TITLE: Sequencing-based Neutralization Assay for Influenza A Virus
AUTHORS: Andrea N. Loes, Rosario Araceli L Tarabi, Jesse Bloom

[DESCRIPTION]
Traditional neutralization assays for influenza virus test a single viral strain against a single serum sample in each measurement. Here we describe a sequencing-based approach for neutralization assays that can measure the titers of serum samples against many viruses at once using the same serum volume and workflow of a traditional neutralization measurement. This method relies on incorporating a nucleotide barcode into the hemagglutinin genomic segment of the influenza virus, pooling many barcoded viruses together, and then using Illumina sequencing to read out the neutralization of all of the viruses in the pool at once. Here we provide the step-by-step protocol for running these assays with serum samples. 

[STEPS]
SECTION: (DAY 1) Determine your plate setup
9. Different plate setups may be used depending on how many samples of sera you are testing, and how many dilutions you wish to run for each serum. For example, you could perform serial dilutions of serum down the plate vertically, running up to 12 serum samples with 7 different dilutions (plus a no-serum control) per serum sample.  Or you could perform dilutions across the plate horizontally, running 8 serum samples with 11 different dilutions per serum-sample (plus a no-serum control). Additional plate set up designs are also feasible to meet the needs of your particular project.

Pictured below is an example plate setup for 12 serum samples with 7 concentrations of serum

 

Pictured below is another example plate setup (8 serum samples with 11 concentrations of serum).

 

SECTION: (DAY 1) Set up incubations of serum and virus
12. Pick an initial serum dilution factor for your assay: this will be the highest serum concentration tested, and other wells will contain serial dilutions of this concentration. You also need to choose what dilution factor to use as you dilute the serum from one well to the next. For example, you might start with an initial dilution of 1:20 and perform 3-fold dilutions down the plate. Recall that the RDE treated sera has already been diluted 1:4 in the steps above.
 

SECTION: (BEFORE DAY 1) RDE-treat &amp; heat-inactivate sera
4. Prepare RDE solution, by adding 20 mL  into one receptor destroying enzyme (RDE) bottle (VWR, Cat. No. 370013). Replace stopper and gently invert to mix. Sterile filter the solution through a 0.22 um filter prior to use. Aliquots of this RDE solution can be stored at -20 C for future use, if needed. 
SECTION: (DAY 1) Set up incubations of serum and virus
13. Add influenza growth media (Opti-MEM supplemented with 0.1% heat-inactivated FBS, 0.3% bovine serum albumin, 100 µg per mL of calcium chloride, 100 U per mL penicillin, and 100 µg
per mL streptomycin) to your plate such that all rows contain either 50 uL or the volume needed for your initial dilution of sera. 

An example is shown below, using the setup described above where 8 serum samples are run horizontally across the plate. In this example, we use an initial dilution of serum of 1:20. As the serum has been diluted 1:4 during RDE treatment and will be diluted 1:2 with virus in a later step, you'd need to dilute the RDE-treated sera 2:5 for the initial dilution in the plate to obtain a final dilution of 1:20.

For this example, you would add 45 µL to the wells in your initial dilution column and 50 µL  in every other well:

 






SECTION: (DAY 1) Set up incubations of serum and virus
14. Add 30 µL to each well in column 2. This leaves you with a 1:10 sera dilution of the original serum (a 2:5 dilution of RDE-treated serum that was already diluted 1:4 during RDE treatment) in 75uL total volume in the wells in column 2.

SECTION: (DAY 1) Set up incubations of serum and virus
15. Perform serial 3-fold dilutions, pipetting 25 µL from column 2 into column 3, and mixing by pipetting up and down several times. Repeat this step with column 3 into column 4 and so forth until you get to row 12, after which you will pipette the residual 25 uL from this column directly into bleach. After this step, you will have completed the serial 3-fold dilutions across the plate. You should still have no serum in column 1, and all 96 wells should now contain 50uL total volume.


SECTION: (DAY 1) Set up incubations of serum and virus
16. The appropriate amount of virus library needed for each plate is experimentally determined as the amount of virus that can be added to the number of cells per well (50,000 MDCK-SIAT1 cells as described below) and still be in the linear range of viral transcriptional output changing with viral dilution. (See note for more detail). As 50 µL of virus will be added to each well, we first dilute the virus library that the appropriate amount of virus is in each 50 µL aliquot, and then add 50 µL to each well (including the control, no serum row of wells).


