Chapter 15 HIV-1 Isolation from Infected Peripheral Blood Mononuclear Cells Stefania Dispinseri, Elisa Saba, Elisa Vicenzi, Neeltje A. Kootstra, Hanneke Schuitemaker, and Gabriella Scarlatti Abstract Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and not in tumor derived cell lines. The procedure involves culture of PBMCs from an infected patient with phytohemagglutinin (PHA)-stimulated PBMC from seronegative donors, which provide susceptible target cells for HIV replication. HIV can be isolated from the bulk population of PBMCs or after cloning of the cells to obtain viral biological clones. Viral production is determined with p24 antigen (Ag) detection assays or with reverse transcriptase (RT) activity assay. Once isolated, HIV-1 can be propagated by infecting PHAstimulated PBMCs from healthy donors. Aliquots from culture with a high production of virus are stored for later use. Key words Isolation, HIV-1, PBMCs, Cell culture
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Introduction The best human immunodeficiency virus 1 (HIV-1) isolation procedure involves the cocultivation of infected peripheral blood mononuclear cells (PBMCs) from a patient with PBMCs from seronegative donors. HIV-1 isolation from patient plasma is also possible, but it is only successful when the samples have high viral load. On the contrary, isolation from patient PBMCs can be achieved even when few infectious viruses are present [1]. The use of primary PBMCs has several advantages for the viral isolation procedure. First of all, the use of donor PBMCs instead of cell lines decreases the possibility of selective pressure, which may avoid selective retrieval of viral variants with specific phenotype [2]. Second, donor PBMCs provide antigenic stimulation of patient PBMCs inducing the expression of interleukin-2 (IL-2) receptors on the surface of T-lymphocytes of these subjects, and as
Elisa Vicenzi and Guido Poli (eds.), Human Retroviruses: Methods and Protocols, Methods in Molecular Biology, vol. 1087, DOI 10.1007/978-1-62703-670-2_15, © Springer Science+Business Media, LLC 2014
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consequence such cells become susceptible to the growth stimulatory effect of exogenous IL-2 added to the medium during virus isolation. Third, seronegative PBMCs represent target cells for HIV infection. Donor PBMCs may vary in susceptibility to viral infection in vitro, and thus, a mixture of PBMCs obtained from two or more donors is preferable. In addition, the potentially inhibitory effects of the subject’s CD8 positive T cells in the sample are diluted out by using PBMCs in excess. In some cases this may enhance virus isolation. Viral production is determined by p24 antigen (Ag) detection assays either in the culture supernatant or intracellularly by detecting the number of p24 Ag-positive cells. Several sensitive ELISA are commercially available or in-house assay, can be performed to measure the levels of p24 Ag in the culture supernatant (see Note 1). An alternatively approach can be based on the determination of the reverse transcriptase (RT) activity in the culture supernatant. The RT assay measures the activity of the HIV RT enzyme present in supernatants of HIV-infected cultures. As the RT enzyme is virionassociated and it is not found as a free protein, the results of an RT assay are generally assumed to reflect the cellular production of complete virions. Unlike the p24 Ag capture assay with reduced crossclade reactivity, the RT activity can be used to quantify the amount of any lentivirus in culture (e.g., all HIV-1 subtypes and HIV-2). HIV RT is Mg2+ dependent and converts genomic RNA in proviral DNA. Using a detergent, the virion is broken and the RT is released. The reaction mixture consists of a poly(A) RNA template and oligo d(T) primer and (α 32P) 2′-Deoxythymidine 5′-Triphosphate, Sodium (dTTP) as substrate for the action of the HIV RT.
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Materials HIV-1 is a class 3 human pathogen and infectious virus should be handled in a biosafety level 3 laboratory. All waste should be disposed by autoclaving and liquid waste should be inactivated, for example, by detergent treatment (i.e., bleach). All the reagents should be warmed at 37 °C or room temperature before use.
2.1 Reagents and Equipment for PBMCs Isolation
1. Cells from HIV-seropositive individual. 2. Healthy blood donors or buffy coats. 3. Phosphate-buffered saline without calcium and magnesium (PBS; Lonza). 4. Lympholyte-H (Cederlane). 5. Phytohemagglutinine (PHA; concentration 5 μg/ml.
