Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

CAUTION: A number of influenza viruses circulating in the human population (e.g., H1, H3) are biosafety level 2 class pathogens that must be handled with care and proper personal protective equipment. Handling of viruses must be approved by the Institutional Review Board. The following protocol was approved by the Institutional Review Board at Mount Sinai.

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 and Neutralization 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 mAb 7B2 (head-specific), 6F12 (stalk-specific)²³ 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 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 HA and 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)²³.
2. Microneutralization assay
   1. Plate Madin Darby Canine Kidney (MDCK) cells at a density of 2 x 10⁴ cells/well in a tissue culture treated 96-well plate and incubate at 37 °C and 5% CO₂ for 17 to 19 h.
      NOTE: The cells can also be allowed to adhere to the bottom of the well for a minimum of 4 h before use.
   2. In a separate 96-well plate, perform seven 3-fold serial dilutions of the human mAb 4D05⁵, CR9114¹⁷ or isotype IgG control, at a starting concentration of 200 µg/mL in 1X Minimal Essential Medium (MEM) supplemented with tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (1 µg/mL) (Figure 2).
      NOTE: Row A should contain 75 µL of diluted antibody (starting concentration of 200 µg/mL). Serially dilute (3-fold) down the plate by transferring 25 µL from Row A to Row B, and so forth. Rows A to H should have a final volume of 50 µL. There is no need to change tips in between dilution transfers.
   3. Dilute the virus stock (reassortant virus expressing the HA and the NA of A/Shanghai/1/13 with the internal segment of A/Puerto Rico/8/34) to a 100 of 50% tissue culture infectious dose (TCID₅₀)/50 µL in 1x MEM supplemented with TPCK-treated trypsin (1 µg/mL)²⁴. Add 50 µL/well of diluted virus to the antibody preparations (step 1.2.2). Add 50 µL of 1X MEM to the uninfected cell control wells.
   4. Incubate the virus-antibody mixtures in a 37 °C incubator (with 5% CO₂) for 1 h.
      NOTE: The virus-antibody mixtures should have a total volume of 100 µL: 50 µL of antibody dilution (step 1.1.2) and 50 µL of virus containing 100 TCID₅₀ (step 1.2.3).
   5. Aspirate the media in the wells and add the entire 100 µL of virus-antibody mixtures to corresponding wells.
      NOTE: The aspiration is done using an 8-channel aspirator adapter attached to a vacuum. Alternatively, an 8- or 12-well multichannel micro-pipette may be used to manually aspirate. All aspiration during the inoculum removal or washes is done from the highest concentration of antibody to the lowest, without the need to change tips.
   6. Wash the monolayer with 200 µL of 1x PBS. Aspirate 200 µL of 1x PBS (as in step 1.2.5). Repeat the washing once more for a total of two washes.
   7. Infect the monolayer of MDCK cells by adding the entire 100 µL/well of virus-antibody mixtures (from step 1.2.4) on the monolayer and infect/incubate at 37 °C (with 5% CO₂) for 1 h.
   8. During the infection, in a separate 96-well plate, prepare another set of antibody dilutions. Add 150 µL of 100 µg/mL of the respective antibody in Row A and 100 µL of 1x MEM supplemented with TPCK-treated trypsin (1 µg/mL) in Rows B to H. Perform 3-fold dilutions by transferring 50 µL from Row A to Row B, and so forth, down to Row H. Discard the last 50 µL from Row H. The total volume for each well should equal 100 µL. Set aside.
      NOTE: The antibodies are diluted in 1x MEM supplemented with TPCK-treated trypsin (1 µg/mL).
   9. Aspirate the virus-antibody inoculum from the monolayer in step 1.2.7 and replenish with the entire 100 µL/well of the appropriate antibody dilution prepared in step 1.2.8.
      NOTE: If a well contained a final antibody concentration of 100 µg/mL during the infection (step 1.2.7), then the replenishing media should also contain a final antibody concentration of 100 µg/mL (step 1.2.8).
   10. Incubate for 24 h in a 37 °C incubator (with 5% CO₂).
   11. Aspirate the media from the 96-well plates and wash with 200 µL/well of 1x PBS three times.
   12. Fix the cells with 100 µL of cold 80% acetone for 1 h at -20 °C.
       NOTE: The 80% acetone solution is diluted in double distilled (dd) H₂O (e.g., 80 mL of 100% acetone plus 20 mL of ddH₂0). The 80% acetone solution can be chilled on ice before use.
   13. Wash the cells with 200 µL/well of 1X PBS three times.
   14. Block the plates with 200 µL/well of 5% milk diluted in 1X PBS and incubate plates at RT for 1 h.
   15. Add 100 µL of biotinylated anti-influenza A nucleoprotein (NP) primary antibody diluted 1:2,000 in 1x PBS/1% bovine serum albumin (BSA) and incubate plates at RT for 1 h.
       NOTE: For influenza B viruses, an influenza B virus-specific anti-NP antibody should be used.
   16. Wash the plates with 1x PBS three times.
   17. Add 100 µL of streptavidin-horse radish peroxidase (HRP) conjugate antibody diluted 1:3,000 in 1x PBS/1% BSA and incubate the plates at RT for 1 h.
   18. Wash the plates with 200 µL of 1x PBS three times.
   19. Add HRP substrate reagent at 100 µL/well and incubate in the dark at RT.
       NOTE: The incubation time should be optimized before the addition of the acidic stopping buffer (below). Generally speaking, 15 to 30 min is sufficient.
   20. Quench the reaction with 50 µL/well of 5 M HCl.
       CAUTION: 5M HCl is a highly corrosive reagent that can cause damage to eyes, skin and mucous membranes. Addition of this reagent should be done under a vented hood with proper personal protective equipment.
   21. Read the plates at 492 nm and subtract the background (uninfected cells) wells.
   22. Calculate the percentage inhibition with the following formula:
       
