Antibodies targeting GII genotypes of human norovirus

Abstract

The disclosure provides antibodies that can block and / or neutralize multiple norovirus GII genotypes, e.g., two or more, or three or more genotypes selected from norovirus GII genotypes GII.2, GII.3, GII.4, GII.6, GII.12, GII.17 and GII.14; methods of producing the antibodies; and methods of treating a patient exposed to norovirus or infected with norovirus using such antibodies.

Description

PATENT Attorney Docket No.090402-1508623-002510PC Client Ref. No. UNC 24-0085 / UTA 8348 GEO ANTIBODIES TARGETING GII GENOTYPES OF HUMAN NOROVIRUS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No. 63 / 656,263, filed June 5, 2024, which is incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under Grant No. AI148260 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] Human norovirus is the leading cause of viral acute gastroenteritis globally, causing an estimated >700 million infections and tens of thousands of deaths annually, primarily in young children and the elderly1. At least four norovirus genogroups infect humans2. Viruses of genogroup II (GII) cause >90% of infections, with variants of the GII.4 genotype accounting for 50-80% of outbreaks3. Global norovirus disease surges occur regularly in all age groups, corresponding with the periodic emergence of a new GII.4 variant4-6. However, the rarity of repeat infections with the same genotype or GII.4 variant in young children and the lack of widespread symptomatic infections in adults during non-GII.4 emergence years indicate that infection can induce long-term broadly protective immunity, supporting the feasibility of disease mitigation through vaccination7,8.

[0004] The norovirus major capsid protein VP1 undergoes self-assembly to form virus-like particles (VLPs) consisting of 90 copies of VP1 dimers that are phenotypically and immunologically indistinguishable from native norovirus virions9. Each VP1 comprises an interior shell (S) and a protruding (P) domain, which is further divided into the hypervariable, immunodominant P2 subdomain and the less variable yet immune-subdominant P1 subdomain. 79710041V.1Due to challenges in cultivating norovirus in vitro until the recent development of human intestinal enteroid (HIE) culture10, the measurement of ligand-binding blockade antibodies has served as a surrogate neutralization assay11,12. Notably, these surrogate neutralizing antibodies (nAbs) have the potential to be an immune correlate of protection due to association with protective responses in unvaccinated subjects13,14,15.

[0005] To date, five norovirus vaccines are in clinical development. Included in all vaccines is a GII.4 immunogen, either presented as a GII.4 VLP or as an adenovirus-based vector encoding GII.4 VP116. One of the intramuscular VLP vaccines has been demonstrated to elicit short-term broad blockade antibody responses to heterologous GII virus not included in the vaccine12,17. This finding suggests that the breadth of serum neutralization and protection could be broadened by the rational design of next-generation immunogens capable of eliciting antibodies that recognize conserved epitopes among divergent GII genotypes, including GII.4 variants. However, there are significant hurdles to vaccine development, including extensive antigenic diversity with > 35 distinct norovirus genotypes and a limited understanding of immune mechanisms of cross-protection.

[0006] A few human monoclonal antibodies (mAbs) and their corresponding neutralizing epitopes have been identified6,18-21. Broad GII.4 ligand-binding blockade human mAbs, designated NVB 716,19and A143120, having been previously reported that bind to less surface- exposed, conserved epitopes, F and I, respectively. However, the recent emergence of the GII.4 Hong Kong (HK) 2019 variant, characterized by antigenic changes in at least six known epitopes, including site I,22underscores the need to assess whether the known conserved epitopes can elicit nAbs that provide pan-GII.4 protection against emerging variants, or if additional support residues within the expanded epitope F or I are required to achieve this breadth. Thus far, no human mAbs have been reported to neutralize HK 2019.

[0007] Several mAbs have been reported to neutralize norovirus by modulating virus particle stability and dynamics18,21,23,24. The M4 nanobody broadly neutralized GII.4 viruses from 2012, 2009, and 2006 variants by inducing particle disassembly, a mechanism similarly observed by the nanobody Nano-8524. The NORO-320 mAb cross-neutralized GII.4 Sydney (SY) 2012 and 79710041V.1GII.17 as a Fab despite having low potency to both viruses. This mAb was further shown to induce virus particle aggregation with unknown mechanism.

[0008] The identification of highly conserved epitopes capable of eliciting circulating human broadly neutralizing mAbs (bNAbs) that potently neutralize not only globally dominant GII.4 strain but also clinically important genotypes, such as GII.3 and GII.2.3,25and (ii) a mechanistic understanding of cross-GII genotype neutralization by humoral immune response at the molecular level are important aspects for further norovirus vaccine development. BRIEF SUMMARY OF ASPECTS OF THE DISCLOSURE

[0009] The present disclosure provides neutralizing antibodies that neutralize norovirus genotypes, e.g., two or more genotypes of clinically important norovirus strains. Thus, in one embodiment, the disclosure features an antibody that neutralizes virus from at least two norovirus GII genotypes, wherein the antibody comprises a VH region comprising an HCDR1 sequence GF(N / T)F(A / G)DYA, an HCDR2 sequence IRS(N / K)(A / T)YGG(A / T)T, and an HCDR3 sequence (S / T)RVGKD(F / Y)GDGLFD(S / Y), and a VL region comprising an LCDR1 sequence SSDVG(N / S)YNL, an LCDR2 sequence EG(S / Y), and an LCDR3 sequence C(L / S)YAG(S / R)SLWV. In some embodiments, the antibody neutralizes norovirus genotype GII.4 and at least two other norovirus genotypes. In some embodiments, the antibody neutralizes norovirus of genotype GII.4, GII.3, and GII.2. In some embodiments, the antibody comprises a VH region comprising an HCDR1 sequence GFNF(G / A)DYA an HCDR2 sequence IRSNTYGGAT, and an HCDR3 sequence SRVGKDFGDGLFDS; and a VL region comprising an LCDR1 sequence SSDVGNYNL, an LCDR2 sequence EGY, and an LCDR3 sequence CLYAGRSLWV. In some embodiments, the antibody comprises the HCDR1 sequence GFNFGDYA, the HCDR2 sequence IRSNTYGGAT, the HCDR3 sequence SRVGKDFGDGLFDS, the LCDR1 sequence SSDVGNYNL, the LCDR2 sequence EGY, and the LCDR3 sequence CLYAGRSLWV. In some embodiments the VHregion has at least 95% identity to SEQ ID NO:3; and the VL region has at least 95% identity to SEQ ID NO:4. In some embodiments the sequence variation occurs in the framework. In some embodiments, the antibody comprises a VH region comprising SEQ ID NO;3 and a VL region comprising SEQ ID NO:4. 79710041V.1

[0010] In other embodiments, the neutralizing antibody comprises the HCDR1 sequence GFNFGDYA, the HCDR2 sequence IRSNTYGGAT, the HCDR3 sequence SRVGKDFGDGLFDS, the LCDR1 sequence SSDVGNYNL, the LCDR2 sequence EGY, and the LCDR3 sequence CLYAGRSLWV. In some embodiments, the VHregion has at least 95% identity to SEQ ID NO:7, 8, or 9. In some embodiments, the sequence variation occurs in the framework. In other embodiments, the antibody comprises the HCDR1 sequence GFNFADYA, the HCDR2 sequence IRSNTYGGAT, the HCDR3 sequence SRVGKDFGDGLFDS, the LCDR1 sequence SSDVGNYNL, the LCDR2 sequence EGY, and the LCDR3 sequence CLYAGRSLWV. In some embodiments, the VH region has at least 95% identity to SEQ ID NO:10. In some embodiments, the sequence variation occurs in the framework. In some embodiments, the VHcomprises SEQ ID NO:7, 8, 9, or 10. In embodiments, the VLregion comprises an LCDR1 sequence SSDVG(N / S)YNL, an LCDR2 sequence EG(S / Y), and an LCDR3 sequence C(L / S)YAG(S / R)SLWV. For example, in some embodiments, the VL comprises an LCDR1 sequence SSDVGNYNL, an LCDR2 sequence EGY, and an LCDR3 sequence CLYAGRSLWV.

[0011] In other embodiments, a neutralizing antibody of the present disclosure comprises a VHregion comprising the HCDR1 sequence GFTFGDYA, the HCDR2 sequence IRSKAYGGTT, the HCDR3 sequence TRVGKDYGDGLFDY, the LCDR1 sequence SSDVGSYNL, the LCDR2 sequence EGS, and the LCDR3 sequence CSYAGSSLWV; and / or comprises a VH region having at least 95% identity to SEQ ID NO:5; and a VL region having at least 95% identity to SEQ ID NO:6. In some embodiments, the VHregion comprises SEQ ID NO:5; and the VLregion comprises SEQ ID NO:6.

[0012] In a further aspect, the disclosure features an antibody that neutralizes two or more norovirus genotypes, wherein the comprises a VHregion comprising an HCDR1 sequence GIILNTYY, an HCDR2 sequence ISASSQTI, and an HCDR3 sequence ATNDGYGGRSRRGSSYYHYGMNF; and a VL region comprising an LCDR1 sequence QPITTF, an LCDR2 sequence ATS, and an LCDR3 sequence QQTYSAPFT; and / or the antibody comprises a VH region having at least 95% identity to SEQ ID NO:1 and a VL region having at least 95% identity to SEQ ID NO:2. In some embodiments, the sequence variation 79710041V.1occurs in the framework. In some embodiments, the antibody neutralizes norovirus of genotype GII.4, GII.3, and GII.2. In some embodiments, the VH region comprises SEQ ID NO:1 and the VLregion comprises SEQ ID NO:2.

[0013] In a further aspect, the disclosure features an isolated nucleic acid or expression vector encoding an antibody heavy and / or light chain variable region of an antibody as described herein, e.g., in the preceding four paragraphs; and host cells comprising the nucleic acid or expression vector. In some embodiments, the host cell comprises a nucleic acid encoding the VHregion and a nucleic acid encoding the VL region of an antibody described herein. In some embodiments, the disclosure further features a method of producing an antibody, the method comprising culturing the host cell under conditions in which the antibody is expressed; and optionally, purifying the antibody.

[0014] In additional aspect, the disclosure features a pharmaceutical formulation comprising a neutralizing antibody of the present disclosure, e.g., as described in the preceding paragraphs; or a pharmaceutical formulation comprising one or more expression vectors encoding an antibody comprising the VHregion and VLregion of the present disclosure.

[0015] The another aspect, the disclosure additionally features a method of preventing or inhibiting a GII genotype norovirus infection, the method comprising administering a pharmaceutical formulation as described herein, e.g., in the preceding paragraph, to a subject that has been exposed to norovirus or is or is infected with norovirus. In some embodiments, administration reduces the viral load in the patient.

[0016] In a further aspect, the disclosure features a method of detecting a norovirus the method comprising contacting a sample suspected of comprising norovirus with an antibody as described herein. In some embodiments, the sample is obtained from a subject that has a norovirus infection or has been exposed to norovirus. In some embodiments, the sample is a bodily fluid or a fecal sample. 79710041V.1BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A-H. Proteomic deconvolution of anti-GII.4 SY 2012 serum IgG repertoires in two subjects with broad GII.4 or cross-GII nAb serum responses. Half-maximal inhibitory dilution (ID50) serum blockade titers in subject A (A) and B (B). Mean ID50titer and 95% confidence intervals (CI) on day 1 (black) and day 29 (blue) are shown. Dashed line, the limit of detection. The red triangle indicates the vaccine immunogen, GII.4 SY 2012. (C) Ig-Seq and BCR- Seq experimental strategy for identification of circulating serum IgG antibodies binding to GII.4 SY 2012 VLP or to GII.3 VLP. (D and E) The serological antibody repertoire recognizing GII.4 SY 2012 in subject A (D) and GII.4 SY 2012 or GII.3 in subject B (E). Each row represents individual clonotypes. Representative mAbs recombinantly expressed for select clonotypes are shown by the row name. In the heatmap, the color scheme displays the relative amount of each clonotype at each time point. Clonotypes below the limit of detection by LC-MS / MS at a specific time point are depicted in gray. See also Figure 5 (F and G) Relative amount of pre-existing versus emergent anti-GII.4 SY 2012 antibody clonotypes on day 29 for subject A (F) and subject B (G). (H) Relative amount of GII.3+GII.4 cross-reactive (CR) in the anti-GII.4 SY 2012 repertoire on day 1 and day 29 for subject B. The size of each stacked bar corresponds to the serum abundance of each clonotype (F–H).

[0018] Figure 2A-H. Antigenic domains of Class I and Class II GII.4 bNAbs. (A) Ligand- binding blockade of GII.4-specific mAbs from subject A towards a time ordered panel of GII.4 variants. IC50, half-maximal inhibitory concentration of mAbs. White box: IC50> 4 μg / ml. (B) bNAbs binned by the ligand-blocking potency (yellow) against a panel of GII.4 epitope mutant VLPs. (C) The ligand-blocking potency of bNAbs to GII chimeric VLPs comprising a GII.3 backbone with grafted conserved GII.4 nAb antigenic sites F, I and / or NERK motif. White box: IC50 > 6 μg / ml. (D and E) HDX-MS analysis of GII.4 SY 2012 VP1 P domain incubated with VX1 (D) or VX6 (E). (F) The two different linear peptides protected by VX1 (dark orange) or VX6 (green) in HDX-MS were color-coded onto the GII.4 SY 2012 P dimer structure (PDB 4WZT). (G) Class I and Class II bNAbs neutralized GII.4 SY 2012 [P16] virus in HIE cell culture assay (upper panel). The mean and standard error of the mean (SEM) were shown. The bottom-panel summarizes the HIE neutralization titer (IC50, ng / ml). (H) Binding sites for Class I (black circle) and Class II (white circle) bNAbs were color-coded onto the GII.4 SY 2012 P 79710041V.1dimer structure (PDB 4WZT, side view). P, pre-existing mAb. E, emergent mAb. For statisticaltest, the pair-wise Student’s t-test was performed for a given exposure time (D and E; *, p 0.05;**, p 0.01; ***, p 0.001; ****, p 0.0001).

[0019] Figure 3A-E. Serum identification of cross-GII neutralizing bNAbs and the evolutionary trajectory of VX22 clonal lineage. (A) Ligand binding blockade of recombinant mAbs from subject B. White box, IC50> 4 μg / ml for GII.4, >10 μg / ml for other GII. (B) Neutralization of VX22 (left panel) and VX20 (right panel) against GII.4 SY 2012 [P16], GII.2, and GII.3 viruses in HIE assay. Each sample was tested in triplicate, and the mean and SEM were shown. (C) IC50 values for each of the GII viruses tested. (D) Phylogenetic tree showing the estimated evolutionary trajectory of clonotype ID 1055 for which recombinant VX22 was selected as representative mAb. The tree was built using day 8 BCR-Seq database. Each tip corresponds to different VH sequences clustered within the clonotype, and the tip diameter is proportional to the number of reads observed. Circular tips indicate that tryptic CDRH3 peptides from a specific VH read were detectable in the day 29 serum by LC-MS / MS (Ig-Seq), whereas triangular tips indicate the absence of tryptic CDRH3 peptides. The VX22 VH tree tip is colored red. (E) Gaining of cross-GII blockade potency and breadth as B cells evolve from unmutated common ancestor (UCA) to VX22 or towards different representative intermediate nodes. White box IC50 >16 μg / ml.

[0020] Figure 4A-G. The crystal structure of VX22 in complex with GII.4 SY 2012 P domain reveals VLP disassembly as the mechanism of neutralization. (A) Crystal structure of VX22 Fab bound to GII.4 SY 2012 P domain (PDB: 8VKX). The asymmetric unit of the crystal contained a single Fab bound to one P domain protomer, with the P domain dimer generated by crystallographic symmetry. (B) The epitope of VX22, shown in orange, consists of two major loops spanning residues 479–484 and 509–513. Additional residues involve Glu235, Asn307, and Asn309. (C) The main interaction between Asn479 and Thr482 (D) or Pro510 (D) with VX22. (E) Negative stain-electron micrographs and 2D class averages of GII.4c VLP without (left) or with (right) the presence of VX22 Fab. After incubation with VX22, fewer intact VLPs were visible, and the P domains of the VLPs showed a high degree of structural mobility. Scale bar, 100nm. (F) Comparison of epitopes among VX22, NORO-320 (PDB: 7JIE) 79710041V.1and A1227 (PDB: 6N81) on the VP1 P domain dimer, with the protomers colored as light and dark grey. The surface of VX22, NORO-320, and A1227 epitopes are colored purple, gold, and green, respectively. (G) Overlay of VX22 (purple) and NORO-320 (gold) binding to GII.4 norovirus VP1 P domain. Both mAbs use distinct angle of approach, with VX22 binding closer to the S domain.

[0021] Figure 5A-C. The bottom-up proteomic analysis of pre- and post-vaccination IgG serological repertoires before and after vaccination. (A) The serological repertoire specific to GII.4 SY 2012 VLP before (on day 1) and after (on day 29) vaccination in subject A. (B and C) In subject B, the serological repertoire binding to GII.4 SY 2012 (B) or GII.3 VLPs (C) were analyzed at each time point. Antibodies binding to GI.1 VLP were depleted from the subject B sera. Each x tick represents different clonotypes. Yellow shade indicates the day 29 clonotypes, accounting for 90% of the entire repertoire by abundance (referred to as D90 diversity index; A– C). Recombinant mAbs selected from clonotypes were shown in the x-axid tick labels. The full clonotype IDs were omitted for visual clarity. See also Tables 2 and 3.

[0022] Figure 6A-E. H gene usage and SHM analysis of serum clonotypes identified in the anti-GII.4 SY 2012 serum repertoire. (A and C) The fraction of pre-existing or emergent clonotypes grouped based on different VH gene usages for subject A (A) or subject B (C). Each bar or pie chart is color-coded by different VH gene usages. Color schemes are consistent between different bar / pie charts (A and C). The center of pie chart represents the number of clonotypes detected within that group, while the relative fraction of each pie chart corresponds to the relative amount of each clonotype as determined detected by LC-MS / MS. (B) Comparison of VH somatic hypermutation (SHM) between pre-existing and emergent clonotypes in subject A. Quartile lines (25th, 50th, and 75th) are displayed in the violin plot. Clonotypes identified in the anti-GII.4 SY 2012 serum antibody repertoires were included in the analysis (A–C). (D) The concordance of VX1 blockade potency and the whole sera nAb potency in subject A. Orange line connects the day 29 serum ID50 titers (left y-axis), and blue line connects the IC50 titers of VX1 (right y-axis) measured across different GII.4 variants. (E) Correlation between the blockade potency of VX1 (x-axis) and the polyclonal serum-neutralizing titer (y-axis) on day 29 across various GII.4 variants. For statistical test, Mann-Whitney U tests were used for unpaired 79710041V.1comparisons (B). The Pearson correlation with a simple linear regression model was conducted using log10 transformed dataset (E). The correlation coefficient (r) with 95% confidence interval, p-value, and the R squared were shown (E).

[0023] Figure 7A-B. Mapping of Class I bNAbs to antigenic domains. (A) Pre-existing (P) and emergent (E) bNAbs were tested for neutralization potency in a surrogate assay against a panel of GII.4 VLPs representing viruses identified in an immune-compromised patient between day 1 (P3.D1) and day 683 (P3.D683) post first sample collection and a sub-panel of VLPs based on the introduction of admixtures of mutations that occurred between P3.D1 and P3.581 or GII.4 FH 2002 for 581.F5 into the P3.581 backbone.19Residue changes between sample collection points are denoted by fill color changes. (B) To bin mAbs into antigenic domains, first, the IC50 difference between the in vivo viruses was recorded (shaded yellow). Second, the IC50 difference between P3.D581 and the most different D581 mutant was recorded (shaded green). Residues common between the two panels defined the key binding domains for the bNAbs.

