Frequent Use of the IgA Isotype in Human B Cells Encoding Potent Norovirus-Specific Monoclonal Antibodies That Block HBGA Binding

Abstract

Noroviruses (NoV) are the most common cause of non-bacterial acute gastroenteritis and cause local outbreaks of illness, especially in confined situations. Despite being identified four decades ago, the correlates of protection against norovirus gastroenteritis are still being elucidated. Recent studies have shown an association of protection with NoV-specific serum histo-blood group antigen-blocking antibody and with serum IgA in patients vaccinated with NoV VLPs. Here, we describe the isolation and characterization of human monoclonal IgG and IgA antibodies against a GI.I NoV, Norwalk virus (NV). A higher proportion of the IgA antibodies blocked NV VLP binding to glycans than did IgG antibodies. We generated isotype-switched variants of IgG and IgA antibodies to study the effects of the constant domain on blocking and binding activities. The IgA form of antibodies appears to be more potent than the IgG form in blocking norovirus binding to histo-blood group antigens. These studies suggest a unique role for IgA antibodies in protection from NoV infections by blocking attachment to cell receptors.

Fig 1. Screening supernatants of EBV-transformed B cell cultures from two NoV-challenged subjects.

B cell culture supernatants were added to replicate microtiter plates coated with NoV VLP and probed with a mixture of (i) a mixture of anti-human (κ + λ; to determine the total number of binders), or (ii) anti-human IgG (γ-specific; to determine IgG frequency), or (iii) anti-human IgA (α-specific; to determine the IgA frequency) secondary antibodies. Blocking assay was done as described in Methods. The number of binding (A450 >1.5) and blocking (A450 <2.1) were counted and percent distribution among binders and blockers was calculated. Distribution of IgG (red) or IgA (blue) classes of antibodies that bound to NoV VLP (A) or blocked VLP—glycan interaction (B) is shown.

Fig 2. Binding and blocking characteristics of purified monoclonal antibodies.

Purified IgG (red) or IgA (blue) antibodies were tested for binding to NV VLP in ELISA (A) or for blocking VLP—glycan interaction (B). Each of the IgG antibodies bound to VLPs with lower EC₅₀ values than IgA antibodies, while in contrast the concentrations needed for blocking were similar for IgG and IgA. The blocking of murine mAb 8812 is shown in black.

Fig 3. Specificity of human mAbs.

The binding (mean absorbance at 450 nm ± SD) of purified mAbs at 20 μg/mL to VLPs representing homologous virus (NoV GI.I) or heterologous human NoVs of different genotypes (A) or antigens representing wild-type or mutant recombinant capsid proteins of homologous virus (B) were assessed by ELISA to evaluate genotype specificity and to infer the subdomain of major capsid protein bound by anti-norovirus mAbs. The data shown in each figure summarizes the results from 2 independent experiments. (C) The ligand specificity of mAb-mediated inhibition of NoV VLP binding to a panel of its glycan ligands (H1, H2, H3, tri-A or Le(y)) was evaluated in HBGA blocking assay for three mAbs, 2L8, 3I23 and 5I2.

Fig 4. Nature of epitopes recognized by anti-norovirus mAbs.

Norovirus VLPs were resolved on SDS-PAGE gels under (A) nonreducing, nondenaturing, or (B) reducing, denaturing conditions and the membranes were probed with anti-norovirus mAbs. All the human antibodies, and the murine mAb 8812, bound to conformational epitopes, while denatured VLP were bound only by mAb 3901. Arrowhead in panel B indicates VP1. (C) Antibodies were binned into competition-binding groups in ELISA as described in Methods. Most of the antibodies seem to compete for the same or spatially proximate epitopes. The asymmetric nature of competition suggests subtle factors such as the angle of approach of the antibodies seem to have an effect on competition.