Fluorescence-barcoded cell lines stably expressing membrane-anchored influenza neuraminidases

Abstract

The discovery of broadly protective antibodies to the influenza virus neuraminidase (NA) has raised interest in NA as a vaccine target. However, recombinant, solubilized tetrameric NA ectodomains are often challenging to express and isolate, hindering the study of anti-NA humoral responses. To address this obstacle, we established a panel of 22 non-adherent cell lines stably expressing native, historical N1, N2, N3, N9, and NB NAs anchored on the cell surface. The cell lines are barcoded with fluorescent proteins, enabling high-throughput, 16-plex analyses of antibody binding with commonly available flow cytometers. The cell lines were at least as efficient as a Luminex multiplex binding assay at identifying NA antibodies from a library of unselected clonal IgGs derived from human memory B cells. The cell lines were also useful for measuring the magnitude and breadth of the serum antibody response elicited by experimental infection of rhesus macaques with influenza virus. The membrane-anchored NAs are catalytically active and are compatible with established sialidase activity assays. NA-expressing K530 cell lines therefore represent a useful tool for studying NA immunity and evaluating influenza vaccine efficacy.

Keywords: Barcode; Barcoding; Binding assays; ELLA; Flow cytometry; Fluorescent protein; Immunoassay; NA-Star assay; Sialidase; Vaccine.

FIG 1.. Standard NA mAbs brightly and specifically label K530-NA cell lines.

Shown are flow cytometry histograms depicting the binding of recombinant IgG versions of standard NA mAbs to K530 cell lines expressing membrane-anchored NAs. Each row corresponds to a monoclonal cell line stably expressing a single type of NA. Each column corresponds to the binding profile for a single mAb. MAbs were incubated with pooled cell lines comprising Option 1 or Option 2, and the resultant data were concatenated into a single figure.

FIG 2.. K530-NA cell lines perform as well as a Luminex assay for identifying NA mAbs.

A) Luminex assay results showing the median fluorescence intensity (MFI) of antigen binding for clonal IgG-containing culture supernatants from Bmem isolated from donor T3. Bovine serum albumin (BSA), insulin, keyhole limpet hemocyanin (KLH), chicken ovalbumin (OVA), and tetanus toxoid (TT) serve as binding-specificity controls. Short, red, horizontal lines in each column denote the limit of detection (LOD), which was calculated as six standard deviations above the mean signal produced by cultures containing no B cells. B) Results of a high-throughput, flow cytometry-based screen using K530-NA cell lines. Each symbol depicts the geometric mean fluorescence intensity (geoMFI) of clonal IgG binding to a K530-NA cell line. The dashed, horizontal line denotes the threshold for binding. Each symbol in (A) and (B) represents a single, clonal IgG-containing culture supernatant. Clonal IgGs of interest are denoted with uniquely colored symbols.

FIG 3.. Use of K530-NA cells to determine the binding breadth of newly identified NA mAbs.

Shown are flow cytometry histograms depicting the binding of recombinant IgG versions of NA mAbs from donors T1, T2, or T3 to K530 cell lines expressing membrane-anchored NAs, as in Fig. 1.

FIG 4.. Analysis of serum IgGs elicited by infection of rhesus macaques with H3N2 influenza virus.

A) Pre-immune (day 0) and immune (day 27 or 28) blood plasma from three rhesus macaques (6451, T651, T771) infected with A/Aichi/2/1968 (H3N2) influenza virus was incubated with K530-NA cell lines. The degree of IgG labeling of each cell line was determined by flow cytometry, as in Fig. 1. Vertical, black lines denote the threshold for specific labeling of cell-surface NA, determined according to the labeling intensity observed for K530 cells expressing no NA (top row). B) Fold-change in the fluorescence intensity of plasma IgG labeling of selected K530-N2 cell lines, after either infection with H3N2 virus (Fig. 4A) or immunization with recombinant H3 HA (Supplemental Fig. 3B). Fold-change was calculated as the ratio of the geometric mean fluorescence intensity (geoMFI) resulting from labeling with the immune plasma IgG, divided by the geoMFI resulting from labeling with pre-immune plasma IgG. Each symbol represents a single animal. The difference in group means was analyzed by two-tailed t-test with Welch’s correction, as described in Materials and Methods.

FIG. 5.. NA catalytic activity of K530-NA cell lines.

A) The NA-Star assay was used to determine the sialidase activity of selected K530-NA cell lines in the presence of serially diluted, NA-binding or control rIgGs. B) The enzyme-linked lectin assay (ELLA) was used to measure the sialidase activity of selected K530-NA cell lines in the presence of serially diluted rIgGs. Error bars represent mean ± S.D.