[0024] Figure 8A-C. Development of GII chimeric VLPs to identify binding domains for Class II bNAbs. (A) GII Chimeric VLPs developed for mapping highly conserved GII.4 nAb binding sites were designed based on known binding residues of two GII.4 broadly blocking or neutralizing mAbs, NVB 716,19and A143120, and their surrounding support residues. Predicted antigenic domains in singles or combinations were recombined into a GII.3 backbone. Yellow blocks denote residues found in GII.4 FH 2002 that differ from GII.3. (B) GII chimeric VLPs that retained PGM binding and blockade potency with NVB 71 (Site Fp, residues 327 and 404), A1431 (Site I, residues 402, 403, and 504-506) or GII.42002.G5 (undefined conserved binding site) were included in further studies. Based upon NVB 71 reactivity, site F was expanded to include residues 254-257 along with 327 and 404. (C) Expanded site F combined with site I (named GII.3 / GII.4 P7) gained blockade potency for NVB 71, A1431, and 2002.G54. Multiple combinations of antibody binding residues and the NERK particle conformation-regulating motif19,11improved the potency of all three mAbs, indicating that Class II bNAb potency is mediated by both antigen-binding residues and antibody access to binding sites. 79710041V.1

[0025] Figure 9. HDX-MS identification of linear P domain epitopes targeted by VX1 or VX6. The hydrogen-deuterium exchange for each of the P domain peptides as a function of exposure times (10 sec, 1 min, 1 hr and 4 hrs). P domains were incubated with VX1 (blue) orVX6 (green). The reference (red) stands for P domain only. Statistical analysis wasperformed using paired student t-tests for comparison between the two peptide stateswithout or with incubation with mAbs. *, p 0.05; **, p 0.01; ***, p 0.001; ****, p 0.0001;blue asterisk for VX1–reference comparison; green asterisk for VX6–reference comparison.

[0026] Figure 10A-B. Intra-clonal phylogenetic tree of clonotype ID 1055. (A) Evolutionary pathway of VH sequences clustered within clonotype ID 1055. The long genetic distance between VH UCA and the first Node 35 was omitted in the tree plot for visual clarity. The horizontal axis represents the estimated number of substitutions per codon site calculated by IgPhyML.29The intermediate node IDs and the tree tip IDs are labeled on the tree. The tree tip diameter corresponds to the BCR read counts for each VH read. Circular tips indicate the Ig-Seq identification of tryptic CDRH3 peptides in the day 29 serum. Triangular tips indicate the VH reads whose CDRH3 peptides are not detectable in the day 29 serum despite VH reads sequenced by BCR-Seq. The VX22 tip is colored red. (B) Antibody blockade of ligand binding curves of UCA and other clonal members of clonotype ID 1055. Each GII VLP was incubated with decreasing concentrations of mAb. The percent control binding compared to no Ab pretreatment was graphed and fit with a log inhibitor normalized dose-response sigmoidal curve fit. The markers and whiskers represent the mean and standard error of the mean.

[0027] Figure 11A-D. Alignment of VH and VL sequences in clonotype ID1055. (A) Sequence alignment of VH sequences clustered within clonotype ID 1055. Each read ID corresponds to tree tips labeled in Figure 10A. VX22 VH read ID is 1481726. (B) Sequence alignment of VL reads paired with the heavy chains. The VL reads are conserved across the VH evolutionary pathway on day 8 time point. Note that few nucleotide mismatches at the 5’ beginning of the framework region1 were due to mutations introduced by the multiplexed forward primers, specifically the hVL2-fwd-OE forward primer53, used in the amplification of IGLV2-derived germline genes rather than being biologically introduced during the course of B cell affinity maturation. The hVL2-fwd-OE forward primer binding site is shown at the 5’ end of 79710041V.1the VL read. VX22 VL read ID is 560294. (C) The VH sequence alignment of Nodes 35, 38, 47, 52, and VX22 against VH UCA predicted by IgPhyM19. Node 39 was not tested, as the nucleotide mutations in this node resulted from degenerate codons, therefore translating identical amino acid residues as those observed in Node 38. (D) Sequence alignment of VX22 VL against VL UCA. The box indicates the CDRH3 (A) or CDRL3 (B) tryptic peptides identified in the day 29 serum eluate by Ig-Seq. The V, (D), and J gene information is displayed at the top of the sequences (C and D).

[0028] Figure 12. Sequence alignment of GI and GII P domains. Regions with interactions with VX22, NORO-320, glycan ligand, and bile acid (for GII.3) are indicated by different colored bars. Sequence alignment was performed using Cluster Omega version 1.2.4 with graphics from ESPript version 3.0. DETAILED DESCRIPTION Terminology

[0029] As used in herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules, and the like.

[0030] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0031] As used herein, the term "antibody" means an isolated or recombinant immunoglobulin binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and specifically covers a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity. 79710041V.1

[0032] As used herein, “V-region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4. The heavy chain V-region, also referred to herein as VH or VH, is a consequence of rearrangement of a V-gene (HV), a D-gene (HD), and a J-gene (HJ), in what is termed V(D)J recombination during B-cell differentiation. The light chain V-region, also referred to herein as VL or VL, is a consequence of rearrangement of a V-gene (LV) and a J-gene (LJ).

[0033] As used herein, “complementarity-determining region (CDR)” refers to the three hypervariable regions in each chain that interrupt the four "framework" regions established by the light and heavy chain variable regions. The CDRs are the primary contributors to binding to an epitope of an antigen. The CDRs of each chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also identified by the chain in which the particular CDR is located. Thus, for example, a VHCDR3 (HCDR3) is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VLCDR3 (LCDR3) is the CDR3 from the variable domain of the light chain of the antibody in which it is found.

[0034] The amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol.227, 799- 817; Al-Lazikani et al., J.Mol.Biol 1997, 273(4)). Definitions of antigen combining sites are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219–221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268–9272 (1989); Martin, et al, Methods Enzymol., 203, 121–153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, 79710041V.1Oxford, 141–1721996). Reference to CDRs as determined by Kabat numbering are based, for example, on Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g.,Chothia and Lesk J. Mol. Biol.196:901-917 (1987)). For example, CDRs according to the Kabat numbering system occur at about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the VL region, and at about 31-35 (CDR1), 50-65 (CDR2) and 95- 102 (CDR3) in the VH when numbered in accordance with the Kabat numbering system (Kabat et al, supra). In another example, residues form a “hypervariable loop” (e.g., about residues 24- 34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the VL, and about residues 26-32 (CDR1), 52- 56 (CDR2) and 95-101 (CDR3 in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, supra). In some embodiments the antibody has symmetrical insertions at one or more of the following points: 28, 36 (CDR1), 63, 74-75 (CDR2) and 123 (CDR3) in the VL, and 28, 36 (CDR1), 63, 74-75 (CDR2) and 123 (CDR3) in the VH when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657-670 (2001)). Sequences that participate in antigen binding may also be identified using on-line tools such as Paratome (e.g., Kunik et al., PLoS Comput Biol (2012) 8:e1002388). As used herein, a “CDR” may refer to CDRs defined by any of these numbering approaches or by a combination of approaches.

[0035] An "Fc region" refers to the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, e.g., for human immunoglobulins, “Fc” refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc may include the J chain. For IgG, Fc comprisesimmunoglobulin domains C 2 and C 3 and the hinge between C 1 and C . It is understood inthe art that the boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, using the numbering according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The term "Fc region" may refer to this region in isolation or this region in the context of an antibody or antibody fragment. "Fc 79710041V.1region " includes naturally occurring allelic variants of the Fc region as well as modified Fc regions, e.g., that are modified to modulate effector function or other properties such as pharmacokinetics, stability or production properties of an antibody.

[0036] The term “equilibrium dissociation constant” abbreviated (KD), refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1M-1). Equilibrium dissociation constants can be measured using any method. Thus, in some embodiments antibodies of the present disclosure have a KDof less than about 50 nM, typically less than about 25 nM, or less than 10 nM, e.g., less than about 5 nM or than about 1 nM and often less than about 10 nM as determined by surface plasmon resonance analysis using a biosensor system such as a Biacore®system performed at 37°C. In some embodiments, an antibody of the present disclosure has a KD of less than 5 x 10-5M, less than 10-5M, less than 5 x 10-6M, less than 10-6M, less than 5 x 10-7M, less than 10-7M, less than 5 x 10-8M, less than 10-8M, less than 5 x 10-9M, less than 10-9M, less than 5 x10-10M, less than 10-10M, less than 5 x 10-11M, less than 10-11M, less than 5 x 10-12M, less than 10-12M, less than 5 xless than 10-13M, less than 5 x 10-14M, less than 10-14M, less than 5 x 10-15M, or less than 10-15M or lower as measured as a bivalent antibody. In the context of the present invention, an “improved” KDrefers to a lower KD. In some embodiments, an antibody of the present disclosure has a KD of less than 5 x 10-5M, less than 10-5M, less than 5 x 10-6M, less than 10-6M, less than 5 x 10-7M, less than 10-7M, less than 5 x 10-8M, less than 10-8M, less than 5 x 10-9M, less than 10-9M, less than 5 x10-10M, less than 10-10M, less than 5 x 10-11M, less thanless than 5 x 10-12M, less than 10-12M, less than 5 x 10-13M, less than 10-13M, less than 5 x 10-14M, less than 10-14M, less than 5 x 10-15M, or less than 10-15M or lower as measured as a monovalent antibody, such as a monovalent Fab. In some embodiments, an anti-tumor antibody of the present disclosure has KDless than 100 pM, e.g., or less than 75 pM, e.g., in the range of 1 to 100 pM, when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore®system performed at 37°C. In some embodiments, an anti-tumor antibody of the present disclosure has KD of greater than 100 pM, e.g., in the range of 100-1000 pM or 500-1000 pM when measured by surface plasmon resonance analysis using a biosensor system such as a Biacore®system performed at 37°C. 79710041V.1

[0037] A "neutralizing anti-norovirus antibody" as used herein refers to an antibody that can prevent norovirus from initiating and perpetuating an infection in a host and / or in target cells in vitro. In the present disclosure, such neutralizing antibodies block binding of GII capsids to ligand. In some embodiments, the present invention provides neutralizing monoclonal human antibodies that are capable of binding to multiple norovirus GII genotypes. In certain embodiments, the 50% (IC50) inhibitory concentration of the neutralizing antibody may be less than about 0.0001 g / ml, less than about 0.001 g / ml, less than about 0.01 g / ml, less thanabout 1 g / ml, less than about 5 g / ml, or less than about 10 g / ml; or may be less than about 20g / ml, or less than about 50 g / ml, or less than about 100 g / ml; and is defined as the antibody concentration required to neutralize about 50% of the input virus in the neutralization assay.

[0038] The term "monovalent molecule" as used herein refers to a molecule that has one antigen-binding site, e.g., a Fab or scFv.

[0039] The term "bivalent molecule" as used herein refers to a molecule that has two antigen- binding sites. In some embodiments, a bivalent molecule is a bivalent antibody or a bivalent binding fragment thereof. In some embodiments, a bivalent molecule is an IgG. In general monoclonal antibodies have a bivalent basic structure. IgG and IgE have only one bivalent unit, while IgA and IgM consist of multiple bivalent units (2 and 5, respectively) and thus have higher valencies. This bivalency increases the avidity of antibodies for antigens.

[0040] The terms "monovalent binding" or "monovalently binds to" as used herein refer to the binding of one antigen-binding site to its antigen.

[0041] The terms "bivalent binding" or "bivalently binds to" as used herein refer to the binding of both antigen-binding sites of a bivalent molecule to its antigen. In some embodiments, both antigen-binding sites of a bivalent molecule share the same antigen specificity.

[0042] The term "valency" as used herein refers to the number of different binding sites of an antibody for an antigen. A monovalent antibody comprises one binding site for an antigen. A bivalent antibody comprises two binding sites for the same antigen. 79710041V.1

[0043] The term “avidity” as used herein in the context of antibody binding to an antigen refers to the combined binding strength of multiple binding sites of the antibody. Thus, “bivalent avidity” refers to the combined strength of two binding sites.

[0044] The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identity over a specified region, e.g., the length of the two sequences, when compared and aligned for maximum correspondence over a comparison window or designated region. Alignment for purposes of determining percent amino acid sequence identity can be performed in various methods, including those using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity the BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.215:403-410 (1990). Thus, for purposes of this invention, BLAST 2.0 can be used with the default parameters to determine percent sequence identity.

[0045] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a VH region polypeptide “corresponds to” an amino acid in the VHregion of SEQ ID NO:1 when the residue aligns with the amino acid in SEQ ID NO:1 when optimally aligned to SEQ ID NO:1. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.

[0046] A “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, hydrophobicity, and / or size of the side group chain is maintained. Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino 79710041V.1acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) large aliphatic nonpolar amino acids Val, Leu and Ile; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro; (viii) aliphatic amino acids Val, Leu, Ile, Met and Cys; and (ix) small hydroxyl amino acids Ser and Thr. Reference to the charge of an amino acid in this paragraph refers to the charge at physiological pH.

[0047] The terms “nucleic acid” and “polynucleotide” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide, and combinations thereof. The terms also include, but is not limited to, single- and double-stranded forms of DNA. In addition, a polynucleotide, e.g., a cDNA or mRNA, may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non- naturally occurring nucleotide linkages. The nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.

[0048] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A “vector” as used here refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0049] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid 79710041V.1molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0050] "Isolated nucleic acid encoding an antibody or fragment thereof" refers to one or more nucleic acid molecules encoding antibody heavy or light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0051] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Thus, a host cell is a recombinant host cells and includes the primary transformed cell and progeny derived therefrom without regard to the number of passages.

[0052] As used herein, "therapeutic agent" refers to an agent that when administered to a patient suffering from a disease, in a therapeutically effective dose, will cure, or at least partially arrest the symptoms of the disease and complications associated with the disease. Neutralizing and Blocking Anti-Norovirus Antibodies

[0053] The present disclosure provides neutralizing antibodies that are capable of neutralizing virus from two or more, typically three or more, norovirus G.II genotypes. Such antibodies are generally referred to in this disclosure as “broadly neutralizing antibodies” for convenience.

[0054] As used herein, "broadly neutralizing antibodies" refers to antibodies that neutralize virus from multiple norovirus genotypes in a neutralization assay. Broadly neutralizing activity of an antibody can be determined by evaluating neutralization (e.g., the ability to infect human intestinal epithelia using an organoid culture) against a panel of noroviruses, which in the context of this disclosure, includes genotype GII noroviruses. The 50% inhibitory concentration (IC50) is the concentration of antibody that reduces infection by at least 50% compared to infection (or binding) in the absence of antibody, or in the presence of a negative control antibody after background is subtracted. In some embodiments, neutralizing activity can also be measured as a function of the area under the positive portion of the neutralization curve. Breadth and potency 79710041V.1are two typical measures that may be employed to characterize an antibody’s neutralizing activity. Breadth is the proportion of tested viruses with IC50 scores that fall below an IC50 cutoff value for neutralizing (or binding) activity. Potency can be calculated using the geometric mean IC50(see, e.g., Hraber et al., J Virol.88:12623-43, 2014; Rademeyer, ).

[0055] For purposes of this disclosure, blockade antibody assays using multiple GII Genotypes serve as a surrogate for broadly neutralizing activity. Blockade antibody assays can be performed, e.g., as described by Lindesmith, et al., J. Virol.88, 8826–8842, 2014 in which virus like particles for multiple GII genotypes are provided, treated with antibody, and incubated with ligand. The amount of bound VPS is quantified and the inhibitory dose IC50 determined.

[0056] In some instances, a broadly neutralizing antibody of the present disclosure may neutralize at least three, or more, norovirus GII genotypes. In certain embodiments, the 50% inhibitory concentration of the neutralizing antibody may be less than about 0.0001 g / ml, lessthan about 0.001 g / ml, less than about 0.01 g / ml, less than about 1 g / ml, less than about 5g / ml, for virus from each of the GII genotype to which the antibody blocks binding. In some embodiments, the 50% inhibitory concentration may be less than about 10 g / ml, less than about 20 g / ml, less than about 50 g / ml, or less than about 100 g / ml for at least one genotype neutralized by the virus. VH and VL regions

[0057] In some embodiments, an anti-norovirus antibody of the present invention has one, two, or three CDRs of a VH sequence as shown in Table 1. In some embodiments, the VH region comprises 1 or 2 substitutions in a CDR1, CDR2, and / or CDR3 relative to the corresponding CDR1, CDR2, or CDR3 sequence shown in Table 1. In some embodiments, the anti-norovirus antibody comprises a VHcomprising a CDR3 sequence (S / T)RVGKD(F / Y)GDGLFD(S / Y), e.g., SRVGKDFGDGLFDS or TRVGKDYGDGLFDY. In some instances, the anti-norovirus further comprises a VL comprising a CDR3 sequence C(L / S)YAG(S / R)SLWV, e.g., CSYAGSSLWV. In other embodiments the anti-norovirus antibody comprises a VH comprising a CDR3 sequence ATNDGYGGRSRRGSSYYHYGMNF, In some instances, the antibody further comprises a VL comprising a CDR3 sequence QQTYSAPFT. 79710041V.1

[0058] In some embodiments, an anti-norovirus antibody of the present invention has one, two, or three CDRs of a VL sequence as shown in Table 1. In some embodiments, the VL region comprises 1 or 2 substitutions in a CDR1, CDR2, and / or CDR3 relative to the corresponding CDR1, CDR2, or CDR3 sequence shown in Table 1.

[0059] In some embodiments, an anti-norovirus binding domain of the disclosure comprises at least one, two, or all three CDRs of a heavy chain variable amino acid sequence of SEQ ID NO:1 as defined by Kabat; and / or comprises at least one, two, or all three CDRs of a light chain variable amino acid sequence of SEQ ID NO:2 as defined by Kabat.

[0060] In some embodiments, anti-norovirus binding domain of the disclosure comprises at least one, two, or all three CDRs of a heavy chain variable amino acid sequence of SEQ ID NO:3 as defined by Chothia; and / or comprises at least one, two, or all three CDRs of a light chain variable amino acid sequence of SEQ ID NO:4.

[0061] In some embodiments, a VH region of the present disclosure comprises an HCDR1 sequence GF(N / T)F(A / G)DYA, an HCDR2 sequence IRS(N / K)(A / T)YGG(A / T)T, and an HCDR3 sequence (S / T)RVGKD(F / Y)GDGLFD(S / Y); or a variant in which one or two amino acids are substituted, in HCDR1, HCDR2, and / or HCDR3. In some embodiments, the VHregion comprises an HCDR1 sequence GFNF(G / A)DYA, an HCDR2 sequence IRSNTYGGAT, and an HCDR3 sequence SRVGKDFGDGLFDS. In other embodiments, the VHregion comprises an HCDR1 sequence GFNFGDYA, an HCDR2 sequence IRSNTYGGAT, the HCDR3 sequence SRVGKDFGDGLFDS. In some embodiments, the antibody comprises an HCDR1 sequence GFTFGDYA, an HCDR2 sequence IRSKAYGGTT and an HCDR3 sequence TRVGKDYGDGLFDY. A VH region of the present paragraph is typically paired with a VL region comprising an LCDR1 sequence SSDVG(N / S)YNL, an LCDR2 sequence EG(S / Y), and the LCDR3 sequence C(L / S)YAG(S / R)SLWV; or a variant thereof in which one or two CDRs, e.g., LCDR1 or LCDR3 comprises one or two amino acid acids are substituted. In some embodiments, the VL region comprises an LCDR1 sequence SSDVGNYNL, an LCDR2 sequence EGY, and an LCDR3 sequence CLYAGRSLWV. In some embodiments, the VL 79710041V.1region comprises an LCDR1 sequence SSDVGSYNL, an LCDR2 sequence EGS, and an LCDR3 sequence CSYAGSSLWV.

