Characterization of Novel Cross-Reactive Influenza B Virus Hemagglutinin Head Specific Antibodies That Lack Hemagglutination Inhibition Activity

Abstract

Humoral immune responses to influenza virus vaccines in elderly individuals are poorly adapted toward new antigenically drifted influenza virus strains. Instead, older individuals respond in an original antigenic sin fashion and produce much more cross-reactive but less potent antibodies. Here, we investigated four influenza B virus hemagglutinin (HA) head specific, hemagglutination inhibition-inactive monoclonal antibodies (MAbs) from elderly individuals. We found that they were broadly reactive within the B/Victoria/2/1987-like lineage, and two were highly cross-reactive with B/Yamagata/16/1988-like lineage viruses. The MAbs were found to be neutralizing, to utilize Fc effector functions, and to be protective against lethal viral challenge in a mouse model. In order to identify residues on the influenza B virus hemagglutinin interacting with the MAbs, we generated escape mutant viruses. Interestingly, escape from these MAbs led to numerous HA mutations within the head domain, including in the defined antigenic sites. We observed that each individual escape mutant virus was able to avoid neutralization by its respective MAb along with other MAbs in the panel, although in many cases binding activity was maintained. Point mutant viruses indicated that K90 is critical for the neutralization of two MAbs, while escape from the other two MAbs required a combination of mutations in the hemagglutinin. Three of four escape mutant viruses had increased lethality in the DBA2/J mouse model. Our work indicates that these cross-reactive antibodies have the potential to cause antigenic drift in the viral population by driving mutations that increase virus fitness. However, binding activity and cross-neutralization were maintained by a majority of antibodies in the panel, suggesting that this drift may not lead to escape from antibody-mediated protection.IMPORTANCE Understanding the immune response that older individuals mount to influenza virus vaccination and infection is critical in order to design better vaccines for this age group. Here, we show that older individuals make broadly neutralizing antibodies that have no hemagglutination-inhibiting activity and are less potent than strain-specific antibodies. These antibodies could drive viral escape from neutralization but did not result in escape from binding. Given their different mechanisms of action, they might retain protective activity even against escape variants.

Keywords: hemagglutination; influenza B; monoclonal antibodies.

FIG 1

HI-inactive MAbs broadly bind B/Victoria and B/Yamagata lineage viruses. (A) A maximum likelihood tree composed of approximately 200 influenza B virus HA protein sequences from both the B/Victoria/2/1987-like (green) and B/Yamagata/16/1988-like (purple) lineages was built. Viruses used in ELISAs are labeled. (B) Minimum binding concentrations of each MAb for B/Victoria/2/1987-like (V) and B/Yamagata/16/1988-like (Y) lineage viruses.

FIG 2

HI-inactive MAbs are neutralizing and active in an ADCC reporter assay. HI-inactive MAbs were characterized using vaccine strains B/Malaysia/2506/2004 and B/Brisbane/60/2008. (A) Neutralization curves for each MAb as determined by a plaque reduction assay (n = 2). (B) IC₅₀ values for plaque reduction assays of each MAb. (C) ADCC reporter activity curves for each MAb (n = 2). MAb CR9114 was used as a positive control. (D) AUC values for ADCC reporter activity.

FIG 3

Antibodies show ranges in protectiveness against lethal challenge in the DBA2/J mouse model. DBA2/J mice (n = 5) were given 3 mg/kg of each MAb 3 h prior to a lethal challenge with B/Malaysia/2506/2004. Data are shown in red for mice vaccinated with AG13-3F04, in green for AG5-E04, in orange for AG10-A05, in blue for AG13-3C02, and in gray for the irrelevant IgG control MAb. (A) The percentage of initial body weight was measured daily for 14 days. (B) Survival curves.

FIG 4

Characterization of MAb EMVs. (A) Location of each HA mutation on the structure of B/Brisbane/60/2008 (PDB ID 4FQM) (6), visualized using PyMOL software. The major antigenic sites of the head domain—the 120 loop (blue), the 150 loop (green), the 160 loop (teal), and the 190 helix (red)—are indicated on the structure (30). Mutations K90E, G164E, and T214A are located within antigenic sites. (B) (Top) Immunofluorescence of MAb binding to EMVs. MDCK cells were infected at an MOI of 1 and were stained using 50 μg of the antibody indicated at the top. Images are representative of binding from two IF experiments. (Bottom) A corresponding heat map describes the percentage of luminescence for each image (with luminescence for B/Brisbane/60/2008 set to 100%). (C) The neutralization of each EMV by each MAb was determined using a plaque reduction assay. The fold change was calculated by comparing the IC₅₀ value for each EMV to that for the wild-type virus B/Brisbane/60/2008. Higher fold changes indicate stronger escape phenotypes. The maximum concentration of MAb used to determine IC₅₀ values was 100 μg/ml.

FIG 5

Positive selection and conservation of B/Victoria/2/1987-like viruses. (A) Selection values (ω) for each residue of the influenza B virus HA head domain. Amino acid positions (residues 57 to 308) are listed along the x axis, while ω values are on the y axis. Residues mutated in EMVs are marked with a red letter “X.” (B) Sequence logo (created by WebLogo) illustrating the amino acid substitutions at each residue of the HA head domain. Residues where escape mutations were present are boxed in red.

FIG 6

Impact of HA point mutations on MAb neutralization. The neutralization of rescued point mutant viruses by each HI-inactive MAb was determined by a plaque reduction assay (n = 2). The fold change was determined by comparing the IC₅₀ value for each rescued mutant virus to that of the rescued virus carrying wild-type B/Brisbane/60/2008 HA. Higher fold changes indicate stronger escape phenotypes.