CVI Accepts, published online ahead of print on 26 November 2014 Clin. Vaccine Immunol. doi:10.1128/CVI.00520-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Mapping Broadly Reactive Norovirus Genogroup I and II Monoclonal

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Antibodies

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Sue E. Crawford1, Nadim Ajami1, Tracy Dewese Parker1, Noritoshi Kitamoto2,

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Katsuro Natori3, Naokazu Takeda3, Tomoyuki Tanaka4 , Baijun Kou1, Robert L. Atmar 1,5

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, and Mary K. Estes1,5

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Houston, TX, 77030, USA; 2School of Human Science and Environment, University

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of Hyogo, Hyogo 670-0092, Japan; 3Department of Virology II, National Institute of

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Infectious Diseases, Shinjuku, Tokyo,162-8640, Japan; 4Sakai City Institute of Public

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Health, Sakai, Osaka, 590-0953, Japan; 5 Department of Medicine, Baylor College of

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Medicine, Houston, TX 77030

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Running title: Epitope common to Noroviruses

Department of Molecular Virology and Microbiology, Baylor College of Medicine,

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*Corresponding Author. Mailing address: Department of Molecular Virology and

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Microbiology, Baylor College of Medicine, One Baylor Plaza, Mail Stop BCM-385,

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Houston, TX 77030. Phone: (713) 798-3585. Fax: (713) 798-3586. E-mail:

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[email protected].

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ABSTRACT

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Noroviruses are responsible for most acute nonbacterial epidemic outbreaks of

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gastroenteritis worldwide. To develop cross-reactive monoclonal antibodies (MAbs)

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for rapid identification of genogroup I (GI) and genogroup II (GII) noroviruses (NoVs)

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in field specimens, mice were immunized with baculovirus-expressed recombinant

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NoV virus-like particles (VLPs). Nine MAbs to the capsid protein were identified that

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detected both GI and GII NoV VLPs. These MAbs were tested in competition

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enzyme-linked immunosorbant assays (ELISA) to identify common epitope

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reactivities to GI and GII VLPs. Patterns of competitive reactivity placed these MAbs

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into two epitope groups (1 and 2). MAbs NV23 and NS22 in group 1 and F120 in

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group 2 were mapped to a continuous region in the C-terminal P1 sub-domain of the

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capsid protein. This domain is within regions previously defined to contain cross-

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reactive epitopes in GI and in GII viruses suggesting common epitopes are clustered

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within the P1 domain of the capsid protein. Further characterization in the

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accompanying manuscript by Kou et al., revealed that MAb NV23, in epitope group 1,

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was able to detect GI and GII viruses in stool. Inclusion of the GI and GII cross-

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reactive MAb NV23 in antigen detection assays may facilitate the identification of GI

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and GII human noroviruses in stool samples as the causative agent of outbreaks and

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sporadic cases of gastroenteritis worldwide.

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INTRODUCTION

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Noroviruses (NoVs) are the major cause of acute, epidemic nonbacterial

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gastroenteritis in adults and children in both developing and industrialized countries

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(1-3). In the United States, NoVs cause 19-21 million cases each year (4, 5). NoV

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outbreaks have been identified in children (6), the elderly (7), military personnel (8,

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9), immunocompromised individuals (10), restaurant patrons (11, 12), travelers to

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developing countries (13, 14), passengers of cruise ships (15), residents of

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healthcare facilities such as nursing homes (16, 17) and hospitals (18), and other

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populations housed in close quarters (19). The increasing incidence of NoV infections

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emphasizes the need to quickly detect and identify the causative agent because

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early diagnosis of NoV infection can be crucial in the effective control of outbreaks

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and decrease secondary attack rate (20).

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Currently, only one immunoassay, the RIDASCREEN Norovirus ELISA (3rd

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generation), is available for NoV diagnosis in the USA and this assay is only

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approved to be used in outbreak settings due to its low sensitivity of detection. The

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difficulty in developing broadly detecting NoV diagnostics is due to the diversity of

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NoV strains. NoVs are classified based on phylogenetic analysis of the viral capsid

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(VP1) gene into six genogroups (G1-GVI). Viruses within genogroups in GI, GII and

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GIV cause human infections. Genogroups are further subdivided into genotypes and

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there are at least 9 GI and 22 GII genotypes, respectively (21, 22). The amino acid

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sequence diversity is 45% between genogroups (22).

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Clear relationships between genotypes and antigenicity have not yet been

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determined due to the lack of a cultivation system.