[0062] In some embodiments, a VH region of the present disclosure comprise an HCDR1 sequence GIILNTYY, an HCDR2 sequence ISASSQTI, and an HCDR3 sequence ATNDGYGGRSRRGSSYYHYGMNF, or a variant thereof in which one or two amino acids in HCDR1 or HCDR2, and / or one, two, or three amino acids in HCDR3 are modified, e.g. substituted. In typical embodiments, a VHregion as described in the present paragraph is typically paired with a VL region comprising an LCDR 1 sequence QPITTF, an LCDR2 sequence ATS; and an LCDR3 sequence QQTYSAPF; or a variant thereof in which one or two amino acids of the LCDR1, LCDR2, and / or LCDR2 is modified, e.g., substituted. Table 1 CDR sequences defined by IMGT.79710041V.1

[0063] In some embodiments, an anti-norovirus antibody binding domain of the present disclosure comprises a heavy chain variable region having at least 70%, 75%, 80%, or 85 % identity to the VHamino acid sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 8, 9, or 10. In some embodiments, the anti-norovirus antibody has at least 90%, 91%, 92%, 93%, or 94% identity to the VHamino acid sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 8, 9, or 10. In some embodiments, the anti-norovirus antibody has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of the variable region sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 8, 9, or 10. In some embodiments, such an anti-norovirus antibody variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO:1, 3, 5, 7, 9, or 10, e.g., as determined by IMGT or Kabat. In some embodiments, the VH comprises an HCDR1, HCDR2, and HCDR3 of an antibody as shown in Table 1, or comprises an HCDR1 sequence GF(N / T)F(A / G)DYA, an HCDR2 sequence IRS(N / K)(A / T)YGG(A / T)T, and an HCDR3 sequence (S / T)RVGKD(F / Y)GDGLFD(S / Y); and has at least 90%, 91%, 92%, 93%, or 94% identity to the VH amino acid sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 8, 9, or 10, where the variation in sequence occurs only in the framework regions. In some embodiments, the VH comprises an HCDR1, HCDR2, and HCDR3 of an antibody as shown in Table 1; or comprises an HCDR1 sequence GF(N / T)F(A / G)DYA, an HCDR2 sequence IRS(N / K)(A / T)YGG(A / T)T, and an HCDR3 sequence (S / T)RVGKD(F / Y)GDGLFD(S / Y) and has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the VHamino acid sequence of the variable region sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 8, 9, or 10, where the variation in sequence occurs only in the framework regions. In some embodiments, the anti-norovirus antibody of the present disclosure comprises an HCDR1 sequence GF(N / T)F(A / G)DYA, an HCDR2 sequence IRS(N / K)(A / T)YGG(A / T)T, and an HCDR3 sequence (S / T)RVGKD(F / Y)GDGLFD(S / Y); or an HCDR1, HCDR2, and HCDR3 of an antibody as shown in Table 1; and has 1, 2, 3, 4, or 5 amino acid substitutions in an least one FR1, FR2, FR3, or FR4 sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, or 10. .

[0064] In some embodiments, the anti-norovirus antibody of the present disclosure that comprises a VHas set forth in the preceding two paragraph comprises a VLregion having at least 79710041V.170%, 75%, 80%, or 85 % identity to the VL amino acid sequence of any one of SEQ ID NOS: 2, 4, or 6. In some embodiments, the anti-norovirus antibody has at least 90%, 91%, 92%, 93%, or 94% identity to the VLamino acid sequence of any one of SEQ ID NOS: 2, 4, or 6. In some embodiments, the anti-norovirus antibody has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VLamino acid sequence of the variable region sequence of any one of SEQ ID NOS: 2, 4, or 6.. In some embodiments, such an anti-norovirus antibody variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO:2, 4, or 6, e.g., as determined by IMGT or Kabat. In some embodiments, the VL comprises an LCDR1, LCDR2, and LCDR3 of an antibody as shown in Table 1, or comprises an LCDR1 sequence SSDVG(N / S)YNL, an LCDR2 sequence EG(S / Y), and an LCDR3 sequence C(L / S)YAG(S / R)SLWV; and has at least 90%, 91%, 92%, 93%, or 94% identity to the VLamino acid sequence of any one of SEQ ID NOS: 2, 4, or 6, where the variation in sequence occurs only in the framework regions. In some embodiments, the VLccomprises an LCDR1, LCDR2, and LCDR3 of an antibody as shown in Table 1, or comprises an LCDR1 sequence SSDVG(N / S)YNL, an LCDR2 sequence EG(S / Y), and an LCDR3 sequence C(L / S)YAG(S / R)SLWV; and has at least has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the VL amino acid sequence of the variable region sequence of any one of SEQ ID NOS: 2, 4, or 6, where the variation in sequence occurs only in the framework regions. In some embodiments, an anti-norovirus antibody of the present disclosure comprises an LCDR1, LCDR2, and LCDR3 of an antibody as shown in Table 1, or comprises an LCDR1 sequence SSDVG(N / S)YNL, an LCDR2 sequence EG(S / Y), and an LCDR3 sequence C(L / S)YAG(S / R)SLWV; and has 1, 2, 3, 4, or 5 amino acid substitutions in an least one FR1, FR2, FR3, or FR4 sequence of any one of SEQ ID NOS: 2, 4, or 6.

[0065] In some embodiments, a norovirus antibody binding domain of the present disclosure comprises a VH comprising having at least 95% identity to SEQ ID NO:1, where the variation occurs in an FR region as defined by IMGT; and a VL having at least 95% identity to SEQ ID NO:2, where the variation occurs in an FR region as defined by IMGT. In some embodiments, the norovirus antibody binding domain comprises a VH comprising SEQ ID NO:1 and a VL comprising SEQ ID NO:2 79710041V.1

[0066] In some embodiments, a norovirus antibody binding domain of the present disclosure comprises a VH comprising having at least 95% identity to SEQ ID NO:3. where the variation occurs in an FR region as defined by IMGT; and a VLhaving at least 95% identity to SEQ ID NO:4, where the variation occurs in an FR region as defined by IMGT. In some embodiments, the norovirus antibody binding domain comprises a VHcomprising SEQ ID NO:3 and a VLcomprising SEQ ID NO:4.

[0067] In some embodiments, a norovirus antibody binding domain of the present disclosure comprises a VH comprising having at least 95% identity to SEQ ID NO:5, where the variation occurs in an FR region as defined by IMGT; and a VL having at least 95% identity to SEQ ID NO:6, where the variation occurs in an FR region as defined by IMGT. In some embodiments, the norovirus antibody binding domain comprises a VH comprising SEQ ID NO:5 and a VL comprising SEQ ID NO:6.

[0068] In some embodiments, the norovirus antibody binding domain comprises a VH having at least 95% identity to SEQ ID NO:7, 8, 9, or 10, where the variation occurs in an FR region as defined by IMGT; and a VLhaving at least 95% identity to SEQ ID NO:4, where the variation occurs in an FR region as defined by IMGT. In some embodiments, the norovirus antibody binding domain comprises a VHcomprising SEQ ID NO:7, 8, 9, or 10; and a VLcomprising SEQ ID NO:4.

[0069] In some embodiments, the norovirus antibody binding domain comprises a VHhaving at least 95% identity to SEQ ID NO:7, 8, 9, or 10, where the variation occurs in an FR region as defined by IMGT; and a VL having at least 95% identity to SEQ ID NO:6, where the variation occurs in an FR region as defined by IMGT. In some embodiments, the norovirus antibody binding domain comprises a VH comprising SEQ ID NO:7, 8, 9, or 10; and a VL comprising SEQ ID NO:6.

[0070] In some embodiments, the disclosure provides an antibody that selectively binds the VP1 capsid P-domain recognized by an antibody comprising a VHregion comprising SEQ ID NO:3 and a VLregions comprising SEQ ID NO:4. 79710041V.1

[0071] In some embodiments, a broadly neutralizing antibody as described herein is modified to remove residues that can be liabilities for production, safety and the like. Desired antibody properties comprise: (1) suitable for a standard platform (expression, purification, formulation); (2) high yield; (3) low heterogeneity (glycosylation, chemical modification, etc.); (4) consistent manufacturability (batch-to-batch, and small-to-large scale); (5) high stability (years in liquid formulation), e.g., minimal chemical degradation, fragmentation, and aggregation; and (6) long PK (in vivo half life), e.g., no off-target binding, no impairment of FcRn recycling, and stability. Considerations for modification of antibodies to improve production and / or safety include, but are not limited to, the presence of a free cysteine, N-linked glycosylation, the presence of an N- terminal glutamate residue, abnormal net charge (see, e.g., Sharma et al, Proc. Natl. Acad. Sci. USA 111:18601-18606, 2014); hydrophobic patches or patches of the same charge, residues subject to proteolysis, asparagine deamidation, aspartate isomerization, lysine glycation, and methionine and tryptophan oxidations.

[0072] Another aspect for consideration in preparing modifications to an antibody is reducing risk of clinical immunogenicity in therapeutic applications. One approach to try to minimize this is to mutate various positions to corresponding to a germline residue. This approach applies for germline genes IGHV, IGHJ, IGKV, IGKJ, IGLV, and IGLJ, and accounts for all of the variable heavy (VH) and variable light (VL) regions except for part of H-CDR3. Alternatively in silico predictions of immunogenicity, such as the prediction of T cell epitopes, or use in vitro assays of immunogenicity, such as ex vivo human T cell activation can be employed. In some instances, CDR residues may be substituted. In other embodiments, framework residues are substituted. Antibody formats

[0073] In a further aspect of the invention, an anti-norovirus antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, antibody. In one embodiment, an anti-norovirus antibody of the present disclosure is an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or F(ab')2fragment. For a review of certain antibody fragments, see Hudson et al. Nat. Med.9: 129-134 (2003). Antibody fragments can be made by various 79710041V.1techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.

[0074] "Antibody fragments" comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody. Examples of antibody fragments or antibodies comprising antibody fragments include Fab, Fab', F(ab')2, Fv (including scFv), Fd, and Fd' antibodies formats; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and bispecific or multispecific or multivalent antibodies formed from antibody fragments, including bi-specific T-cell engager (biTE) fusion proteins having two scFvs, e.g., from different antibodies; bispecific antibodies that target two epitopes on the same antigen, or two different antigens and the like, or an antigen-specific binding fragment that is combined with a binding domain that binds to and modulates activity of a molecule on a leukocyte, such as a T-cell receptor or NK cell receptor. Such molecules include CD3 and Fc receptor molecules, among others. In some embodiments, an antibody fragment of an antibody of the present disclosure may be a component of an Fc fusion protein. Structures of such antibodies fragments are known. For example, a Fab fragment, is a monovalent ferment originally identified by papain digestion, comprising a VL, CL, VH,and CH1 domain; a Fab' fragment is a Fab fragment additionally having one or more cysteine residues at the C-terminus of the CH1 domain; an Fd fragment has VH and CH1 domains; an Fd' fragment has VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; an Fv fragment has a VL and VH domains of a single antibody; F(ab')2 fragments are bivalent fragments that have two Fab' fragments linked by a disulfide bridge at the hinge region; “diabodies” have two antigen binding sites, with a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain; “linear antibodies” contain a pair of tandem Fd segments (VH-CH1-VH- CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions.

[0075] In some embodiments an anti-norovirus antibody in accordance with the present disclosure is a in a monovalent format. Alternatively, the antibody may be a bivalent or multi- valent antibody. In some embodiments, the anti-norovirus antibody, e.g., an antibody fragment, may be incorporated into a bispecific or multi-specific antibody. For example, in some 79710041V.1instances, the antibody may comprise an antibody of the present disclosure that neutralizes viruses of two or more, preferably three or more, Norovirus GII genotypes; and another anti- norovirus antibody that targets additional genotypes. Fc regions

[0076] In another embodiment, the antibody is a substantially full length antibody comprising an Fc region, e.g., an IgG antibody or IgA antibody. In some instance an antibody comprising a VHregion and VLregion of the present disclosure comprises an IgA region, such as a human IgA1 or IgA2. In some instance an antibody comprising a VHregion and VLregion of the present disclosure comprises an IgG region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region sequence. Fc engineering

[0077] An anti-norovirus antibody of the present disclosure comprises an Fc region, which as described herein, may be a variant Fc region engineered to alter one or more functional properties of the antibody, such as extending serum half-life and / or modulating effector function, including complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity. Furthermore, an antibody of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. For purposes of describing amino acids present in an Fc region, the positions are numbered using EU index numbering where the designation “position number X” means that X is an amino acid that is present at the indicated position. For purposes of describing mutations in an Fc region, the designation “Xposition numberY” means that Y is an amino acid that is substituted for X relative to a reference sequence at the indicated position. For example, L234A, means that A is substituted for an L that occurs in a reference Fc region sequence at position 234. Similarly, the designation “-“ when coupled with a position number, e.g., “position number-“, refers to a deletion, relative to a reference sequence, at the indicated position in the Fc region. For example, “236-“ indicates that the residue at position 236 is deleted in an Fc region relative to a reference Fc region sequence. 79710041V.1

[0078] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-norovirus antibody, e.g., to increase stability and / or modulate effector function. An Fc region variant may thus comprise a human Fc region sequence, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region sequence, or a human IgA1 or IgA2 Fc region, that comprises at least one amino acid modification, such as a substitution, compared to a native Fc region sequence. An Fc region variant may also comprise further modifications. Accordingly, in some embodiments, the Fc region variant can comprise at least 80% identity, or at least 85%, at least 90%, or at least 95% identity to a native human Ig regions, e.g., an IgG1, IgG2, IgG3, or IgG4 region, or IgA1 or IgA2 region, and comprises specific Fc region modifications that influence effector function. For example, in some embodiments, an anti-norovirus antibody amy comprise a human IgG1 Fc region comprising amino acid modifications L234A and L235A to reduce effector function. IN some embodiments, a human IgG1 may comprises amino acid modifications M252Y, S254T, T256E to enhance antibody half-life.

[0079] In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody or antibody having an altered glycosylation pattern can be made. Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen or, if made in the Fc region, to influence effector function. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Generation of antibodies

[0080] Anti-norovirus antibodies as disclosed herein are commonly produced using vectors and recombinant methodology well known in the art (see, e.g., Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, Current Protocols in Molecular Biology editions through May 1, 2024). Reagents, cloning vectors, and kits for genetic manipulation are available from commercial vendors. Accordingly, in a further 79710041V.1aspect of the invention, provided herein are isolated nucleic acids encoding a VH and / or VL region, or fragment thereof, of any of the anti-norovirus antibodies as described herein, or variants thereof; vectors comprising such nucleic acids and host cells into which the nucleic acids are introduced that are used to replicate the antibody-encoding nucleic acids and / or to express the antibodies. Such nucleic acids may encode an amino acid sequence containing the VLand / or an amino acid sequence containing the VHof the anti-norovirus antibody (e.g., the light and / or heavy chains of the antibody). In some embodiments, the host cell contains (1) a vector containing a polynucleotide that encodes the VL amino acid sequence and a polynucleotide that encodes the VH amino acid sequence, or (2) a first vector containing a polynucleotide that encodes the VL amino acid sequence and a second vector containing a polynucleotide that encodes the VHamino acid sequence.

[0081] In a further aspect, the invention provides a method of making an anti-norovirus antibody as described herein. In some embodiments, the method includes culturing a host cell as described in the preceding paragraph under conditions suitable for expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium). In some embodiments, the host cell is a hybridoma.

[0082] Suitable vectors containing polynucleotides encoding antibodies of the present disclosure, or fragments thereof, include cloning vectors and expression vectors. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and / or may carry genes for a marker that can be used in selecting clones containing the vector. Examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1 plasmids, pCR1, RP4, phage DNAs, and shuttle vectors. These and many other cloning vectors are available from commercial vendors.

[0083] Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure. The expression vector may replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors 79710041V.1include but are not limited to plasmids and viral vectors, including adenoviruses, adeno- associated viruses, retroviruses, and any other vector.

[0084] Suitable host cells for expressing an anti-norovirus antibody as described herein include both prokaryotic or eukaryotic cells. For example, anti-norovirus antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. Alternatively, the host cell may be a eukaryotic host cell, including eukaryotic microorganisms, such as filamentous fungi or yeast, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern, vertebrate, invertebrate, and plant cells. Examples of invertebrate cells include insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells. Plant cell cultures can also be utilized as host cells.

[0085] In some embodiments, vertebrate host cells are used for producing anti-norovirus antibodies of the present disclosure. For example, mammalian cell lines such as a monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol.36:59,1977; baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.23:243-251, 1980 monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; and FS4 cells may be used to express anti-norovirus antibodies. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp.255-268, 2003. 79710041V.1

[0086] A host cell transfected with an expression vector encoding an anti-norovirus antibody of the present disclosure, or fragment thereof, can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptides may be secreted and isolated from a mixture of cells and medium containing the polypeptides. Alternatively, the polypeptide may be retained in the cytoplasm or in a membrane fraction and the cells harvested, lysed, and the polypeptide isolated using a desired method.

[0087] In some embodiments, provided herein is a method of generating variants of an anti- norovirus antibody as disclosed herein. Thus, for example, a construct encoding a variant CDR, e.g., that retains the binding and neutralization activity of the parent antibody, but one or more modifications, e.g., an amino acid substitution, deletion or addition, relative to a CDR presented of a heavy chain, and / or a light chain in Table 1 can be modified and an antibody comprising the modified heavy and / or light chain tested for binding and / or neutralization activity. Such an analysis can also be performed with one or more frameworks regions and an antibody having the desired activity can then be selected. Norovirus Detection Methods / Anti-Norovirus Antibody Conjugates

[0088] In some embodiments, an antibody of the present disclosure can be used in immunoassays to detect norovirus antigens for any purpose, including quantitative detection of virus levels in a sample, purification of norovirus, monitoring of viral load, assessment of vaccine quality and the like. In some embodiments, an anti-norovirus as described herein is used to detect norovirus in a sample is from a subject that has symptoms of norovirus infection and / or is at risk for norovirus infections, e.g., has been exposed to norovirus or individuals infected with norovirus. In some embodiments, the sample is a fecal sample, blood sample, e.g., serum or plasma or any other bodily fluid that may contain norovirus. In some embodiments, an anti- norovirus antibody described herein may be used to detect norovirus in a sample, such as a water sample, beverage sample, food sample, and / or a sample obtained and processed from a surface suspected of begin contaminated with norovirus. Illustrative assays employ an anti-norovirus antibody include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), fluoroimmunoassays, chemiluminescent assays, bioluminescent assays. Various assay formats 79710041V.1such as sandwich immunoassays, competitive immunoassays, lateral flow assays, western blot- type assays, immunohistochemistry, flow cytometry and radioimmunoassays, and immunoprecipitation assays may be employed for detecting norovirus antigen.

[0089] In a further aspect, an anti-norovirus antibody of the present disclosure may be conjugated or linked to therapeutic and / or imaging / detectable moieties. For example, the anti- norovirus antibody may be conjugated to a detectable marker or a therapeutic agent. Methods for conjugating or linking antibodies are well known in the art. The moiety may be linked to the antibody covalently or by non-covalent linkages.

[0090] In some embodiments, the antibody may be linked to a radionuclide, an iron-related compound, a dye, a fluorescent agent, or an imaging agent. In some embodiments, an antibody may be linked to agents, such as, but not limited to, metals; metal chelators; lanthanides; lanthanide chelators; radiometals; radiometal chelators; positron-emitting nuclei; microbubbles (for ultrasound); liposomes; molecules microencapsulated in liposomes or nanosphere; monocrystalline iron oxide nanocompounds; magnetic resonance imaging contrast agents; light absorbing, reflecting and / or scattering agents; colloidal particles; fluorophores, such as near- infrared fluorophores.

[0091] In another aspect, the disclosure provides a kit comprising one or more anti-norovirus antibodies of the present disclosure. In still further embodiments, the present disclosure concerns immunodetection kits for performing any assay to detect the present of norovirus using the antigen. Such kits comprises at least one antibody of the present disclosure. Additional kit components can comprises detection reagents, including for examples, a secondary antibody, a further antibody for detection, reagents, reagents for detecting a signal, e.g., enzyme detection assays for enzyme-linked assays, buffers, control reagents, and the like. Pharmaceutical Compositions

[0092] In a further aspect, provided herein are pharmaceutical compositions for administration of an anti-norovirus antibody of the present disclosure to a mammalian subject, preferably a human, that is infected with norovirus or is at risk of norovirus infection, e.g., for example by exposure to norovirus or being present in a household or other closed environment in which 79710041V.1other people are infected, in an amount and according to a schedule sufficient to prevent norovirus infection or reduce viral load in the subject. Such compositions may comprise an anti- norovirus antibody or a polynucleotide encoding the antibody, and a pharmaceutically acceptable diluent or carrier. In some embodiments, the polynucleotide encoding the antibody may be contained in a plasmid vector for delivery, or a viral vector. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the antibody. As used herein, a "therapeutically effective dose" or a "therapeutically effective amount” refers to an amount sufficient to prevent, cure, or at least partially arrest norovirus infection or symptoms of norovirus infection and its complications. A therapeutically effective dose can be determined by monitoring a patient’s response to therapy. Typical benchmarks indicative of a therapeutically effective dose include amelioration of symptoms of the disease in the patient, including, for example, reduction in viral load and increases in lymphocyte numbers. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health, including other factors such as age, weight, gender, administration route, etc. Single or multiple administrations of the antibody will be dependent on the dosage and frequency as required and tolerated by the patient.

