NOROVIRUS ANTIBODIES

Abstract

The present invention relates to a binding polypeptide specifically binding to the amino acid sequence W-V-N-X.sup.1-F-Y-X.sup.2 (SEQ ID NO: 1), wherein X.sup.1 represents any amino acid, preferably Q or P, and wherein X.sup.2 represents any amino acid, preferably, T or S in a norovirus polypeptide. The present invention further relates to polynucleotide encoding a binding polypeptide of the present invention an to a host cell comprising the same or the polynucleotide of the invention. The present invention further relates to a method of detecting the presence of a norovirus capsid polypeptide in a sample and to kits, devices, and uses making use of the binding peptide of the invention.

Claims

1-15. (canceled)

16. A binding polypeptide specifically binding to the amino acid sequence W-V-N-X.sup.1-F-Y-X.sup.2 (SEQ ID NO:1) wherein: (a) X.sup.1 represents any amino acid, preferably Q or P, and (b) X.sup.2 represents any amino acid, preferably, T or S, in a norovirus polypeptide.

17. The binding polypeptide of claim 16, wherein the binding polypeptide is an antibody.

18. The binding polypeptide of claim 16, wherein the binding polypeptide is a single-domain antibody (VHH).

19. The binding polypeptide of claim 16, wherein the binding polypeptide specifically binds to a sequence selected from: TABLE-US-00003 (i)  (SEQ ID NO: 2) F-X.sup.6-X.sup.5-X.sup.4-X.sup.3-W-V-N-X.sup.1-F-Y-X.sup.2, (ii)  (SEQ ID NO: 3) X.sup.53X.sup.52...X.sup.3-W-V-N-X.sup.1-F-Y-X.sup.2;  and/or (iii)  (SEQ ID NO: 4) X.sup.53X.sup.52...X.sup.8-F-X.sup.6-X.sup.5-X.sup.4-X.sup.3-W-V-N-X.sup.1-F-Y-X.sup.2, wherein X.sup.3 to X.sup.52 represent any amino acid and wherein X.sup.53 represents a non-charged amino acid, preferably, L, P, M, Q, or N.

20. The binding polypeptide of claim 16, wherein the binding polypeptide competes in binding to a capsid polypeptide of a norovirus genogroup II (GII) with a VHH encoded by SEQ ID NO:5.

21. The binding polypeptide of claim 16, wherein the binding polypeptide comprises the complementarity determining regions (CDRs) GSIFSIYA (SEQ ID NO:6), ISSGGGTN (SEQ ID NO:7), and KREDYSAYAPPSGS (SEQ ID NO:8).

22. The binding polypeptide claim 16, wherein the binding polypeptide comprises an amino acid sequence essentially having the amino acid sequence of SEQ ID) NO:9.

23. The binding polypeptide of claim 16, wherein the norovirus polypeptide is a norovirus capsid polypeptide.

24. A host cell comprising the binding polypeptide according to claim 16.

25. A kit for diagnosing, preventing or/and treating a norovirus infection, comprising the binding polypeptide of claim 16 in a housing.

Description

FIGURE LEGENDS

[0100] FIG. 1: A) Direct ELISA. of norovirus virus like particles (VLPs) with nano-85. Nano-85 detected GII.10 VLPs at a dilution of 64,000, GII.4 and GII.12 VLPs at a dilution of 32,000 and GI.1 VLPs at a lower dilution of 4,000. X-axis: dilution, Y-axis: Absorption. B) As in A), but indirect ELISA.

[0101] FIG. 2: A. Cartoon view of nano-85 bound to the lower region of the GII.10 P domain. B. Stereo view of A). A network of interactions is responsible for tight binding.

[0102] FIG. 3: A) amino acid sequence of a single-domain antibody of the present invention; CDR sequences are underlined; B) exemplary nucleic acid sequence encoding the single-domain antibody of A).

[0103] FIG. 4: Electron microscopy of GII.10 norovirus virus-like particles pretreated at an approx. 1:1 molar ratio with different antibodies; samples were applied to EM grids and stained with 4% uranyl acetate: A) GII.10 VLPs untreated, B) GII.10 VLPs pretreated with 5B18 IgG, C) GII.10 VLPs pretreated with Nano-85, and D) GII.10 VLPs pretreated with Nano-25.

[0104] FIG. 5: Electron microscopy of GII.4 norovirus virus-like particles pretreated at an approx. 1:1 molar ratio with Nano-85; samples were applied to EM grids and stained with 4% uranyl acetate: A) GII.4 VLPs untreated, B) GII.4 VLPs pretreated with Nano-85.

