Self-assembly of the recombinant capsid protein of a swine norovirus into virus-like particles and evaluation of monoclonal antibodies cross-reactive with a human strain from genogroup II

Abstract

Noroviruses (NoVs) are responsible for the majority of gastroenteritis outbreaks in humans. Recently, NoV strains which are genetically closely related to human genogroup II (GII) NoVs have been detected in fecal specimens from swine. These findings have raised concern about the possible role of pigs as reservoirs for NoVs that could infect humans. To better understand the epidemiology of swine NoVs in both the swine and the human populations, rapid immunoassays are needed. In this study, baculovirus recombinants were generated to express the capsid gene of a swine NoV GII genotype 11 (GII.11) strain which self-assembled into virus-like particles (VLPs). Subsequently, the purified VLPs were used to evoke monoclonal antibodies (MAbs) in mice. A panel of eight promising MAbs was obtained and evaluated for their ability to bind to heterologous VLPs, denaturated antigens, and truncated capsid proteins. The MAbs could be classified into two groups: two MAbs that recognized linear epitopes located at the amino-terminal half (shell domain) of the swine NoV GII.11 VLPs and that cross-reacted with human GII.4 NoV VLPs. The other six MAbs bound to conformational epitopes and did not cross-react with the human GII.4 VLPs. To our knowledge, this is the first report on the characterization of MAbs against swine NoVs. The swine NoV VLPs and the MAbs described here may be further used for the design of diagnostic reagents that could help increase our knowledge of the prevalence of NoV infections in pigs and the possible role of pigs as reservoirs for NoVs.

FIG. 1.

Expression of swine NoV VLPs. (A) H5 cells were infected with wt or SwNV1 recombinant baculovirus, and equal amounts of infected cell lysates were analyzed by SDS-10% PAGE and Coomassie blue staining. Molecular weight markers (MW; in 10³ Da) are given on the left. (B) Electron microscopy of negatively stained purified swine NoV VLPs. Purified particles were analyzed for protein content by SDS-10% PAGE and stained with Coomassie brilliant blue (inset). Scale bar, 100 nm.

FIG. 2.

(A) Reactivities of the MAbs with SwVA34 and human GII.4 (HuGII.4) NoV VLPs. The wells of ELISA plates were coated with equal amounts of purified SwVA34 or human GII.4 VLPs, and serial dilutions (threefold) of the eight hybridoma cell culture supernatants were used to detect antigens (OD, optical density). (B) Reactivities by Western blotting of MAbs SwNV3A10 and SwNV6E11 against SwVA34 and human GII.4 capsid proteins. Purified VLPs were resolved by SDS-10% PAGE and transferred to membranes which were reacted with the indicated hybridoma cell culture supernatants. Molecular weight markers (MW; in 10³ Da) are given on the left.

FIG. 3.

(A) Analysis of MAb SwNV3A10 binding to the fragments of VP1. H5 cells were infected with recombinant baculoviruses expressing the swine NoV full-length VP1 or the protein domains: N/S, P, and P2. Equal amounts of infected cell lysates were analyzed by SDS-12% PAGE and Coomassie blue staining. Arrows indicate the positions of the recombinant polypeptides N/S and P2. Molecular weight markers (MW; in 10³ Da) are given on the left. (B) Reactivity in Western blots of MAb SwNV3A10 against the VP1 fragments. Infected cell lysates were resolved by SDS-12% PAGE and transferred to membranes, which were reacted with the hybridoma cell culture supernatant of MAb SwNV3A10. Molecular weight markers (MW; in 10³ Da) are given on the left.

FIG. 4.

Reactivities of MAbs SwNV3A10 and SwNV2C1 by flow cytometry. H5 insect cells infected with recombinant baculoviruses expressing the indicated NoV capsid proteins were incubated with hybridoma cell culture supernatants plus a R-phycoerythrin-conjugated anti-mouse antibody and analyzed by flow cytometry. The dashed line corresponds to the results for cells infected with wt baculovirus. HuGII.4, human GII.4.