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Keywords:

  • cross-reactivity;
  • monoclonal antibody;
  • sapovirus

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1–7], 7 GII, [GII.1–7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus-like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty-five hybrid clones producing MAbs were obtained. Twenty-four MAbs were characterized by ELISA, according to their cross-reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross-reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup-specific; and (iii) those reactive in a genotype-specific manner. Further analysis of three broadly cross-reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV.

List of Abbreviations: 
CBB

Coomassie brilliant blue

DAB

diaminobenzidine

EM

electron microscopy

G

genogroup

HRPO

horseradish peroxidase

MAb

monoclonal antibody

NoV

norovirus

OD

optical density

OPD

ortho-phenylenediamine

ORF

open reading frame

P domain

protrusion domain

TPBS

Tween 20 phosphate-buffered saline

S domain

shell domain

SaV

sapovirus

Sf9

Spodoptera frugiperda

VLP

virus-like particles

VP1

capsid protein

WB

western blotting

Sapovirus, a member of the family Caliciviridae, causes gastroenteritis in humans and is a significant public health problem (1–5). SaV was originally identified by EM of fecal specimens obtained during a gastroenteritis outbreak (6, 7).

The SaV capsid is composed of 90 dimers of capsid protein (VP1) (8). SaV has a ∼7.5 kb genome of single-stranded positive-sense RNA that is predicted to encode two or three ORFs. The functions of proteins encoded by ORF2 and ORF3 are unknown. However, ORF1 encodes nonstructural proteins and VP1 (9, 10). VP1 is likely produced by cleavage of the ORF1 polyprotein by viral protease or by translation from subgenomic RNA (3′-coterminal with the virus genome), or both (11, 12). A tripeptide, MEG, conserved among human SaV strains, is probably the putative VP1 start on the subgenomic RNA. VP1 expressed from the putative subgenomic RNA or putative VP1-encoding construct in insect or mammalian cells self-assembles into virus-like particles that are morphologically similar to native SaV (12, 13–20). SaV VP1 has an apparent molecular mass of 60 kDa (11, 12, 21). Based on their complete VP1 sequences, SaVs are classified into at least five genogroups: GI, GII, GIII, GIV and GV. GI, GII, GIV and GV infect humans, and GIII infects porcine species (9). Human SaVs can be further separated into 16 genetic clusters (seven GI [GI.1–7], seven GII, [GII.1–7], one GIV and one GV) (22).

Because there is no cell-culture system or small-animal model for human SaV, SaV VLPs have been used as models of SaV virion for immunogenic, antigenic and structural studies. The capsid proteins of human SaVs have high antigenic diversity (16, 17, 20, 21, 23). However, little information is available about whether specific regions of the VP1 are important for antigenic specificity, and whether type-specific and/or cross-reactive epitopes are present in SaVs.

Monoclonal antibodies are powerful tools for the study of viruses and proteins. A panel of MAbs against SaV VLPs would be valuable for antigenic and structural analysis as well as useful for developing new diagnostic assays for human SaV. In this study, we established such a panel of MAbs broadly cross-reactive to all human SaV genogroups, GI, GII, GIV and GV, as well as MAbs specific to either genogroups or genotypes.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Generation of recombinant baculoviruses