[0105] FIG. 6: Inhibition of binding to porcine gastric mucin (PGM) by antibodies of the invention. GII.10 VLPs A) or GII.4 VLPs B) were pre-incubated with serially diluted Nano-85 and added to plates coated with PGM. The bound VLPs were detected with polyclonal antibody to GII.10 or GII.4 VLPs. The percentage (%) inhibition was calculated by dividing the OD490 of VLP-Nanobody complex by the average OD490 of VLPs alone, which was set to 100% binding. X-axis: nanobody concentration (μg/ml), Y-axis: % inhibition.

[0106] FIG. 7: Improved binding of Nano-85 muteins to genogroup V norovirus. Experimental conditions were as described for FIG. 1A.; X-axis: nanobody concentration (μg/ml), Y-axis: Absorption at 490 nm.

[0107] FIG. 8: Superposition of Nano-85 onto the cryo-EM structure of GII.10 VLP. Six Nano-85-P domain (black spheres) complexes were positioned onto the VLP (light grey) in order to show the possible clashes in the two-fold axes. The space available suggests Nano-85 is able to bind at all possible P dimers simultaneously.

EXAMPLES

Example 1: Materials and Methods

VLP Production

[0108] The capsid gene of Norovirus GII.10 Vietnam026 (AF504671), GII.12 Hiro (AB044366), GI.1 Norwalk virus (AY502016), and GII.4 NSW-2012 (AFV08795) was cloned into a baculovirus expression system as previously described (Hansman, G. S., L. T. Doan, T. A. Kguyen, S. Okitsu, K. Katayama, S. Ogawa, K. Natori, N. Takeda, Y. Kato, O. Nishio, M. Noda, and H. Ushijima. 2004. Detection of norovirus and sapovirus infection among children with gastroenteritis in Ho Chi Minh City, Vietnam. Arch Virol 149:1673-1688; Lin, C. M., F. M. Wu, H. K. Kim, M. P. Doyle, B. S. Michael, and L. K. Williams. 2003. A comparison of hand washing techniques to remove Escherichia coli and caliciviruses under natural or artificial fingernails. J Food Prot 66:2296-2301). VLPs were harvested at five days post infection. The supernatant was pelletized and applied to a 15-45% sucrose ultracentrifugation gradient (Beckmann SW40-Ti rotor) for 2 h at 4° C. Fractions were confirmed using EM and homogenous particles were pooled and concentrated to 2-10 mg/ml.

P Domain Production

[0109] The P domain of GII.10 (Vietnam026), GII.12 (Hiro), GII.4 (Saga-2006), and GII.4 (NSW-2012) was produced as previously described (Hansman, G. S., C. Biertumpfel, I. Georgiev, J. S. McLellan, L. Chen, T. Zhou, K. Katayama, and P. D. Kwong. 2011. Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability. Journal of virology 85:6687-6701). Briefly, the P domain was cloned in expression vector pMal-c2X (New England Biolabs) and transformed into BL21 cells. Transformed cells were grown at 37° C. in LB medium for 2 h. Expression was induced with IPTG (0.75 mM) at OD.sub.600 of 0.6 for 18 h at 22° C. Cells were harvested by centrifugation and disrupted by sonication. A His-tagged fusion-P domain protein was purified from a Ni-NTA column and digested with HRV-3C protease (Novagen) overnight at 4° C. The cleaved P domain was separated on the Ni-NTA column and dialyzed in gel filtration buffer (GFB, 0.35 M NaCl and 2.5 mM Tris (pH 7.4)) overnight at 4° C. The P domain was further purified by size exclusion chromatography with a Superdex-200 column and stored in GFB at 4° C.

Nanobody Production

[0110] A single alpaca was injected subcutaneously on days 0, 7, 14, 21, 28 and 35 with ˜115 μg GII.10 VLP protein per injection (VIB Nanobody Service Facility, at Vrije University Brussel, Belgium). A VHH library was constructed and screened for the presence of antigen-specific nanobodies. A VHH library of about 10.sup.8 independent transformants was obtained. Three consecutive rounds of panning were performed on solid-phase coated with GII.10 VLPs (20 μg/well). Totally, 143 individual colonies were randomly selected. Crude periplasmic extracts were analyzed using ELISA for the presence of antigen specific Nanobodies. Forty-seven colonies were positive and nucleotide sequencing revealed these represented 35 different Nanobodies that belonged to 17 distinct groups based on sequence alignments. In this study, Nano-85 was examined.

Expression and Purification of Nanobody Proteins.