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Expression of the 3’ end of the genome by using the recombinant baculovirus

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system results in the formation of virus-like particles (VLPs) that are structurally and

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antigenically similar to the native virion (23-25). The major capsid protein VP1 is

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structurally divided into the shell (S) domain that forms the internal structural core of

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the particle, and the protruding (P) domain, which is exposed on the outer surface of

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the particle (23). The P domain is further subdivided into the P1 subdomain (residues

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226 to 278 and 406 to 520 for GI.1 Norwalk virus) and the P2 subdomain (residues

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279 to 405 for GI.1 Norwalk virus) (23). P2 represents the most exposed surface of

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the viral particle and is involved in cellular histoblood group antigen (HBGA) binding

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(26-28).

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In spite of x-ray crystallographic knowledge of several noroviruses, information

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is just beginning to emerge defining specific regions of the capsid protein containing

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cross-reactive epitopes. Most information on the antigenic characteristics of NoVs

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comes from the study of monoclonal antibodies (MAbs) generated to VLPs from both

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GI and GII viruses (27, 29-40). The majority of these MAbs are genogroup-specific

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and only recognize viruses closely related to the immunogen used to generate the

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MAb. The present study analyzed cross-reactive MAbs that recognize epitopes on

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viruses in both genogroups GI and GII that may be useful in the development of

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improved diagnostic assays to detect NoVs.

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MATERIALS AND METHODS

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Development and characterization of monoclonal antibodies. MAbs were

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isolated as previously described (33). A panel of 9 MAbs (NV23, NV37, NV3, NV57,

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NV7, NS22, NS941, F8, and F120) were generated to NoV VLPs. MAbs NV23,

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NV37, and NV3 hybridomas were previously derived from spleen cells of mice

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immunized orally with recombinant Norwalk virus [NV, GI.1; sequence accession

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number M87661 (25, 41)] VLPs, while MAbs F8 and F120 hybridomas were obtained

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from spleen cells of mice immunized orally with recombinant Kashiwa 47 virus [KAV,

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GII.13; AB078334 (33)] VLPs. MAbs NV57 and NV7 hybridomas were obtained from

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spleen cells of mice immunized orally with NV VLPs. MAbs NS22, and NS941

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hybridomas were obtained from spleen cells of mice immunized orally with a mixture

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of NV and recombinant Snow Mountain Agent [SMA, GII.2 (31)] VLPs. Two

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previously characterized MAbs, NV3901 and NS14, were also used in this study (35).

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The binding reactivity of these MAbs was characterized by direct enzyme-

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linked immunosorbant assay (ELISA) against a panel of 4 GI VLPs and 7 GII VLPs

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(33). The GI VLPs used were GI.1 Norwalk/1968 (NV; M87661), GI.4 1643/2008

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(GQ413970), GI.6 TCH-099/2003 (KC998959), and GI.7 TCH-060/2003 (JN005886).

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The GII VLPs used were GII.2 Snow Mountain/1976 (SMA; AY134748) (31) and

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TCH-560/2002 (KC998960), GII.3 TCH-577/2004 (KF006265), GII.4 HOV TCH-

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186/2002 (EU310927) (35) and Grimsby/1995 (GRV; AJ004864) (42), GII.6 E99-

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13646/1999 (GU930737), GII.7 TCH-134/2003 (KF006266), GII.12 E00-13842/2000

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(KF006267), and GII.17 Katrina/2005 (DQ438972). Purified NoV particles (100 µl of 1

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µg/ml in 0.01 M PBS, pH 7.4) were coated onto 96-well polyvinyl chloride plates

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(Dynatech, Chantilly, VA) overnight at 4oC. The plates were blocked with 200 µl 5%

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BLOTTO (Carnation nonfat milk) in PBS for 2 hours at 37oC. After the plates were

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washed three times with PBS containing 0.05% Tween 20 (PBS-T), serial two-fold

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dilutions beginning at 1:100 in 0.5% BLOTTO of each MAb was added to each well

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and the plates were incubated for 1 h at 37oC. The plates were washed five times

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with PBS-T and bound antibody was detected by addition of 100 µl of goat anti-

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mouse IgG-HRP (1:3000 dilution in 0.5% BLOTTO, Sigma-Aldrich, St. Louis, MO) for

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1 h at 37oC. The plates were then washed five times with PBS-T and developed with

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100 µl of TMB Microwell Peroxidase Substrate System (A:B=1:1, KPL, Gaithersberg,

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MD) for 10 min at room temperature. The reaction was stopped by the addition of 100

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µl of 1 M H3PO4 and the optical density (OD) at 450 nm was read with a SpectraMAX

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M5 plate reader (Molecular Devices, Sunnyvale, CA). Non-coated wells were used as

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negative controls. The positive cutoff threshold was calculated as the mean of the

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negative control wells plus three times the standard deviation of the negative control

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wells.

