Infant antibody and B-cell responses following confirmed pediatric GII.17 norovirus infections functionally distinguish GII.17 genetic clusters

Figures

• 

  Figure 1. Co-circulation of antigenically distinct GII.17 clusters in young children. (A).Phylogenic analysis of the N-terminal and shell region of ORF2 (252 bp) of seven norovirus-positive AGE stools from the cohort and known isolates. (B) Phylogenetic analysis for full capsid sequence (1,629 bp) from GII.17 cluster I (ID_132) or cluster IIIb (ID_263) infections. (C) P2 domain amino acid alignment for ID_132 and ID_263 sequences against cluster I, II, IIIa, and IIIb reference sequences (Genbank IDs indicated). Amino acid identity represented by dots (·) and deletions by dashes (–). (D) Serum IgG against GII.17 VLPs representing clusters I, II, and IIIb were measured by EIA pre– and post–GII.17 infection in seven infants with symptomatic GII.17 infections. Fold increases pre- versus post-infection are presented. Dashed line, no change in titer. (E) Serum blockade antibody titers against cluster I and IIIb GII.17 VLPs were measured pre– and post–GII.17 infection in seven children with symptomatic GII.17 infections. Fold increases preversus post-infection are presented. Dashed line, no change in titer.

• 

  Figure 2. GII.17 blockade antibody responses in young children are genotype-specific. (A) The fold change between blockade antibody titers before and after GII.17 infection was determined for GII.17, GI.3, GII.2, GII.4 Sydney, GII.6, and GII.12. For each donor, VLP for the GII.17 cluster with the highest post-infection blockade titer was used in the assay. Marker, donor; bar, GMT; error bars, 95% confidence intervals; dashed line, no change in titer; **, P < 0.001 relative to heterotypic responses by Dunn’s multiple comparisons test. (B) Correlation analysis of GII.17 titer compared with titers to other genotypes: Spearman correlation, P > 0.05 comparing GII.17 titers to those for each heterotypic genotype. Dashed line, limit of detection.

• 

  Figure 3. Characterization of NVA.1, a human mAb to a variant-specific blockade antibody epitope of GII.17 cluster IIIb. (A) Memory B cells (MBCs) from donor 434 were immortalized and cultured with IL-21 and CD40L-L cells as polyclonal 50 cell per well cultures in 60 wells on each of three plates for 3 weeks and screened for IgG/IgA binding to GII.17, GII.4 2012 Sydney, and GII.12 VLPs by EIA. The assay positive cutoff was optical density (OD) ≥ 0.5. Positive controls were normal human serum (NHS), and negative controls included secondary Abs alone and tissue culture medium. (B) Supernatants from six polyclonal GII.17–positive MBC cultures (plate and well indicated, e.g., 1C6 is plate 1, well C6) were tested across a dilution range for blockade activity against cluster IIIb GII.17 VLP binding to pig gastric mucin (PGM). The inverse of the dilution to achieve ID50 dilution is indicated in the legend. From each of these polyclonal cultures, monoclonal B cells were sorted, grown, and retested for GII.17-binding activity. Paired IGH/IGL sequencing from GII.17-binding monoclonals revealed multiple copies of an IgA clone (named NVA.1) and an IgG clone (NVG.1) originating from a polyclonal…

• 

  Figure 4. NVA.1-like cluster IIIb–specific antibodies are a dominant component of the serum antibody response to GII.17 cluster IIIb following infection. Plates coated with GII.17 cluster IIIb VLP were incubated with decreasing concentrations of NVA.1 (IgA) or NVA.1G (IgG) mAb before addition of sera from cluster IIIb–infected children with blockade activity. Sera was added at the blockade ID50 titer, and bound serum IgA (, gray symbols) or IgG (, blue symbols) was detected by anti-human IgA or anti-human IgG and the lowest concentration of mAb that inhibited at least 50% of serum binding compared with that of no added mAb was determined. Full binding curves are shown in Figure S6 . ND, not determined on the basis of low serum binding of IgA at the ID50. Dashed line, assay cutoff for BOB activity. Data represent the average of three independent experiments.

• 

  Figure 5. GII.17-cluster IIIb blocking serum and mAbs exhibit neutralization activity in human intestinal enteroid cultures. Single-dose neutralization of GII.17 cluster IIIb in HIE. Each sample was tested at a single concentration (serum diluted 1:100 and mAb at ~1 µg/mL) against a fixed virus concentration (100 TCID50). Virus control (no antibody) was included. Norovirus replication was assessed by RT-qPCR at 3 days post-infection (dpi). Results are expressed as genomic copies per well.