Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

The overall goal of this method is to provide a working protocol on how to elucidate the epitope of a neutralizing monoclonal antibody. This method can help answer key questions in immunology and virology fields that can help define host virus interactions, develop therapeutics and diagnostic tools, all of which can aid in the design of vaccines. The main advantage of this technique is that is can be performed with basic tissue culture and molecular biology techniques without special instruments or equipment.

Demonstrating the procedure will be Mark Bailey, an MD, PhD student from Peter Palese's laboratory. Begin this procedure with characterization of antibodies based on hemoagglutination inhibition, or HI, and neutralization activities as described in the text protocol. Neutralizing antibodies that have HI activity are further analyzed by first preparing four dilutions of the antibody of interest in increasing concentrations in one times PBS in a volume of 100 microliters per dilution.

Next, prepare a virus stock of one million plaque-forming units per milliliter in one times PBS in a 400-microliter volume. Then, mix 100 microliters of one million plaque-forming units per milliliter of virus with 100 microliters of each antibody dilution or 100 microliters of one 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 the incubation, sacrifice the virus-infected embryonated eggs by placing them at four degrees Celsius for a minimum of six 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. To generate escape variants against neutralizing antibodies that lack HI activity, the virus must be passaged in the presence of increasing amounts of antibody.

Plate MDCK cells in a six-well plate at a density of one million cells per well. Incubate the cells for a minimum of four hours in a 37-degrees-Celsius incubator with 5%carbon dioxide. Meanwhile, dilute the virus stock to one million plaque-forming units per milliliter.

Prepare a single dilution of antibody at 0.02 milligrams per milliliter in one times MEM with TPCK-treated trypsin in a 250-microliter volume. Use higher antibody concentrations for all following passages. Mix 250 microliters of diluted virus with 250 microliters of diluted antibody, or 250 microliters of one times MEM as the no-antibody control.

Incubate the virus antibody mixture for 30 minutes in a 37-degrees-Celsius incubator with 5%carbon dioxide. Following incubation of the cells, aspirate the media using a glass Pasteur pipette and wash the monolayer of cells with one milliliter of one times PBS. Add 500 microliters of the mixtures into the wells before incubating in a 37-degrees-Celsius incubator with 5%carbon dioxide for one hour.

After one hour, supplement the wells with two milliliters of one times MEM with TPCK-treated trypsin. Check the cells at 48 hours post-infection for signs of the cytopathic effect, or CPE, on the microscope, or perform a hemoagglutination assay to detect a viral growth. If there is growth CPE in the culture supplemented with antibody, harvest the supernatant in multiple cryotubes.

Label the tubes with the passage number and store them at minus 80 degrees Celsius. Save 100 microliters of the supernatant to infect a fresh monolayer of MDCKs with two milliliters of one times MEM supplemented with TPCK trypsin and antibody. Remember to include a no-antibody control for every passage.

Increase the concentration of the antibody in each successive passage until virus growth is still viable, even with the final concentration of 0.6 milligrams per milliliter of antibody. Freeze the multiple vials of supernatant of each passage and store at minus 80 degrees Celsius. Continue to the isolation and analyses of the escaped variants as detailed in the text protocol.

Vaccine-induced antibodies isolated from individuals vaccinated with the candidate H7N9 influenza A vaccine were used to generate escape mutant variants. Escape mutant mapping revealed that many of the antibodies recognized critical residues in distinct locations on the viral HA.Each residue, indicated in red, represents the location of critical amino acids required for efficient binding of a monoclonal antibody, while the majority of the HI-positive antibodies have escaped mutant residues near previously reported antigenic sites of the H7HA. The HI-negative antibodies generated escape mutants with point mutations in the stock region.

Once mastered, this technique can be applied to elucidate the structural determinants of protection against other pathogens. This technique is not only limited to generating escape variants of monoclonal antibodies, but also against antiviral compounds. After watching this video, you should have a good understanding of how to elucidate the binding epitope of monoclonal antibodies by generating escape variants in vitro.

While attempting this procedure, it's important to remember to be cautious when working with viruses and use appropriate personal protection equipment.