[0093] Various pharmaceutically acceptable diluents, carriers, and excipients, and techniques for the preparation and use of pharmaceutical compositions will be known to those of skill in the art in light of the present disclosure. Illustrative pharmaceutical compositions and pharmaceutically acceptable diluents, carriers, and excipients are also described in Remington: The Science and Practice of Pharmacy 20th Ed. (Lippincott, Williams & Wilkins 2012). In particular embodiments, each carrier, diluent or excipient is "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not injurious to the subject. Often, the pharmaceutically acceptable carrier is an aqueous pH-buffered solution. Some examples of materials which can serve as pharmaceutically-acceptable carriers, diluents or excipients include: water; buffers, e.g., phosphate-buffered saline; sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; 79710041V.1oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0094] The pharmaceutical composition can be formulated for any suitable route of administration, including for example, parenteral, intrapulmonary, intranasal, or local administration. Parenteral administration can include intramuscular, intravenous, intraarterial, intraperitoneal, oral or subcutaneous administration. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration and has a concentration of antibody of 10-100 mg / ml, 10-50 mg / ml, 20 to 40 mg / ml, or about 30 mg / ml. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous injection and has a concentration of antibody of 50-500 mg / ml, 50-250 mg / ml, or 100 to 150 mg / ml, and a viscosity less than 50 cP, less than 30 cP, less than 20 cP, or about 10 cP. In some embodiments, the pharmaceutical compositions are liquids or solids. In particular embodiments, the pharmaceutical compositions are formulated for parenteral, e.g., intravenous, subcutaneous, or oral administration.

[0095] The formulation of and delivery methods of pharmaceutical compositions will generally be adapted according to the site and the disease to be treated. Formulations include those in which the antibody is encapsulated in micelles, liposomes or drug-release capsules (active agents incorporated within a biocompatible coating designed for slow-release); ingestible formulations; formulations for topical use, such as creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays.

[0096] In some embodiments, e.g., for parenteral administration, the antibodies or antigen- binding fragments thereof are formulated in a unit dosage injectable form (solution, suspension, 79710041V.1emulsion) in association with a pharmaceutically acceptable, parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used.

[0097] The dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection, the characteristics of the subject, and the subject's history. In particular embodiments, the amount of antibody or antigen-binding fragment thereof administered or provided to the subject is in the range of about 0.1 mg / kg to about 50 mg / kg of the subject's body weight. Depending on the type and severity of the infection, in certain embodiments, about 0.1 mg / kg to about 50 mg / kg body weight (e.g., about 0.1-15 mg / kg / dose) of antibody or antigen-binding fragment thereof may be provided as an initial candidate dosage to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of the therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.

[0098] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is provided to the subject in combination with one or more additional therapeutic agents used to treat norovirus infection. In certain embodiments, a method for treating or preventing a norovirus infection in a mammal, e.g., a human, having or at risk of having the infection is provided, comprising administering to the human a therapeutically effective amount of an antibody as disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of one or more (e.g., one, two, three, one or two, or one to three) additional therapeutic agents. In one embodiment, a method for treating a norovirus infection in a human having or at risk of having the infection is provided, comprising administering to the human a therapeutically effective amount of an antibody as disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of one or more (e.g., one, two, three, one or two, or one to three) additional therapeutic agents.

[0099] In some embodiments, two or more neutralizing anti-norovirus antibodies, e.g., as described in the present disclosure, may be administered to the subject. In some embodiments, 79710041V.1the two or more antibodies may have different neutralization capabilities, i.e., they exhibit a different neutralization profiles for different GII norovirus genotypes, as compared to each other. In some embodiments, the antibody may be administered with another anti-norovirus therapeutic antibody.

[0100] In some embodiments, an additional therapeutic agent may be an anti-norovirus agent. Such therapeutic agents include agents to maintain hydration, anti-nausea medications, and the like.

[0101] In certain embodiments, when an antibody of the present disclosure as described herein is combined with one or more additional therapeutic agents as described above, the components of the composition are administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

[0102] “Co-administration” of an antibody as disclosed herein with one or more additional therapeutic agents generally refers to simultaneous or sequential administration of an antibody or fragment thereof disclosed herein and one or more additional therapeutic agents, such that therapeutically effective amounts of the antibody or fragment thereof disclosed herein and one or more additional therapeutic agents are both present in the body of the patient.

[0103] Co-administration includes administration of unit dosages of the antibody disclosed herein before or after administration of unit dosages of one or more additional therapeutic agents, for example, administration of the antibody within seconds, minutes, or hours of the administration of one or more additional therapeutic agents. For example, in some embodiments, a unit dose of an antibody disclosed herein is administered first, followed within seconds or minutes by administration of a unit dose of one or more additional therapeutic agents. Alternatively, in other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed by administration of a unit dose of an antibody within seconds or minutes. In some embodiments, a unit dose of an antibody disclosed herein is administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more additional therapeutic agents. In other embodiments, a unit dose of one or more additional 79710041V.1therapeutic agents is administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of the antibody.

[0104] The combined administration may be co-administration, using separate pharmaceutical compositions or a single pharmaceutical composition, or consecutive administration in either order, wherein there is optionally a time period while both (or all) therapeutic agents simultaneously exert their biological activities. Such combined therapy may result in a synergistic therapeutic effect. In certain embodiments, it is desirable to combine administration of an antibody of the invention with another antibody directed against another antigen associated with the norovirus infectious agent.

[0105] As described herein, the antibody may also be administered by gene therapy by administration of a nucleic acid comprising one or more polynucleotides encoding the antibody. In certain embodiments, the polynucleotide encodes an scFv. In particular embodiments, the polynucleotide comprises DNA, cDNA or RNA. In certain embodiments, the polynucleotide is present in a vector, e.g., a viral vector

[0106] The following examples are offered for illustrative purposes, and are not intended to limit the invention. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same results. Examples—Identification of Neutralizing Antibodies from Vaccinated Subjects

[0107] This disclosure describes use of high-resolution liquid chromatography with tandem mass spectrometry (LC-MS / MS)-driven proteomics (Ig-Seq), rigorous biochemical and MS- based epitope mapping assays, and structural analysis to quantitatively characterize the circulating serum IgG repertoire of two norovirus experimental vaccine recipients specifically selected based on the breadth of serum nAb responses either (1) across GII.4 variants or (2) across GII genotypes. In the first subject, vaccination back-boosted highly abundant serum antibody clonotypes targeting epitopes conserved among rapidly evolving GII.4 variants spanning from 1987 to the most recent 2019 strains. In the second subject, we identified recalled bNAbs with unprecedented cross-GII neutralization potency and determined the B cell evolutionary trajectory that led to the acquisition of cross-GII neutralization breadth. An epitope 37 79710041V.1shared among divergent GII genotypes was further characterized at atomic resolution. Our study highlights that immunogens designed to improve the presentation of these sites may provide broad protection across different norovirus genotypes and durability against viral evolution within the globally dominant GII.4 genotype. Select vaccinees demonstrate broad GII.4 or cross-GII nAb serum responses.

[0108] Healthy participants between 18-49 years of age and a BMI between 17-35 were recruited to participate in a study of an oral norovirus vaccine candidate (ClinicalTrials.gov, NCT03897309). The vaccine candidate was based on recombinant, non-replicating adenovirus type 5 that was dried, tableted, and enteric coated. Two of those subjects were immunized with a single oral dose of an adenovirus 5 vector-based candidate at 5e10 IU carrying the VP1 capsid gene of GII.4 Sydney (SY) 2012 (Figures 1A and 1B). These subjects were selected for their unusually broad serum blockade titers and were thus studied in detail. In subject A, vaccination significantly boosted serum nAb titers to all tested GII.4 variants circulating from 1987 to 2019 (3.5- to 68-fold) on day 29 compared to the pre-vaccination sera on day 1 (Figure 1A). Serum from subject B had exceptional cross-genotype nAb breadth across seven GII and two GI genotypes on day 1 before vaccination, which were modestly boosted (2.0- to 3.6-fold) on day 29 following vaccination (Figure 1B). In both donors, the greatest fold increase in nAb titer was observed against the GII.4 SY 2012 vaccine strain, whereas the highest blockade titer was to ancestral GII.4 variants (Figures 1A and 1B), suggesting that immune imprinting likely impacts the response to mucosal norovirus vaccination in adults, similarly to what has been observed following intramuscular vaccination and natural infection.12,20,22The GII.4 SY 2012-specific serological repertoire is dominated by a few recalled, cross- reactive clonotypes.

[0109] We determined the compositions of antigen-specific plasma IgG clonotypes and their respective relative abundance in the serum by using Ig-Seq proteomics approach (Figure 1C).26–28Affinity chromatography with immobilized GII.4 SY 2012 VLPs was employed to purify the antigen-specific fraction of serum antibodies as reported previously.20Ig-Seq analysis of subject A demonstrated an oligoclonal and polarized anti-GII.4 serological repertoire, with the top five 79710041V.1most abundant clonotypes constituting more than 60% of the anti-GII.4 post-vaccination repertoire by abundance (determined by LC-MS / MS XIC peak area; Figures 1D and S1A). Interestingly, three of the top five most abundant clonotypes used the same IGHV1-18*01, IGHJ5*02, and IGKV3-11*01 germline genes (Tables S1 and S3). A comparison of pre-and post-vaccination serum repertoire showed that the vaccination back-boosted antigen-specific clonotypes that were already detectable in the day 1 serum before vaccination (Figure 1D). Consequently, the vaccine-boosted antibodies collectively comprised 65% of the post- vaccination serum responses (Figure 1F). Among the recalled antibodies, IGHV1-18 usage was again prevalent, with four IGHV1-18 pre-existing clonotypes comprising more than 50% fraction of the entire serum repertoire on day 29 (Figure 6A). Approximately 30% of the serological repertoire post-vaccination consisted of clonal lineages that were not detectable in the day 1 affinity eluates and are hitherto referred to as “emergent” serum antibodies (Figure 1F). The emergent antibodies showed a significantly higher level of variable heavy chain somatic hypermutation (VH SHM) compared to pre-existing antibodies (p=0.037; Figure 6B), implying that the emergent clonotypes were also likely derived from memory B cells.

[0110] We analyzed the serum antibody repertoire of subject B using a strategy designed to reveal antibodies with broad GII ligand-binding blockade breadth. Building on our previous finding that cross-GI and GII binding mAbs have no blockade nor neutralization against GII.4 virus,20here we hypothesized that depleting GI-binding antibodies could facilitate the identification of cross-GII bNAbs.20Thus, the serum from donor B was first depleted of GI.1- specific antibodies and was subsequently used to purify antibodies that bind to either GII.3 or to GII.4 SY 2012 by affinity chromatography with the respective immobilized VLPs (Figure 1C). Antibody clonotypes detected in both the GII.4 and GII.3 serum repertoires are defined as GII.3 and GII.4 cross-reactive (GII.3+GII.4 CR).

[0111] Vaccination in subject B significantly increased the relative abundance of GII.3+GII.4 CR clonotypes by more than 3-fold on day 29 compared to day 1, which collectively accounted for over 75% of the anti-GII.4 serum response on day 29 based on the relative amount deduced from the XIC area (Figures 1E and 1H). Notably, a substantial fraction of GII.3+GII.4 CR antibodies were back-boosted by vaccination, as indicated by the top four most abundant 79710041V.1clonotypes on day 29 (Figures 1E and 1G). This suggests that the serum responses of subject B are likely a result of recall responses from memory B cells elicited by earlier exposures. Unlike subject A, subject B showed no sign of IGHV1-18 VH gene usage in the day 29 anti-GII.4 repertoire (Table S2). Instead, IGHV4-59 and IGHV3-48 were prevalent, constituting nearly 70% of the post-vaccination serum response (Figure 6C). Consistent with our previous report on serum repertoires to GII.4 consensus (GII.4c) VLP,20the highly skewed VH gene usage in both subjects, particularly among the recalled antibodies, could potentially indicate the presence of germline-encoded motifs that preferentially recognize GII.4 or other GII genotypes. Biochemical epitope mapping of broad-GII.4 neutralizing mAbs reveals two different classes of antigenic landscapes: Class I and Class II.

[0112] For subject A, thirteen mAbs, comprising approximately 70% of the anti-GII.4 serum repertoire by abundance, were recombinantly expressed and characterized for blockade potency and breadth against a panel of time-ordered GII.4 variants (Table S3). Among these, ten mAbs displayed ligand binding blockade activity to at least one GII.4 variant with an IC50 lower than the limit of detection (4 μg / ml), while none of these mAbs neutralized non-GII.4 strains. Of note, VX1 was selected from the top-most abundant clonotype comprising 44% of the post- vaccination repertoire (Figure 1D), and its ligand-binding blockade potency significantly correlated with the whole serum nAb titer to various GII.4 variants that were neutralized by VX1 (p=0.003, Figures 6D and 6E). This suggests that the serum nAb landscape of subject A is substantially driven by a single dominant clonotype represented by VX1.

[0113] We categorized GII.4 neutralizing serum mAbs into two classes (Figure 2A): Class I mAbs neutralized multiple GII.4 variants except for HK 2019, a prospective strain that recently appeared after the GII.4 SY 2012 vaccine strain.22In contrast, Class II mAbs neutralized ligand binding of diverse GII.4 variants tested, including HK 2019, with IC50 values ranging from 0.02 g / ml to 1.1 g / ml. Although the emerging variant HK 2019 has not yet spread as widely as historical strains, it has features favoring global spread, such as marked divergence in dominant nAb antigenic sites coupled with diverse ligand binding.22Therefore, the neutralization of HK 79710041V.12019 by Class II bNAbs suggests that these mAbs recognize site(s) more conserved across GII.4 variants than Class I bNAbs.

[0114] To biochemically define the antigenic residues impacting the blocking potency of Class I and Class II GII.4 bNAbs, we took advantage of a panel of epitope mutant VLPs with amino acid substitutions covering most major antigenic sites (Figure 7A).19 By tracking common residues among mutant VLPs with changes in nAb potency (Figure 7B), we determined that four of six bNAbs – VX1, VX7, VX9, and VX14 – in Class I mapped to key residues bridging surface-exposed variable antigenic sites AE, AG, and AGE, while one mAb, VX3, mapped to site E (Figure 2B). The antigenic site recognized by VX5 could not be defined using current sets of mutant VLPs.

[0115] To identify the Class II epitopes, we engineered an additional panel of GII chimeric VLPs comprised of a non-binding GII.3 backbone transplanted with combinations of GII.4 residues known to be conserved nAb sites based on mAbs NVB 71 (Siteor A1431 (Site I)20and surrounding residues hypothesized to mediate neutralization of human mAbs, either as binding sites or through allosteric effects on epitope presentation (Figures 8A–8C). By using the GII.3 / GII.4 chimeric VLPs, we determined that three of the four Class II bNAbs, VX6, VX11, and VX13, along with NVB 71, mapped to a domain comprised of expanded antigenic site F (residues 254-257, 327, 404) (Figure 2C). VX10, with relatively weak neutralization of GII.4 required an expanded region (GII.3 / GII.4 P7) composed of both epitopes F and I (402, 403, 504- 506) to gain neutralization potency (Figure 2C). The chimeric GII VLP-ligand blocking was improved for VX6, VX11, and A1431 by the addition of the NERK motif (residues 234, 310, 316, 484, and 493), a region outside the antibody epitopes thought to impact Ab access to conserved, partially occluded epitopes, along with site F residues, through allosteric effects (Figure 2C).11,19Therefore, these findings suggest that, in addition to antibody binding residues, access to binding residues through VLP conformational changes also determines the neutralization potency for Class II bNAbs. HDX-MS experiments reveal P domain peptides targeted by Class I and Class II bNAbs. 79710041V.1

[0116] By performing hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, we further confirmed the neutralizing epitopes for VX1 and VX6, representing Class I and Class II bnAbs, respectively. Across four exposure time points, we observed significant reduction in deuterium uptake upon the VX1 binding to a GII.4 SY 2012 P domain peptide spanning residues 287–298, mapping to site A residues 294 and 296–298 (Figures 2D and 2F). Three additional peptides overlapping this region show a similar degree of reduction (Figure 9), suggesting that these linear residues likely serve as a direct contact epitope for VX1. Likewise, VX6 binding significantly reduced the deuterium exchange in a peptide spanning residues 250–257, mapping to site F linear residues 254–257 (Figures 2E and 2F). VX6 also induced a low degree of protection from deuterium uptake in distant peptides spanning residues 222–233 or 287–298 (Figure 9). We speculate that these residues are allosterically impacted upon VX6 binding to site F. Moreover, deuterium exchange in peptides containing a single neutralizing epitope residue remained unaffected by VX1 or VX6 binding, suggesting that the biochemical mutation introduced at this residue reduced blockade potency (Figures 2B and 8B), likely by inducing changes in the local topology of VLPs rather than directly interfering mAb binding. Together, HDX-MS and biochemical assays support our findings of neutralizing epitopes targeted by Class I and Class II bNAbs. Class I and Class II bNAbs neutralize the replication of the GII.4 SY 2012 virus in HIE.

[0117] Next, we selected four representative mAbs – VX1 and VX14 from Class I, and VX6 and VX10 from Class II – to determine whether the serologically relevant bNAbs binding to different ligand binding blockade epitopes also neutralize GII.4 virus ex vivo in the HIE norovirus culture system. All four mAbs potently reduced the genomic copies of GII.4 SY 2012 [P16] virus, with IC50 ranging from 2.5 ng / ml to 55 ng / ml (Figure 2G). Together, our results reveal multiple antigenic landscapes for broadening GII.4 neutralizing responses, which include (i) highly accessible residues that lie across antigenic sites (Class I) and (ii) residues that are less surface-exposed but highly conserved across GII.4 norovirus capsid protein (Class II; Figure 2H). 79710041V.1Serum identification of bNAbs with exceptional cross-neutralization breadth across divergent GII genotypes.

[0118] Sixteen mAbs from subject B, comprising 60% of the anti-GII.4 serum IgG repertoire, were recombinantly expressed for biochemical characterization (Table S3). Among these, five mAbs, detected at low abundance in serum, showed weak binding by enzyme immunoassay (EIA) and did not block GII.4 SY 2012 when tested at 4 μg / ml; consequently, they were not further characterized. The remaining eleven mAbs successfully blocked ligand binding to GII.4 SY 2012 (Figure 3A). Remarkably, two GII.3+GII.4 CR mAbs, VX22 and VX20, identified in the serum repertoire with moderate and high abundance, respectively (Figure 1E), blocked ligand binding of six additional GII genotypes, including GII.2, GII.3, GII.6, GII.12, GII.17, and GII.14 (for VX22) (Figures 3A and 10B). The HIE neutralization assay confirmed that both mAbs potently reduced the genomic copies of GII.4 SY 2012 [P16] virus, with IC50 values as low as 23 ng / ml and 1.1 ng / ml for VX20 and VX22, respectively, and inhibited GII.2 and GII.3 virus replication (Figures 3B and 3C). To our knowledge, VX20 and VX22 are the only human mAbs discovered so far with GII.2, GII.3, and GII.4 cross-neutralization potency and ligand-binding blockade breadth across seven different GII genotypes. VX22 showed superior GII.4 neutralization potency compared to VX20 (Figures 3A–3C), warranting further in-depth study. Antibody evolution trajectory analysis reveals the acquisition of cross-GII neutralization breadth after heterologous genotype infection.