DNA fragments corresponding to the putative subgenomic RNA region of the genome (approximately 2.3 kb in length) of GI.6 Nichinan (GenBank accession number AB455803 [24]), GII.3 20082029 (AB630068 [22]), GII.3 D1711 (AB522391 [25]), GII.3 Kushiro5 (AB455793 [26]), GII.3 Nayoro4 (AB455794 [26]), GII.4 Kumamoto6 (AB429084 [1, 22]), GII.7 20072248 (AB630067 [22]), and GIV Yakumo8 (AB455795 [26]) were amplified by PCR with KOD-Plus-DNA polymerase (Toyobo) as previously described (27). A forward primer (5′-CAGATCTGCAGCGGCCGCATGGAGGN8–10[N indicates strain specific sequence]-3′) included a NotI site (underlined), and a common reverse primer (5′-GTCCCAGGAAAGGATCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′) included a BamHI site (underlined). The amplified fragments were cloned into NotI- and BamHI-digested baculovirus transfer vector pVL1392 (Orbigen, San Diego, CA, USA) with an In-Fusion Advantage PCR Cloning Kit (Takara, Shiga, Japan), according to the manufacturer's protocol. Escherichia coli HST08 premium competent cells (Takara) were used to transform and propagate the transfer plasmid. Sequencing analysis confirmed the consensus sequence of each strain. An insect cell line derived from Sf9 (Riken Cell Bank, Tsukuba, Japan) was co-transfected with a linearized wild-type Autographa californica nuclear polyhedrosis virus DNA (Baculo-Gold, BD Bioscience, Franklin Lakes, NJ, USA) and the transfer vectors carrying the human SaV putative subgenomic RNA region by the lipofectin-mediated method as specified by the manufacturer (Gibco BRL, Gaithersburg, MD, USA).

Expression and purification of human sapovirus virus-like particles

For larger scale expression of the SaV capsid proteins, BTl-Tn-5B1–4 (Tn5), an insect cell line derived from Trichoplusia ni (Invitrogen, San Diego, CA, USA), was infected with recombinant baculoviruses at a multiplicity of infection of 10 and incubated for 7 days at 26°C, as previously described (28). Eight novel VLPs derived from GI.6 Nichinan, GII.3 20082029, GII.3 D1711, GII.3 Kushiro5, GII.3 Nayoro4, GII.4 Kumamoto6, GII.7 20072248 and GIV Yakumo8 were purified as follows. Intact cells, cell debris, and progeny baculoviruses were removed by centrifugation at 10,000 g for 60 min. The supernatant was then centrifuged at 174,899 g for 3 hr in a Beckman SW32-Ti rotor rotor (Beckman Coulter, Fullerton, CA, USA). The resulting pellet was resuspended in EX-CELL 405 Serum Free medium (SAFC Biosciences, Lenexa, KS, USA) at 4°C overnight and the debris removed by centrifugation at 10,000 g for 30 min at 4°C. The supernatant was then centrifuged at 154,000 g for 2 hr in a Beckman TLA55. The resulting pellet was resuspended in EX-CELL 405 Serum Free medium (SAFC Biosciences) at 4°C overnight, and the debris removed by centrifugation at 10,000 g for 5 min at 4°C. After mixing with 2.1 g of CsCl in MilliQ water, the sample was centrifuged at 148,862 g for 24 hr at 10°C in a Beckman SW55-Ti rotor. After fractionation (20 × 250 μL each), each aliquot was diluted with EX-CELL 405 medium, and centrifuged at 154,000 g for 2 hr at 4°C in a Beckman Coulter TLA55 rotor. The resulting pellet was resuspended in EX-CELL 405 medium. Seven VLPs derived from GI.1 Mc114 (AY237422 [27]), GI.5 Yokote1 (AB253740 [17]), GII.2 Mc10 (AY237420 [10]), GII.3 C12 (AY603425 [10]), GII.3 Syd53 (DQ104360 [29]), GIV Syd3 (DQ104357 [29]) and GV NK24 (AY646856 [30]) were expressed and purified as previously described (14, 16, 17, 20).

Preparation of monoclonal antibodies

The PAI myeloma cell line (kindly provided by M. Kotani, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) was cultured in Dulbecco's modified Eagle's medium with 15% FCS. The MAbs were prepared essentially as previously described (31, 32), with minor modifications. Briefly, BALB/c mice were primed intraperitoneally with 1–10 μg of purified VLPs (GI.1 Mc114, GI.5 Yokote1, GI.6 Nichinan, GII.3 Syd53, GIV Syd3, or GV NK24 VLPs) per mouse, in the presence of adjuvant. The mice received booster inoculations four times at 1 week intervals, the final injection of antigen being administered i.v. Three to four days after the final injection, the animals were killed and cells from their spleens fused with the myeloma cells. The culture medium of the hybridomas that resulted from successful fusions was screened for reactivity by ELISA. ELISA plates were coated with VLPs as described below. Positive hybridomas were cloned by limiting dilution and antibody-producing clones were grown and stored in liquid nitrogen until used for further tests. Finally, ascites fluid was prepared by injecting the hybridomas into pristane-primed mice and used to provide the MAbs for this study. The isotype and subclass of each MAb were determined by ELISA with anti-mouse subtype MAbs (Cappel Laboratories, West Chester, PA, USA) or an IC kit (IsoQuick, Sigma, Saint Louis, MO, USA).