   23. If an antibody has neutralization activity (and no HI activity), proceed to step 2.2.
       NOTE: Antibodies that lack in vitro neutralization activity will lack HI activity.

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 3A)
   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^24,25.
   8. Perform the hemagglutination assay, as described above^24,26. 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 assay²⁴ (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.
2. Protocol 2: HI-negative/Neutralization-positive antibodies (Figure 3B)
   NOTE: In order to generate escape variants against neutralizing antibodies that lack HI activity, the virus must be passaged in the presence of increasing amounts of antibody.
   1. Plate MDCK cells in a 6-well plate at a density of 1 x 10⁶ cells/well and incubate for a minimum of 4 h in a 37 °C incubator (with 5% CO₂).
   2. Dilute the virus stock to 10⁶ PFU/mL or the virus from previous passage in 1x MEM with TPCK-treated trypsin (1 µg/mL) in a 500 µL volume.
   3. Prepare a single dilution of antibody (0.02 mg/mL for the original passage or higher for all following passages) in 1x MEM with TPCK-treated trypsin in a 250 µL volume.
   4. Mix 250 µL of diluted virus with 250 µL of diluted antibody (+antibody) or 250 µL of 1x MEM (no antibody control).
   5. Incubate the virus-antibody mixture for 30 min in a 37 °C incubator (with 5% CO₂).
   6. Aspirate the media using a glass Pasteur pipette and wash the monolayer of cells with 1 mL of 1X PBS.
   7. Add 500 µL of the mixtures into the wells and incubate in a 37 °C incubator (with 5% CO₂) for 1 h.
   8. After 1 h, supplement the wells with 2 mL of 1x MEM with TPCK-treated trypsin (1 µg/mL).
   9. Check the cells at 48 h post-infection for signs of cytopathic effect (CPE) on the microscope or perform a hemagglutination assay to detect viral growth²⁶.
   10. If there is gross CPE in the cultures supplemented with antibody, harvest the supernatant in multiple cryo-tubes, label with the passage number and store at -80 °C.
   11. Save 100 µL of the supernatant to infect a fresh monolayer of MDCKs with 2 mL of 1x MEM supplemented with TPCK-trypsin and antibody. Remember to include a no antibody control for every passage.
       NOTE: Increase the concentration of the antibody by two-fold (or at the discretion of the researcher) in the next passage (every two days).
   12. Increase the concentration of the antibody in each successive passage until virus growth is still viable even with a final concentration of 0.6 mg/mL of antibody. Freeze the multiple vials of supernatant of each passage and store at -80 °C.
       NOTE: The no antibody control is crucial in verifying the growth of virus from one passage to another. If there is gross CPE in the no antibody control, but no CPE in the +antibody group, this indicates that the concentration of the antibody was too high and no escape variants were generated. If there is gross CPE in the no antibody control, but only moderate CPE in the +antibody group, this indicates the presence of potential escape variants. In the next passage, increase the passage volume to 200 µL of supernatant and maintain the concentration of antibody to increase the likelihood of generating escape variants.

3. Isolation of Escape Variants Through Plaque Purification

1. Plate MDCK cells in a 6-well plate at a density of 1 x 10⁶ cells/well and incubate for a minimum of 4 h in a 37 °C incubator (with 5% CO₂).
2. Dilute the antibodies in 1x MEM with TPCK-treated trypsin starting at 300 µg/mL in a volume of 250 µL and mix with 250 µL of corresponding escape mutant virus. Virus passaged in the absence of antibody should also be plaque purified.
3. Aspirate the media from the cells, wash with 1x PBS three times and add the entire 500 µL of the virus-antibody mixture (step 3.2).
4. Incubate the plates for 1 h in a 37 °C incubator (with 5% CO₂) making sure to rock back and forth every 10 min to prevent drying of the monolayer.
5. Aspirate the virus-antibody mixtures and replenish the wells with overlay agar media containing corresponding amounts of antibody (300 µg/mL; step 3.2).
6. Incubate the plate for 40-44 h in a 37 °C incubator (with 5% CO₂).
7. Circle the visible plaques with a blue- or black-colored marker to facilitate plaque picking.
8. Pick four plaques for each escape mutant virus-antibody combination, as well as wild-type viruses that were passaged in MDCK cells or eggs in the absence of antibody.
9. Resuspend the plaque in 100 µL of 1x PBS.
10. Inject the entire 100 µL of plaque purified escape mutant virus into 10-day old SPF embryonated chicken eggs.
11. Incubate the eggs for 40-44 h in a 37 °C incubator (without 5% CO₂).
12. Perform a hemagglutination assay to confirm the presence of virus (step 1.1).