 

SECTION: (DAY 1) Set up incubations of serum and virus
17. Incubate plate of virus and serum 37 °C 60 min 

SECTION: (DAY 1) Set up incubations of serum and virus
11. Thaw virus and RDE-treated sera Room temperature 30 min 

SECTION: (DAY 1) Add cells to plate
18. After the virus-serum mix has been incubated for 35 min of the 60 min incubation time, prepare cells to be added to the plate. First, aspirate media off of a confluent 100 mm plate of MDCK-SIAT1 cells. Wash the cells once with 2 mL. Then, treat cells with 2 mL , incubating at 37 °C  until cells disassociate from the plate (this typically takes around 5-10 min, since MDCK-SIAT1 cells are highly adherent). Once the cells are mostly dissociated, pipetting gently can help dissociate the remainder.

SECTION: (DAY 1) Add cells to plate
20. Inactivate the trypsin by adding 4 mL , washing the cells off the plate and resuspending in this media. Transfer the resuspended cells to a conical tube. Centrifuge 300 x g, 3 min , then aspirate the supernatant. Wash cells once with 5 mL , and then resuspend cells in 5 mL . Use a cell counter to determine cell concentration, and dilute cells to 1e6 cells/mL with more influenza growth media. You will need about 5.5 mL of cells at 1e6 cells/ml in influenza growth media for each 96-well plate that you wish to run.

SECTION: (DAY 1) Add cells to plate
21. Once the plate with the serum and the virus library has been incubating for 1 hour total, add 50 µL to each well.
SECTION: (DAY 1) Add cells to plate
22. Return the plate containing cells, serum, and virus library to the 5% CO2 incubator. Incubate at 37 °C for 960 min. This time allows non-neutralized viruses to infect cells and transcribe viral RNA. 

SECTION: (DAY 2) cDNA synthesis
24. Approximately 15 hours into the incubation period of the cells and virus, start thawing reagents from the iScript Select Synthesis Kit (BioRad) needed to perform cDNA synthesis (5x reaction mix, nuclease-free water, GSP enhancer, gene-specific primer, iScript enzyme mix) and a 200 pM aliquot of the RNA spike-in control. Most of the reaction mix components can be thawed Room temperature but the the enzyme mix and the RNA spike-in control should be thawed on ice.
SECTION: (DAY 2) cDNA synthesis
25. When your RNA spike-in control is thawed, mix 55 µL into 5445 µL, thereby diluting the spike-in control 1:100 for a final concentration of 2 pM. You will end up with 5.5 mL total volume of lysis buffer using these specific amounts, which is sufficient for one plate. Adjust the above amounts according to how many plates of neutralization assays you set up.
SECTION: (DAY 2) cDNA synthesis
26. When your cDNA synthesis reagents have thawed, prepare a cDNA synthesis mastermix. We use an 18 µL final reaction volume for each well, typically preparing sufficient mastermix for 100 reactions per plate to allow excess prior to distributing the the mastermix to PCR tubes. For one plate, your mastermix can be prepared like so:

400 µL 
900 µL 
200 µL 
200 µL 
100 µL 

Please adjust these amounts according to how many plates of neutralization assays you are running.

Aliquot 18 µL into 8-strip PCR tubes or a 96-well PCR plate.


 


SECTION: (DAY 2) cDNA synthesis
27. 
 

SECTION: (DAY 2) cDNA synthesis
28. Remove the supernatant from the plate using a multichannel pipette, taking care not to disturb the cells and pipette this directly into bleach solution.  
SECTION: (DAY 2) cDNA synthesis
29. Wash the infected cells once with 150 µL  per well. This wash step removes any residual virus from the supernatant and improves the cell lysis efficiency. Take care not to disturb the adhered cells when performing this wash step. When removing this liquid, pipette directly into bleach, and rinse tips with bleach solution. Examine the wells of the plate to ensure that residual liquid is completely removed from the wells of the plate prior to adding lysis buffer. This is essential as if residual liquid remains, this will dilute the spike-in control and result in noise in the measurements collected with this assay.

SECTION: (DAY 2) cDNA synthesis
30. Add 50 µL (with the RNA spike-in control added) to each well. Wait 2-5 minutes for cells to lyse. Examine wells under the microscope to determine that cell lysis has occurred. This lysis buffer is gentle, expect that nuclei will remain adhered to the plate, though the cell borders will be less defined following lysis.
SECTION: (DAY 2) cDNA synthesis
31. After 2-5 minutes, transfer lysate to a new 96-well PCR plate so that the lysis of cells will not continue.
SECTION: (DAY 2) cDNA synthesis
32. Add 2 µL to the strip tubes (or 96-well PCR plate) containing 18 µL

SECTION: (DAY 2) cDNA synthesis
33. Transfer the strip tubes (or 96-well PCR plate) containing cDNA synthesis reaction mixes to the thermocycler with the lid heated to 60 °C 

Set the following protocol:
SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
34. During cDNA synthesis, prepare Round 1 PCR mixes.
 