All animal procedures conformed to the Animal Handling and Ethical Regulations of the University of Hyogo and the provisions of the Declaration of Helsinki. This research project was approved by the Ethics Committee of the University of Hyogo.

Enzyme-linked immunosorbent assay

An indirect ELISA, with slight modifications, was used to screen and characterize the MAbs (31). Briefly, 96-well microplates (Nunc-immune plate, Nunc, Roskilde, Denmark) were coated with 100 ng of  VLPs/well in 50 μL of PBS (pH 7.2) overnight at 4°C. The plates were washed with TPBS and blocked with 5% skim milk in PBS for 1 hr at 37°C. The MAbs (ascites, appropriate dilution, 50–100 μL) were added and incubated for 1 hr at 37°C. After washing with TPBS, 50 μL of a 1:2000 dilution of HRPO–conjugated goat anti-mouse immunoglobulin G (IgG), IgM, or IgA (Cappel Laboratories) was added to each well and incubated for 1 hr at 37°C. After washing, 50 μL of OPD-H2O2 (0.5 mg of ortho-phenylenediamine/mL, 0.002% H2O2, 0.1 M citrate-phosphate buffer, pH 5.5) was added, incubated for 10–20 min, and the optical densities measured at 490 and 655 nm with a Microplate Reader (Model 550, Bio-Rad, Richmond, CA, USA).

To further characterize the epitopes recognized by these MAbs, a competitive indirect ELISA was performed as previously described (33, 34) with a slight modification. Briefly, VLP was used to coat 96-well microplates overnight at 4°C at a concentration of 50–100 ng/well in PBS (pH 7.2). In separate tubes, MAbs at a concentration of 5–500 ng/mL (depending on the VLPs used for the coating) were added to decreasing concentrations of competitor VLP (10, 1, 0.1 and 0.01 μg/mL) in PBS (pH 7.2) containing 1% skim milk, and then incubated overnight at 4°C. As a control, MAb without competitor VLP was included in each plate. The VLP-coated plates were washed and blocked with 5% skim milk for 1 hr at 37°C, 100 μL of each of the VLP-MAb reaction mixtures was added to duplicate wells, and the plates were incubated for 2 hr at 37°C. The reactivity of the antibody to the competitors was expressed as B/B0, where B is the amount of antibody bound to the coating antigen in the presence of the competitor, and B0 is the amount in the absence of the competitor.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

The VLPs (0.5–1 μg per track) were suspended in electrophoresis sample buffer containing 1% SDS, 10% 2-mercaptoethanol, 50 mM Tris-HCl (pH 6.8), 0.0025% phenol red, and 10% glycerol. The samples were boiled for 2 min, then subjected to 10% SDS-PAGE (catalog no. EMP-8020; 1 mm thick, 8 cm long, 7 cm wide; Iwaki, Tokyo, Japan) at a constant current of 20 mA for 1.5–2 hr. The gels were stained with 0.1% CBB (Sigma) or silver staining kit (Ez stain Silver, Atto, Tokyo Japan).

Immunoblotting

Western blotting analysis was performed as previously described (31, 35) with slight modifications. Briefly, after electrophoresis, the gel was transferred electrophoretically to a nitrocellulose membrane (0.45 μm pore size, Millipore, Bedford, MA, USA) in a semidry transfer (EPM-8460; Iwaki) at a constant current of 70 mA for 2–3 hr. The strips were prepared and incubated overnight at room temperature with the MAbs (ascites fluid) at a dilution of 1:500–1000. The blots were incubated with a 1:2000 dilution of HRPO-conjugated goat anti-mouse IgG, IgM, and IgA (Bio-Rad) for 1 hr at 37°C. The strips were soaked in a solution of DAB (0.5 mg/mL, 3–′diaminobenzidine, 0.001% H2O2, 50 mM Tris-HCl buffer, pH 6.0) to detect the antigen-antibody complexes on the strips.