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Competition ELISA. Two methods of competition ELISA were used. For the first

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competition ELISA, MAbs were biotinylated using an Antibody Biotinylation Kit

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(American Qualex Manufactures, San Clemente, CA). Briefly, MAbs (5 mg/ml) were

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dialyzed against 1:10 dilution of carbonate buffer overnight. A long chain N-

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hydroxysuccinimide ester biotin (NHS-LC-Biotin) solution (0.5 mg biotin/0.5 ml

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distilled water) was added to the protein solution to a final concentration of 74 µg

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biotin/mg protein and gently shaken for 1 hour at room temperature. The reaction

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mixture was then passed over a Sephadex G25 column. The biotin conjugate was

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eluted off the column using PBS buffer, concentrated, and stored at 4°C. Microtiter

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plates (Sumitomo Bakelite Co Ltd., Tokyo, Japan) were coated overnight at 4°C with

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50-500 ng/well of GI.1 NV or GII.4 GRV (42) VLPs in 100 µl of 0.05 M carbonate

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bicarbonate buffer (pH 9.6). Each well was washed twice with PBS-T and blocked

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with 5% BLOTTO in PBS-T for at least 1 hour at room temperature. In separate

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tubes, optimized concentrations of biotinylated MAb (which produced an OD of 0.5 to

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2.0 when no competitor MAb was present) were added to competitor MAb diluted

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(×64, ×256, ×1024, ×4096, and ×16,384) in PBS (pH 7.2). Following two washes with

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PBS-T, 100 µl of the MAb mixtures was added to duplicate wells, and the plates were

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incubated for 2 hours at 37°C. After washing four times with PBS-T, 50 µl of a 1:1000

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to 1:2000 dilution of HRP-conjugated streptavidin (Cosmo Bio Co., LTD., Tokyo,

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Japan) was added to all wells and reacted for 2 hours at 37°C. After washing, 50 µl

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of o-phenylenediamin-H2O2 (0.5 mg/ml o-phenylenediamin, 0.002% H2O2, 0.1 M

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citrate-phosphate buffer, pH 5.5) was added as a substrate and developed for 10

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minutes. The color reaction was stopped by the addition of 50 µl of 2 N H2SO4. The

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optical density (OD) at 405 nm and 630 nm was determined. The OD average of

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duplicates was calculated and the percentage of competition or enhanced binding

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was determined for all competitor MAb concentrations (×64, ×256, ×1024, ×4096 and

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×16384) based on the value of the PBS control. The value of biotinylated MAb

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binding to coating VLPs in the absence of competitor MAb was defined as zero.

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A second method from Hale et al. (29) was used to analyze competition

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between MAbs NV3901, NS14 and NV23. MAbs were purified using protein G

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columns (Pierce, Rockford, IL) according to the manufacturer’s instructions. One

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MAb, at a concentration of 2 μg/ml in 0.05 M carbonate bicarbonate buffer, pH 9.6,

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was used to coat flat-bottomed polyvinylchloride microtiter plates (Dynatech

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Laboratories, Alexandria, VA) overnight at 4°C. In separate tubes, a constant

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concentration of NV or HOV VLPs (based on each MAb to give an OD450 between 0.5

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–0.6) was added to decreasing concentrations of competitor MAb (5, 1, 0.5, 0.1, and

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0.05 μg/ml) in 0.5% BLOTTO in PBS and incubated overnight at 4°C. A control of

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VLPs in 0.5% BLOTTO without competitor was included in each plate. The antibody-

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coated microtiter plates were washed three times with PBS-T and blocked with 5%

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BLOTTO for 1 hour at 37°C. Following six washes with PBS-T, each of the VLP-MAb

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reaction mixtures was added to triplicate wells, and the plates were incubated for 2

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hours at 37°C. After washing six times, a 1:5000 dilution of rabbit anti-NV VLP or

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rabbit anti-HOV hyperimmune serum in 0.5% BLOTTO was added to each well, and

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the plates were incubated for 1 hour at 37°C. Following incubation, plates were again

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washed, and a 1:5000 dilution of goat anti-rabbit IgG conjugated to horseradish

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peroxidase (Sigma, St. Louis, MO) was added. Plates were incubated for an

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additional hour, and washed again. To develop the ELISA, 100 μl of 3,3’,5,5’-

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tetramethylbenzidine (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) was

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added to each well, and the color reaction was stopped by the addition of 100 μl of 1

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M H3PO4. The optical density at 450 nm was read, and the average of triplicates was

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calculated. The percentage of competition was determined for the competitor MAb

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concentrations based on the value of the PBS control, where the value of the VLP

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binding to coating MAb in the absence of competitor MAb was defined as zero.