4. Extraction of Viral RNA and Analysis of HA Sequence Variation

1. Extract the viral RNA from 200 µL of escape mutant virus allantoic fluid with a mono-phasic solution of phenol and guanidine isothiocyanate.
   CAUTION: Phenol is a volatile liquid reagent that can cause cough, shortness of breath and moderately irritate the skin by contact.
2. Amplify the HA segment from the viral RNA with the use of a reverse transcriptase and gene-specific primers for the influenza A HA segment²⁷.
   NOTE: The universal primers for influenza B viruses described in Table 2 amplify both the HA (~1,800 bp) and the NA (~ 1,500 bp)²⁸.
3. Resolve the RT-PCR product in a 1% agarose gel and cut out the correctly sized band (~1,800 bp).
4. Gel extract the PCR product using a silica-membrane based purification procedure and send the cDNA out for sequencing.
5. Identify the amino acid residue required for antibody binding by differentiating the mutations found on putative escape variants and passaged wild-type virus due to either cell culture adaptation or immunological selection.
6. Clone the PCR product containing the wild-type or escape variant HA into a pCAGGs expression vector (NotI and NheI).
7. Binding of the antibody to the escape variant HA can be assessed with one of two options (or both) described below.

5. Antibody Binding Analyses of Escape Variants

1. Immunofluorescence
   1. Plate 293T cells at a density of 2 x 10⁴ cells/well in a 96-well plate and incubate in the 37 °C incubator (with 5% CO₂) for 24 h.
   2. Transfect the cells with 0.10 µg/well of the pCAGGS plasmids encoding the escape mutant HA, virus passaged HA, and wild-type HA (unpassaged) with the use of a transfection reagent.
   3. Incubate the 96-well plates for 48 h in 37 °C incubator (with 5% CO₂).
   4. Fix with 100 µL of 0.5% paraformaldehyde for 15 min at RT.
      CAUTION: Paraformaldehyde is a volatile liquid reagent that can cause cough, shortness of breath and moderately irritate the skin by contact. It has been designated as a potential human carcinogen. Addition of the reagent should be done in a vented chemical hood.
   5. Wash with PBS 1x three times. Block with 5% milk in 1x PBS for 1 h at RT.
   6. Wash with 1x PBS three times. Incubate with 5 µg/mL of antibody of interest for 1 h at RT.
   7. Incubate with 100 µL of secondary antibody (anti-human or anti-mouse Alexa 488) at a dilution of 1:2,000 in 1x PBS/1% BSA for 1 h at RT in the dark.
   8. Wash with 1x PBS three times. Observe the cells on a fluorescent microscope for positive or negative staining.
2. Fluorescence-activated Cell Sorting (FACS)
   1. Plate 293T cells at a density of 2 x 10⁵ cells/well in a 6-well plate and incubate at 37 °C (with 5% CO₂) for 24 h.
   2. Transfect the cells with 0.50 µg/well with pCAGGS plasmids encoding the escape mutant HA, virus only passaged HA, and wild-type HA with the use of a transfection reagent.
   3. Incubate the 6-well plates for 48 h at 37 °C (with 5% CO₂).
   4. After 48 h, aspirate the growth media and wash with 1x PBS two times gently (making sure that the monolayer is undisturbed).
   5. Harvest the transfected 293T cells with 500 µL of FACS buffer (1x PBS/2% fetal calf serum).
      NOTE: FACS buffer should be pre-chilled at 4 ºC before use.
   6. Centrifuge the harvested 293T cells at 300 x g for 5 min at 4 °C.
   7. Aspirate the FACS buffer, and resuspend with 200 µL of FACS buffer containing mAbs of interest (final concentration of 1 to 5 µg/mL). Incubate at RT for 20 min.
   8. Centrifuge the cells at 300 x g for 5 min at 4 °C. Wash two times with 500 µL of FACS buffer. Aspirate carefully with a glass Pasteur pipette as to not disturb the pellet.
   9. Resuspend with 200 µL of FACS buffer containing secondary antibody conjugated to Alexa 488 (final dilution of 1:1,000). Incubate in the dark at 4 °C for 20 min.
   10. Centrifuge the cells at 300 g for 5 min at 4 °C. Wash two times with 500 µL of FACS buffer and carefully aspirate the wash buffer.
   11. Resuspend in 500 µL of FACS buffer and assess binding of mAbs and/or polyclonal sera to cells transfected with HA by FACS.
       NOTE: Remember to include a no mAb/polyclonal sera control as well as an untransfected sample in the experiment.