Immunogenetic Mechanisms Driving Norovirus GII.4 Antigenic Variation

Abstract

Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs) characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987–1997), contemporary (2004–2009), and broad (1987–2009). NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294–298 and 368–372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393–395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing epitopes and consequently, antibody-driven receptor switching; thus, protective herd immunity is a driving force in norovirus molecular evolution.

Human Sera Collected between 1979 and 2010 Possess Blocking-Antibody Titers to Pandemic GII.4 Noroviruses Isolated over Three Decades

“…Most interesting from a biological point of view, and perhaps explaining the observed differences in blocking titers found in this study, the alignment also revealed that the GII.4 NoV strains used in this study have major changes in antigenic epitopes A (amino acids [aa] 294, 296 to 298, 368, and 372), D (aa 393 to 395), and E (aa 407, 412, and 413), as shown in Table 3 (6,7,18,(37)(38)(39). Previous studies have reported epitope A to be highly immunodominant and suggested it was the target of blocking antibodies against early strains (7,38).…”

“…Moreover, there are no amino acid differences between the two strains in site E (Table 3). Short-lived NoV immunity following experimental challenge (15,16), together with an epochal GII.4 evolution pattern, indicates NoV immunity is more complex (17)(18)(19). How long immunity against symptomatic norovirus infection lasts is a question under debate.…”

“…VLP-PGM serum blocking experiments were performed essentially as previously described (18). Briefly, Thermo Scientific MaxiSorp 96-well plates were coated with pig gastric mucin (Sigma-Aldrich; 10 g/ml in PBS, pH 7.3) for 4 h at room temperature and subsequently blocked overnight at 4°C with PBS containing 5% dry milk and 0.05% Tween.…”

Rhesus enteric calicivirus surrogate model for human norovirus gastroenteritis

“…Based on studies with G2.4 human NoVs it was suggested that antigenic changes facilitating escape from herd immunity may also drive changes in HBGA binding affinities and lead to altered population susceptibility Lindesmith et al, 2012). To model this hypothesis we recently generated neutralization escape mutants by repeatedly subculturing ReCVs under constant serotype-specific polyclonal neutralizing antibody pressure (unpublished data).…”

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

“…A number of other studies have identified norovirus-specific monoclonal antibodies (mAbs) and single chain variable domains (VHH or Nanobodies) that could block norovirus VLP binding to HBGAs [20,[23][24][25][26][27]. However, most of these antibodies and Nanobodies are genotype specific, which limits their therapeutic potential [28].…”