Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

Summary

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

Abstract

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

Introduction

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing continual flu outbreaks in poultry and wild birds, accounting for enormous socio-economical losses in the global poultry industry. Sometimes, humans also get infected with it, faced with a high fatality rate^1,2. However, HPAI virus research is often hindered, given that it cannot be handled outside of biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses are safe enough to practice in biosafety level 2 laboratories.

H5 HPAI pseudoviruses belong to chimeric viruses consisting of surrogate virus cores, lipid envelopes with the surface glycoproteins from influenza viruses, and reporter genes. Pseudovirus cores are usually derived from lentiviral human immunodeficiency virus (HIV), retroviruses such as murine leukemia virus (MLV), and vesicular stomatitis virus (VSV)³. Specifically, the HIV-1 packaging system is widely used to produce influenza pseudovirus, where the primary genes provided are gag and pol. The HIV gag gene expresses core proteins. The pol gene expresses the integrase and reverse transcriptase, both of which are necessary for the expression of the reporter gene in transduced cells. Mimicking the genome of the surrogate virus, the reporter gene is embraced into the pseudovirus core in RNA form. The reporter gene will express the protein in host cells. The gene expression levels of reporter genes can be used to measure pseudovirus infection efficiency^3,4. The primary reporter is firefly luciferase to measure the relative luminescence units (RLU) or relative luciferase activity (RLA) in transduced cells. Other reporters such as lacZ, Gaussia, and Renilla luciferase are also used, only to a lesser extent⁵.

Pseudoviruses are ideal tools to study neutralizing antibodies against H5 HAPI viruses. To measure the neutralizing antibody potency, pseudovirus neutralization (PN) assays⁶ are used.Hemagglutinin (HA) and neuraminidase (NA) are glycoproteins on the surface of the influenza A virus^7,8. The HA is composed of a globular head domain for receptor binding and a stem domain for membrane fusion. The NA protein has the sialidase activity to facilitate virus release^7,8. A PN assay can measure neutralizing antibodies directed to HA proteins. Neutralizing antibodies directed to the head and stem region of HA can also be detected by viral attachment and entry assays. Compared with wild-type viruses, pseudovirus neutralization experiments have more sensitive detection values, can be safely handled in a Level 2 biosafety laboratory, and are generally easier to operate in practice.

This protocol presents in detail the procedures and critical steps of H5 HPAI pseudovirus preparations and PN assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays. In this study, the A/Thailand/1(KAN)-1/2004(TH) strain from H5N1 HPAI viruses was used as an example. To obtain the immune sera used in assays, this protocol selected the HA protein originating from the TH strain as the immunogen to immunize mice.

Protocol

All pseudovirus-related experimental operations were performed under ABSL2 condition in the Institut Pasteur of Shanghai Chinese Academy of Sciences (IPS, CAS). Animal experiments were performed based on Institutional Animal Care and Use Committee-approved animal protocols of IPS, CAS.

1. Pseudovirus packaging with Calcium-phosphate transfection

1. Make single-cell suspensions of HEK293FT cells (9 x 10⁵ per mL) in complete DMEM medium (Table 1). Add 10 mL of the single-cell suspensions to a T75 flask, incubate the cells in a 37 °C, 5% carbon dioxide (CO₂) incubator for 20 h before the transfection.
   NOTE: HEK293FT low passage (<20 passages) is recommended. Ensure that the cell monolayer is 80-90% confluent.
2. Replace the medium with 10 mL of fresh complete medium containing chloroquine (100 µM) 2 h before transfection.
3. Mix the reagents and plasmid DNA as shown in Table 2 and Figure 1: ddH₂O, pCMV/R-HA, pCMV/R-NA, pHR-Luc, pCMV Δ 8.9, 2.5 M CaCl₂, and 2x HEPES buffer. Pipette up and down 5 times gently, incubate the mixture at room temperature (RT) for 20 min.
4. Transfer the mixture into the medium above the cells, and rock the flask gently. Incubate the cells in the cell incubator for 15 h.
5. Replace the medium with 15 mL of fresh complete DMEM medium. Incubate the cells in a 37 °C, 5% CO₂ incubator for 65 h.
   NOTE: The color of the medium will be light orange or slightly yellow, and at least 80% of the cell should show the cytopathic effect (CPE) under the inverted light microscope.
6. Harvest the supernatant, and centrifuge it at 2095 x g for 20 min at 4 °C. Collect the supernatant, aliquot it, and store it at -80 °C.

