a vaccine, for the prevention and/or treatment of norovirus infection.

6. The composition of claim 5, wherein the pharmaceutical composition further comprises one or more adjuvants selected from an aluminum adjuvant, a TRL adjuvant, or a saponin adjuvant;

preferably, the aluminium adjuvant is selected from one or more of aluminium hydroxide, aluminium phosphate and aluminium sulphate; more preferably, the aluminum adjuvant is aluminum hydroxide;

preferably, the TRL adjuvant is TRL9 adjuvant; more preferably, the TRL9 adjuvant is a CPG adjuvant; further preferably, the nucleotide sequence of the CPG adjuvant is selected from one of SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11;

preferably, the saponin adjuvant is QS21 or Iscom adjuvant.

7. The composition of claim 5 or 6, wherein the norovirus infection is gastroenteritis, preferably viral acute gastroenteritis.

8. An immunoassay kit comprising the composition of any one of claims 1 to 4;

preferably, the kit is a kit for detecting norovirus.

9. Use of a composition according to any one of claims 1 to 7 or an immunoassay kit according to claim 8 for the preparation of a product,

(1) a medicament for the prevention and/or treatment of norovirus infection;

(3) a kit for diagnosing norovirus infection; or

(4) Immunogens for the development of norovirus antibodies.

10. Use according to claim 9, wherein the norovirus infection is gastroenteritis, preferably viral acute gastroenteritis.

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