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Homotypic competition was included as a positive control for all coating MAbs.

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Cloning, expression and analysis of norovirus fusion proteins. Characterization

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of the GST-norovirus fusion protein deletion mutants has been described previously

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(35). Numbering of the constructs indicates the N-terminal (first) and C-terminal (last)

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norovirus residue contained within a construct. NoV fusion proteins were expressed

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in BL-21 cells (Novagen, Madison WI) and purified using glutathione-sepharose 4B

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beads (Amersham Pharmacia Biotech, Piscataway, NJ).

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Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose

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membrane (Hybond-C, Amersham Pharmacia Biotech). The proteins were detected

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using a polyclonal goat anti-GST antibody (Amersham Pharmacia) at a dilution of

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1:8000, a mouse hyperimmune anti-NV VLP serum at a dilution of 1:5000, or a rabbit

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hyperimmune anti-HOV VLP serum at a dilution of 1:5000 in 0.5% BLOTTO. MAb

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ascites were used for detection at a dilution of 1:1000. All secondary antibodies used

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were conjugated to horseradish peroxidase (Sigma, St. Louis, MO). Membranes were

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developed by chemiluminescence using the Western Lightning detection reagent

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(Perkin-Elmer Life Sciences, Inc., Boston, MA) following the manufacturer’s protocol.

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RESULTS

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Monoclonal antibodies to norovirus VLPs that recognize both genogroup I and

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genogroup II viruses can be separated into two epitope groups. We previously

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reported the characterization of MAbs generated against NoV VLPs with respect to

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their reactivity to GI and GII VLPs by ELISA or western blot (33). MAbs NV23, NV37,

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and NV3, obtained from mice administered GI.1 NV VLPs perorally, and F8 and

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F120, obtained from mice administered GII.13 KAV VLPs perorally, recognized both

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GI [GI.1 NV by ELISA and GI.1 NV and Seto 124 (SeV; AB031013), GI.2 Funabashi

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258 (FUV; AB078335) and G1.4 Chiba 407 (CV; AB022679) by western blot] and GII

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[GII.2 SMA, GII.4 GRV and Narita 104 (NAV; AB078336), GII.6 Ueno 7K (UEV;

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AB078337), GII.12 Chitta 76 (CHV; AB032758), and GII.13 KAV by ELISA] VLPs

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(33). Additional MAbs, NV57 and NV7, were obtained from mice administered NV

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VLPs perorally, and NS22 and NS941, were obtained from mice administered NV

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and SMA VLPs perorally. Subsequent characterization of the reactivity to a larger

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panel of GI and GII VLPs by ELISA (Table 1) showed each MAb recognized at least

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a subset of both GI and GII VLPs by ELISA suggesting each MAb detects a common

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epitope in both GI and GII VLPs.

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To determine whether the MAbs detect similar or different epitopes in GI and

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GII VLPs, each of these MAbs was then tested in competition ELISAs with GI.1 NV

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and GII.4 GRV VLPs to define them into epitope groups (Fig. 1). In competition

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ELISAs with GI.1 NV VLPs, homotypic competition for the 9 MAbs was found to vary

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between 10 and 90% at the highest concentration (×64, about 500 µg/ml) of

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competitor antibody used in the ELISAs (Fig. 1A, biotinylated MAb NV23, NV37,

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F120 and F8 shown). MAb NV23, NV37, NV3, NV57, NV7, NS22, or NS941 each

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showed 80% or greater competition with itself and these other 6 MAbs (Fig. 1A, top

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panels for biotinylated MAb NV23 or NV37). When MAb F8 or F120 was used as the

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competitor MAb, only 50 to 60% competition was observed (at x64 concentration) for

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biotinylated MAb NV23, NV37, NV3, NV57, NV7, NS22, or NS941 (Fig. 1A, top

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panels for biotinylated MAb NV23 or NV37).

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A reduced level of competition was seen when MAbs NV23, NV37, NV3,

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NV57, NV7, NS22 and NS941 were used as competitor MAbs with biotinylated MAbs

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F120 or F8 as compared to homotypic competition (Fig. 1A, bottom panels), although

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to a lesser extent than seen when F8 or F120 were used as the competitor MAb

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against these MAbs (Fig. 1A, top panels). This difference may be due to a

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conformational change in the epitope recognized by the antibody leading to a

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reduced affinity in binding. Alternatively, the epitopes may overlap such that one MAb

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causes steric hindrance while the other may not. Similar competition patterns were

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obtained when GII.4 GRV VLPs were used as a coating antigen with NV23 and F120

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(Fig. 1B, left and right panels, respectively).