Sigma-Aldrich):
final
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6. Complete medium: RPMI-1640 supplemented with 10 % fetal bovine serum (FBS; Lonza), and 1 % penicillin streptomycin (Pen/Strep; Lonza). 7. Sterile 10 ml pipettes. 8. Sterile 25 ml pipettes. 9. Sterile 50 ml plastic tubes. 10. Electric pipettor. 11. Humidified 37 °C incubator with 5 % CO2. 12. Centrifuge. 13. Sterile flask. 2.2 Reagents for Viral Isolation from PBMCs
1. Three days PHA-stimulated healthy blood donor PBMCs. 2. PBMCs from infected patient. 3. Complete medium: RPMI-1640 supplemented with 10 % FBS and 1 % Pen/Strep. 4. IL2-medium: RPMI-1640 supplemented with 10 % FBS, 1 % Pen/Strep, and 200 U/ml recombinant Interleukin 2 (IL-2; Chiron). 5. Sterile 5 ml pipettes. 6. Sterile T25 flask. 7. Electric pipettor. 8. Humidified 37 °C incubator with 5 % CO2. 9. Centrifuge.
2.3 Reagents for Isolation of Biological Clonal Viral Variants from PBMCs
1. Three days PHA-stimulated healthy blood donor PBMCs. 2. IF medium: IMDM (Iscoves Modified Dulbecco’s Medium, BioWhittaker) supplemented with 10 % FBS and 1 % Pen/ Strep. 3. IL-2 medium: IMDM supplemented with 10 % FBS, 1 % Pen/ Strep, 10 U/ml IL-2, Ciprofloxacin (5 μg/ml), and polybrene (5 μg/ml). 4. Heparin (Leo Pharma). 5. Polybrene (Sigma). 6. Trisodium citrate dihydrate (TNC, Merck). 7. Phosphate-buffered saline (PBS, BioWhittaker). 8. FBS (Hyclone). 9. rIL-2 (Chiron Benelux). 10. Penicillin (Pen) and Streptomycin (Strep) (Gibco BRL). 11. Ciprofloxacin (Bayer).
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12. 0.2 % Triton X-100 (Merck) solution: PBS supplemented with 0.2 % Triton X-100. Addition of a dye makes it easy to recognize Triton inactivated samples. 13. MT-2 cells (ATCC, CRL-9096). 14. Sterile 10 ml pipettes. 15. Sterile 25 ml pipettes. 16. Sterile 15 and 50 ml plastic tubes. 17. Pasteur pipettes. 18. 96 wells flat bottom microtiter plates. 19. Pipette (0.5–10 μl; 2–20 μl; 20–200 μl) 20. Multi channel pipette (50–200 μl). 21. Sterile pipette tips. 22. Humidified 37 °C incubator with 5 % CO2. 23. Centrifuge. 2.4 Reagents for HIV-1 p24 Ag Staining
1. Fix & Perm Kit (Invitrogen). 2. Anti-p24 antibody: FITC-conjugated KC57 monoclonal antibody (Beckman Coulter). 3. Paraformaldehyde solution: 4 % paraformaldehyde (PFA) (Sigma). To prepare 4 % PFA solution, dissolve 1 g of PFA powder in a final volume of 25 ml of PBS. Heat at 56 °C until the powder is completely dissolved. Bring the solution at room temperature and pH to 7.2. Store at 4 °C.
2.5 Reagents for Reverse Transcriptase Detection Assay
1. RT cocktail: 60 mM Tris–HCl pH 7.8, 75 mM KCl, 5 mM MgCl2, 0.1 % Nonidet P40, 1 mM EDTA, 5 μg/ml poly A (Amersham Pharmacia Biotech, Inc.), 0.162 μg/ml oligo dT: pd(T)12–18 (Invitrogen). 2. 0.5 M Dithiothreitol (DTT). 3. (α32P) dTTP (PerkinElmer). 4. DE81 paper (Whatman). 5. SSC 20×: 3 M NaCl and 0.3 M Na Citrate pH 7.0. 6. 1450 MicroBeta PLUS and Cassette for 32P (1450-118).
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Methods
3.1 Isolation of PBMCs from Healthy Blood Donor Buffy Coats
1. PBMCs are isolated from buffy coats from two or more different healthy blood donors. PBMCs from each donor are isolated and stimulated separately. 2. Prepare two 50 ml tubes for each buffy coat and transfer 20 ml of buffy coat in each tube. Dilute the buffy coats 1:1 with PBS.