Sequence analysis

To confirm the sequences of the panel of plasmids used in this study, nucleotide sequence analysis was performed with a Big Dye Terminator (version 3.1) Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Tokyo, Japan) and an automated sequencer, the 3130 Genetic Analyzer (Applied Biosystems). Nucleotide sequences were assembled with the program Sequencher, version 4.10.1 (Gene Codes, Ann Arbor, MI, USA). Nucleotide and amino acid sequences were analyzed with GENETYX Mac software, version 16.0.4 (Genetyx, Tokyo, Japan).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Expression of novel human sapovirus virus-like particles

Sapovirus capsid proteins were expressed in an insect cell, Tn5, and VLPs were purified by CsCl equilibrium density gradient centrifugation as described in Materials and Methods. EM analysis of eight purified SaV VLPs from GI.6 Nichinan, GII.3 20082029, GII.3 D1711, GII.3 Kushiro5, GII.3 Nayoro4, GII.4 Kumamoto6, GII.7 20072248 and GIV Yakumo8 showed cuplike surface depressions and almost homologous particles with diameters of approximately 41–43 nm (Fig. 1). The morphology of these recombinant particles is very similar to those we have observed in previous studies (14, 16, 19, 20).

image

Figure 1. Electron micrographs of novel SaV VLPs. VLPs derived from (a) SaV GI.6 Nichinan, (b) GII.3 20082029, (c) GII.3 D1711, (d) GII.3 Kushiro5, (e) GII.3 Nayoro4, (f) GII.4 Kumamoto6, (g) GII.7 20072248 and (h) GIV Yakumo8 have diameters of 41–43 nm. Purified VLPs were stained with 4% uranyl acetate (pH 4) and examined by an electron microscope (TEM-1400; JOEL, Japan) operating at 80 kV. Scale bars = 100 nm.

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Isolation of sapovirus monoclonal antibodies

BALB/c mice were immunized intraperitoneally with purified SaV VLPs and their spleen cells fused with PAI myeloma cells. Sixty-five hybrid clones producing MAbs were obtained from six different recombinant human SaV VLPs from GI.1 Mc114, GI.5 Yokote1, GI.6 Nichinan, GII.3 Syd53, GIV Syd3 or GV NK24. Twenty-four MAbs were obtained from mouse ascites and classified into five groups, according to their patterns of ELISA reactivity with 15 VLPs (GI.1 [n= 1], GI.5 [n= 1], GI.6 [n= 1], GII.2 [n= 1], GII.3 [n= 6], GII.4 [n= 1], GII.7 [n= 1], GIV [n= 2], and GV [n= 1]) (Table 1). The MAbs were grouped as follows: MAbs cross-reacting with all GI, GII, GIV and GV (group A, n= 8); GI-specific or type-specific MAbs (group B, n= 7); GII-specific or type-specific MAbs (group C, n= 3); GIV-specific MAbs (group D, n= 2); and GV-specific MAbs (group E, n= 4).

Table 1.  Reactivities of monoclonal antibodies with 15 SaV VLPs in ELISA
GroupMAbImmunogenIsotypeReactivitya
GIbGIIGIVGV 1 NK24
1c Mc1145 Yokote16 Nichinan2 Mc103 C123 200820293 D17113 Syd533 Kushiro53 Nayoro44 Kumamoto67 200722481 Syd31 Yakumo8
  1. aReactivities: +++, strong (OD ratio of sample/PBS > 10); ++, moderate (5–9); +, weak (1–4); –, negative (< 1); NT, not tested.