[0119] Next, we aimed to elucidate the antibody evolutionary pathway leading to VX22. Thus, we reconstructed the phylogenetic tree for clonotype ID 1055 using IgPhyML29 and defined the unmutated common ancestor (UCA) that served as the initial precursor for the clonotypic lineage (Figures 3D, 10A, and 11A). Considering that this particular clonotype contained the same VL sequences on day 8 (with a VL SHM of 5.5%; Figure 11B), we consequently expressed two versions of the UCA: UCA1, comprising the unmutated germline genes for both VH and VL, and UCA2, using the germline VH paired with the same mutated light chain conserved across VH reads (Figures 11C and 11D). Interestingly, the “fully germline” UCA1 blocked ligand binding to GII.12 but to none of the other GII genotypes tested at 8 μg / ml (Figure 3E). However, 79710041V.1when the unmutated VH of UCA1 was paired with the conserved VX22 light chain in UCA2, the resulting antibody not only had improved GII.12 blocking potency but also gained GII.3 blockade, indicating that the light chain alone contributes to GII.3 blockade.

[0120] Additionally, we expressed representative antibodies with VH sequences corresponding to intermediate nodes in the phylogenetic tree that either led to VX22 or to mAbs that evolved from a divergent branch of the clonotype (Figure 3D). The VH SHMs from the germline heavy chain to the earliest intermediate Node 35 conferred the most significant improvement in cross- GII breadth, expanding the ligand-binding blockade to GII.4 SY 2012, GII.14, GII.2, and GII.6 (Figure 3E). Two additional heavy chain mutations accumulated between Node 35 and Node 38 (Lys78Arg and Asp86Asn; Figure 11C) result in further expansion of blockade breadth to include GII.17, following the pattern of VX22. Antibodies derived from tree Nodes 47 and 52, which were identified in the BCR repertoire but were not detected in the day 29 serum, maintained cross-GII nAb breadth (Figure 3E). Overall, in line with the high serum titer to GII.12 observed on day 1 in subject B and the high blocking potency of VX22 to GII.12 (Figures 1B and 3A), our results suggest that the ancestor of VX22 likely originated from an earlier GII.12 infection. Subsequent infection(s) likely resulted in a mutated VL that acquired neutralization breadth to GII.3, and later, affinity maturation of the heavy chain resulted in an unprecedented breadth of VX22 and other members of the clonotype. The relatively shallow slope for VX22 blockade curve to GII strains other than GII.4 SY 2012 (Figure 10B) suggests that antibody access to the VX22 epitope may be partially occluded for these genotypes as reported previously.11 Crystallographic analysis of VX22 in complex with GII.4 VP1 P domain reveals a highly conserved cross-GII neutralizing epitope.

[0121] Next, we solved the 3.35Å crystal structure of VX22 Fab bound to the P domain of VP1 from GII.4 SY 2012 (Figure 4A and Table 5). VX22 buried 583 Å2 of the lateral surface area of the P1 subdomain. The epitope recognized by VX22 was focused on two loops, spanning residues 479–484 and 509–513 of VP1 (Figure 4B), both of which are well-conserved across divergent GII genotypes (Figure 12). Within loop 479–484, Asp100A (CDRH3) and Tyr91 79710041V.1(CDRL3) of the VX22 heavy and light chain, respectively, electrostatically interact with Asn479 and Thr482 on the P1 subdomain of VP1 (Figure 4C). The second loop, spanning 509–513, contributes to the main bonding through a proline-aromatic interaction between Pro510 of VP1 and Tyr30 (CDRL1) of the VX22 light chain (Figure 4D). The neighboring Pro511, despite lacking direct contacts, is likely responsible for the correct positioning of Pro510 engaging with VX22 (Figure 4B). Finally, Arg94 (CDRL3) of the VX22 light chain formed a salt bridge with Glu235, Asn307 and Asn309 of GII.4 VP1 (Figure 4B) at the edge of the contact interface surrounded by solvent. Given that none of these three residues are conserved in non-GII.4 genotypes (Figure 12), it is likely not a critical interaction mediating cross-GII neutralization breadth, although the loss of salt bridge due to mutations at these sites could potentially lower the neutralization potency for non-GII.4 viruses as we observed in the HIE assay (Figure 3E).

[0122] As the VX22 epitope is distant from the ligand binding sites located at the apex of the P2 subdomain, we next asked whether VX22 neutralizes the GII.4 virus by impacting virus particle integrity. Negative staining EM showed that, in the absence of VX22 Fab, a high number of intact VLP was observed as expected, maintaining consistent size and symmetry (Figure 4E). After a brief incubation with VX22 Fab, however, fewer intact particles were observed, and a substantial number of smaller intermediate proteins. likely remnants from disassembled VLPs, appeared in the background (Figure 4E). The 2D class-averaged images of the few remaining VLPs showed poorly defined P domains (Figure 4E), suggesting structural flexibility in these regions in the presence of VX22. The disassembly of VLPs may not be surprising, given that VX22 Fab approaches the epitope from an upward angle, with the constant region projecting downward towards the virion interior. This binding mode could potentially cause steric clash with the interior S domain (Figures 4F and 4G). Together, our structural data suggests that the mechanism of VX22 neutralization involves virus particle disassembly, a finding that can be leveraged for designing future vaccine immunogens capable of conferring broad protection across GII genotypes Summary of antibody identification

[0123] Norovirus vaccines currently in clinical development were designed based on VP1 or VLPs from historically circulating viruses, either in mono or bivalent formulations, as an 79710041V.1immunogen. One of these vaccine platforms conferred short-term, cross-genotype protection in adults primarily by boosting antibodies elicited by earlier exposures.12,17Compared to adults, however, infants who have limited immune history typically elicit genotype-specific nAbs that lack broad neutralization breadth.7Moreover, viral strains first encountered during early childhood heavily imprint serum responses, favoring strains from exposures very early in life, even after subsequent infections with new variants or different genotypes.22,30These observations indicate the complexity of inducing broadly protective nAbs through norovirus vaccination and further underscore the need for improved immunogen design capable of eliciting bNAbs in all ages. This is particularly crucial in populations with a severe burden of disease, including children, the elderly, and the immunocompromised with prolonged norovirus infection.1,31Here, by analyzing, in molecular detail, the serological repertoires of two subjects carefully selected based on their bulk serum neutralization profiles, we identified epitopes that lead to broad protection within or across divergent GII genotypes.

[0124] First, we found that oral immunization with an adenovirus-vectored vaccine robustly back-boosted broad GII.4 neutralizing mAbs that blocks not only historical and contemporary GII.4 strains but also the prospective HK 2019, which has an antigenic profile indicative of potential global spread.22Four bNAbs – VX1, VX14, VX6, and VX10 – showed a robust inhibition of GII.4 SY 2012 virus replication in HIE. Within Class I, VX1, demonstrated high SY 2012 neutralization potency in HIE, comparable to that of A1431 (IC50; VX1, 2.5 ng / ml; A1431, 6.9 ng / ml). As quantified by LC-MS / MS, more than 40% of the serum fraction on day 29 was comprised by a single clonotype from which VX1 was selected as a representative mAb, and notably, the VX1 blockade potency closely mirrors the whole serum nAb titers to a multitude of GII.4 variants circulating between 1987 to 2019. This finding suggests that the serological response following vaccination can be mediated by a single dominant clonotype imprinted by earlier infection(s).

[0125] Among Class I bNAbs, VX14 showed a strong preference for CW 1987 strain in the ligand blockade, aligning with a relatively lower neutralization to vaccine SY 2012 virus (IC50: 55 ng / ml) compared to VX1. This indicates that VX14 might have been elicited in response to infection by CW 1987 or a highly similar strain, leading to immune imprinting toward historical 79710041V.1strains. Considering that VX1 and VX14 mapped to surface-exposed epitopes near the ligand binding region (sites AE and site AGE, respectively), both mAbs likely neutralize the virus by exerting steric hindrance to the GII.4-ligand binding interaction. Several mAbs that bridge across different antigenic sites have been reported previously, which may also reflect the overlapping nature of antigenic sites on the repetitive structure of VLPs.20,32Alternatively, it can be speculated that not all multiple bridging sites are directly bound by Class I mAbs. Instead, certain residues may allosterically modulate the local topology of the capsid protein, affecting the blocking capacities of Class I mAbs. Another interesting observation is that VX1 and VX14 use the same IGHV1-18*01, IGHJ5*02, and IGKV3-11*01 genes and have identical CDRH3 and CDRK3 lengths while using distinct CDRH3 and CDRK3 amino acid residues. This finding potentially suggests that germline-encoded motifs may play an important role in recognizing Class I epitopes, as has been reported by multiple studies revealing convergent evolution of antibody repertoires to recurrent epitopes following influenza, Epstein-Barr, Ebola, or SARS- CoV-2 viral infections or immunizations.33–40The continued profiling of VH and / or VL gene stereotyped signatures commonly observed after norovirus exposures will aid in assessing the qualities of antibody clonotypes induced by infection or clinical vaccine immunogens.

[0126] To precisely map the binding epitope of Class II bNAbs, we created GII chimeric VLPs presenting expanded regions of previously reported conserved GII.4 antigenic sites and their associated support motifs (NERK).19Within Class II, VX6 and VX10, constituting 5.2% and 0.6% of the post-vaccination serum repertoire, respectively, neutralized GII.4 SY 2012 virus by targeting the less surface-exposed F+NERK (GII.3 / GII.4 P5) or F+I (GII.3 / GII.4 P7) antigenic domains. In subject A, the expanded site F is immunodominant over site I, and more importantly, there was subtle yet significant difference among Class II bNAbs binding to site F. Specifically, VX6 and VX11 require the NERK motif to bind site F, whereas VX13 can bind to site F in the null GII.3 backbone without the need for the NERK motif. Similarly, we have previously reported on NVB 71 (site F) and A1431 (site I) whose ligand-binding blockade is dependent on the NERK motif. Given that sites F, I, and NERK are all located distal to the apex of the particle and the ligand binding site, virus neutralization by Class II bNAbs is likely mediated via an allosteric effect on VLP structural dynamics rather than direct inhibition of access to the ligand 79710041V.1binding site. Further structural analysis is needed to understand the mechanisms by which Class II mAbs impact conformational flexibility in the context of virus particles and to verify whether NERK residues form a direct contact site for Class II bNAbs.

[0127] From subject B, we identified two serologically abundant mAbs, VX20 and VX22, capable of blocking ligand binding of at least seven GII genotypes, including GII.2, GII.3, GII.4, GII.6, GII.12, GII.17 and GII.14 (for VX22). As tested in the HIE assay, these two antibodies were further shown to neutralize the replication of at least three GII viruses from GII.4, GII.3, and GII.2 genotypes, particularly with robust potency against the vaccine-matched virus (GII.4 SY 2012, IC50; VX20, 23 ng / ml; VX22, 1.1 ng / ml). Considering that GII viruses are the most dominant genogroup responsible for community outbreaks and chronic infections in immunocompromised individuals,3,41VX20 and VX22 are promising lead drug candidates for prophylaxis and therapeutic treatment of norovirus-associated illness.

[0128] By tracing the B cell evolutionary trajectory that led to VX22, we found evidence that this antibody lineage was likely elicited in response to an earlier GII.12 or related infection and subsequently gained potency toward heterogeneous genotypes following repeated exposures. Of note, the initial breadth was associated with SHM of the VL chain, which was subsequently “fixed” at least among the day 8 circulating B cells. Mutations in the VH chain consequently led to the marked expansion of neutralization breadth, as observed by VX22 and its intermediate nodes. A rather surprising finding from the VX22 clonal analysis is the variation in the shape of the ligand blockade curve among different GII genotypes. This suggests differences in the dynamics of VLP structures across distinct GII genotypes, which could ultimately affect the blocking kinetics of VX22 clonal members for each of the GII viruses differentially. Overall, the developmental pathway of VX22 supports the feasibility of broadening cross-GII neutralization breadth through vaccinations in subjects with pre-existing immunity. It remains to be determined whether mucosal vaccine delivery to the small intestine employed by this candidate oral vaccine impacts the affinity maturation of VX22 clonal lineage. Further, the question of how this clone continues to evolve longitudinally remains to be further studied, given that germinal center responses have been reported to persist for several months following viral infection or vaccination.42,4379710041V.1

[0129] Lastly, our crystallography analysis revealed that VX22 laterally binds to the P1 subdomain proximal to the S domain. Interestingly, the VX22 epitope partially overlaps with that of A122720and NORO-32018. All three mAbs interact with Arg484 within the NERK motif11and are in close proximity to the dimeric interface of VP1 dimers. However, unexpectedly, the functional outcomes with respect to viral neutralization vary significantly among the three, ranging from a lack of neutralization (A1227)20or a weak cross-GII.4 and GII.17 HIE neutralization activity, which is also dependent on the antibody isotype and the presence or absence of the Fc domain (NORO-320), to a potent cross-GII neutralization breadth (VX22). Upon closer inspection of the structure, it is evident that the angle of VX22 Fab binding is oriented upward towards the epitope, located approximately 20° below that of NORO-320, making it susceptible to steric clash with the surrounding domains in the VLP. Indeed, we confirmed that CH1 constant region of the VX22 Fab makes steric clashes not only with the neighboring P domains but also with the S domain when overlaid onto the resting structure of VLPs. In contrast, the NORO-320 Fab approaches the epitope at a downward angle and buries a surface area twice as large (1,278 Å2vs 583 Å2) as VX22 by extending its contact area to the upper region of the dimeric interface of the other capsid protomer. This distinct mode of interactions may explain VLP disassembly following VX22 incubation. It remains to be determined whether changes in pH, temperature, or the presence of chelating agents, conditions which were previously reported to modulate particle breathing,23,44impact bNAbs binding or bNAbs-driven virus particle dynamics. Overall, the diverse set of epitope footprints, revealed by our exhaustive molecular, biochemical, and structural analyses, lay the groundwork for designing novel norovirus immunogens capable of stimulating and clonally expanding B cells specific to further insights into distinct mechanisms to achieve broad serum neutralization potency, including steric or allosteric blocking of virus-ligand interactions and virus particle disassembly.

[0130] VH and VL sequences of mAbs in this study are deposited in GenBank (NCBI) with accession numbers PP582219–PP582276. 79710041V.1MATERIALS AND METHODS FOR ANTIBODY IDENTIFICATION. Human subject selection and sampling.

[0131] Coded samples from select donors with serum and peripheral blood mononuclear cells(PBMCs) available for exploratory analyses were provided by Vaxart, Inc (ClinicalTrials gov,NCT03897309). Participants provided written informed consent before enrollment and trial participation. Sera were screened for the breadth of nAb in a surrogate neutralization assay to identify donors for molecular-level serological characterization. Two donors were selected for detailed studies due to exceptionally high GII.4 nAb breadth (subject A) and GII nAb breadth (subject B). Both donors received a single dose of the Vaxart oral candidate vaccine based on an adenovirus 5 vector-based platform carrying the VP1 capsid gene of GII.4 SY 2012 and a dsRNA adjuvant. Subject A is a 28-year-old female African American participant who received vaccination in June 2019. Subject B is a 37-year-old female white subject who received vaccination in March 2019. The vaccine was administered as oral tablet on day 1. Sera were collected on day 1 before vaccination and on day 29 after vaccination, and peripheral blood mononuclear cells (PBMCs) were collected on day 8 after vaccination. The use of coded samples was categorized as non-Human Subjects Research by the University of North at Carolina Chapel Hill (19-0226). The IRB of the University of Texas at Austin relied upon a single IRB approved by UNC-Chapel Hill. All sera and PBMCs were received coded with no link back to donoridentification. Sera were heat-inactivated for 30 minutes (min) at 56 C before use in immuneassays. Cell lines.

[0132] Baby hamster kidney cell line (ATCC CCL-10) is derived from five unsexed 1-day-oldhamsters and cultured in 5% CO2 at 37 C in Minimum Essential Medium with Earles salts and L-glutamine supplemented with 7% fetal Clone II (Hyclone), non-essential amino acids, sodium pyruvate and Antibiotic-Antimycotic.47HIE cells were kindly provided by Dr. Mary K. Estes, Baylor College of Medicine, Houston, Texas. Adult (female) secretor positive jejunal HIE cultures (J2 cell line) were grown as undifferentiated 3D cultures as described previously with minor modifications.10,48Briefly, HIEs were recovered from liquid nitrogen, suspended in 20 μl of Matrigel (Corning), plated in a single well of a 24 well plate, and grown as 3D cultures in 500 79710041V.1μL IntestiCult™ Organoid Growth Medium (Human) (STEMCELL™ Technologies) supplemented with 10 mM Y-27632 (Sigma-Aldrich). Media was refreshed every other day. Highly dense 3D cultures were passaged once per week. To passage, HIE were taken up in ice- cold complete medium without growth factors (CMGF-), broken up by repeating pipetting cycles, suspended in Matrigel (Corning), and plated in 24 well plates. CMGF- is composed of Advanced Dulbecco Modified Eagle Medium (DMEM) / F12 supplemented with 1% GlutaMAX, 1% Penicillin / Streptomycin, 1% 1M HEPES. To prepare differentiated monolayers, single cell suspension was required. Undifferentiated 3D cultures were taken up in ice-cold 0.5mM EDTA and broken up by repeating pipetting cycles. After centrifugation (200 x g for 5 min at 4°C), single cell suspension was obtained upon treatment of the HIE with 0.05% Trypsin / 0.5mM EDTA (Invitrogen) for 3–4 min at 37°C. Trypsin was then inactivated by adding CMGF- / 10% FBS. Cell suspensions were prepared by pipetting up and down and passing the cells through a 40 mm cell strainer. The cells were pelleted for 5 min at 400 x g, suspended in IntestiCult™ Organoid Growth Medium (Human) (STEMCELL™ Technologies) supplemented with 10 mM Y-27632 (Sigma-Aldrich) and plated as undifferentiated monolayers in collagen IV (Sigma- Aldrich) pre-coated 96 well plates. After 24 h, culture medium was replaced with differentiation medium to induce cell differentiation. Differentiation media is composed of equal volumes of CMGF- and IntestiCult™OGM basal medium (STEMCELL™ Technologies). Cells were differentiated for 4 days. Media was refreshed every other day. Sf9 (ATCC CRL-1711) is a female pupa ovarian cell line derived from a fall armyworm and was cultured at 27°C in ESF 921 media supplemented with 5% heat inactivated FBS. Expi293F cell line (Thermo Fisher Scientific) is derived from female human embryonic kidney 293 cells and maintained at 37°C and 5–8% CO2 using Expi293™ Expression Medium (Thermo Fisher Scientific). METHOD DETAILS VLP production.

[0133] ORF2 genes were inserted directly into the Venezuelan equine encephalitis virus 3526 replicon vector pVR21 for the production of VLPs in baby hamster kidney cells (ATCC CCL- 10).47Synthetic ORF2 were produced by Bio Basic Inc (MARKHAM) without internal Apa1, Asc1, and Not1 restriction sites for insertion into pVR21 and with optional codon optimization for human expression.30mRNA was generated using the mMESSAGE mMachine T7 51 79710041V.1transcription kit (Invitrogen). To harvest VLPs, cells were lysed with 1% Triton X-100 in PBS plus cOmplete™ EDTA-free protease inhibitor cocktail (Roche) at ~27 hours (hrs). Cell lysates were then layered on to a 40% sucrose in PBS cushion. VLPs were purified into the cushion by ultracentrifugation at 120,000 x g for 75 min and stored at -80°C. VLP integrity was confirmed by ligand and antibody binding and visualization of negatively strained particles of ~40 nm by electron microscopy. Surrogate neutralization assay.

[0134] Blockade antibody assays were performed as described previously.11VLPs were pre- treated with 2–3 fold dilutions of serum or mAbs for 1 hr and transferred to ligand-coated plates for 1 hr. The bound VLPs were detected by rabbit anti-VLP antisera (Cocalico Biologicals). Pig gastric mucin (PGM) (10ug / mL) was the ligand source for all VLP except GII.2 and GII.12, for which human type B saliva (1 / 500 dilution) was the ligand source.7Inhibitory dose 50% titers (ID50for serum and IC50for mAbs) and 95% confidence intervals (95% CI) were determined from log inhibitor normalized dose-response sigmoidal curve fits using GraphPad version 10.0.0.11All mAbs were tested at 4 μg / ml for initial reactivity screening. Negative responses were assigned a titer outside the limit of detection (highest titer tested) for visualization on heatmaps. Virus neutralization assay using human intestinal organoid.