[0111] The Nanobodies were cloned into a pHEN6C expression vector and grown in E. coli WK6 cells overnight at 28° C. Expression was induced with 1 mM IPTG at OD.sub.600=0.7. Nanobodies were extracted from periplasm and the supernatant collected. Nanobodies were separated on a Ni-NTA column and purified by size exclusion chromatography using a Superdex-200 column as previously described (9). Nanobodies were concentrated to 2-5 mg/ml and stored in GFB.

ELISA Experiments

[0112] Nanobody reactivity against VLPs and P domains were measured using a direct ELISA as previously described (Hansman, G. S., R. Guntapong, Y. Pongsuwanna, K. Natori, K. Katayama, and N. Takeda. 2006. Development of an antigen ELISA to detect sapovirus in clinical stool specimens. Arch Virol 151:551-561). Briefly, microtiter plates were coated with 2 μg/ml of VLPs (GII.10, GII.12, and GII.4) or 7 μg/ml GII.10 P domain. VLPs were diluted in PBS (pH 7.4), which preserved their structural integrity (Hansman, G. S., D. W. Taylor, J. S. McLellan, T. J. Smith, I. Georgiev, J. R. Tame, S. Y. Park, M. Yamazaki, F. Gondaira, M. Miki, K. Katayama, K. Murata, and P. D. Kwong. 2012. Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle. Journal of virology 86:3635-3646). Nanobodies were serially diluted in PBS from a starting concentration of ˜10 μM, and then 100 μl was added to triplicate wells. The His-tagged-Nanobodies were detected with a secondary HRP-conjugated anti-His IgG. For sandwich ELISA, plates were coated overnight with commercially produced monoclonal antibodies (ViroStat, USA). VLPs (GII.10, GII.12, GII.4, and GI.1) were added for 1 h at 37° C., and then detected as described above. Detection of norovirus virions from clinical specimens was also performed using the sandwich ELISA with ˜1 μM GII.4 specific monoclonal antibody as capture and ˜1 μM Nano-85 as detector. A detection limit was set at 0.15 for all experiments, which was ˜3 times the value of the (PBS only) negative control.

Purification and Crystallization of Norovirus P Domain and Nanobody Complexes

[0113] The P domain and Nanobody were mixed in a 1:1.4 molar ratio and incubated at 25° C. for ˜90 min. The complex was purified by size exclusion chromatography using a Superdex-200 column and concentrated to 2.8 mg/ml. Complex crystals were grown using hanging-drop vapor diffusion method at 18° C. GII.10 P domain and Nano-85 crystals were grown in 0.2 M calcium acetate, 18% (w/v), PEG8000, and 0.1 M sodium cacodylate (pH 6.5); GII.10 P domain and Nano-25 crystals were grown in 20% (w/v) PEG3350, and 0.2 M ammonium dihydrogen phosphate; Saga-2006 GII.4 P domain and Nano-85 crystals were grown in 10% (w/v) PEG8000 and 0.1 M HEPES (pH 7.5); and NSW-2012 GII.4 P domain and Nano-85 crystals were grown in 10% (w/v) PEG8000 and 0.1 M HEPES (pH 7.5). Prior to flash-freezing in liquid nitrogen, crystals were transferred to a cryoprotectant containing the mother liquor in 30% ethylene glycol.

Data Collection, Structure Solution, and Refinement

[0114] X-ray diffraction data were collected at the European Synchrotron Radiation Facility, France at beamlines BM30A and ID23-1 and processed with XDS (Kabsch, W. 1993. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Cryst 26:795-800). Structures were solved by molecular replacement in PHASER (McCoy, A. J., R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni, and R. J. Read. 2007. Phaser crystallographic software. Journal of Applied Crystallography 40:658-674). The GII.10 P domain was solved using molecular replacement with GII.10 P domain (PDB ID 3ONU) and a previously determined Nanobody (PDB ID 3P0G) as search models. Structures were refined in multiple rounds of manual model building in COOT (Emsley, P., B. Lohkamp, W. G. Scott, and K. Cowtan. 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486-501) and refined with PHENIX (Adams, P. D., P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, and P. H. Zwart. 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213-221) Structures were validated with Procheck (Morris, A. L., M. W. MacArthur, E. G. Hutchinson, and J. M. Thornton. 1992. Stereochemical quality of protein structure coordinates. Proteins 12:345-364) and Molprobity (Chen, V. B., W. B. Arendall, 3rd, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral, L. W. Murray, J. S. Richardson, and D. C. Richardson. 2010. MolProbity: all-atom structure validation for macromolecular crystallography. Acta crystallographica. Section D, Biological crystallography 66:12-21).