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3. Prepare four 50 ml tubes (two tubes for each buffy coat) and add 10 ml of Lympholyte-H. 4. Add the diluted buffy coat on top of the Lympholyte-H in each corresponding tube. 5. Centrifuge the tubes for 30 min at 561 × g without brake at room temperature. 6. Remove the ring fraction on top of the Lympholyte-H with a sterile pipet and add two rings together in one clean 50 ml tube. Supplement the ring fractions with PBS to 50 ml. 7. Centrifuge the tubes for 10 min at 389 × g at room temperature with brake. 8. Discard the supernatant and resuspend the cells in 50 ml PBS. 9. Centrifuge the tubes for 10 min at 249 × g at room temperature with brake. 10. Discard the supernatant and resuspend the cells in 50 ml PBS (see Note 2). 11. Centrifuge the tubes for 10 min at 140 × g at room temperature with brake (see Note 3). 12. Discard the supernatant and resuspend the cells in 30 ml complete medium. 13. To activate the cells, count the cells and transfer 1 × 106 PBMC/ml in 25 or 75 ml flask with complete medium + 5 μg/ml PHA. 14. Incubate at 37 °C, 5 % CO2 for 2–4 days (best 3 days). 3.2 Isolation of HIV-1 from Patients’ PBMCs
1. Mix 2 × 106 patient cells with 15 × 106 healthy blood donor PBMCs in the T25 flask in a total culture volume of 8 ml of IL2-medium. 2. Incubate the flask in the incubator at 37 °C with a humidified atmosphere and 5 % CO2 for 3–4 days. 3. On the third or fourth day, discard one-third to two-thirds volume of supernatant and replace fresh IL2-medium. 4. At day 7, test the presence of virus in an aliquot of supernatant using a p24 Ag capture ELISA or the RT activity assay. 5. Remove two-thirds volume of the culture supernatant and add 2–3 × 106 fresh, 2–4 days PHA-stimulated donor PBMCs to the remaining cells in the flask. Add IL2-medium up to 8 ml. 6. Incubate the flask in the incubator at 37 °C with a humidified atmosphere and 5 % CO2 for 3–4 days. 7. Repeat steps 3–5. Test supernatant for HIV productive infection each time prior to medium change or fresh PHAstimulated PBMCs addition (see Note 4). 8. When the culture supernatant is positive, collect the viruscontaining supernatant and aliquot in 1.5 ml cryovials to be stored at −80 °C.
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Fig. 1 Scheme for the isolation of biologically clonal HIV-1 variants
3.3 Isolation of Biological Clonal Viral Variants from PBMCs
1. Dilute 2–3 day PHA-stimulated donor PBMCs in IL-2 medium (see Note 5) to a cell density of 1 × 106/ml. 2. Resuspend patient PBMCs in IL-2 medium at a cell density of 5 × 104, 10 × 104, 20 × 104, and 40 × 104 cells/ml. 3. Seed the patient cell suspension in 96-well microtiter plates at 100 μl/well (32–48 wells per cell concentration). 4. Add 100 μl PHA-stimulated healthy donor PBMCs (1 × 105 cells/well). Wrap culture plates in plastic wrap to reduce evaporation. 5. Culture the cells in an incubator at 37 °C, with a humidified atmosphere and 5–10 % CO2 for 7 days. 6. On the seventh day, transfer one-third volume (65 μl) of culture supernatant (by multichannel pipette) to a new 96-well plate and inactivate the supernatant by addition of 65 μl of 0.2 % Triton X-100 and incubating for at least 30 min (see Fig. 1). 7. Test the supernatant for the presence of virus using an ELISA detecting p24 Ag (see Note 3). Inactivated culture supernatant samples for p24 Ag can be stored overnight at 4 °C or for longer periods of time at −20 °C. 8. Resuspend the culture and transfer one-third volume (65 μl) of culture (by multichannel pipette) to a new 96-well plate.
