Despite the role of noroviruses as major etiological agents of acute viral gastroenteritis, research on vaccines has historically been hampered by the lack of a permissive cell line or robust animal model (18
). Currently, recombinant VLPs serve as a tool to study the antigenic structure of noroviruses (23
) and are in development as vaccine candidates (13
). Yet there are many unanswered questions regarding the effective design and development of norovirus vaccines, such as the identification of antigenic sites involved in virus neutralization and protection from disease.
Over the last decade, GII.4 noroviruses have been the most common cause of gastroenteritis outbreaks in the United States and Europe (19
). In 2002, the Farmington Hills cluster of GII.4 viruses displaced older variants of GII.4 globally and became an important cluster from an evolutionary and epidemiological perspective (39
). In order to further elucidate the antigenic properties of this genetic cluster, we developed and characterized a panel of six IgG MAbs against the capsid protein of MD2004-3, a Farmington Hills-like strain.
In concordance with evidence that norovirus genogroups present major antigenic differences (20
), none of the MAbs detected VLPs from genogroups GI and GIV. Two (A3 and B15) of the six MAbs were cross-reactive with different GII VLPs, and the remaining four MAbs reacted with one (A10, B11, and B12) or two (A6) GII.4 VLPs. Interestingly, the epitopes of five of the MAbs mapped within the P domain, and that of one (B15) mapped within the S domain. Several cross-reactive MAbs have been described for noroviruses, and epitopes for most of them have been mapped within the most conserved regions of the norovirus capsid protein (C-terminal P or S domain). As suggested previously, cross-reactive MAbs could potentially be used in diagnostic assays (3
); however, Oliver et al. (45
) described a cross-reactive MAb that recognized a linear epitope in the S domain of the VLPs from bovine noroviruses that did not detect native virions in fecal samples from experimental animals. They proposed that because the S domain forms the innermost domain of the capsid, the epitope is not accessible in native virions. In the 5-fold axis of the GII.4 capsid, there is a small surface-exposed region of the S domain that is conserved among GII strains (data not shown). Although it is possible that B15 might recognize this exposed region, it showed low reactivity with native MD145-12 virions in a 10% stool suspension. Together, these results suggest that certain S domain epitopes may not be optimal for detection of small quantities of viruses in stool, even though they are shown to be highly cross-reactive with purified VLPs.
The blocking of norovirus HBGA binding sites by sera from immunized animals or infected humans has been used as a surrogate for a norovirus neutralization assay (26
). Importantly, it was recently shown that the ability of sera to block VLP-HBGA interaction correlates with protection against infection in NV-vaccinated chimpanzees and against illness in infected human volunteers (8
). Five of the six MAbs described here could block the interaction of MD2004-3 VLPs with two synthetic HBGA carbohydrates (H3 and B), and as expected, their recognition epitopes mapped within the P domain. The S domain-specific MAb B15 did not block the interaction of MD2004-3 VLPs with carbohydrate H3, consistent with the likely internal location of its epitope in the capsid. Our unexpected finding that MAb B15 caused partial blocking of MD2004-3 VLPs with carbohydrate B (and not H3) suggests that binding of some antibodies in the S domain might also influence capsid interactions with certain carbohydrates.
Recently, two binding sites for MAbs developed against VLPs from different GII.4 strains were described. One site comprised a cluster of seven residues (i.e., residues 294 to 298, 368, and 372) (11
), while the other included three residues (i.e., residues 407, 412, and 413) (37
). Importantly, these residues were shown to interact with HBGA-blocking MAbs, suggesting that they may be associated with protective immunity (50
). We used the various reactivity patterns of our panel of GII.4 MAbs against the VLPs to identify putative residues involved in determining their specificity and designed MD2004-3 capsids carrying mutations in those residues. Allen et al. (2
) reported that after introduction of point mutations into recombinant GII.4 capsids, they failed to obtain recombinant VLPs for one of the four designed constructs, suggesting that the introduction of certain mutations can be structurally unfavorable for assembly. We showed that several mutant capsids engineered in this study reacted with the conformational MAbs and therefore acquired the correct folding. However, two of the MAbs (A6 and A10) in our study lost their reactivity when residues Ala 294 and Asp 295 were mutated to Gly and Ile, respectively, confirming that residues 294 and 295 are involved in the formation of an important antigenic site (1
). Thus, these data are consistent with the involvement of this epitope in diversification of GII.4 strains and in immune responses against norovirus infection.