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The 9 MAbs were classified into two groups according to their competition

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ELISA patterns as follows: group 1 (NV23, NV37, NV3, NV57, NV7, NS22 and

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NS941) and group 2 (F8 and F120). The results obtained suggest the 7 MAbs of

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group 1 recognize the same or almost identical epitope. Group 2 MAbs likely

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recognize a similar or closely overlapping epitope with group 1.

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Groups 1 and 2 monoclonal antibodies recognize an epitope within the C-

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terminal P1 domain. To identify the binding site(s) for the epitope groups 1 and 2

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cross-reactive MAbs, a subset of MAbs from each epitope group was tested by

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western blot analysis for their ability to recognize deletion mutants of the GII.4 HOV

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VP1 capsid protein. From epitope group 1, MAbs NV23 and NS22 were mapped

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because these MAbs were identified to react with GI, GII and GIV VLPs by both

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direct ELISA and by capture ELISA in the companion paper by Kou et al. Both NV23

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and NS22 produced the same pattern of recognition (Fig. 2) and detected amino

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acids 453-495 but failed to detect amino acids 473-540, which defined the binding

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region for these monoclonal antibodies as contained within GII.4 HOV amino acids

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453–472 (corresponding to GI.1 NV amino acids 437-457). Although similar to NV23

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and NS22, each of the other MAbs from epitope group 1 (NV3, NV7, NV37, NV57

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and NV941, and epitope group 2 F8) detected the minimal HOV 453-495 construct

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(Fig. 3) but these were not further mapped because they lacked reactivity to VLPs in

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a capture ELISA. Analysis of MAb NV23 binding to GI.1 NV deletion mutants

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confirmed that NV23 bound to an overlapping sequence within the NV capsid protein

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with the minimal binding region, based on available constructs, comprised of amino

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acids 406-466 (Fig. 4).

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Western blot analysis with the GII.4 HOV VP1 capsid protein deletion mutants

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was performed to characterize the binding site of the epitope group 2 MAb F120.

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Similar to the epitope group 1 MAbs, the epitope group 2 MAb F120 also detected

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the GII.4 HOV amino acids 453-495 and failed to detect amino acids 473-540

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indicating the epitopes for groups 1 and 2 map to amino acids 453-472 (Fig. 5).

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NoV monoclonal antibodies bind to distinct sites of the norovirus capsid

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protein. The location of the domain that epitope group 1 MAbs NV23 and NS22 and

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epitope group 2 MAb F120 detect is shown in the crystal structures of VP1 from GI.1

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NV (amino acids 437-457) and GII.4 2004 variant (amino acids 453-472) P domains

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(Fig. 6A, top left and right panels, respectively). Previous work by our group identified

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the epitopes for MAbs NV3901 and NS14, respectively (35). MAb NV3901 is specific

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for GI viruses and mapped to NV amino acids 454-520 and specifically to E472 that

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forms a salt bridge with K514 (35). MAb NS14 recognizes GII viruses and mapped to

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HOV amino acids 473-495. Alignment of the binding sites of each of these MAbs

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shows that all of the MAbs recognize overlapping or adjacent regions of the NoV

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capsid proteins (Fig. 6B). Alignment of the epitope group 1 and group 2 MAb binding

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domain from the test VLPs used in this and the companion study of Kou et al. shows

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a number of conserved amino acids with only the alanine completely conserved

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within this domain (Fig. 6C).

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MAb NV23 is broadly reactive and detects GI and GII strains of virus (43),

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whereas NV3901 and NS14 are GI- and GII-specific, respectively. To further

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characterize MAb NV23 and determine whether the binding site for NV23 is distinct

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from the sites for NS14 and NV3901, we performed competition ELISAs using MAbs

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NV23 (Fig. 7A), NS14 (Fig. 7B) and NV3901 (Fig. 7C). Because we found that the

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VLP conformation changes upon direct coating onto ELISA plates, we performed

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competition ELISAs by capturing the VLPs using MAbs. Neither MAb NV3901 nor

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NS14 competed for VLP binding to MAb NV23 when used as either the coating or

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competitor antibody (Fig. 7). However, NV23 did show some level of competition with

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NV3901 (although not greater than 50%), which suggests NV23 may inhibit NV3901

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binding as a result of steric hindrance. These results confirm that MAbs NV23, NS14

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and NV3901 recognize distinct epitopes within the C terminal P1 domain of VP1.