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9. Add 1 × 105 freshly prepared 2–3 day PHA-stimulated healthy donor PBMCs (steps 12–15) in 135 μl IL-2 medium to each well. Wrap culture plates in plastic wrap to reduce evaporation. 10. Incubate microcultures in an incubator at 37 °C, with a humidified atmosphere and 5–10 % CO2. 11. Add 2 × 104 MT-2 cells in a total volume of 100 μl IF medium per well to the remaining one-third culture volume. Wrap culture plates in plastic wrap to reduce evaporation (see Note 6). 12. Incubate the MT-2 microcultures in an incubator at 37 °C, with a humidified atmosphere and 5–10 % CO2. 13. Check p24 Ag-positive microcultures for syncytia at day 4 and 7 after the onset of the culture by microscopy (Fig. 1). 14. Repeat steps 6–13 every week. Continue cultures for 4–5 weeks. 15. Calculate the frequency of infected PBMCs using the formula for Poisson distribution (u = −lnF0, in which F0 is the fraction of negative cultures). When fewer than 33 % of wells seeded with a certain concentration are positive, the viruses in one well are thought to originate from one infected cell. However, if possible, it is better to pick the virus variants only from concentrations with fewer than 10 % positives. 3.3.1 Anticipated Results
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The number of clonal biological HIV-1 variants that is obtained by the protocol described here is highly variable and depends largely on the viral load of the patient at the moment the PBMC sample was taken. Indeed, a significant association between the viral RNA load in serum and the frequency of productively infected cells present in patient PBMCs has been demonstrated [3]. Also the time between the start of the coculture and detection of the first biological HIV-1 isolates is highly variable and is associated with the stage of the disease of the patient at the moment the PBMC sample was taken. When a PBMC sample from a patient in a late stage of the disease is taken, fast replicating HIV-1 variants can often be detected as early as at day 7. In contrast, isolation of slow replicating HIV-1 or HIV-2 variants from long-term nonprogressors may take 28–35 days of microculture and involve additional steps such as depletion of CD8 cells and/or stimulation of patient PBMCs before virus isolation [4]. SI or CXCR4-using HIV-1 variants develop relatively late in the course of infection in approximately half of the individuals infected with subtype B HIV-1. A relation between the viral load and the virus phenotype has also been demonstrated. The highest viral loads are observed in patients with SI variants.
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The increase in viral load in patients with SI variants is mainly due to the selective expansion of the SI HIV-1 load, although it is important to note that the NSI variants remain present throughout the disease course, even after the emergence of SI variants. In the described procedure the presence of SI or X4-variants in the patient sample is monitored by cocultivation of the primary PBMC cultures with MT-2 cells [5]. 3.4 HIV-1 p24 Ag Staining
1. Resuspend cells suspension with FACS buffer to a final concentration of 10 × 106 cells/ml. Place 100 μl of cell suspension into FACS tubes (Becton Dickinson) (see Note 7). 2. Resuspend the pellet with 100 μl of Buffer A (Fixation Medium from Fix & Perm Kit). Vortex gently to mix, and incubate for 15 min in the dark at room temperature. 3. Wash with 1 ml of FACS buffer. Centrifuge for 5 min at 249 × g and aspirate the supernatant. 4. Resuspend the pellet with 100 μl of Buffer B (Permeabilization Medium from Fix & Perm Kit) containing 0.5 μl of FITCconjugated monoclonal antibody against p24 Ag (KC57). Vortex each tube and incubate for 20 min in the dark at room temperature. 5. Wash with 1 ml of FACS buffer. Centrifuge for 5 min at 249 × g and aspirate the supernatant. 6. Resuspend pellet with 100 μl of FACS buffer for immediate analysis. Vortex gently to mix. 7. It is possible to add 100 μl of storage samples in the dark at 4 °C for maximum 24 h.
3.5 RT Activity Assay
1. Prepare RT cocktail in advance and store in aliquots at −20 °C. ~ 32P) 2. Immediately before use, add 8 μl 0.5 M DTT and 1 μl (◻ dTTP to 1 ml of RT cocktail. 3. Dispense 25 μl/well in a 96-well plate. 4. Add 5 μl of culture supernatant. 5. Incubate the plates at 37 °C in a plexiglass box for 2–3 h. 6. Two hour incubation is generally sufficient for the RT reaction to take place. Radioactivity incorporated into the reaction product will increase linearly with incubation time. Prolonged incubation times may thus be desirable if (α 32P) dTTP is 3 weeks old, however, background counts will increase with prolonged incubation. 7. After incubation with the RT cocktail, any infectious HIV will be rendered noninfectious by the NP-40 detergent in the mix. 8. While the plates are incubating, stamp a sheet of Whatman DE81 paper using an ink stamp that produces a pattern reflecting the format of the 96-well plate.