2. Detection of the HA, NA and HIV-1 p24 protein expression of influenza pseudovirus

1. Detect the HA protein expression by Hemagglutinin (HA) assay.
   1. Add 50 µL of PBS into columns 2-12, rows A, B, and C of a 96-well round-bottom plate.
      NOTE: Row A, B, and C belong to 3 independent replication tests, and column 12 is used as the negative control.
   2. Add 100 µL of pseudovirus into the wells of column 1. Transfer 50 µL from column 1 into Column 2, mix well by pipetting up and down 5 times, perform the two-fold dilutions until the last column 11, and discard the extra 50 µL from column 11.
   3. Add 50 µL of 0.5% erythrocyte into each well, pipette it up and down gently twice. Incubate the plate for 30-60 min at RT or until the erythrocytes of the negative control form the regular spots at the bottom of the well.
   4. Observe and record HA titers of the pseudovirus.
      NOTE: HA unit of the pseudoviruses is the highest dilution factor of the virus that can cause 100% hemagglutination of the red blood cells.
2. Detect HA and NA protein expression by the western-blot assay.
   NOTE: All the western-blot steps are performed according to the manual of Molecular cloning (4^th edition).
3. Detect the HIV-1 p24 protein expression by the HIV-1 p24 ELISA kit (Table of Materials).
   1. Add 50 µL of the lysis buffer into 450 µL of pseudovirus sample. Mix it thoroughly.
   2. Add 300 µL of the wash buffer to each well of the microplate. Strike the plate inverted to remove the wash buffer completely. Repeat the wash 5 times.
   3. Add 200 µL of the standard and samples to each well separately. Incubate them at 37 °C overnight or for 2 h.
   4. Aspirate the contents of the plate and wash the plate as described in step 2.3.2.
      NOTE: Leave one well of the assay plate empty to use as a substrate blank.
   5. Add 200 µL of the p24 detector antibodies to each well. Incubate them at 37 °C for 1 h. Aspirate and wash the plate as described in step 2.3.2.
   6. Add 100 µL of the streptavidin-Peroxidase working solution to each well, and incubate them at 37 °C for 30 min. Aspirate and wash the plate as described in step 2.3.2.
   7. Add 100 µL of the substate working solution to each well, including the substrate blank well, and incubate them at 37 °C for 30 min.
      NOTE: Blue color will develop in the wells.
   8. Add 100 µL of the stop buffer to each well.
      NOTE: The blue color will change to yellow immediately.
   9. Read the optical density at 450 nm within 15 min. Record the p24 titers of the pseudovirus

3. Pseudovirus titration

1. Make single-cell suspensions of MDCK cells (5 x 10⁴ per mL) in complete DMEM medium, add 1250, 2500, 5000, 10000, 20000, and 40000 cells to different wells of a 96-well flat-bottom plate. Incubate the cells in a 37 °C, 5% CO₂ incubator for 20 h.
2. Take out the pseudovirus from the -80 °C refrigerator, thaw it on the ice, and vortex the pseudovirus.
3. Add 6 HAU (6x HA units) pseudoviruses to each well of 96-well round-bottom plate and replenish each well with complete DMEM up to 120 µL.
4. Pipette up and down the mixture 5 times to mix thoroughly. Incubate the mixture plate in a 37 °C, 5 % CO₂ incubator for 1 hour.
5. Transfer 100 µL of the mixture to the cells in the 96-well plate. Put the cell culture plate back into the incubator for 48 h, 60 h, and 72 h after virus infection.
6. Take the plate out of the incubator and perform the luciferase assay (Table of Materials).
7. Remove the medium from the wells of the plate carefully. Rinse the cells with 200 µL of PBS and remove the PBS as much as possible.
   NOTE: Flip the plate and discard the supernatant by reverse slapping it on absorbent paper. If the testing cell line could not firmly attach to the bottom, remove the medium aspiration carefully.
8. Add 50 µL of the lysis buffer to each well, and rock the culture plate several times. Store the plate at -80 °C overnight.
   NOTE: Mix 1 volume of 5x lysis buffer with 4 volumes of water to make the 1x lysis buffer. Equilibrate the 1x lysis buffer to RT before use. Rock the plate to ensure that the cells are covered with the lysis buffer completely. The single freeze-thawprocess can help the cell be lysed completely.
9. Take the plate out, equilibrate at RT for 2 h.
   NOTE: The cell should be lysed completely.
10. Rock the plate several times gently, and transfer all the cell lysates to opaque 96-well plates.
11. Add 50 µL of the substrate to each well, rock the plate several times gently, and mix substrate and lysis buffer thoroughly.
12. Measure the light produced within 5 min using a luminometer. Record the luciferase titers of the pseudovirus.
    ​NOTE: When a proper substrate is added, light is emitted as a by-product via a chemical reaction in which luciferin is converted to oxyluciferin by the luciferase enzyme. The amount of light produced is proportional to the amount of the luciferase enzyme.