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DISCUSSION

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Noroviruses are a major cause of sporadic cases and epidemic outbreaks of

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gastroenteritis. To effectively implement infection control measures following

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outbreaks, sensitive and rapid diagnostic assay are needed to identify the causative

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agents of viral gastroenteritis. Containment of outbreaks relies on rapid diagnosis;

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identification of causative agent, within 3 days versus 4 or more days following the

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first case, resulted in outbreak containment an average of 6 days sooner (7.9

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compared to 15.4 days) (20). Many currently available immunologic reagents used to

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study noroviruses are genotype-specific, which limits the usefulness to identify

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antigenically distinct viruses. Broadly genogroup cross-reactive antibodies are

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needed for rapid identification of NoVs in field specimens. Identification of cross-

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reactive epitopes on the NoV capsid proteins also provides important information

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regarding the antigenic characteristics of these viruses.

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The present work extends our previous report (33, 44) that oral immunization

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with different VLPs can generate NoV cross-reactive MAbs. These MAbs were found

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to compete for binding on GI.1 and GII.4 VLPs and MAbs NV23, NS22 and F120

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map to the C-terminal P1 sub-domain of the capsid protein that includes amino acids

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453-472 in the GII.4 HOV sequence. Although the competition ELISA segregated

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MAbs NV23 and NS22 into epitope group 1 and F120 into epitope group 2, these

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MAbs map to the same region of the capsid suggesting the epitopes for these MAbs

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are overlapping but not the same domain and may have competed for binding due to

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steric hindrance. A full understanding of this observation may be obtained by

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structural studies of VLP-Fab complexes, which is outside the scope of this

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manuscript.

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Interestingly, the amino acids 453-472 domain overlaps or is adjacent to the

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region we previously described that contains epitopes for genogroup specific MAbs

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NV3901 (GI-specific) and NS14 (GII-specific) (35). The epitopes for NV23

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characterized in this study and for NV3901 and NS14 appear to be distinct based on

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competition ELISAs. This C-terminal domain, while not any more highly conserved

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than the P1 sub-domain overall, contains conserved residues suitable to act as NoV

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cross-reactive epitopes (Fig. 6C). For the MAbs mapped in this study, the domain

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analogous to amino acids 453-472 in the GII.4 HOV structure are amino acids 437-

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457 in the GI.1 NV structure (26) (Fig 6A). In the context of the NV VLP structure, the

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surface-exposed residues in this domain can be seen within the 5-fold axis in the P1

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subdomain (23) (Fig. 6A bottom panel, surface-exposed residues labeled in

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magenta). Interestingly, the conserved glutamic acid/glutamine at position 448

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(numbered according to the NV sequence) is not surface exposed. However, the

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proline at position 451 (numbered according to the NV sequence) is conserved in all

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GI and GII sequences with the exception of the GII.7 virus strain. Modeling the

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threonine residue in GII.7 in place of the proline in the context of the GII.4 2004

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variant P domain indicates that the threonine is retained on the surface of the

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structure. The remainder of surface exposed residues following position 451

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(numbered according to the NV sequence) are surface exposed in both the NV and

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GII.4 2004 variant structures (Fig. 6A, top panel) suggesting this domain may be

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responsible for the common GI and GII reactivity.

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333

The nine MAbs described in this study detected both GI and GII VLPs by

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direct ELISA when the VLPs were coated onto ELISA plates in PBS. However, Kou

335

et al., observed an increase or decrease in reactivity to a subset of VLPs in the direct

336

ELISA with MAbs NV3 and NV57 when the VLPs were plated in water (43).

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Differences in GI.1 and GII.4 attachment efficiency in the presence of phosphate has

338

been previously described and was attributed to differential responses of the unique

339

arrangement of exposed amino acid residues on the capsid surface of each VLP

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strain, which may explain the different reactivity of MAbs to VLPs plated in water

341

versus PBS (45). Furthermore, the reactivity of these MAbs differed in a capture

342

ELISA compared to the reactivity in the direct ELISA. The reactivity in the capture

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ELISA correlated with analysis by surface plasmon resonance, which revealed that

344

NV23 and NS22 bound GI and GII VLPs with high affinity, while NV57 only bound GII

345

test VLPs. In contrast, NV37 bound only GI.1 VLPs and NV3 did not bind any test

346

VLP. Additionally, F120 and F8, similar to the epitope group 1 MAbs NV3, NV7,

347

NV37, NV57 and NS941, do not detect GI or GII VLPs in a capture ELISA indicating

348

these MAbs interact with amino acids that are not surface exposed on VLPs in

349

solution but interact with amino acids that are exposed due to binding to the ELISA

350

plate. These results support that the conformation of the VLP changes, either due to

351

the buffer used to plate the VLPs or to direct coating of VLPs onto the wells in ELISA

352

plates, thus exposing residues that are buried within the VLP in solution.