  2. bGenogroup

  3. cGenotype (22)

A5C9Nichinan  IgG1+++++++++++++++++++++++++++++++++++++++++++++
 1A1Nichinan  IgG1+++++++++++++++++++++++++++++++++++++++++++++
 5C1Nichinan  IgG1+++++++++++++++++++++++++++++++++++++++++++++
 6C4Nichinan  IgM+++++++++++++++++++++++++++++++++++++++++++++
 8127Yokote1  IgG1++++++++++++++++++++++++++
 6D3Nichinan  IgG1+++++++++++++NT++++++++++++++
 3D2Nichinan  IgG1++++++++++++NT+++++++++
 4G7Nichinan  IgG1+NT+++++++NT+++++++
B616Yokote1  IgG1+++++++
 1325Mc114  IgG1++
 5001Mc114  IgG1++NTNTNTNTNTNTNTNT
 4357Mc114  IgG1++NTNTNTNTNTNTNTNT
 627Yokote1  IgG1++NTNTNTNTNTNTNTNT
 7F8Nichinan  IgM++NT
 1F2Nichinan  IgM++NT
C1803Syd53  IgG3++++++++++++++++++
 8083Syd53  IgG3NT++++NTNT++NTNTNTNT
 1015Syd53  IgG2b+++++++++++
D819Syd3  IgG1NTNTNTNTNTNTNT++++
 806Syd3  IgG1NTNTNTNTNTNTNT++++
E1496NK24  IgG1+++
 155NK24  IgG1+++
 4971NK24  IgG1NTNTNTNTNTNTNTNT+++
 1052NK24  IgG1NTNTNTNTNTNTNTNT+++
 anti-SaV serum  +++++++++++++++++++++++++++++++++++++++++++++
 PBS  

Monoclonal antibodies cross-reactive with heterologous genogroups and genotypes.

Eight group A MAbs showed binding to all GI, GII, GIV and GV VLPs examined in this study, although their reactivity to each VLP was different. Four MAbs (namely, 5C9, 1A1, 5C1 and 6C4) reacted consistently and strongly with VLPs from different genogroups and genotypes when the OD ratios between samples and PBS were greater than 10. On the other hand, another four MAbs (8127, 6D3, 3D2 and 4G7) reacted either strongly or moderately (OD ratio: 5–9) with GI, GIV and/or GV VLPs, and weakly (OD ratio 1–4) with GII VLPs in ELISA (Table 1). Among these eight MAbs, three (5C9, 1A1 and 8127) were further confirmed by WB analysis to react with GI, GII, GIV and GV SaV VP1 with an apparent molecular mass of about 60 kDa (Fig. 2). These results indicate that SaV capsid protein carries at least one epitope common to GI, GII, GIV and GV SaV. Protein bands smaller than 60 kDa are likely truncated VP1 as previously described, though direct evidence is lacking (14).

image

Figure 2. SDS-PAGE of purified SaV VLPs and western blots. VLPs from (a, b) GI (GI.5 Yokote1) and (c, d) GI.6 Nichinan, GII (20072248), GIV (Yakumo8) or GV (NK24) were separated by SDS-PAGE. The proteins were stained with (a) CBB or transferred to a nitrocellulose sheet which was then incubated with (b) MAbs 8127, (c) 5C9 or (d)1A1. Lane M, molecular mass markers (kDa).

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Genogroup- or genotype-specific monoclonal antibodies

Seven group B MAbs reacted exclusively with GI VLPs, but not with any other VLPs from GII, GIV and GV SaVs. MAb 616 bound to three GI SaV VLPs (GI.1 Mc114, GI.5 Yokote1 and GI.6 Nichinan) in a genogroup-specific manner, whereas MAbs 1325, 5001, 4357, 627, 7F8 and 1F2 bound to VLPs in a genotype-specific manner (Table 1). Three group C MAbs (1803, 8083 and 1015) showed reactivity only with GII VLPs. MAb 1803 reacted to all GII VLPs (GII.2, -3, -4, and -7) examined, but not to GI, GIV and GV VLPs. On the other hand, MAb 1015 reacted with all GII.3 VLPs, but not with GII.2, GII.4, GII.7, GI, GIV and GV VLPs, demonstrating that this MAb is likely to be GII.3-specific (Table 1). Two group D MAbs (819 and 806) were specific to GIV (Syd3 and Yakumo8) VLPs and did not bind to GI, GII and GV VLPs. Four group E MAbs (1496, 155, 4971 and 1052) were specific to GV VLPs (Table 1).

