Antigenic Mapping of the Recombinant Norwalk Virus Capsid Protein Using Monoclonal Antibodies

Abstract

Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV capsid protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the capsid protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K capsid protein, suggesting that the C-terminal half of the capsid protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the capsid protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus capsid proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.

Characterization of Monoclonal Antibodies Generated against Norwalk virus Gil Capsid Protein Expressed in Escherichia coli

“…From these findings, the following 2 points can be concluded: (i) MAbs against self-assembled rVLPs tend to recognize discontinuous epitopes, while MAbs against soluble rNV protein recognize continuous epitopes and (ii) major epitopes of MAbs against rVLPs are located mainly at the C-terminal domain, while those of MAbs against soluble rNV are located mainly at the N-terminal domain. The epitope distribution of MAbs raised against rVLPs was analyzed for the N-terminal domain (NV68 residues 1-227) and C-terminal domain (NV68 residues 228-530) (7). A detailed analysis of each MAb reaction region should be performed for their MAbs and compared with the present findings.…”

High Efficiency Cross‐Reactive Monoclonal Antibody Production by Oral Immunization with Recombinant Norwalk Virus‐Like Particles

“…Cross-reactivities of the MAbs with a variety of VLPs were tested by using VLPs-coated ELISAs as described previously (11). A single dose of rNV VLPs used for oral immunization produced broadly crossreactive MAbs not only against genogroup I but also 885 genogroup II VLPs (Table 4).…”

“…P3 or PAI myeloma cells were cultured in Dulbecco's Modified Eagles' Medium (DMEM) in the presence of 15% fetal calf serum (FCS). In experiment I, splenocytes(1-2ϫ10 6 cells/ml) were fused with P3 or PAI cells at a ratio of 2:1 as described previously (11). After cloning in medium supplemented with hypoxanthine-aminopterinthymidine, the cells were cultured with DMEM containing 15% FCS.…”

“…In order to confirm cross-reactivity of the MAbs, ELISA assays were performed as previously described (11). Briefly, both genogroup I and genogroup II VLPs including rNV, Seto 124 (r124), Chiba 407 (rCV) and Funabashi 258 (r258) for genogroup I, Snow Mountain agent (rSMA), Glimsby (rGV), rMX, Kashiwa 47 (r47), Narita 104 (r104), Chitta 76 (r76) and Ueno 7K (r7K) for genogroup II and rSapovirus were coated using the same procedures as above.…”

“…Although the resulting MAbs were useful to characterize the native virus, they only recognized closely-related Norwalk like virus (NLV) strains (11), and the yields of MAbs were low. NV is naturally spread by the fecaloral route and sera from adults cross-react by ELISA with many NoVs in the Caliciviridae family (27), and rNV VLPs have been shown to induce mucosal and systemic antibody responses when delivered orally to mice or volunteers without adjuvant (2,3,9).…”