353

The presence of broadly cross-reactive epitopes on VLPs was demonstrated

354

by the reactivity of polyclonal antiserum as well as monoclonal antibodies generated

355

against NoV VLPs that detected both GI and GII VLPs (29, 33, 35, 46-48). Additional

17

356

evidence that the C-terminal P domain contains cross-reactive epitopes is the

357

identification of monoclonal antibody MAb14-1. This MAb, which detects 15

358

recombinant virus-like particles GI.1, GI.4, GI.8, and GI.3 and GII.1-7, GII.12-14, and

359

GII.16, genotypes [renumbered according to Zheng et al. (22) and Kroneman et al.

360

(21)], was mapped to a conformational epitope involving amino acids 418 to 426 and

361

526 to 534, regions surrounding amino acids 453-472 identified as the site for the

362

cross-reactive MAbs characterized in this study (Fig. 6B) (48). Additionally, the

363

crystal structure of monoclonal antibody 5B18, which is currently in use in a

364

commercial norovirus ELISA detection kit (Denka Seiken, Japan) and binds

365

numerous GII genotypes but not GI NoVs, was found to interact with amino acids

366

Val433, Glu496, Asn530, Tyr533, Thr534, and Leu535 on the GII.10 P domain (30).

367

However, this region of the protruding domain was determined to be occluded in the

368

crystal structure and led to the suggestion that NoV particles are capable of extreme

369

conformational flexibility to allow this monoclonal antibody to bind to its epitope (30).

370

The only norovirus diagnostic immunoassay currently approved for use in the

371

United States is the RIDASCREEN Norovirus 3rd Generation Antigen EIA (enzyme-

372

linked immunosorbent assay). However, it is currently approved for use only in

373

outbreak settings because it lacks sensitivity (49-52). The increasing evidence that

374

noroviruses are undergoing antigenic variation highlights the need for diagnostic

375

assays that detect broadly cross-reactive epitopes (53-55). We have identified a

376

broadly cross-reactive epitope in the C terminal P1 domain that is surface exposed;

377

the majority of these residues are dissimilar between human norovirus strains

378

suggesting that this region may remain invariant while other domains of the virus

18

379

undergoing antigenic variation. Therefore, inclusion of MAb NV23 in antigen

380

detection assays may facilitate the identification of GI and GII human noroviruses in

381

stool samples as the causative agent of outbreaks and sporadic cases of

382

gastroenteritis worldwide.

383

19

384

ACKNOWLEDGMENTS

385 386

This work was supported by Public Health Service grants NIH R01 AI38036 and P01

387

AI57788, training grant T32 A107471 (T.D.P.), and P30 CA125123 that funds the

388

Recombinant Protein and Monoclonal Antibody Production Shared Resource at

389

Baylor College of Medicine from the National Institute of Allergy and Infectious

390

Disease, and the John S. Dunn Research Foundation (to RLA). M.K.E. is named as

391

an inventor on patents related to cloning of the Norwalk virus genome and she, TNT

392

and NK receive royalties from commercialization activities through Baylor College of

393

Medicine for NoV monoclonal antibody reagents.

394 395

20

396

FIGURE LEGENDS

397 398

Figure 1. Competition ELISAs for biotinylated MAbs using GI.1 and GII.4 VLPs.

399

Biotinylated monoclonal antibodies (A) NV23, NV37, F120 and F8 or (B) NV23 and

400

F120 were mixed with competitor monoclonal antibodies NV23 (−●−), NV37 (−○−),

401

NV3 (−▲−), NV57 (−Δ−), NV7 (−■−), NS22 (−□−), NS941 (−♦−), F8 (--●--), and F120

402

(--○--) and then used to detect (A) GI.1 NV or (B) GII.4 VLPs bound to microtiter

403

plates. The 50% cutoff for significant competition is indicated by the dotted line.

404

Homotypic and heterotypic competition were observed.

405 406

Figure 2. Mapping epitope group 1 monoclonal antibodies NV23 and NS22. (A) HOV

407

VLPs, GST and purified GST-tagged HOV capsid protein deletion mutants containing

408

the indicated residues were analyzed by western blot with anti-GST antiserum,

409

polyclonal rabbit anti-HOV VLP antiserum, MAbs NV23 or NS22 as indicated below

410

each blot. (B) Schematic representation of the location of the constructs relative to

411

the full-length VP1 protein and a summary of recognition by NV23 and NS22. GST,

412

purified GST protein; HOV VLPs, purified Houston virus GII.4 VLPs.