Epitopes recognized by cross-reactive monoclonal antibodies

Three MAbs classified as group A (5C9, 1A1 and 8127) had broad reactivity to VLPs derived from all human SaVs genogroups (GI, GII, GIV and GV). The specificity of these MAbs was further examined by competitive ELISA. When GI.6 Nichinan VLP was used as the coating antigen, binding of MAb 5C9 was completely inhibited. In addition, binding of MAb 5C9 was similarly blocked by three heterotypic VLPs (GII.7 20072248, GIV Yakumo8 and GV NK24) (Fig. 3a). Similar results were obtained when GII.7 20072248 VLP was used as the coating antigen. Binding of MAb 5C9 was completely inhibited by homotypic GII.7 VLP and also by three heterotypic VLPs, GI.6 Nichinan, GIV Yakumo8 and GV NK24 (Fig. 3b). The results were similar when two other VLPs, GIV Yakumo8 and GV NK24, were used as the coating antigens (data not shown). Binding of MAb 1A1 to four VLPs (GI.6 Nichinan, GII.7 20072248, GIV Yakumo8 and GV NK24), as well as inhibition by homotypic and heterotypic VLPs was exactly the same as that of MAb 5C9 (Fig. 3c,d), suggesting these two MAbs (5C9 and 1A1) recognize a common epitope. On the other hand, binding of MAb 8127 to GI.5 Yokote1 and GV NK24 VLPs was different. Binding of MAb 8127 to GI VLP was strongly blocked by both homotypic GI VLP and heterotypic GV VLP (Fig. 3e) and binding to GV VLP was similarly inhibited by both homotypic GV VLP and heterotypic GI VLP (Fig. 3f). However, inhibition by two other competitor VLPs, GII.7 20072248 and GIV Yakumo8, was incomplete (Fig. 3e, f), demonstrating that the epitope recognized by MAb 8127 is different from that of MAbs 5C9 and 1A1.

image

Figure 3. Competitive ELISA to differentiate epitope recognition. Microplates were coated with VLPs from (a) GI.6 Nichinan, (b) GII.7 20072248, (c) GIV Yakumo8, (e) GI.5 Yokote1, or (d, f) GV NK24. A reaction mixture of the various concentrations of the competitor VLPs and a MAb was added to the plates and binding of the MAb to the coated VLPs was measured as described in Materials and Methods. The competitor VLPs used in this experiments were ○, GI.6 Nichinan; •, GI.5 Yokote1; X, GII.7 20072248; △, GIV Yakumo8; □, GV NK24. PBS(+) was used as a control without competitor. (a, b) MAbs 5C9, (c, d) 1A1 and (e, f) 8127 were incubated with the competitor VLPs.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

In this study, we established 65 hybridoma cell lines from six mice immunized with six SaV VLPs, and characterized 24 MAbs in detail. These MAbs were classified into group A (MAbs broadly cross-reactive to all GI, GII, GIV and GV strains), and groups B–E (genogroup-specific or genotype-specific MAbs). We also obtained another 27 MAbs specific to particular strains: five to GI.1 Mc114, three to GI.5 Yokote1, five to GII.3 Syd3, five to GIV Syd3, and nine to GV NK24 (data not shown). In addition, 14 MAbs were positive by ELISA but negative by WB. We did not further characterize these 41 MAbs in this study.