For one sample, the mix is as follows:
1.5 µL 
1.5 µL 
25 µL 
17 µL 
5 µL 
= 50 µL 

The following mix is enough to prepare for 96 samples:
150 µL 
150 µL 
2500 µL 
1700 µL 
=4500 µL 

Aliquot 45 µL per well in a 96-well PCR plate.
Add 5 µL . Seal the 96-well PCR plate with a PCR plate seal that is appropriate for thermal cycling and cold storage, such as (#MSF1001, BioRad).


SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
35. Set the PCR plate containing Round 1 PCR reactions in thermocycler with the lid heated to 100 °C 
Set the following protocol:

SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
35.1. Initial denaturation
95 °C 2 min 

SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
35.2. Denaturation
95 °C 20 s 

SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
35.3. Annealing
56 °C 10 s 

SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
35.4. Extension
70 °C 20 s 

SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
35.5. Repeat previous three PCR cycle steps for denaturation, annealing, and extension 19 more times
SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
35.6. Final extension
70 °C 2 min 

SECTION: (DAY 2) Round 1 PCR: adding overlap for indexing primers
35.7. Hold 10 °C 

*This is a potential stopping point, samples could be stored a -20 °C overnight, if needed before progressing to the next step of the assay.

SECTION: (DAY 2) Round 2 PCR: adding indices
37. Prepare mix for Round 2 PCR. For one sample, the reaction mix is as follows:
2.4 µL 
20 µL 
16.6 µL 
1 µL 
= 40 µL 


We prepare a mastermix of KOD and H2O and apply this first to the plate, then add the primers. The following mix is enough to prepare for 96 samples:
2000 µL 
1660 µL 
= 3660 µL 

Transfer 36.6 µL into each well of a 96-well PCR plate.

Add 2.4 µL (each mix of primers should match the number assigned to your sample).

Add 1 µL . 

Seal the 96-well PCR plate with a PCR plate seal that is appropriate for thermal cycling and cold storage.
SECTION: (DAY 2) Round 2 PCR: adding indices
38. Set 96-well PCR plate containing the Round 2 reaction mixes in thermocycler with the lid heated to 100 °C 
Set the following protocol:
SECTION: (DAY 2) Round 2 PCR: adding indices
38.1. Initial denaturation
95 °C 2 min 
SECTION: (DAY 2) Round 2 PCR: adding indices
38.2. Denaturation
95 °C 20 s 
SECTION: (DAY 2) Round 2 PCR: adding indices
38.3. Annealing
66 °C 10 s 
SECTION: (DAY 2) Round 2 PCR: adding indices
38.4. Extension
70 °C 20 s 
SECTION: (DAY 2) Round 2 PCR: adding indices
38.5. Repeat previous three PCR cycle steps for denaturation, annealing, and extension 19 more times
SECTION: (DAY 2) Round 2 PCR: adding indices
38.6. Final extension
70 °C 2 min 
SECTION: (DAY 2) Round 2 PCR: adding indices
38.7. Hold 10 °C 

*This is a potential stopping point, samples could be stored a -20 °C overnight, if needed before progressing to the next step of the assay.
SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
40. Prepare a 1% agarose gel. This can be done during Round 2 PCR.
SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
41. Pool Round 2 PCR products at equal volume (enough so you have at least ~200uL total volume). 

*We pool using a multichannel, first pooling all columns of the plate into a single 8-strip (5 uL from each sample), then pooling all 8 of the pools for each row of the plate. This will result in total volume of 480 uL per plate. It is not necessary to prepare this total volume for sequencing, only approximately ~100 uL needs to be gel extracted and bead purified for sequencing, but we have found it useful to pipette a larger volume (5 uL as opposed to 2 uL) per sample when pooling to ensure that all samples are included in the final pool. 
SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
42. Run 100 µL on your gel from step 33 at 85 V 40 min . We often run 30-40 µL of the sample per well on the agarose gel (with loading buffer), this results in 3-4 lanes of sample to be cut out and processed for sequencing.

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
43. 
 

Cut the band out of the gel, and extract the DNA using a Nucleospin Gel Extraction Kit (740609, Takara), or alternate gel extraction kit. Transfer the eluate to a strip tube for magnetic bead cleanup with AMPure XP SPRI Reagent (Beckman Coulter).
SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
44. Perform a magnetic bead cleanup on your sample using AMPure XP SPRI Reagent to remove residual salts from the gel extraction:
SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
44.1. Add 2X the volume of your sample of AMPure XP SPRI Reagent (i.e. if you eluted in 40 uL add 80 uL Ampure XP beads).