The identification and characterization of viral B cell epitopes have been important in establishing the roles of different antigenic sites in protection (5
). Using overlapping fragments of the norovirus capsid protein, Yoda et al. (67
) have shown the presence of three continuous antigenic sites for a GII.3 strain (NV36 virus), with two in the S domain and one in the P domain. Because synthetic short peptides or overlapping fragments of the VP1 protein generally cannot be used to map discontinuous epitopes, competitive ELISAs have been employed widely in the topological mapping of MAbs that recognize conformational epitopes. Thus, using competitive ELISAs, Hale et al. (22
) detected the presence of five antigenic sites in the capsid from NV (GI.1 strain), and the binding domain from one site was determined by mutagenesis analyses (46
). Cross-competition assays using the panel of MAbs described here showed the presence of at least four antigenic sites in the capsid of GII.4 noroviruses, with two of these overlapping (). Using GI/GII chimeric VP1 proteins, we identified the binding domains of the four antigenic sites. Specifically, one unique antigenic site (site I) and two overlapping sites (sites IIa and IIb) were detected in the P domain, while site III was mapped in the S domain. MAbs A6 and A10 share the same antigenic site (IIa), consistent with their loss of reactivity when residues 294 and 295 from the capsid were subjected to mutagenesis. MAbs B11 and B12 recognize overlapping epitopes in the same antigenic site (IIb), although competition assays and reactivity patterns with different VLPs suggest that these MAbs map to different residues.
The ability of the pool of six MAbs to efficiently block the binding of a high-titer hyperimmune serum raised against GII.4 VLPs suggests that the major antigenic sites of the capsid are represented in the MAb panel. Interestingly, when combinations of three MAbs representing each of the three antigenic sites described for the P domain were examined, the blocking effect was similar to that of the pool of six MAbs. Thus, these data suggest that the three antigenic sites described here are likely immunodominant sites of the GII.4 norovirus capsid. It will be important to examine whether serum antibodies from immune individuals block the binding of these MAbs. If so, epitope blocking assays employing these MAbs might serve as a surrogate for virus neutralization.
Three possible mechanisms can be involved in the blocking of the HBGA binding site: (i) direct blocking, (ii) steric interference, or (iii) conformational changes that affect the structure. The fine mapping of MAbs A6 and A10 in conjunction with the fact that the other conformational MAbs, which mapped to different antigenic areas, did not significantly lose their reactivity after incubation with competitor MAbs (i.e., A3 and B15) provides evidence that steric interference is the mechanism by which these MAbs block the HBGA binding site of GII.4 noroviruses. Importantly, MAbs directed against the P2 domain have been shown to neutralize murine norovirus (MNV) infection (30
), and one MNV MAb has been shown to neutralize infection by covering the outer surface of the P2 domain without causing any apparent conformational changes in the capsid protein (30
). Thus, because our GII.4 MAbs bind to multiple epitopes of the P2 domain and block HBGA-VLP interactions, they may prove useful in defining functional antigenic sites of human noroviruses in structural studies.
Norovirus vaccines based on VLP formulations have gained special interest because they have been shown to induce both systemic and mucosal immune responses when administered intranasally or orally (27
). Recently, El-Kamary et al. reported data from phase I clinical trials in which NV VLPs were administered intranasally to humans, with monophosphoryl lipid A (MPL) and chitosan as adjuvants (13
). No vaccine-related serious adverse events were reported, and high IgG and IgA titers developed when 100 μg of VLPs was administered. In addition, Bok et al. (8
) have shown that chimpanzees vaccinated intramuscularly with GI VLPs, but not GII VLPs, were protected against infection with GI Norwalk virus, confirming the idea that multivalent vaccines may be required for protection against currently circulating noroviruses. In this regard, the S/P (S domain from one genogroup and P domain from another) chimeric norovirus VLPs might present the advantage of inducing broadly reactive immune responses and thus may constitute a new platform for norovirus VLP-based vaccines. It has been shown that the CD4+
epitopes are located in different domains of VLPs, depending on the norovirus strain (41
). Thus, even though it is believed that most B cell neutralizing epitopes might be located in the P domain of the capsid, the presence of T cell epitopes in the S domain suggests that vaccination with S/P chimeric VLPs might enhance the immune response and cross-protection against different genogroups.
In summary, we report the generation and characterization of a new panel of MAbs directed toward epitopes on the capsid of GII.4 norovirus, the predominant norovirus genotype. The VP1 domain specificity of each MAb was established with a panel of VLPs and GI/GII chimeric VLPs, and the ability to block HBGA binding corresponded to specificity for the P domain. Modeling and mutagenesis studies established that evolution in the norovirus capsid protein may affect both antigenic and carbohydrate recognition phenotypes, consistent with a link between HBGA blocking and the potential neutralization activity of antibodies. Further characterization of the reagents developed in this study may facilitate the establishment of surrogates of protection against norovirus illness and may provide new approaches to development of candidate vaccines.