[0135] Adult secretor positive jejunal HIE cultures (J2 cell line) were grown as undifferentiated 3D cultures, and differentiated monolayers were prepared as described previously.48Serial dilutions of each mAb were prepared in complete medium without growth factors (CMGF- :Advanced DMEM / F12 , 1% GlutaMAX, 1% penicillin / streptomycin, 1% 1 M HEPES) supplemented with 500 μM sodium glycochenodeoxycholate (GCDCA) plus 50 M ceramide and pre-incubated with 100 TCID of each virus for 1hr at 37°C and 5% CO2. CR9114 influenza mAb was used as a negative control. CDC Human Research Protection Office determined the study as public health non-research; therefore, human subject regulations did not apply. Confluent 4-day old differentiated HIE monolayers were inoculated with 100 μl of each mAb- virus mixture in duplicate. After 1 hr incubation at 37°C and 5% CO2, monolayers were washed 79710041V.1twice, and 100 μL of differentiation medium (containing 500 μM GCDCA plus 50 μM ceramide) was added to each well. For each set of infections, one plate was immediately frozen at -70°C and a duplicate plate was incubated at 37°C, 5% CO2for 24 h and frozen at -70°C. Three technical replicates for each mAb-virus mixture dilution were performed. Viral RNA was extracted from cultures (cells and media) at 1 hr post-infection (hpi) and 24 hpi using the KingFisher instrument and MagMAX - 96 Viral RNA Isolation Kit (Applied Biosystems) according to the manufacturer’s instructions. Norovirus RNA was detected by GI / GII TaqMan real-time RT-PCR.49Standard curves were generated using 10-fold serial dilutions of GII.4 SY 2012 RNA transcripts. Neutralization was expressed as a percentage of reduction in viral genome copies when compared to a no-antibody-control within each assay. Neutralization IC50 titer was determined from log-transformed, non-linear, dose-response sigmoidal curves using GraphPad version 10.0.0. Structural Homology modeling.

[0136] Antigenic domains were mapped onto the surface of the GII.4 SY 2012 P domain dimer (PDB 4wzt) using the PyMOL Molecular Graphics System, Version 2.4.1 Schrödinger, LLC. VH-only BCR-Seq.

[0137] 1 ml of PBMCs collected on day 8 was spun down at 300 x g for 10 min and resuspended in 1 ml of TRIzol™ Reagent (Thermo Fisher Scientific) for RNA isolation. The cell lysate was added to a 5PRIME Phase Lock Gel tube (QuantaBio).0.2ml of chloroform per 1ml TRIzol was added, and after 2 min of incubation at room temperature (r.t.), the Phase Lock Gel tubes were centrifuged at 12,000 x g for 10 min at r.t. for phase separation. RNeasy®Mini Kit (Qiagen) was used to purify RNA from the top aqueous phase according to the manufacturer’s protocol. RNA was eluted in 30 μl of RNase-free H2O.

[0138] To synthesize the first-strand cDNA, a total of 500 ng of template RNA was mixed with 1 μl of 50 μM Oligo d(T) 20 primer (Thermo Fisher Scientific) and 1 μl of 10 mM dNTP mix. The final reaction volume was brought up to 13 μl by adding RNase-free H2O. The oligo d(T) primer was annealed to the template RNA at 65°C for 5 min. To prepare the reverse transcriptase (RT) reaction, the following reagents were added to the primer-annealed RNA: 4 μl of 5 x SSIV 79710041V.1buffer, 1 μl of 0.1 M DTT, 1 μl of RNAse inhibitor, and 1 μl of SuperScript® IV Reverse Transcriptase (Thermo Fisher Scientific). The RT reaction was carried out by incubating the mixture at 50°C for 10 min, followed by an inactivation step at 80°C for 10 min. To remove template RNA, 1μl of RNaseH was added to the first-strand cDNA and incubated at 37°C for 20 min.

[0139] To generate VH amplicons, IgG + IgA or IgM master mix were prepared separately by adding 400 μl of H2O, 50 μl of 10x FastStart™ High Fidelity Reaction Buffer, 12.5 μl of 10 μM VH-only forward primer mix,5012.5 μl of (i) 10 μM IgG + IgA or (ii) 10 μM IgM constant region reverse primer mix (IgG rev: SGATGGGCCCTTGGTGGARGC; IgA rev: GGCTCCTGGGGGAAGAAGCC; IgM rev: GGTTGGGGCGGATGCACTCC), 10 μl of 10 mM dNTP, and 5 μl of FastStart™ High Fidelity enzyme (Sigma-Aldrich). After transferring the 50 μl master mix to a no-template control PCR tube, 9 μl of cDNA template was added to the remaining master mix and split into 9 x 50 μl reactions. Bulk VH-only amplicons were amplified using the following PCR conditions: 95°C for 2 min; [92°C for 30 sec, 50°C for 30 sec, 72°C for 1 min] x 4 cycles; [92°C for 30 sec, 55°C for 30 sec, 72°C for 1 min] x 4 cycles; [92°C for 30 sec, 63°C for 30 sec, 72°C for 1 min] x 22 cycles; 72°C for 7 min. The amplified PCR product was concentrated in 15 μl of H2O using a DNA Clean & Concentrator kit (Zymo Research). The eluate was run on a 1% agarose gel to extract IgG + IgA (400 base pair) or IgM (500 base pair) amplicon. After gel purification (Zymo Research), VH amplicons were eluted in 15 μl of H2O and submitted to the Genome Sequencing and Analysis Facility (GSAF) at The University of Texas at Austin (UT Austin) for Illumina MiSeq 2 x 300 base pair (bp) Next-generation sequencing. Natively paired VH:VL BCR-Seq.

[0140] VH:VL paired amplicon library was prepared by emulsifying single cells using flow- focusing device (FFD), followed by an emulsion-based overlap extension (OE) RT-PCR and nested PCR as described previously.51,52Two millions of day 8 PBMCs were resuspended in 20 ml PBS and single-cell isolated into emulsion droplets containing 19 ml of cell lysis buffer (100 mM Tris-HCl pH7.5, 500 mM lithium chloride, 10 mM EDTA, 1% lithium dodecyl sulfate, and 5 mM dithiothreitol) and 1 ml of magnetic poly(dT) beads (Thermo Fisher Scientific). The 54 79710041V.1poly(dT) beads were rescued from the emulsion by adding hydrated ether (Sigma-Aldrich). Subsequently, the rescued beads were resuspended in a pre-chilled RT-PCR master mix containing the following reagents as described previously:53115 μl of 10 μM APEX common reverse primer mix, 57.5 μl of 10 μM IgGAMKL constant region reverse primer mix, 9.2 μl of 12.5 μM human VH framework region 1 (FR1) OE forward primer mix, 14.9 μl of 7.7 μM human VL FR1 OE forward mix, 28.75 μl of ultrapure BSA (Thermo Fisher Scientific), 287.5 μl of 10x concentrated RTX buffer (600 mM Tris-HCl pH8.4, 250 mM (NH4)2SO4, 100 mM KCl, and 10 mM MgSO4), 57.5 μl of 0.2 mg / ml exonuclease-deficient version (N210D) of reverse transcription xenopolymerase (RTX)54, 115 μl of SUPERase•In™ RNase Inhibitor (Thermo Fisher Scientific), 57.5 μl of 10mM dNTP (New England Biolabs), 575 μl of 5 M Betaine (Sigma-Aldrich), and 1557.2 μl of H2O. The beads were then added to a DT-20 dispersing tube (IKA) pre-filled with ice-cold 9 ml emulsion oil and re-emulsified using emulsion-dispersing spinner at 600 rpm for 5 min. The emulsion mixture was dispensed in a 96-well PCR plate, 100 μl per well. The OE RT-PCR reaction was carried out using the following conditions: 68°C for 30 min; 94°C for 2 min; [94°C for 30 sec, 60°C for 30 sec, and 68°C for 2 min] x 25 cycles; 68°C for 7min. After the reaction, emulsions were broken by centrifugation at 16,000 x g for 30 sec followed by removing upper mineral oil phase. An equal volume of hydrated ether was added, vortexed twice, and centrifuged again at 16,000 x g for 30 sec. After removing the ether layer, this step was repeated twice to fully break the emulsions. Finally, any residual ether was completely discarded and evaporated by placing the tubes in a chemical fume hood for 20 min followed by a speed-vacuuming using Vacufuge plus complete system (Eppendorf) for 45 min at r.t. Magnetic poly(dT) beads were pelleted from the sample, and the aqueous layer containing DNA amplicons was PCR-purified using PCR purification kit (Zymo Research). DNA was eluted in 2 x 15 μl H2O. To optimize the nested PCR conditions, three different test reactions were prepared, each with 2 μl of OE RT-PCR product, 1 μl of 10 μM nested primer mix,530.5 μl of 10 mM dNTP, 5 μl of 10 x DreamTaq buffer, 0.5 μl of DreamTaq™ Hot Start DNA Polymerase (Thermo Fisher Scientific), and 41 μl of H2O for each reaction. The nested PCR reaction was carried out using the following conditions: 94°C for 2 min; [94°C for 30 sec, 62°C for 30 sec, and 72°C for 20 sec] x 25, 30, or 35 cycles; 72°C for 7 min. The quality of PCR products was compared for each PCR cycle by running on a 1% agarose gel. The cycle number 55 79710041V.1that yields clear single band at ~800bp without any off-target amplifications or any smears around the target size were selected as an optimal cycle number for subsequent large, nested PCR. The 4 times scaled-up large, nested PCR reactions were then performed using the cycle number selected and the same PCR conditions. The nested PCR products were concentrated in 20 μl H2O and gel extracted as described above.

[0141] To attach Illumina adaptor sequences, five different reactions were prepared, each with different combinations of primer sets as follows: (a) hIgGA and IgKL primers, (b) hIgGA and Linker_VH primers, (c) IgM and IgKL primers, (d) IgM Linker_VH primers, and (e) IgKL and Linker_VL primers as described previously.51,52,53For each reaction, 30 ng of nested PCR products were mixed with 25 μl of NEBNext® High-Fidelity 2x PCR Master Mix (New England Biolabs), 4 μl of 10 μM primer 1, and 4 μl of 10 μM primer 2, and the final volume was adjusted to 50 μl with H2O. Each reaction was amplified using different conditions as follows: 98°C for 30 sec; [98°C for 10 sec, 62°C for 30 sec, and 72°C for varying extension time - 33 sec for (a), 20 sec for (b), 33 sec for (c), 20 sec for (d), and 20 sec for (e) x varying PCR cycles, specifically 8 cycles for (a) and (b), and 5 cycles for (c), (d), and (e)]; 72°C for 7 min. The PCR product from each reaction was purified and concentrated in 20 μl H2O. Subsequently, 50 ng of PCR product from each reaction was mixed with 25 μl of NEBNext® High-Fidelity 2 x PCR Master Mix, 6 μl of forward primer (5’- AATGATACGGCGACCACCGAGATCTACACGACGACTCGTCGGCAGCGTC-3’), and 6 μl of reverse primers containing unique Illumina adaptor sequences with a final volume adjusted to 50 μl with H2O. PCR reaction was performed as follows: 98°C for 30 sec; [98°C for 10 sec, 62°C for 30 sec, and 72°C for varying times depending on the reaction (a) 33 sec, (b) 20 sec, (c) 33 sec, (d) 20 sec, and (e) 20 sec] x 8 cycles; and 72°C for 7 min. PCR products were concentrated in 20 μl H2O, and the reaction (a), (b), (c), (d), and (e) yielded 1100bp, 600bp, 1100bp, 600bp, and 550bp of DNA gel bands, respectively. After gel extraction, each amplicon was eluted in 30 μl H2O. Finally, 15 μl eluate from each reaction was submitted to the GSAF at UT Austin for sequencing on the Illumina MiSeq platform, with 2 x 300bp paired-end reads and a minimum of 106reads for each reaction. 79710041V.1Bioinformatic analysis of BCR-Seq.

[0142] Bioinformatic analysis of BCR-Seq was performed as reported previously.28,52,55,56To summarize, the R1 and R2 paired-end FASTQ files from bulk VH-only sequencing were quality- filtered and R1-R2 merged using PEAR version 0.9.6 software with default settings except following changes: the minimum overlap size, 10 bp; the maximum possible length, 700bp; the minimum possible length, 50 bp; the maximum proportions of uncalled bases, 1 bp; the number of threads to use, 4.57The R1 and R2 stitched VH-only sequences were aligned against IMGT V, D, and J germline genes using MiXCR software version 2.1.6.58Any VH sequences with at least 2 read counts were clustered into a set of unique clonotypes using a single-linkage hierarchical clustering algorithm based on >90% identity, measured by Levenshtein distance, across complementarity-determining region 3 heavy chain (CDRH3) amino acid sequences. It is important to note that the clonotype IDs from bulk VH-only sequencing were used to index the clonotype information for CDRH3 peptides identified by LC-MS / MS. Paired-end FASTQ files from the natively paired VH: VL sequencing (reaction (a)-(e) described above) were quality- filtered using Trimmomatic software version 0.27 with the following settings: 5bp sliding window; cutting when the average quality per base drops below 20; removal of reads below 100 bp.59Similarly, filtered reads were IMGT V, D, and J annotated using MiXCR, and the resulting R1 and R2 files were stitched together based on the same read ID. Any unproductive reads that contain stop codon or truncated reads were removed, and the resulting productive VH:VL paired reads with a minimum of 2 reads were subjected for a centroid-based UCLUST clustering (USEARCH software version 10.0.240) to perform BCR clustering based on >90% identity across CDRH3 nucleotide sequences.60Sample processing for Ig-Seq (bottom-up LC-MS / MS).

[0143] To deconvolute antigen-specific serum IgG repertoires, we used bottom-up liquid chromatography-tandem mass spectrometry (LC-MS / MS) guided by a donor-specific MS search database comprising >106bulk VH transcripts and single-cell paired VH: VL BCR reads (see BCR-Seq method above) sequenced from peripheral blood mononuclear cells (PBMCs) on day 8 as described previously (Figure 1C).20,27,281ml of serum collected on day 1 (pre-vaccination) or day 29 (post-vaccination) was diluted with an equal volume of PBS and applied to 1.5 ml of 57 79710041V.1hydrated Protein G agarose plus 50% slurry resin (Thermo Fisher Scientific) packed into a Pierce™ Disposable Column (Thermo Fisher Scientific). After washing the column with 30 ml of PBS, serum IgG antibodies were eluted with 10 ml of 100 mM glycine-HCl, pH 2.7, and the eluate was immediately neutralized using 1 ml of 1M Tris-HCl pH 8.0. Eluates were concentrated using Vivaspin® Turbo 4, 10 kDa (Sartorius) and centrifuged at 4000 x g for 10 min until the buffer was fully exchanged to PBS. The final volume was adjusted to 1 ml using PBS. Samples were digested into F(ab)2 fragments by adding 100 μg IdeS FabRICATOR (Genovis) per 5mg IgG at 37°C for 2 hrs.

[0144] To immobilize VLPs, 50 mg of NHS-Activated dry agarose (Thermo Fisher Scientific) was dissolved into 0.5 mg of VLPs resuspended in 1x PBS pH 7.0 and incubated at 4°C overnight on an end-to-end tube rotator. The next day, the resin with conjugated VLPs was packed into Pierce™ Centrifuge Column 0.8 mL (Thermo Fisher Scientific) and washed with 2 column volumes (c.v.) of PBS. Any empty active sites in the NHS resin were blocked with 1ml of 1M ethanolamine pH 8.2 at room temperature (r.t.) for 20 min. After blocking, the column was washed with 10 c.v. of 2 M Urea pH 7.0 by centrifuging at 1000 x g for 30 sec to remove any non-immobilized particles from the resin. VLP affinity column was then immediately washed with 20 c.v. of PBS pH 7.0 by centrifuging at 1000 x g for 30 sec. Subsequently, F(ab)2 fragments were added to the VLP affinity column and incubated at 37°C for 1.5 hr using end-to- end rotator.

[0145] Following the antibody-antigen association step, flow-through was collected by centrifugation at 1000 x g for 30 sec. Subsequently, affinity column was washed with 12 c.v. of PBS pH 7.0. The bound F(ab)2 fragments were eluted with 360 μl of 1% formic acid (Thermo Fisher Scientific) at 1000 x g for 30 sec. The elution step was repeated six times. For each elution, 30 μl of eluate was immediately neutralized with 20 μl of 1 M Tris-HCl, pH 8.0. Eluates, flow-through, and pre-column fractions were tested on enzyme immunoassays (EIA) coated with 1 μg / ml VLPs. This confirmed the depletion of antigen-specific F(ab)2 fragments in the flow- through and the presence of antigen-specific F(ab)2 in the eluates. To remove 1% formic acid, all eluates were speed-vacuumed using Vacufuge plus complete system (Eppendorf) at 45°C for 1.5 hr in aqueous mode until 5 μl of final volume was left. After speed-vacuuming, eluates were combined into a single tube, and 0.2 v / v 1 M Tris pH 8.5 was added to the sample. The final pH 79710041V.1was adjusted to 7.0, and the final volume was adjusted to 50 μl by adding MS grade H2O (Fisher Scientific). If the final volume exceeds 50 μl after pH adjustment, downstream sample processing protocol were scaled up accordingly. For subject A, affinity chromatography with immobilized GII.4 SY 2012 VLP was used. For subject B, flow-through was collected from affinity chromatography with immobilized GI.1 VLP to deplete GI.1-binding F(ab)2. The GI.1 flow-through was subsequently divided into two fractions – one fraction for affinity pull down with immobilized GII.4 SY 2012 VLP and the other fraction for affinity pull down with immobilized GII.3 VLP.

[0146] Next, (a) 10 μg of flow-through in 50 μl H2O and (b) all eluate materials in 50 μl H2O were denatured using an equal volume (50 μl) of 100% 2,2,2-trifluoroethanol (Sigma-Aldrich). Samples were disulfide-bond reduced with 1.2 μl of 500mM Bond-Breaker TCEP solution (Thermo Fisher Scientific) at 55°C for 1 hr in a dry shaker. After reduction, samples were alkylated by adding 3 μl of 550 mM iodoacetamide (Sigma-Aldrich) at r.t. for 30 min in dark. Samples were diluted to a final volume of 1 ml by adding 892 μl of 40 mM Tris-HCl pH 8.0. For each sample, 2 μg of MS grade Trypsin gold (Promega) was added and incubated at 37°C for 4 hrs. The digestion reaction was quenched by adding 10 μl of 100% formic acid. Samples were speed-vacuumed at 45°C for 3 hrs until less than 20 μl was left and then resuspended in 50 μl 0.1% formic acid. Samples were submitted to the UT Austin Center for Biomedical Research Support Biological Mass Spectrometry Facility (RRID: SCR_021728) for protein identification by LC-MS / MS using Thermo Ultimate 3000 RSLCnano UPLC coupled to Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer. Sample run method for LC-MS / MS.

[0147] Prior to LC-MS sample injection, peptides were either manually or robotically desalted using Millipore C18 ZipTip Pipette Tips following the manufacturer’s protocol. The desalted, dried peptides were re-suspended in 0.1% formic acid and transferred to the LC instrument auto- sampler. Samples were loaded onto a C18 trap column with triplicate injections (2 cm × 75 μm inner diameter) for pre-concentration and desalting, which was then transferred to a C18 analytical column (25 cm × 75 μm inner diameter) packed with 3 μm material (Thermo Acclaim 79710041V.1PepMap 100). A gradient from 5 to 45 % mobile phase B (0.1% formic acid in acetonitrile) eluted peptides by increasing hydrophobicity over 100 min with a total run time of 120 min. MS data profile in positive mode was acquired in the orbitrap with settings: 3 sec cycle time, 120,000 resolution, scan range 400-1600 m / z, maximum injection time of 60 milliseconds, and 60% radio frequency (RF) lens value. A minimum signal intensity of 5E3 was required to trigger a data- dependent scan with charge states 2 to 6 inclusive. The dynamic exclusion properties were set to allow each precursor to be selected up to two times before being added to the dynamic exclusion list, with additional properties of + / - 25 ppm and 30 sec exclusion. A targeted mass exclusion list containing the m / z values for 88 IgG peptides was included. Centroid MS / MS data were acquired in the linear ion trap using quadrupole isolation followed by HCD fragmentation with the following settings: 1.6 m / z isolation window, stepped HCD collision energies (27, 31, and 35%), and rapid ion trap scan rate with dynamic maximum injection time mode. MS search workflow.