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
45.1. Load strip of tubes onto magnetic block. Incubate 5 min .

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
45.2. Pull off liquid while being careful to avoid disrupting magnetic beads. Wash twice, using  150 µL  each time (again, taking care to not disrupt the magnetic beads). Extract all liquid from tube and wait for pellet to start drying.

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
45.3. When the pellet is dry (but not so dry that it has started to crack,  approximately 10 min ) resuspend in 40 µL . Remove strip of tubes from magnetic block and repeatedly pipette the liquid up and down to thoroughly mix the beads into the elution buffer. Incubate atRoom temperature for 5 min .

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
45.4. Place strip tubes on magnetic block, incubate 5 min .

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
45.5. Once the beads are adhered to the magnet, remove the residual liquid from the beads, careful not to disturb the beads, and transfer this liquid to new 1.5 uL tube.
SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
46. Quantify total DNA concentration, for this we use a Qubit. We typically obtain 40 µL of sample at 0.005 µg/µL 

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
47. Label, appropriately dilute, and submit sample for sequencing as determined by the sequencing service you are using. We use Illumina NextSeq for these runs (a P1 run for 1 plate and a P2 should be used for 2-4 plates). We submit the 181 bp amplicon for sequencing with a 50 bp read length. We aim to obtain an average coverage of between 500,000-1,000,000 reads/well. This is likely higher coverage than necessary, however, by over-sequencing we allow for variability in loading of the different wells, which is helpful given that we are not performing any sample normalization prior to pooling. 
SECTION: (BEFORE DAY 1) RDE-treat &amp; heat-inactivate sera
3. Thaw sera on ice 

SECTION: (BEFORE DAY 1) RDE-treat &amp; heat-inactivate sera
5. Pipette RDE solution into labeled, externally threaded tubes, such that you can dilute the sera 1:4. For our sera samples, we added 300 µL . Then, added 100 µL.
SECTION: (BEFORE DAY 1) RDE-treat &amp; heat-inactivate sera
6. To removes residual sialic acids in serum incubate serum and RDE at 37 °C 150 min 
SECTION: (BEFORE DAY 1) RDE-treat &amp; heat-inactivate sera
7. Then, to heat-inactivate the RDE and serum, incubate at 56 °C 30 min 

SECTION: (BEFORE DAY 1) RDE-treat &amp; heat-inactivate sera
8. Move samples to ice, and then store heat-inactivated and RDE-treated serum samples at -80 °C until you are ready to begin the neutralization assays.
SECTION: (DAY 1) Set up incubations of serum and virus
10. 
 

SECTION: (DAY 2) cDNA synthesis
33.1. Incubate at 42 °C 60 min 
SECTION: (DAY 2) cDNA synthesis
33.2. Inactivate enzymes at  85 °C 5 min 
SECTION: (DAY 2) cDNA synthesis
33.3. Hold at 4 °C 

*This is a potential stopping point, samples could be stored a -20 °C overnight, if needed before progressing to the next step of the assay.

SECTION: Barcoded influenza HA library
1. 
 

SECTION: (BEFORE DAY 1) RDE-treat &amp; heat-inactivate sera
2. 
 

SECTION: (DAY 1) Add cells to plate
19. 
 

SECTION: (DAY 2) cDNA synthesis
23. 
 

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
39. 
 

SECTION: (DAY 2) Pooling &amp; Preparing for Sequencing
45. Incubate Room temperature 5 min .
SECTION: (DAY 2) Round 2 PCR: adding indices
36. 
 

SECTION: Data Analysis
48. Following demultiplexing of the sequencing run, sequencing data is analyzed using the modular analysis pipeline developed by the Bloom lab for processing high-throughput sequencing-based neutralization assays. This pipeline is available at https://github.com/jbloomlab/seqneut-pipeline

See associated manuscript for a detailed description of this analysis method. 

Briefly, this pipeline takes in the FASTQ files from a sequencing run, calculates the counts for each barcoded viral variant and the RNA spike-in control in each well. The ratio of barcoded viral variant to spike-in control in each well containing serum is normalized to the ratio of barcoded viral variant to spike-in control in the no-serum control well to calculate a fraction infectivity. Then, neutralization titers are computed by fitting Hill-curve style neutralization curves to the fraction infectivity values using the neutcurve package; see the documentation for the details of these curves. The titers represent the reciprocal serum dilutions at which half the viral infectivity is neutralized. For more details, please see the description at https://github.com/jbloomlab/seqneut-pipeline