Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Neutralizing antibodies that have HI activity are further analyzed by first preparing 4 dilutions of the antibody of interest in increasing concentrations in 1 times PBS in a volume of 100 microliters per dilution. Next, prepare a virus stock of 1 million plaque-forming units per milliliter in 1 times PBS in a 400 microliter volume.

Then, mix 100 microliters of 1 million plaque-forming units per milliliter of virus with 100 microliters of each antibody dilution or 100 microliters of 1 times PBS. Incubate the samples for one hour in a 37 degrees Celsius incubator with 5% carbon dioxide.

After vortexing briefly, inject 200 microliters of each mixture into specific pathogen-free embryonated chicken eggs. Then, incubate the eggs at 37 degrees Celsius without carbon dioxide for 40 to 44 hours. Following incubation, sacrifice the virus-infected infected embryonated eggs by placing them at 4 degrees Celsius for a minimum of 6 hours. Next, harvest the allantoic fluid from the eggs as previously described.

Finally, confirm the escape variants by performing the HI assay as described in the text protocol.