Precise characterization of norovirus (Norwalk-like virus)-specific monoclonal antibodies with broad reactivity

Abstract

We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.

FIG. 1.

Purification of recombinant proteins. Fusion proteins were purified from E. coli lysates by using Ni-agarose or Q-Sepharose column chromatography. The purity of each stretch was analyzed by SDS-PAGE. Lane 1, marker; lane 2, TRX (1 μg); lanes 3 to 14, recombinant fusion stretches S9 (1 μg), S10 (1 μg), S11 (0.5 μg), S12 (0.8 μg), S13 (0.8 μg), S14 (0.6 μg), S15 (1 μg), S16 (1 μg), S17 (1 μg), A1 (0.8 μg), A2 (0.8 μg), and A3 (1 μg), respectively.

FIG.2.

(a) Reactivities of MAbs toward three 21-amino-acid stretches. The reactivities of MAbs 1B4 and 1F6 against fragments F2-1, F2-2, and F2-3 were measured by ELISA and are expressed as percentages. The optical density (OD) of TRX was subtracted from that of each sample. The OD of rNV36 was considered to be 100%, and the percentage of reactivity was calculated as (OD of sample) × 100/(OD of rNV36). (b) Relationship of the 40-amino-acid fragment 2 with the three 21-amino-acid stretches used in the ELISA for which results are shown in panel a. The underlined amino acid sequences are the unique part of fragment 2 (other parts overlap with proximal fragments). (c) Comparison of the reactivities of MAbs 1B4 and 1F6 toward various rNV capsid proteins. Under “Category” on the left, “Epitope” indicates that the 11-amino-acid stretch S1, which is part of the rNV36 whole capsid protein, is the epitope of the two MAbs. NV36 (MX) had been used for generating these two MAbs, so it was used as a positive control in this experiment. Modified stretches (S2 to S6) were constructed by slight modifications from the original data available in GenBank. GI proteins (S7 to S12) were constructed based on GenBank data for GI type NV strains. Recombinant fragment 00-013 (80 amino acid residues) was constructed from the NV clinical isolate 00-013. GII proteins (S13 to S17) were constructed based on GenBank data for GII type NV strains. A1 is a 5-amino-acid stretch that is conserved among NV strains. Artificial stretches (A2 and A3) are artificially constructed fragments that are supposed to have a conformation completely different from those of clinical NV strains. Representative strain names, where available, are given, as are amino acid sequences of the positions equivalent to those of S1 in each peptide. The reactivities of 1B4 and 1F6 were examined by ELISA, and the strength of each reaction was expressed as a percentage as described for panel a. Accession numbers of the NV strains listed in this figure are as follows: for KU8GI, AB067547; for Desert Shield (DS), U04469; for Stav/95/Nor, AF145709; for BS5, AF093797; for Southampton (SOU), L07418; for KU24aGI, AB067549; for KU4bGI, AB058536; for Camberwell, AF145896; for Hawaii, U07611; for KU17GII, AB058556; for KU44GII, AB058581; and for Alphatoron98, AF195847.

FIG. 3.

(a) Comparison of the reactivities of MAbs 1B4 and 1F6 toward various rNV capsid proteins. Reactivities were measured by ELISA and are expressed as percentages. The optical density (OD) of TRX was subtracted from that of each sample. The OD of rNV36 was considered to be 100%, and the percentage of reactivity was calculated as (OD of sample) × 100/(OD of rNV36). Strain types MX, LD, Amsterdam, SMA, and Aichi'76 are categorized as GII, while types NV68 and CV are categorized as GI. (b) Sequence alignment of the various rNV capsid proteins used in the ELISA for which results are shown in panel a. Amino acid residues corresponding to fragment 2 are given at the top. The epitope of the two MAbs (QQNIIDPWIMN; boxed) is within fragment 2 in NV36. The amino acid residues located at both edges of the box are considered to be recognition stretches of these two MAbs. Accession numbers of strains in this figure are as follows: Mexico (MX), U22498; Lorsdale (LD), X86557; Amsterdam98, AF195848; SMA, U70059; Gifu'96, AB045603; NV68, NC001959; Chiba (CV), AB022679.