4. Immune sera preparation

NOTE: The immune serum will be used for cell-related experiments, and the experimental operation should be carried out under aseptic conditions.

1. Prepare 12 eight-week-old female BALB/c mice. Divide equally the mice into two groups.
   NOTE: One group was DDV group and the other was the negative control group.
2. DDV group immunization: Prime twice intramuscularly with 100 µg of a codon-optimized DNA plasmid encoding A/Thailand/(KAN-1)/2004 (TH) HA protein at week 0 and week 3 and boost once intraperitoneally with 512 HAU TH virus-like particles (VLP) at week 6.
3. Control group immunization: Prime twice intramuscularly with 100 µg empty vector plasmid DNA at week 0 and week 3 and boost once intraperitoneally with HIV-1 gag VLP at week 6.
4. Collect the mouse blood 2 weeks after the last immunization.
   NOTE: Here, the mice were anesthetized with pentobarbital sodium (65 mg/kg) at the time of blood collection. After blood collection from the submandibular vein, the mice were euthanized immediately with CO₂.
5. Keep the blood at RT for 2 h, then 4 °C overnight. Centrifuge at 900 x g for 10 min at 4 °C and collect the supernatant. Inactivate the sera by placing it at 56 °C for 30 min.
6. Store the immune sera in a -80 °C refrigerator for use.
   NOTE: Refer to Supplementary Figure 1 for the flowchart that describes the major procedures of mouse immunization.

5. Pseudovirus neutralization (PN) assay

1. Make single-cell suspensions of MDCK cells (5 x 10⁴ per mL) in complete DMEM medium, and add 100 µL of the cell suspension to each well of a 96-well flat-bottom plate. Incubate the cells in a 37 °C, 5% CO₂ incubator for 20 h.
2. Take out the pseudovirus and sera from the -80 °C refrigerator, thaw it on the ice, and vortex thoroughly.
3. Dilute the sera in the complete medium 20 times, and add 100 µL of complete DMEM medium to the wells of a 96-well round-bottom plate from columns 2-10.
4. Add 200 µL of the diluted sera into the wells of column 2, transfer 100 µL from column 2 to column 3, dilute the sera as 1:20, 1:40, 1:80, etc., to 1:10240 successively from column 2 to column 10, and discard the 100 µL from column 10.
5. Add 100 µL of DMEM complete medium to the wells of column 11 as the negative control.
6. Add 100 µL of pseudoviruses to each well, pipette up and down the mixture 5 times thoroughly, and incubate the mixture plate in a 37 °C, 5% CO₂ for 1 h.
7. Transfer 100 µL of the mixture from the 96-well round-bottom plate to the corresponding well of the 96-well cell culture plate. Put the plate back in the incubator (37 °C, 5% CO₂) for 60 h.
8. Take the plate out of the incubator. Perform the luciferase assay as described in steps 3.7-3.12.

6. Pseudovirus attachment assay

1. Make single-cell suspensions of MDCK cells (4 x 10⁴ per mL) in complete DMEM medium, and add 100 µL cell suspensions to each well in a 96-well flat-bottom plate. Incubate the cells at 37 °C, 5% CO₂ for 20 h.
2. Incubate the pseudovirus with sera in DMEM containing 1% BSA at 4 °C overnight.
   NOTE: The volume of the mixture is 100 µL.
3. Block the MDCK cells with 100 µL per well of DMEM containing 1% BSA at 4 °C for 1 h.
4. Inoculate the pseudovirus and serum mixture (100 µL) onto the MDCK monolayer and incubate it at 4 °C for 2 h.
5. Wash the cell plate 4 times with PBS containing 1% BSA to remove any unbound virus.
6. Lyse the cells with 100 µL of luciferase lysis buffer at RT for 30 min.
7. Quantify the amount of bound virus on the cells with an HIV-1 p24 ELISA kit as described above (section 2.3).

