Isolation of a High Affinity Neutralizing Monoclonal Antibody against 2009 Pandemic H1N1 Virus That Binds at the ‘Sa’ Antigenic Site

Abstract

Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs) have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K D = 2.1±0.4 pM) murine MAb, MA2077 that binds specifically to the hemagglutinin (HA) surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC50 = 0.08 µg/ml). MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 µg/ml) against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic) on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein ‘Sa’ and ‘Sb’ sites were independently mutated, localized the binding site of MA2077 within the ‘Sa’ antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning

“…This also indicates that the mutations identified result in precise disruption of the antibody binding site rather than more general structural effects ( Fig 3 ). The use of yeast display to map antibody epitopes in the HA head domain has been described previously [ 42 , 61 ] however the technology has not yet been successfully used to characterise antibodies binding outside the head nor were these studies conducted in combination with deep sequencing. We have used yeast display and deep mutational scanning for the first time to map cross-neutralising antibodies to the stem region and have shown cross-neutralising nanobodies (R1a-A5, R2b-E8, R2b-D9, R1a-B6, R2a-G8) bind to a highly conserved binding pocket.…”

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

“…Competition ELISA between the antisera raised against the test immunogens and the bnAb CR6261 IgG was carried out as described elsewhere (50). Briefly, 96-well half area plates (Corning Incorporated) were coated with 3 μg pandemic H1N1 A/California/04/2009 rHA (Sino Biological Inc.) in 50 μL PBS and kept overnight at 4°C.…”

“…Twenty-five microliters of antisera were added to each well starting at a 1:100 dilution followed by a threefold serial dilution in PBSB. As a control for nonspecific competition, a previously characterized head-specific neutralizing mAb IgG MA2077 was used (50). Twenty-five microliters of mAb MA2077 was added to each well starting at a concentration of 2 mg/mL followed by threefold serial dilutions.…”

Applications of Yeast Surface Display for Protein Engineering

“…Yeast display has also been applied to engineer and affinity mature numerous antibodies, including antibodies against cholera toxin 41 , FITC 22 , HIV-1 gp120 42 , hemagglutinin surface glycoprotein of the H1N1 virus 43 , HER2/neu 44 , T cell receptors 28 , TNF-α 45 , and a host of other targets (see reference 46 for a review). In a classical demonstration of the technological capabilities afforded by yeast display, a fluorescein binding antibody was engineered with a K D equal to 48 fM and a dissociation rate greater than 1,000-fold lower than the parent antibody 30 , representing one of the strongest protein binding interactions ever engineered and among the strongest found in nature.…”

“…Cells displaying weak-binding proteins are then selected and their encoding DNA is sequenced to identify consensus amino acid sites that substantially influence the affinity of the protein pair, and thus, are suggestive of the binding interface location. These strategies have been applied to map the binding epitopes of EGFR-specific antibodies 95,96 and engineered EGFR-specific scaffold proteins 97 , antibodies against H1N1 43 and H5N1 98 virus hemagglutinin surface glycoprotein, gp120-binding antibodies 99 , and other binding pairs as reviewed elsewhere 100,101 .…”