Group A MAbs are broadly reactive to all GI, GII, GIV and GV SaV VLPs by both ELISA and WB (Table 1 and Fig. 2), indicating that cross-reactive epitope(s) is/are present on the SaV VP1 in these four genogroups. Based on the competition ELISA, the epitopes recognized by MAbs 5C9 and 1A1 seems to be common to them but different to that of MAb 8127 (Fig. 3). In ELISA, the reactivity of three MAbs (5C9, 1A1 and 5C1) was consistent and strong to GI, GII, GIV and GV VLPs. MAbs 8127 reacted strongly with GI and GV VLPs but weakly with GII and GIV VLPs (Table 1). The different reactivities between different genogroups of SaV VP1 partly supports the possibility of distinct epitopes for MAbs 5C9, 1A1 and MAb 8127.

The X-ray crystallographic structure of human SaV has not been reported, but cryo-electron microscopy has revealed structural similarities between human SaV VLPs and NoV VLPs (8). X-ray crystallographic studies of human NoV VLPs has revealed that NoV VP1 has the following two principal domains: a shell (S) domain, and a protrusion (P) domain that is further divided into three subdomains called N-terminal P1, P2 and C-terminal P1 (36). The NoV S domain and P1 subdomains are highly and moderately conserved, respectively. The P2 domain is highly variable among NoV strains (8, 37), this domain is likely to be the key determinant of strain specificity and antigenicity (37). MAbs broadly cross-reactive with NoV VLPs have been described (31, 34, 38–41), these studies demonstrating that the epitopes are located in the S domain (40, 41) or C-terminal P1 domain (38, 42) in the NoV VP1.

Hyper-immune sera raised against human SaV VLPs has revealed distinct antigenicity among different human SaV genogroups and genotypes (15–17, 20, 21, 23, 43). Genogroup- and genotype-specific MAbs (group B–E MAbs) were also isolated in this study. These MAbs will be useful tools for further study of the antigenic determinant of human SaV. Amino acid sequence homology of VP1 among the 15 SaV strains is 28.1% (data not shown). Despite these significant amino acid sequence variations among different genogroups and genotypes of SaV VP1, the predicted S domain is relatively more conserved than the P2 domains (Fig. 4). From the amino acid sequence alignment, the antigenic determinant is likely to be present in the predicted P2 domain in human SaV, and common epitopes may occur in the predicted S or P1 domains, although further experiments are necessary.

image

Figure 4. Amino acid alignment of nine representative SaV VP1 protein sequences. Predicted amino acid sequences of GI.6 Nichinan, GI.1 Mc114, GI.5 Yokote1, GII.2 Mc10, G II.3 Nayoro4, GII.4 Kumamoto6, GII.7 20072248, GIV Yakumo8 and GV NK24 VP1 are shown. Asterisks indicate conserved amino acids among the 15 SaV strains used in this study. Dots indicate identical amino acid residues. The extents of S and P domains indicated in the above sequences were inferred from published information about NoV VP1 (8).

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In conclusion, we have established a panel of MAbs that are reactive with human SaV VLPs in a broad, genogroup-specific or genotype-specific manner. The broadly reactive MAbs are of particular interest as possible reagents for the development of human SaV detection or diagnostic assays (i.e., ELISA or immunochromatography) in clinical settings, because ELISA using hyperimmune sera shows narrow reactivity to specific genogroups (15–17, 21, 23, 43). Because human SaVs have also been detected in clams (25,44), oysters (45) and environmental water (46–51), broadly reactive MAbs may also become valuable tools for concentrating or removing human SaVs from food or environmental specimens.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

We thank Akira Iwakiri (Miyazaki Prefectural Institute for Public Health and Environment Science), Setsuko Iizuka (Shimane Prefectural Institute for Public Health and Environment Science), Seiya Harada (Kumamoto Prefectural Institute for Public Health and Environment Science) and Setsuko Ishida (Hokkaido Institute of Public Health) for providing SaV positive stool specimens. We also thank to Michiyo Kataoka (Department of Pathology, National Institute of Infectious Diseases) for her excellent assistance with electron microscopy. This work was supported in part by grants for Research on Food Safety, as well as Research on Emerging and Re-emerging Infectious Diseases, from the Ministry of Health, Labour, and Welfare of Japan.

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The authors declare no financial or commercial conflicts of interest.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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