[0148] LC-MS / MS proteomics analysis was performed as described previously.27,28,55First, personalized MS search database was created for each donor by concatenating (i) VH and VL reads sequenced by bulk VH-only and paired VH: VL sequencing (BCR-Seq), (ii) GII.4 SY 2012 or GII.3 capsid sequences , (iii) common contaminants list provided by MaxQuant, and (iv) an Ensembl version 73 human protein database. Thermo Proteome Discover software version 1.4 was used to identify the matching experimental spectra against the custom MS search database for each subject. MS search workflow consists of three sequential software: the search begins with the Spectrum Selector, whose output is then fed into the Sequest HT, and ends with the Percolator, which uses the output results from the Sequest HT. For the Spectrum Selector, MS1 was selected as a precursor for the MS2 scan. Any fragment ions ionized by a nanospray with a collision energy <1000 and a positive polarity mode were selected. Collision-induced dissociation was selected as a fragmentation method used to activate the scan. Parameters for the Sequest HT were set with the following conditions: maximum missed cleavage sites of 2 and the peptide length ranging from 6 aa to 144aa. The average precursor mass and the fragment mass were set to False, while the mass tolerance for matching fragment peaks was set to 5 ppm for precursor mass and 0.5 Da for fragment mass. The weight 79710041V.1of a, b, c, x, y, and z ions were set to 0, 1, 0, 0, 1, and 0, respectively. For dynamic modifications, maximum equal or dynamic modifications per peptide were set to 3 or 4, respectively, and the oxidation (+15.9995Da) was selected as a C-terminal dynamic modification. For static modifications, carbamidomethyl (+57.021Da) was selected as a C-terminal static modification. The precursor ions area detector node was set for the entire workflow. To set the parameters for the Percolator node, the maximum delta Cn value was set to 0.05, and the target false discovery rate for high-confidence and medium-confidence peptide hits were set to 0.01 and 0.05, respectively, with a validation based on q-value. CDRH3 peptide spectratyping and clonotype indexing.

[0149] High-confidence peptide-spectrum matches (PSMs) associated with the precursor ions for each peptide were used for CDRH3 spectratyping. Any ambiguous peptides with an average delta mass deviation exceeding 1.5 ppm were excluded. Peptide abundance for each peptide was quantified as the sum of extracted ion chromatography peak area, also known as XIC area. Each VH peptide was assigned and indexed to unique clonotypes. BCR-seq data from bulk VH-only BCR-Seq was used to define the clonotype membership, as described above. To measure the abundance of each clonotype, we summed the XIC areas of CDRH3 peptides that are (i) uniquely associated with a single clonotype and (ii) that have PSMs 2 across triplicate elution injections. Any CDRH3 peptides found in multiple clonotypes or having only 1 PSM detected across triplicate elution injections were excluded in the downstream analysis. Bioinformatic analysis of Ig-Seq serum antibody repertoires.

[0150] To categorize clonotypes into antigen-binding or non-antigen-binding group, we compared the XIC areas in the elution and the flow-through for each clonotype. After comparison, clonotypes for which elution XIC areas are higher than flow-through XIC areas by a factor of was defined as antigen-specific. The adjusted cut-off, , was calculated usingthe following formula:_ ___ , = 5, where _ _ represents the total elutionXIC areas for all CDRH3 peptides, and _ _ represents the total flow-through XIC79710041V.1areas for all CDRH3 peptides. For instance, in scenarios where _ _ for a givenclonotype equals the _ _ , any clonotypes with an _ _ 5-times higher thanthe _ _ are defined as antigen-binding5 . Conversely, inscenarios where the _ _ is 1.26E+10 and the _ _ is 5.13E+09 for a givenclonotype, clonotypes with an _ _ 12.30-times higher than the _ _12.30 were defined as antigen-binding. Any clonotypes that passed theserum collection time point or at least in one affinity pull-down (either GII.3 or GII.4 pull-down, in the case of subject B) were added to the final list of antigen-binding clonotypes. Among these, we defined cross-reactive clonotypes as those that were detectable in both GII.3 and GII.4 affinity column eluates. We further classified antigen-binding clonotypes identified in day 29 serum into two groups: pre-existing and emergent. Pre-existing clonotypes were defined as those that were already detectable on day 1 antigen-specific affinity eluates before vaccination. In contrast, emergent clonotypes were defined as those that were not detectable in the day 1 affinity eluates and only detected on day 29 eluates following vaccination. For downstream analysis, clonotypes for which elution XIC areas are three times higher than the level of detection (LOD, XIC 1.00E+6) are selected.27

[0151] The relative abundance (%) of each clonotype in the anti-GII.4 or GII.3 serum repertoire was determined by dividing the total elution XIC areas for a given clonotype by the total elution XIC areas for all clonotypes detected. Compared to the relative abundance, the relative amount is a normalized relative abundance, which incorporates both the relative abundance and the serum ID50 titer for that specific time point (Figures 1D and 1E) as described previously.28Consequently, the day 29 relative amount is equal to the day 29 serum abundance (day 29 relative amount = day 29 relative abundance x day 29 ID50 titer / day 29 ID50 titer = day 29 relative abundance), whereas the day 1 relative amount is calculated as day 1 relative abundance x day 1 ID50titer / day 29 ID50titer. See Figures 1A and 1B for the serum ID50titer. To compare the molecular features of the serum antibody repertoire, representative VH SHM for each clonotype was determined as a weighted average of the SHM of VH reads clustered within that specific clonotype, with weights based on the read count for each VH read. 79710041V.1VH Phylogenetic analysis of clonotype ID 1055 (VX22).

[0152] The phylogenetic tree of clonotype ID 1055 was built using IgPhyML implemented through the Dowser R package version 1.2.0.61IgPhyML operates the HLP19 substitution model that adapts the context-sensitive feature of SHMs (i.e., hot spot or cold spot biases) and integrates changes in codon frequencies during B cell affinity maturation.29To generate input file for the downstream IgPhyML analysis, all VH reads clustered within the clonotype ID1055 were annotated with VDJ germline genes according to the AIRR standard format using AssignGenes.py script provided by the Change-O R package version 1.3.0.62For each VH read, germline sequence was reconstructed using CreateGermlines.py script (Change-O package) with a -g dmask flag to prevent any potential ambiguous alignment of D-segment. The resulting tsv output file includes “germline_alignment_d_mask” column, which is formatted into data frame object in R version 4.2.2. The data frame was then fed into formatClones function provided by the Dowser R package, which returned a tibble of airrClone objects. Subsequently, getTrees function provided by the Dowser R package was used to execute IgPhyML software using build="igphyml" flag. The IgPhyML phylogenetic tree was built using HLP19 model parameters, including the motifs="WRC_2:0,GYW_0:1,WA_1:2,TW_0:3,SYC_2:4,GRS_0:5", hotness="e,e,e,e,e,e", omega="e,e", and partition=”cf”. Additionally, optimize="tlr" flag was used to optimize tree topology, branch length, and substitution parameters, and --ncdr3 flag was used to remove CDRH3, thereby accurately visualizing VH SHM in the tree. The clonal tree was visualized using plotTrees function provided by the Dowser R package,61along with ggtree R package version 3.4.463. Monoclonal antibody (mAb) expression.

[0153] We expressed recombinant mAbs that are present in the serum with high confidence. To do this, we selected representative mAbs from serum clonotypes for which peptides were identified by LC-MS / MS proteomics with high XIC abundance and high coverage across VH sequences, particularly within the CDR region of the antibody. Paired VH and VL amino acid sequences were codon-optimized and synthesized as gblocks (Integrated DNA Technologies). The gblocks were cloned into pcDNA 3.4 vector encoding human IgG1 Fc constant region (for VH) or kappa or lambda light chain constant region (for VL) depending on the type of light 63 79710041V.1chain gene usage as described previously.56The VH and VL plasmids were mixed at a 1:3 mass ratio and transfected into Expi293F cells according to the manufacturer’s guidelines of Gibco™ Expi293™ Expression System (Thermo Fisher Scientific). Cells were harvested on day 5 after transfection. Monoclonal antibodies were purified from filtered supernatants using Protein G agarose resin (Thermo Fisher Scientific) as described above and buffer-exchanged into PBS using Vivaspin Turbo 4, 10kDa (Sartorius). The quality of expressed mAbs was confirmed by SDS-PAGE using Bolt Bis-Tris Plus 4-12% gels (Thermo Fisher Scientific) and size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva). Hydrogen-deuterium exchange mass spectrometry (HDX-MS).

[0154] HDX-MS experiments were carried out on a Xevo-G2 Qtof (Waters Corporation) with an HDX chamber, a binary pump and an auxiliary pump. The setup in the HDX chamber consisted of a 50 μl injection loop, an online digestion using a POROS™ pepsin column (Affipro s.r.o; Cat. AP-PC-001), an Acquity UPLC BEH C181.7 μm 2.1 x 30.0 mm trapping column (Waters Corporation) and an Acquity UPLC BEH C181.7 μm 1.0 x 100 mm analytical column (Waters Corporation) for separation, and was cooled to 0.5°C. Each injection consisted of 60 pmol P domain for the reference and with 90 pmol antibody, with 3 technical replicates. VX1 and VX6 were incubated with P domain on ice for at least 30 min. D2O reaction mix was made by diluting a 20x PBS solution to 1x using 99.9% D2O (Sigma-Aldrich). Samples were diluted to an end volume of 30 μl using the D2O reaction mix and incubated for 10 sec, 1 min, 1 hr and 4 hrs. The reaction was quenched with 30 μl 6M urea (Merck), 1.5 M thiourea (Sigma-Aldrich), 300 mM TCEP (Sigma-Aldrich) solution in 0.1% formic acid (BioSolve BV), pH adjusted to 2.51. The LC method consisted of a 3 min trapping step followed by a 10 min gradient of milliQ / acetonitrile in 0.1% formic acid (BioSolve BV). Results were processed using DynamX version 3.0.0 (Waters Corporation). Parameters for the peptide coverage were set at minimum peptide length 4, minimum intensity 1000, and file threshold 2 out of 3. Spectra of peptides were reviewed manually. The data was exported and analyzed using Microsoft Excel, applying an independent samples t-test with 4 degrees of freedom, and significance set to p < 0.05. Uptake plots were plotted using GraphPad Prism 10.1.2 (GraphPad Software, LLC) using the exported mean and standard deviation from DynamX. 79710041V.1Structure determination of VX22–P domain structure.

[0155] Fab fragments of VX22 were produced by the insertion of a HRV3C site in the hinge region of the antibody. The modified antibody was expressed by transient transfection in EXPI293F cells for 6 days at 37°C. The supernatant was collected and applied to Protein A resin. The antibody was cleaved on-column with HRV3C protease, and the eluted Fabs were collected and applied to a Superdex S-200 size exclusion column equilibrated in PBS. GII.4 SY 2012 P domain dimers were produced in E coli using pMAL expression system. The expression of MBP fused to GII.4 SY 2012 P domain was induced with 1 mM IPTG, which was then allowed to grow for 16 hours at 22°C. Protein was harvested using amylose resin, and the P domain released by on-column cleavage with HRV-3C. Eluted protein was collected and applied to a Superdex S- 200 column equilibrated in PBS. P domain in complex with Fab was generated by incubation for 1 hr at r.t., followed by another round of gel filtration on the same column. Crystals were grown by the hanging-drop method with the protein complex at 12 mg / ml, mixed with solution composed of 1M LiSO4, 15% PEG 8,000. Crystals were harvested and frozen in well solution supplemented with 12.5% glycerol and 12.5% ethylene glycol. Data was collected at the 22_ID beamline at the APS light source. Data processing was performed using HKL3000, and structure building and refinement using Coot and Phenix. Protein interface analyses were performed with the PISA server.64Antibody numbering follows the Kabat rule. Negative stain microscopy of GII.4 VLP.

[0156] VLPs of GII.4-consensus (GII.4c) were produced by VP1 expression in Sf9 cells using the Bac-to-Bac baculovirus system. Bacmids were produced by transformation of DH10Bac cells with pFastBac vector containing the VP1 insert, and the resulting bacmids were transfected into Sf9 cells. After 2 passages of the virus, Sf9 cells at 3x106were infected with recombinant baculovirus and allowed to express protein for 4 days. The supernatant was harvested, and VLPs were concentrated by ultracentrifugation over an OptiPrep cushion at 75,000 x g for 2 hrs. The cushion was then collected with an equal volume of supernatant, and the sample was spun through a self-formed OptiPrep gradient for 4 hrs at 250,000 x g. The visible band was collected and applied to a Sephacryl S-500 column equilibrated with PBS. Negative stain images were 65 79710041V.1collected by applying sample at 0.1 mg / ml to a glow-discharged CF200-CU carbon support copper grid. Grids were washed and then stained with uranyl acetate and were imaged using FEI Tecnai T20 transmission electron microscopy. QUANTIFICATION AND STATISTICAL ANALYSIS

[0157] Two-sided, non-parametric Mann-Whitney U test was performed using GraphPad Prism version 10.2.1 for unpaired comparisons of VH SHM between the pre-existing and emergent clonotypes. The Pearson’s correlation test and the simple linear regression analysis were performed using GraphPad version 10.2.1 to determine the correlation between the two immune metrics. Pair-wise Student’s t-test was performed using GraphPad version 10.1.2 for comparisons between the reference state and the antibody complex for each time point. The mean and 95% confidence intervals or standard error of the mean were shown. Statistical analyses reported in this study are noted in the legend of each figure and used a threshold for significance p<0.05.

