Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Abstract

Source: Leon, P. E., et al. Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies. J. Vis. Exp. (2017).

This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

Protocol

NOTE: HA-specific antibodies that inhibit viral replication can generally be categorized into i) ones that bind on or adjacent of the receptor binding site on top of the globular head and ii) ones that bind distal of the receptor binding domain, which includes the lateral side of the globular head and the stalk region of the HA. Antibodies that target the receptor binding site prevent the engagement of sialic acid motifs on the surface of target cells and can be measured using a hemagglutination inhibiti…

Representative Results

Figure 1: HI assay. (A) A schematic for the setting up a HI assay to test the activity of two mouse H1-specific mAbs 7B2 (head-specific) and 6F12 (stalk-specific) using a 96-well V-bottom plate, and (B) an example of the results of an HI assay <img alt="Figure 2" src="/files/ftp_upload/22085/22083_Figur…

Materials

Falcon 96-well clear V-bottom plate	Corning, Inc.	353263	Assay plate use for the hemagglutination inhibition assay
96-well V-bottom plate	Nunc	249662	Assay plate used for the hemagglutination assay
Chicken red blood cells	Lampire Biological Laboratories	7201403	Used to assess the ability of influenza virus to agglutinate
reassortant A/California/04/09 (H1)	Palese Laboratory		reassortant virus expressing the HA and NA of A/California/04/09 (H1N1) with the internal segments of A/Puerto Rico/8/34 (H1N1)
reassortant A/Shanghai/1/13 (H7)	Palese Laboratory		reassortant virus expressing the HA and NA of A/Shanghai/1/13 (H7N9) with the internal segments of A/Puerto Rico/8/34 (H1N1)