413 414

Figure 3. Monoclonal antibodies detect HOV amino acids 453-495. GII.4 HOV VLPs

415

and purified GST-tagged HOV capsid construct amino acids 453-495 were analyzed

416

by western blot with polyclonal rabbit anti-HOV VLP antiserum, MAbs NV3, NV37,

417

NS941, NV57, NV7 and F8 as indicated below each blot.

418

21

419

Figure 4. Mapping epitope group 1 MAb NV23. (A) GI.1 NV VLPs and purified GST-

420

tagged NV capsid protein deletion mutants containing the indicated residues were

421

analyzed by western blot with polyclonal rabbit anti-NV VLP antiserum and MAb

422

NV23 as indicated below each blot. (B) Schematic representation of the location of

423

the constructs relative to the full-length VP1 protein and a summary of recognition by

424

NV23.

425 426

Figure 5. Mapping epitope group 2 MAb F120. (A) Purified GST-tagged HOV capsid

427

protein deletion mutants containing the indicated residues were analyzed by western

428

blot with polyclonal rabbit anti-HOV VLP antiserum (top panel), or F120 (bottom

429

panel). (B) Schematic representation of the location of the constructs relative to the

430

full-length VP1 protein and a summary of recognition by F120. GST, purified GST

431

protein; HOV VLPs, purified Houston virus VLPs.

432 433

Figure 6. Location of binding site for epitope group 1 MAbs NV23 and NS22 and

434

epitope group 2 MAb F120. (A) The location of the amino acids that correspond to

435

the epitope binding sites for epitope groups 1 MAbs NV23 and NS22 and 2 MAb

436

F120 on the crystal structure of GI.1 NV [amino acids 437-457, top right panel;

437

Protein Data Bank (PDB) accession number 1IHM] and GII.4 HOV (amino acids 453-

438

472, top left panel; PDB accession number 3SKB) P domain dimer prepared using

439

PyMOL. The ribbon structure for P1 is shown in grey, P2 in blue, and the residues

440

corresponding to the minimal binding domain are indicated in green with the surface-

441

exposed residues in magenta. Bottom panels show (left panel) the NV VLP crystal

22

442

structure (VIPER data base accession number 1IHM) with the A-B dimers in blue, the

443

C-C dimers in green, and the surface-exposed residues in the MAb minimal binding

444

domain in magenta using Chimera. The boxed area (white) is magnified (right panel)

445

and shows the P2 domain surrounding the 5-fold axis with the MAb surface-exposed

446

binding residues in magenta. (B) Alignment of binding sites for cross-reactive MAbs.

447

The binding site for each MAb is shown relative to its position within the P domain.

448

Amino acid numbers for the MAbs characterized in this study and NS14 correspond

449

to GII.4 HOV sequence (GenBank accession number EU310927); amino acid

450

numbers for NV3901 correspond to GI.1 NV sequence (M87661); amino acid

451

numbers for MAb 14-1 correspond to GII.4 1207 sequence (DQ975270); amino acid

452

numbers for 8C7 correspond to GI.1 Norwalk virus (NV 96-908, AB028247); amino

453

acid numbers for 5B18 correspond to GII.10 Vietnam026 (AF504671). (C) Alignment

454

of the MAb-binding domain in the P1 capsid sequences for the indicated virus strains.

455

The amino acid numbering corresponds to each virus strain|GenBank assession

456

number. The minimal binding domain for MAbs NV23, NS22 and F120 on the GI.1

457

NV and GII.4 2004 HOV capsid region are indicated in green with the surface-

458

exposed residues in magenta. The symbols under each column denote (*) identical

459

amino acids, (:) different but highly conserved amino acids, (.) amino acids that are

460

somewhat similar, and ( ) dissimilar amino acids.

461 462

Figure 7. Competition ELISAs between GI-specific (3901), GII-specific (NS14) and GI

463

and GII-specific (NV23) monoclonal antibodies. Competition ELISAs showing

464

competition between monoclonal antibodies (A) NV23, −♦−, (B) NS14, −■−, and (C)

23

465

3901, −▲−. Coating antibody is indicated at the top of the graph, and the competitor

466

MAb in the box. The 50% cutoff for significant competition is indicated by the dotted

467

line.

24

468

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644 645 646

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29

Table 1. Reactivity of MAbs against GI and GII VLPs by ELISA. Reactivity by ELISA Mab NV23 NV37 NV3 NV7 NV57 NS941 NS22 F8 F120

Route Organ Immunogen po SP NV po SP NV po SP NV po SP NV po SP NV po SP NV + SMA po SP NV + SMA po SP KAV po SP KAV Positive reactivity below the limit of detection

GI.1

GI VLPs GI.4 GI.6

GI.7

GII.2

GII.3

GII.4

GII VLPS GII.6 GII.7

GII.12

GII.17