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The anti-GII.4 serum repertoires in subject A.22431 VX1 0.091 44.08218 IGHD3-16 IGHJ5 CARGSVTPPDTLRGRPMGGFDPW 9.5318037 VX5 6.522IGHV1-18 IGHD2-15 IGHJ5 CARESPYCNGRTACGGYDPW 8.8038169 VX6 5.245IGHV4-31 IGHD2-2 IGHJ3 CVKGDFSSSWYRHDVLDLW 9.093271 VX14 0.429 5.001IGHV1-18 IGHD2-15 IGHJ5 CARDRPDIVIGQAPRSLGGYDPW 8.194357 1.036 2.484IGHV1-18 IGHD3-22 IGHJ1 CARTPMTYYYDRIGFYEDW 8.8936948 2.368 IGHV1-2CTKLLPYSQGFDPW 9.7321135 VX7 2.280IGHV4-59CARGPGGVLDFVSHFAGNWFDPW 8.13848 2.096IGHV1-18 IGHD1-1 IGHJ5 CARDRPDLAVMTTAFEQAPRSLGGYDPW 9.34518 0.163 2.039IGHV4-59 IGHD2-15 IGHJ5 CARTALGFRPW 6.1536449 VX8 0.075 2.006IGHV4-59 IGHD6-19 IGHJ6 CARHGGSGYNSYYFGLDVW 5.0423143 VX15 1.844IGHV4-31 IGHD3-10 IGHJ4 CARGYFGGYLTNW 7.083882 0.053 1.819 IGHV3-9 IGHD7-27 IGHJ3 CAKHSRIWGLATSNRKAFDVW 4.9644325 VX11 0.002 1.478IGHV4-31 IGHD6-13 IGHJ4 CARGLFSSSWYREDTLDYW 7.011652 1.318IGHV3-33 IGHD2-2 IGHJ5 CARGPKGFQLLGGLDPW 8.911620 0.114 1.234IGHV4-39 IGHD3-9 IGHJ4 CTRVLTGSISEHW 7.235705 0.062 1.101 IGHV1-3 IGHD3-10 IGHJ2 CAREGWFGKRGYFDLW 3.909439 0.076 1.000IGHV3-30 IGHD3-22 IGHJ4 CAKDFDPYYESTVYCYLRDW 8.801949 0.033 0.837IGHV1-18 IGHD6-19 IGHJ4 CASGIRGWYPDFDYW 8.1422305 0.815IGHV1-18 IGHD6-19 IGHJ5 CARDSPYSSGRGGYDPW 9.193801 0.808IGHV4-31 IGHD6-13 IGHJ3 CARGEFSSSWYRHDVIDVW 6.53117 VX12 0.764IGHV4-39 IGHD3-10 IGHJ5 CARLPLLQWFGERGHFFDSW 9.1441979 VX9 0.723IGHV4-61 IGHD2-2 IGHJ4 CARGFVEAPAAYFDYW 8.943445 0.691IGHV3-23 IGHD1-1 IGHJ4 CARDGYNHIPLDYW 7.86119 0.654IGHV4-39 IGHD5-12 IGHJ4 CARQSGVASTVGPYMKYDFDSW 12.731146 0.625IGHV3-49 IGHD6-6 IGHJ5 CTRRLPFGQFDPW 8.4233880 VX10 0.042 0.587IGHV3-23 IGHD1-14 IGHJ5 CATMNLDPQTRDSNWLDPW 7.5679710041V.142924 0.469IGHV1-18 IGHD3-16 IGHJ5 CARTGVGGVAVPGGVDPW 6.38204 0.391IGHV4-59 IGHD2-15 IGHJ4 CARHGQYCSGGRCYSRIDDW 8.0732609 0.366IGHV5-51 IGHD1-20 IGHJ4 CASPTVNWKDLPDYW 4.2139586 0.360IGHV3-15 IGHD5-12 IGHJ4 CATDLGNGIVEASKWVFVSVFDNW 5.913367 0.357 IGHV3-7 IGHD2-21 IGHJ2 CATERVCGGDCYFVPWYFDLW 4.921641 0.341IGHV1-18 IGHD4-17 IGHJ5 CARDRVKMTRGPIGGLDPW 8.9515117 0.335IGHV1-18 IGHD3-10 IGHJ6 CARDMVKYYGSGIFRGGMDVW 9.144404 0.324IGHV3-30 IGHD4-23 IGHJ3 CTDERGYGGRSRRGFDIW 7.802028 0.014 0.318IGHV4-59 IGHD6-19 IGHJ3 CVRGGRAMLIWKLAFDVW 16.235751 0.027 0.302 IGHV1-2 IGHD6-19 IGHJ5 CARGGTVADTPIELGDRTWFDPW 8.6515141 0.292IGHV1-18 IGHD6-13 IGHJ5 CARDMVRVTPAARGGCDPW 9.6240693 0.289IGHV4-59 IGHD3-16 IGHJ4 CARVSGSYRYDMDYFDHW 6.793 0.038 0.267IGHV4-34 IGHD5-12 IGHJ5 CARGDYSGRRLHLQAYFDPW 7.6637648 0.011 0.231IGHV3-15 IGHD2-15 IGHJ4 CTTDPRHYLFDYW 3.621657 0.218IGHV4-59 IGHD3-10 IGHJ5 CARHKPGSSWSYGSERKGANWFDPW 5.1833066 0.206IGHV3-11 IGHD3-16 IGHJ5 CATNRYDLKSPRGGNWFDPW 10.355352 0.125 0.196IGHV3-23 IGHD3-3 IGHJ4 CVKDRDFDIWSPDRLLDYW 6.405764 0.182IGHV3-13 IGHD1-26 IGHJ4 CARGKSGYVDYW 4.507 0.178IGHV4-59 IGHD3-10 IGHJ5 CARTGILGGVDPW 9.4443074 0.171IGHV3-11 IGHD3-10 IGHJ4 CARADHYGSGSYLFDYW 9.03849 VX13 0.167IGHV4-31 IGHD2-8 IGHJ4 CARDHISGYYFDYW 6.3611251 0.165IGHV3-30 IGHD3-22 IGHJ4 CAKVHLPFYSNAHYGTDFW 7.6943457 0.113 0.159 IGHV3-9 IGHD1-26 IGHJ3 CARKKGEWDLDVFDVW 6.9716392 0.141IGHV3-11 IGHD3-10 IGHJ4 CARDTQRGYSMFSDYL 8.102980 0.012 0.129IGHV3-33 IGHD3-9 IGHJ4 CARDRKPFWTGSYIFEYW 5.9937261 0.127IGHV4-34 IGHD6-13 IGHJ4 CAGGVFSSNWYRAEVIDSW 7.1179710041V.1Table 3. The anti-GII.3 or anti-GII.4 serum antibody repertoire in subject B.79710041V.137657 0.206 0.077 3-23 6-19 J6 CAKTRPVSVYHYYSMDVW 5.14 2343 0.395 0.342 0.068 3-7 3-10 J4 CARANSFDYW 3.49 699 0.080 0.327 0.035 1-69 1-1 J4 CARGAVELERRCHFDYW 4.78 38401 VX84 1.104 0.028 3-11 3-16 J6 CARYARLGDVMGPDVW 11.17 283 VX35 0.093 2.445 0.008 3-30 4-17 J4 CARKFGDYPLDYW 9.70 2876 1.052 0.029 0.003 3-11 2-15 J6 CARERPRSSGLDVW 4.40 1793 VX42 0.088 0.516 1-69 5-24 J6 CARQEEMTKILGMDVW 13.42 303 0.029 1.003 1-18 5-18 J4 CVRSAYNSGLDYW 9.10 2733 1.285 2-5 2-15 J6 CAHRGRDGYSHHDYGLDVW 6.20 1874 0.353 1-18 2-2 J6 CARSQLQLGFYYGMDVW 6.55 13335 0.323 4-39 3-9 J4 CARDSDDILIGWNRFDFW 9.41 3134 4-55 5-18 J4 CAGARGYIYGYAYW - 19511 4-34 6-13 J1 CARLGFSSSWYRAEHFQHW -aRelative amount (%) on day 1 or day 29. The serological repertoire was analyzed by Ig-Seq after depleting GI.1-binding antibodies in the serum 79710041V.1Table 4. Sequence summary of recombinantly expressed mAbs for subject A or subject B.IGH IGH I IGK Subject A VX1 V1- D3- GHJ CARGSVTPPDTLRGRPCQHLGNWPFTF 5.1 18 16 5MGGFDPW11 IGH IGH IGHJ CARES IGK Subject A VX5 V1- D2- PYCNGRTACGG V3CQQRTNWPRGG5 5YD8.8-IGKJ4 3.818 1PW11FIGH IGH Subject A VX6 V4- D6- IGHJ CAKGDFSSSWYRHDFL 39.1IGKIGKJ3 CQQYYNAPFTF 6.931 13DLW V4-1IGH IGH IGHJ CARDRPDI IGK Subject A VX14 V1- D2- VIGQVPRSL GGYDPV3- IGKJ518 15W11 IGH t A VX7 V4- IGH IGHJ C IGK Subjec ARGPGGVLDFVSHFA D3-3 5GNV3-CQQYAVSPITF 59WFDPW20 IGH IGH 6- IGHJ CARHGGSGYN IGK Subject A VX8 V4- D SYYFGL DVWV1-CQQSYNILSF 59 19 39 IGH IGH Subject A VX15 V4- D3- IGHJ CARGYFGGYLTNWIGLV CAAWDDGLRGVL1-4731 10FIGH IGH Subject A VX11 V4- D6- IGHJ CARGLFSSSWYREDTL IGK DY7.031 13W V4-179710041V.1IGH IGH ject B VX38 V3- D2- IGH IGK Sub J 4CARDPCGGDCMDFW 7.4V1- IGKJ4 CLQSDSSPLTF 7.7 74 21 39 IGH IGH Subject B VX16 V1- D5- IGHJ IGK 4IGKJ3 CQTLNSYPRVTF 4.569 12V1-9IGH IGH Subject B VX64 V3- D4- IGHJ CARNGPKYYSNSLQYF IGLV CGTWDSSLNAGV 4QYWIGLJ2 2.430 111-51 FIGH IGH Subject B VX22 V3- D4- IGHJ CSRVGKDFGDGLFDS IGLV 4W7.62-23CCLYAGRSLWVF 5.549 17 IGH IGH Subject B VX39 V4- D1- IGHJ IGK 3CAIYHDRHGFDVWV4-1IGKJ3 CQQYYSTPFTF 3.631 14 IGH Subject B VX18 V3- IGH IGHJ CAKEANKYYIHGWHMF CGTWDVNVKAWV D3-93.530 3DSW FIGH IGH IGHJ CARDC IGK Subject B VX40 V4- TSTTCRIYYYGV1- IGKJ4 CQQSHSTPRTF 4.7 28 D2-2 6MDVW39 IGH IGH VX31 V4- D6- IGHJ CAGAVEV IGK Subject B RRVGNWFDV1- IGKJ4 CQQSYSTPFTF 4.4 59 19 5PW39 IGH IGH IGHJ CARGLSGAPPGGFDS IGLV CGADHASGTNFV Subject B VX23 V3- D7- 510.4 IGLJ3 5.153 27W 9-49 YVFIGH IGH Subject B VX84 V3- D3- IGHJ CARYARLGDVMGPDV IGLV 6W11.21-402.911 16 IGH IGH IGHJ IGK Subject B VX35 V3- D4-4CARKFGDYPLDYW 9.7V3- IGKJ3 CHQSGSSPVTF 5.7 30 17 20 IGH IGH Subject B VX42 V1- D5- IGHJ IGLV 6CARQEEMTKILGMDVW 13.47-4IGLJ3 CLLSYNGPWVF 7.169 246The V, D, and J sequence information of recombinant mAbs representing abundant serum clonotypes detected in subject A or subject B serological repertoire. 79710041V.1Table 5. Crystallography data collection and refinement statistics for VX22–VP1 P domain.Values in parentheses are for the highest-resolution shell 79710041V.1Illustrative Antibody VHand VLSequences: SEQ ID NO:1 VX20_VH EVQLVESGGGLIQRGGSLRLSCEASGIILNTYYMNWVRQPPGKGLEWLAHISASSQTIFY ADSVKGRFVISRDNAKNSLYLDMDSLRVEDTAIYYCATNDGYGGRSRRGSSYYHYGM NFWGQGTTVTVSS SEQ ID NO:2 VX20_VL DIRVTQSPSSLSASVGDRVAITCRASQPITTFLNWYRHKPGEAPQLLIYATSTLQRGVPSR FSGSGSETTFTLAISSLQPEDFATYYCQQTYSAPFTFGPGTKLEIT SEQ ID NO:3 VX22_VH EVQLVESGGGLVQPGRSLRLSCTVAGFNFGDYAMSWVRQAPGKGLEWVGFIRSNTYG GATGYAASVKGRFTISRDDSRNIAYLQMNSLKTEDTALYYCSRVGKDFGDGLFDSWGQ GTLVTVSS SEQ ID NO:4 VX22_VL QSVLIQPASVSGSPGQSITISCTGTSSDVGNYNLFSWYQQKPGKVPKLIIYEGYKRPSGVS DRFSGSKSGNTASLTISGLQAEDEADYSCCLYAGRSLWVFGGGTKVTVL SEQ ID NO:5 UCA_VH EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVGFIRSKAYGG TTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRVGKDYGDGLFDYWGQG TLVTVSS SEQ ID NO:6 UCA_VL QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSSLWVFGGGTKLTVL SEQ ID NO:7 Node 35_VH 79710041V.1EVQLVESGGGLVQPGRSLRLSCTAAGFNFGDYAMSWVRQAPGKGLEWVGFIRSNTYG GATGYAASVKGRFTISRDDSKNIAYLQMDSLKTEDTALYYCSRVGKDFGDGLFDSWGQ GTLVTVSS SEQ ID NO:8 Node 38_VH EVQLVESGGGLVQPGRSLRLSCTAAGFNFGDYAMSWVRQAPGKGLEWVGFIRSNTYG GATGYAASVKGRFTISRDDSRNIAYLQMNSLKTEDTALYYCSRVGKDFGDGLFDSWGQ GTLVTVSS SEQ ID NO:9 Node 47_VH EVQLVESGGGLVQPGRSLRLSCTTAGFNFGDYAMSWVRQAPGKGLEWVGFIRSNTYGG ATGYAASVKGRFTISRDDSRNIAYLQMNSLKADDTALYYCSRVGKDFGDGLFDSWGQG TLVSVSS SEQ ID NO:10 Node 52_VH EVQLVESGGGLVQPGRSLRLSCTTAGFNFADYAMSWVRQAPGKGLEWVGFIRSNTYGG ATGYAASVKGRFTISRDDSRNIAYLQMNSLKADDTALYYCSRVGKDFGDGLFDSWGPG TLVSVSS SEQ ID NO:27 VX20 VHnucleotide sequence encoding SEQ ID NO:1 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGATACAACGAGGGGGGTCCCTGAG ACTCTCCTGTGAAGCCTCTGGAATCATCCTCAATACCTATTACATGAATTGGGTCCG CCAGCCTCCAGGGAAGGGGCTGGAGTGGCTTGCACACATTAGTGCTAGCAGTCAAA CCATATTTTATGCAGACTCTGTGAAGGGCCGATTCGTCATCTCCAGAGACAACGCCA AGAACTCACTGTATTTGGACATGGACAGCCTGAGAGTCGAGGACACGGCTATATATT ACTGTGCGACCAACGACGGTTATGGGGGGCGATCCCGGAGAGGCTCATCCTACTAC CACTACGGCATGAACTTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG SEQ ID NO:28 VX20 VLnucleotide sequence encoding SEQ ID NO:2 GACATCCGGGTGACCCAGTCTCCGTCCTCCCTGTCTGCATCTGTCGGAGACAGAGTC GCCATCACTTGCCGGGCAAGTCAGCCCATTACCACCTTTTTAAACTGGTATCGACAC 79710041V.1AAACCAGGGGAAGCCCCTCAGCTCCTGATCTATGCTACATCCACTTTACAACGTGGA GTCCCATCACGGTTCAGTGGCAGTGGATCTGAGACAACTTTCACTCTCGCCATCAGC AGTCTCCAACCTGAAGATTTTGCGACTTATTACTGTCAGCAGACTTACAGTGCCCCG TTCACTTTTGGCCCGGGGACCAAGCTGGAGATCA SEQ ID NO:29 VX22 VHnucleotide sequence encoding SEQ ID NO:3 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAG ACTCTCCTGTACAGTTGCTGGATTCAACTTTGGTGATTATGCTATGAGTTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAATGGGTAGGTTTCATTAGAAGCAATACTTATGG TGGGGCAACAGGATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATG ATTCCAGAAACATCGCCTACCTACAAATGAACAGTCTGAAGACCGAGGACACAGCC TTATATTATTGCAGTAGAGTGGGGAAGGACTTCGGTGACGGGCTCTTTGACTCATGG GGCCAGGGAACCCTGGTTACCGTCTCCTCAG SEQ ID NO:30 VX22 VL nucleotide sequence encoding SEQ ID NO:4 CAGTCTGTTCTGATTCAGCCTGCGTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCA TCTCCTGCACTGGAACCAGCAGTGATGTTGGGAATTATAACCTTTTCTCCTGGTACC AACAGAAGCCAGGCAAAGTCCCCAAACTCATTATTTATGAGGGCTATAAGCGGCCC TCAGGGGTTTCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACA ATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTCCTGCTGCTTATATGCAGGT CGTAGTCTTTGGGTGTTCGGCGGAGGGACCAAGGTGACCGTCCTA SEQ ID NO:31 UCA VHnucleotide sequence encoding SEQ ID NO:5 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAG ACTCTCCTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGG TGGGACAACAGAATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATG ATTCCAAAAGCATCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCC GTGTATTACTGTACTAGAGTGGGGAAGGACTACGGTGACGGGCTCTTTGACTACTGG GGCCAGGGAACCCTGGTCACCGTCTCCTCA 79710041V.1SEQ ID NO:32 UCA VLnucleotide sequence encoding SEQ ID NO:6 CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACC ATCTCCTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTAC CAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCC CTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGAC AATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCTCATATGCAGG TAGTAGTCTTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO:33 node 35 VHnucleotide sequence encoding SEQ ID NO:7 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAG ACTCTCCTGTACAGCTGCTGGATTCAACTTTGGTGATTATGCTATGAGTTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAATGGGTAGGTTTCATCAGAAGCAATACTTATGG TGGGGCAACAGGATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATG ATTCCAAAAACATCGCCTACCTGCAAATGGACAGTCTGAAGACCGAGGACACAGCC TTATATTATTGCAGTAGAGTGGGGAAGGACTTCGGTGACGGGCTCTTTGACTCATGG GGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO:34 node 38 VHnucleotide sequence encoding SEQ ID NO:8 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAG ACTCTCCTGTACAGCTGCTGGATTCAACTTTGGTGATTATGCTATGAGTTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAATGGGTAGGTTTCATTAGAAGCAATACTTATGG TGGGGCAACAGGATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATG ATTCCAGAAACATCGCCTACCTGCAAATGAACAGTCTGAAGACCGAGGACACAGCC TTATATTATTGCAGTAGAGTGGGGAAGGACTTCGGTGACGGGCTCTTTGACTCATGG GGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO:35 node 47 VHnucleotide sequence encoding SEQ ID NO:9 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTCAG ACTCTCCTGTACAACTGCTGGATTCAACTTTGGTGATTATGCTATGAGTTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAATACTTATGG 79710041V.1TGGGGCAACAGGATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGACG ATTCCAGAAACATCGCCTACCTGCAAATGAATAGTCTGAAGGCCGACGACACAGCC TTATATTATTGTAGTAGAGTGGGGAAGGACTTCGGTGACGGGCTCTTTGACTCCTGG GGCCAGGGAACCCTGGTCAGCGTCTCCTCA SEQ ID NO:36 node 52 VHnucleotide sequence encoding SEQ ID NO:10 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTCAG ACTCTCCTGTACAACTGCTGGATTCAACTTTGCTGATTATGCTATGAGTTGGGTCCGC CAGGCTCCAGGGAAGGGGCTCGAGTGGGTAGGTTTCATTAGAAGCAATACTTATGG TGGAGCAACA 79710041V.1

Claims

WHAT IS CLAIMED IS:

1. An antibody that neutralizes virus from at least two norovirus GII genotypes to cellular ligand, wherein the antibody comprises: a VHregion comprising an HCDR1 sequence GF(N / T)F(A / G)DYA, an HCDR2 sequence IRS(N / K)(A / T)YGG(A / T)T, and an HCDR3 sequence (S / T)RVGKD(F / Y)GDGLFD(S / Y), and a VL region comprising an LCDR1 sequence SSDVG(N / S)YNL, an LCDR2 sequence EG(S / Y), and an LCDR3 sequence C(L / S)YAG(S / R)SLWV.

2. The antibody of claim 1, wherein the antibody neutralizes virus of norovirus genotype GII.4 and at least two other norovirus genotypes, wherein the antibody comprises a VHregion comprising an HCDR1 sequence GFNF(G / A)DYA, an HCDR2 sequence IRSNTYGGAT, and an HCDR3 sequence SRVGKDFGDGLFDS; and a VLregion comprising an LCDR1 sequence SSDVGNYNL, an LCDR2 sequence EGY, and an LCDR3 sequence CLYAGRSLWV.

3. The antibody of claim 2, wherein the VHregion has at least 95% identity to SEQ ID NO:3 and the VLregion has at least 95% identity to SEQ ID NO:

4.

4. The norovirus of claim 3, wherein the antibody neutralizes norovirus of genotype GII.4, GII.3, and GII.

2.

5. The antibody of claim 3, wherein the antibody comprises a VH region comprising SEQ ID NO:3 and a VLregion comprising SEQ ID NO:

4.

6. The antibody of claim 2, wherein: the VH region has at least 95% identity to SEQ ID NO:7, 8, 9, or 10 7. The antibody of claim 6, wherein: the VH region comprises SEQ ID NO:7, 8, 9, or 10.

8. The antibody of claim 1, wherein the antibody comprises a VH region comprising an HCDR1 sequence GFTFGDYA, an HCDR2 sequence IRSKAYGGTT and an 79710041V.1HCDR3 sequence TRVGKDYGDGLFDY; and a VLregion comprising an LCDR1 sequence SSDVGSYNL, an LCDR2 sequence EGS, and an LCDR3 sequence CSYAGSSLWV.

9. The antibody of claim 8, wherein the VHregion has at least 95% identity to SEQ ID NO:5 and the VLregion has at least 95% identity to SEQ ID NO:

6.

10. The antibody of claim 8, wherein the VHregion comprises SEQ ID NO:5 and the VLregion comprises SEQ ID NO:

6.

11. An antibody that neutralizes virus from at least two norovirus GII genotypes to cellular ligand, wherein the antibody comprises: a VHregion comprising an HCDR1 sequence GIILNTYY, an HCDR2 sequence ISASSQTI, and an HCDR3 sequence ATNDGYGGRSRRGSSYYHYGMNF; and a VL region comprising an LCDR1 sequence QPITTF, an LCDR2 sequence ATS, and an LCDR3 sequence QQTYSAPFT.

12. The antibody of claim 11, wherein the antibody neutralizes norovirus of genotype GII.4, GII.3, and GII.

2.

13. The antibody of claim 11, wherein the VH region has at least 95% identity to SEQ ID NO:1 and the VL region has at least 95% identity to SEQ ID NO:

2.

14. The antibody of claim 13, wherein the VHregion comprises SEQ ID NO:1 and the VL region comprises SEQ ID NO:

2.

15. An antibody comprising a VHcomprising a CDR3 sequence (S / T)RVGKD(F / Y)GDGLFD(S / Y).

16. The antibody of claim 15, wherein the VHcomprises a CDR3 sequence SRVGKDFGDGLFDS or TRVGKDYGDGLFDY.

17. The antibody of claim 15 or 16, further comprising a VLcomprising a CDR3 sequence C(L / S)YAG(S / R)SLW. 79710041V.

118. The antibody of claim 21, wherein the VLcomprises a CDR 3 sequence CSYAGSSLWV.

19. An antibody comprising a VHcomprising a CDR3 sequence ATNDGYGGRSRRGSSYYHYGMNF.

20. The antibody of claim 19, further comprising a VL comprising a CDR3 sequence QQTYSAPFT.

21. An isolated nucleic acid encoding the antibody heavy and / or light chain variable region of the antibody of any one of claims 1-14.

22. A host cell comprising the nucleic acid of claim 21.

23. An expression vector comprising the nucleic acid of claim 21.

24. A host cell comprising the expression vector of claim 23.

25. A host cell comprising a nucleic acid encoding the VHregion and a nucleic acid encoding the VLregion of an antibody of any one of claims 1-14.

26. A method of producing an antibody, the method comprising culturing a host cell of claim 22, 24, or 25 under conditions in which the antibody is expressed.

27. The method of claim 26, further comprising purifying the antibody.

28. A pharmaceutical formulation comprising the antibody of any one of claims 1-14.

29. A pharmaceutical formulation comprising one or more expression vectors encoding an antibody comprising the VH region and VL region of any one of claims 1-14.

30. A method of preventing or inhibiting a GII genotype norovirus infection, the method comprising administering a pharmaceutical formulation of claim 28 or 29 to a subject that has been exposed to norovirus or is or is infected with norovirus. 79710041V.

131. The method of claim 30, wherein the method reduces the viral load in the patient.

32. The antibody of any one of claims 1-14 or the pharmaceutical formulation of claim 28 or 29, for use in treating or preventing a norovirus infection in a patient.

33. Use of the antibody of any one of claims 1-14 or the pharmaceutical formulation of claim 28 or 29 in the manufacture of a medicament for treating or preventing a coronavirus infection in a patient.

34. A method of detecting a norovirus the method comprising contacting a sample suspected of comprising norovirus with an antibody of any one of claims 1-14; and detecting whether or not the antibody binds to a norovirus antigen present in the sample.

35. The method of claim 34, wherein the sample is obtained from a subject that has a norovirus infection or has been exposed to norovirus.

36. The method of claim 34 or 35, wherein the sample is a bodily fluid or a fecal sample. 79710041V.1

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