Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

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 inhibition (HI) assay. Antibodies that are HI-negative, such as stalk-specific antibodies, can still inhibit viral replication, but can only be assessed using neutralization assays.

1. Categorizing Antibodies Based on HI Activities

1. HI assay
   1. In a 96-well V-bottom plate, add 25 µL of 1x PBS on columns 2 to 12.
   2. Dilute the monoclonal antibody (mAb) 7B2 (head-specific), 6F12 (stalk-specific) and an isotype control to 100 µg/mL in 1x PBS and aliquot 50 µL of the diluted antibody preparations into column 1. Also, include a no mAb control by adding 50 µL of 1x phosphate-buffered saline (PBS) (Figure 1A).
   3. Perform 2-fold serial dilutions of the antibodies by transferring 25 µL from column 1 to column 2, and so forth. Discard the last 25 µL from column 12 (Figure 1A).
      NOTE: Make sure to include a row of no antibody control.
   4. Dilute the virus stock (a reassortant virus expressing the hemagglutinin (HA) and neuraminidase (NA) of A/California/04/09 with the internal segments of A/Puerto Rico/8/34) to 8 hemagglutination units/25 µL diluted virus stock. Add 25 µL of the diluted virus stock (8 hemagglutination) to each well (Rows A to G).
      NOTE: The antibody and virus mixture should have a final volume of 50 µL with a starting final concentration of 50 µg/mL.
   5. Incubate the plate at room temperature (RT) for 45 min.
   6. For the back-titration row (H), add 50 µL of 1x PBS on wells H2 to H12. Add 100 µL of the 8 hemagglutination units/25 µL to well H1. Serially dilute two-fold by transferring 50 µL from H1 to H2, and so forth. Discard the last 50 µL from well H12. Finally, add 50 µL of 0.5% chicken red blood cells (RBC) to all wells of the 96-well V-bottom plate.
      NOTE: The mAb samples in the assay should have a final volume of 100 µL: mAb (25 µL), virus (25 µL) and RBC (50 µL). The final volume of the no mAb control should contain 25 µL of 1x PBS, 25 µL of virus and 50 µL of RBC.
   7. Incubate the plate at 4 °C for 1 h.
   8. Visually read the plates for HI activity. If there is a positive readout for a particular antibody, proceed to step 2.1 to generate escape variants. If the antibody is HI-negative, proceed below to step 1.2 to assess if the antibody has neutralizing activity in a cell culture assay.
      NOTE: A positive readout of an HI-active mAb is indicated by a dark red RBC pellet in the center of a well (Figure 1B; 7B2) that will form a tear drop when the 96-well V-bottom plate is held at a 45° angle. A negative readout will not form a dark red RBC pellet in the well (Figure 1B; 6F12 and no mAb). The isotype control will also not form a dark red RBC pellet and should look identical to the stalk-specific antibody, 6F12 or the no mAb control sample (Figure 1B).

2. Generation of Escape Mutant Variants

NOTE: Neutralizing antibodies that have or lack HI activity are further analyzed with the specific protocols described below.

1. Protocol 1: HI-positive/Neutralization-positive antibodies (Figure 2A)​
   1. Prepare a virus stock of 10⁶ plaque forming units/milliliter (PFU/mL) in 1x PBS in a 400 µL volume.
   2. Prepare four dilutions of the antibody of interest in increasing concentrations (e.g., 0, 0.5, 0.05 and 0.005 mg/mL) in 1x PBS in a volume of 100 µL per dilution.
      ​NOTE: Wild-type virus should always be passaged in parallel and in the absence of antibody. The sequences of these passaged viruses will assist in distinguishing between adaptation to cell culture conditions and escape mutations.
   3. Mix 100 µL of 10⁶ PFU/mL of virus with 100 µL of each antibody dilution or 100 µL of 1x PBS.
   4. Incubate for 1 h in a 37 °C incubator (with 5% CO₂). Vortex briefly. Inject 200 µL of each mixture into specific-pathogen free (SPF) embryonated chicken eggs.
   5. Incubate the eggs at 37 °C (without CO₂) for 40-44 h.
   6. Sacrifice the virus infected-embryonated eggs by incubating at 4 °C for a minimum of 6 h.
   7. Harvest the allantoic fluid from the eggs, as previously described.
   8. Perform the hemagglutination assay, as described above. If all of the allantoic fluid preparations do not have hemagglutination titers, repeat from step 2 with antibody dilutions ranging from 0.005 mg/mL to 0.00005 mg/mL.
      NOTE: A saturating concentration of an HI-positive antibody may neutralize all the virus particles present. Therefore, it may be necessary to decrease the amount of antibody present in the passaging.
   9. Confirm the escape variants by performing the HI assay (step 1.1).
      NOTE: HI-active antibodies block HA engagement of sialic acid motifs on the target cells. Therefore, a virus in the presence of its cognate antibody loses the ability to agglutinate RBCs (presence of RBC pellet). Theoretically, escape variants of HI-active antibodies can still bind sialic acid motifs even in the presence of its cognate antibody and thus can agglutinate RBCs (no RBC pellet). If the HI of the antibody of interest is still detectable, repeat the protocol from step 2.1.2 with a higher starting concentration of the antibody.