Example 2

Nanobody Binding Specificity

[0115] Nano-85 was analyzed in this study. Initially, the Nanobody binding characteristics were analyzed with GII.10 VLPs and the corresponding P domain (FIG. 1A). Nano-85 detected GII.10 VLPs at a dilution of 64,000 (˜170 pM). A similar binding pattern was also observed with GII.10 P domains, where Nano-85 detected the P domains at dilutions of 17,800. The cross-reactivities of the Nanobody were analyzed using VLPs from other genotypes (GI.1, GII.4 (NSW-2012), and GII.12). Nano-85 detected GII.4 and GII.12 VLPs at a dilution of 32,000 (˜340 pM) and GI.1 VLPs at a lower dilution of 4,000 (FIG. 1A).

[0116] A sandwich ELISA was performed to confirm the binding of Nano-85 to intact particles. Wells were first coated with GII genotype specific monoclonal antibodies and then GII.10, GII.12, and GII.4 (NSW-2012) VLPs were added. Nano-85 detected the captured GII.10, GII.12, and GII.4 VLPs at dilutions of 17,280, 8,640, and 2,160, respectively (FIG. 1B). Twelve GII.4 positive clinical stool specimens were also tested in order to determine the Nanobody diagnostic potential. Nano-85 was able to detect 8 of 12 norovirus GII.4 positive specimens (Table 1). No cross-reactivities were observed with RT-PCR negative control specimens.

TABLE-US-00002 TABLE 1 Detection of norovirus in stool samples Sample 8 17 19 42 47 49 58 59 OD.sub.490 0.11 0.16 0.20 0.14 0.14 0.12 0.23 0.12

[0117] The Nanobody tightly bound to all three P domains with Kds ˜3 to 30 nM.

X-Ray Structures of P Domain and Nanobody Complexes

[0118] In order to identify the Nanobody recognition sites, we determined the X-ray crystal structures of GII.10 P domain in complex with Nano-25 and Nano-85. We also determined the X-ray crystal structures of GII.4 (Saga-2006) P domain Nano-85 complex and GII.4 (NSW-2012) P domain Nano-85 complex in order to better understand cross-reactivity binding interactions at the atomic level. The GII.10 P1 subdomain comprised of residues 222-277 and 427-549, whereas the P2 subdomain was located between residues 278-426 (Hansman, G. S., C. Biertumpfel, I. Georgiev, J. S. McLellan, L. Chen, T. Zhou, K. Katayama, and P. D. Kwong. 2011. Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability. Journal of virology 85:6687-6701). The GII.4 P1 subdomains comprised of residues 224-274 and 418-530, whereas the P2 subdomains were located between residues 275-417. The P1 subdomains compromised of β-sheets and one α-helix, whereas the P2 subdomains contained six antiparallel β-strands that formed a barrel-like structure. The P domains in the complex structures were reminiscent of the apo structures and showed little conformational change. Nano-85 was well refined for most residues and showed a typical immunoglobulin domain fold of other known Nanobody structures (Aline Desmyter, T. R. T., Mehdi Arbabi Ghahroudi, Minh-Hoa Dao Thi, Freddy Poortmans, Raymond Hamers, Serge Muyldermans & Lode Wyns. 1996. Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nature structural biology 3:803-81). As expected, amino acid changes were mostly located in CDR1, CDR2, and CDR3. Nano-85 CDR3 contained a seven amino acid insertion.

Structure of GII.10 P Domain and Nano-85 Complex

[0119] Nano-85 bound with a monomeric interaction at the lower section of the GII.10 P1 subdomain (FIG. 2). The total interface surface area of the GII.10 P domain and Nano-85 was 736 A2 as calculated using PISA software (Krissinel, E., and K. Henrick. 2007. Inference of macromolecular assemblies from crystalline state. Journal of molecular biology 372:774-797). Crystal packing showed alternative binding sites on the GII.10 P domain, however these were less favorable, having an interface surface area less than 360 Å2 and only a few hydrogen bonds. The Nano-85 was held with a network of binding interactions, including hydrogen bonds and hydrophobic interactions. Eight GII.10 P domain residues (Trp528, Asn50, Thr534, Trp528, Leu477, Phe532, and Tyr533) formed direct hydrogen bonds with Nano-85. Four GII.10 P domain residues (Leu477, Phe525, Val529, and Phe532) were formed hydrophobic interactions with Nano-85. One electrostatic interaction was observed between Phe532 of GII.10 P domain and Lys96 of Nano-85.