7. Assessment of viral entry

1. Make single-cell suspensions of MDCK cells (5 x 10⁴ per mL) in complete DMEM medium, and add 100 µL of the cell suspension to each well of a 96-well flat-bottom plate. Incubate the cells at 37 °C, 5% CO₂ for 20 h before the assay.
2. Inoculate the pseudovirus (100 µL) onto an MDCK monolayer in a 96-well plate at 4 °C for 6 h.
3. Wash the cell plate 4 times with PBS containing 1% BSA to remove unbound pseudoviruses.
4. Add 100 µL of the serially diluted immune sera or sera from sham vaccination mice in DMEM containing 1% BSA to the monolayer, and incubate cell at 4 °C for 2 h.
5. Remove the cell supernatant, and wash the cell culture plate 4 times with PBS containing 1% BSA.
6. Add fresh DMEM to the cells and incubate for 60 h in a 37 °C, 5% CO₂ incubator.
7. Measure the relative luciferase activity (RLA) of the cells as described in steps 3.7-3.12.
   NOTE: Viral entry, as indicated by RLA, was expressed as a percentage of the reading obtained in the absence of sera, which was set as 100%.

Results

HA, NA and HIV-1 p24 protein expression of influenza pseudovirus
To identify the viral packaging efficiency, influenza pseudovirus stocks were first detected by HA assay (Figure 2A). HA units per milliliter of influenza pseudoviruses are 643 (Table 3). Western-blot assay and sandwich ELISA assays were used to test HA, NA, and HIV-1 p24 protein expression. Then the ratios of HA unit and the amount of gag p24 for pseudoviruses were calculated. The result...

Discussion

HEK293FT cells are usually used as packaging cells to produce pseudoviruses. Regular mycoplasma detection is essential during the cell culture. Mycoplasma contamination can drastically decrease the pseudovirus yields and sometimes close to zero. Compared with other contaminations, mycoplasma contaminations do not lead to pH value changes or turbidity of the cell culture medium.Even a high concentration of mycoplasma is not visible with naked eyes or under a microscope. Three popular methods of mycoplasma detection are my...

Materials

Name	Company	Catalog Number	Comments
1% Chiken Erythrocyte	Bio-channel	BC-RBC-C001	Reagent
96-well cell culture plates (flat-bottom)	Thermo fisher scientific	167008	consumable material
96-well cell culture plates (round-bottom)	Thermo fisher scientific	163320	consumable material
Allegra X-15R	Beckman coulter	--	Equipment/Centrifuge
BD Insulin Syringes	BD	324910	consumable material
Calcium Chloride Anhydrous	AMRESCO	1B1110-500G	Reagent
chloroquine diphosphate	Selleck	S4157	Reagent
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	12100-046	Reagent
Fetal Bovine Serum	Gibco	16000-044	Reagent
HEK293FT	Gibco	R700-07	Cell line
HEPES FREE ACID	AMRESCO	0511-250G	Reagent
HIV-1 p24 Antigen ELISA	ZeptoMetrix	801111	Reagent kit
Luciferase Assay System Freezer Pack	Promega	E4530	Reagent kit
MDCK.1	ATCC	CRL-2935	Cell line
Microcentrifuge Tubes 1.5 mL	Thermo fisher scientific	509-GRD-Q	consumable material
Nunc Conical Centrifuge Tubes 15 mL	Thermo fisher scientific	339650	consumable material
Nunc Conical Centrifuge Tubes 50 mL	Thermo fisher scientific	339652	consumable material
Nunc EasYFlask 75 cm2	Thermo fisher scientific	156499	consumable material
Penicillin-Streptomycin	Gibco	15140-122	Reagent
Pipette Tips (10 μL)	Thermo fisher scientific	TF102-10-Q	consumable material
Pipette Tips (100 μL)	Thermo fisher scientific	TF113-100-Q	consumable material
Pipette Tips (1000 μL)	Thermo fisher scientific	TF112-1000-Q	consumable material
Serological pipets (5 mL)	Thermo fisher scientific	170355N	consumable material
Serological pipets (10 mL)	Thermo fisher scientific	170356N	consumable material
Trypsin/EDTA	Gibco	25200-072	Reagent
Varioskan Flash	Thermo fisher scientific	--	Equipment/Microplate reader
Water Jacket Incubator	Thermo fisher scientific	3111	Equipment/Cell incubator
Pentobarbital sodium salt	Sigma	57-33-0	Reagent