Structure of GII.4 P Domains and Nano-85 Complexes

[0120] In order to describe Nano-85 binding interactions with other noroviruses, we solved the X-ray crystal structures of two different GII.4 P domains (Saga-2006 and NSW-2012) in complex with Nano-85. Saga-2006 and NSW-2012 P domains had 93% amino acid identity and both had ˜55% amino acid identity with GII.10 P domain. Similar to GII.10, Nano-85 bound at the lower section of the GII.4 P1 subdomain. The total interface surface area of the GII.4 P domains and Nano-85 were ˜736 Å.sup.2. Nano-85 was held with a network of binding interactions similar to GII.10 P domain. Eight GII.4 P domain residues (Trp528, Asn50, Thr534, Trp528, Leu477, Phe532, and Tyr533) formed direct hydrogen bonds with Nano-85. Four GII.4 P domain residues (Leu477, Phe525, Val529, and Phe532) formed hydrophobic interactions with Nano-85. One electrostatic interaction was observed between Phe532 of GII.4 P domain and Lys96 of Nano-85.

Conservation of Nanobody Binding Sites

[0121] The norovirus P domain amino acid sequences of representative GII genotypes was aligned using ClustalX. Six of eight GII P domain residues interacting with Nano-85 were highly conserved among the diverse genotypes: The sequence was WVNQFYT (SEQ ID NO:10) for GII.10, GII.6, and GII.4; WVNQFYS (SEQ ID NO:11) for GII.1, GII.2, GII.5, GII.7, GII.8, and GII.12; WVNPFYT (SEQ ID NO:12) for GII.3. Interestingly, the member of genogroup I included in this study only has four amino acids of the motifs in common with the members of GII (amino acids WV and FY) and is recognized anyway (FIG. 1A).

Superposition of the GII.10 P Domain Nanobody Complex on the GII.10 VLP

[0122] The binding sites of the Nanobodies were located in the lower region of the P domain. Superposition of the X-ray crystal structure of GII.10 P domain Nanobody complexes on the cryo-EM GII.10 VLP showed a discernible Nanobody clash on the particle. Likewise, the X-ray crystal structure of the GII.10 P domain Fab complex showed a similar clash on the cryo-EM GII.10 VLP. Nanobody and Fab binding to the norovirus P domains does not cause any conformational changes in the P domain. However, the hinge region between the S and P domains is expected to allow a certain degree of flexibility of the P domains so that the Nanobody can attach to the occluded site on the particles (Smith, T. J. 2011. Structural studies on antibody recognition and neutralization of viruses. Curr Opin Virol 1:150-156). As shown in FIG. 8, Nano-85 can occupy all possible P dimers simultaneously.

Example 3: Disassembly of Norovirus VLPs by Nano-85

[0123] GII.10 VLPs (FIG. 4) were incubated with the following antibodies: control (no antibody) (FIG. 4A); monoclonal 5B18 IgG (as disclosed in EP 2 757 111 A1, FIG. 4 B), Nano-85 (FIG. 4 C), and Nano-25 (FIG. 4 D). Nano-25 is a nanobody binding to a similar site of the norovirus VLP, but contacting different amino acids of the viral capsid. As shown in FIG. 4, only Nano-85 causes disassembly of the VLPs. FIG. 5 shows results of the analogous experiment using GII.4 VLPs.

Example 4: Inhibition of Norovirus Binding to Porcine Gastric Mucin (PGM) by Nano-85

[0124] Histo-blood group antigens (HBGAs) were found to be the natural binding factor on the cell for noroviruses (Tan et al. (2005), Trends in Microbiology 13(6):285). Moreover, it was found that porcine gastric mucin (PGM) competes with HBGA in binding to norovirus (Tian et al. (2005), Lett Appl Microbiol 41(4):315), which is why binding of norovirus to PGM has been used as a model system for binding of noroviruses to their natural binding factors. Accordingly, binding of norovirus GII.10 and GII.4 VLPs to PGM was determined after preincubation of the VLPs with serial dilutions of Nano-85. As shown in FIG. 6, Nano-85 inhibits norovirus binding to PGM in a concentration-dependent manner, with half-maximal inhibition occurring at a concentration of less than 10 μg/ml.

Example 5: Redesign of Nano-85

[0125] The information underlying FIG. 2 was used to increase binding of Nano-85 to genogroup V (GV) norovirus by modifying Nano-85 CDR regions. Residues A33 and Y100, known to be involved in binding to GII norovirus, were modified to better fit into the binding site of genogroup V norovirus. The binding site on the P domain is unchanged. As shown in FIG. 7, a double A33L/Y100F mutein of Nano-85 shows strongly increased binding to genogroup V norovirus.