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9. Using a multichannel pipette and non-sterile pipette tips, pipette 6 μl of the reaction mixture from each well onto the DE81 paper, being careful not to tear the paper by contact with the pipette tips. 10. Dry the paper. 11. Prepare 1× SSC by diluting the 20× SSC stock solution with H2O. 12. Wash the paper four times by placing in a dish containing enough 1× SSC to cover the paper and rocking on a platform for 5 min/wash. 13. Wash again in 95 % ethanol for 5 min. The wash liquid must be discarded in radioactive-liquid waste. 14. Dry the paper in the incubator for 20 min. The paper is very fragile when wet and care must be taken when picking it up. 15. Expose the filter for autoradiography overnight. 16. Develop the film, and count the filter paper in 1450 MicroBeta PLUS using 32P Cassette (1450-118). The advantage of carrying out autoradiography before quantification is that it is possible to visualize the reaction templates and identify any potential irregularity.
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Notes 1. There are several p24 Ag capture ELISA commercially available. Sensitivity of most of them is usually very high and thus, may require extensive dilutions of the culture supernatant to determine the actual value. For the method at point 3.3, HIV-1 p24 Ag capture ELISA used is provided by the AIDS Vaccine Program (NCI-SAIC Frederick). Alternatively for all methods described here in-house equivalent assays can be performed with commercially available antibodies. For example, Aalto Bioreagents supplies antibodies and provides several alternative protocols for the detection (www.aaltobioreagents.ie). 2. When many erythrocytes are present, these can be removed by cell lysis. Resuspend the cells in 1 ml of sterile water before the last washing step, agitate gently for 20 s, add up to 50 ml PBS, and complete the washing. 3. PBMCs can now be cryopreserved in FCS supplemented with 10 % DMSO. Cryopreserved cells can be stored in liquid nitrogen for more than 10 years. 4. Cultures are kept until positive for p24 Ag at increasing levels for three consecutive times (possibly for two consecutive times p24 must be >2 ng/ml). Otherwise cultures are interrupted at day 28–30.
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5. The IL-2 medium contains polybrene, which is known to enhance HIV-1 infection presumably by facilitating the interaction between negatively charged virus particle and cell membrane of the target cell. 6. Working with primary cells from blood donor and HIV-1infected patients may result in frequent introduction of mycoplasma. Precautions should be taken to prevent infection of other cells or cell lines. It is recommended to add ciprofloxacin to the cultures to inhibit replication of mycoplasma. 7. It is important to include a control of uninfected cells. This control should be stained for p24 Ag detection and used to set the background signal. References 1. van 't Wout AB, Schuitemaker H, Kootstra NA (2008) Isolation and propagation of HIV-1 on peripheral blood mononuclear cells. Nat Protoc 3:363–370 2. Sawyer LS, Wrin MT, Crawford-Miksza L, Potts B, Wu Y, Weber PA, Alfonso RD, Hanson CV (1994) Neutralization sensitivity of human immunodeficiency virus type 1 is determined in part by the cell in which the virus is propagated. J Virol 68:1342–1349 3. Blaak H, de Wolf F, van 't Wout AB, Pakker NG, Bakker M, Goudsmit J, Schuitemaker H (1997) Temporal relationship between human
immunodeficiency virus type 1 RNA levels in serum and cellular infectious load in peripheral blood. J Infect Dis 176:1383–1387 4. van Rij RP, Schuitemaker H (2002) Host genetic factors in the clinical course of HIV-1 infection: chemokines and chemokine receptors. Community Genet 5:88–101 5. Koot M, Vos AH, Keet RP, de Goede RE, Dercksen MW, Terpstra FG, Coutinho RA, Miedema F, Tersmette M (1992) HIV-1 biological phenotype in long-term infected individuals evaluated with an MT-2 cocultivation assay. AIDS 6:49–54
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Introduction The best human immunodeficiency virus 1 (HIV-1) isolation procedure involves the cocultivation of infected peripheral blood mononuclear cells (PBMCs) from a patient with PBMCs from seronegative donors. HIV-1 isolation from patient plasma is also possible, but it is only successful when the samples have high viral load. On the contrary, isolation from patient PBMCs can be achieved even when few infectious viruses are present [1]. The use of primary PBMCs has several advantages for the viral isolation procedure. First of all, the use of donor PBMCs instead of cell lines decreases the possibility of selective pressure, which may avoid selective retrieval of viral variants with specific phenotype [2]. Second, donor PBMCs provide antigenic stimulation of patient PBMCs inducing the expression of interleukin-2 (IL-2) receptors on the surface of T-lymphocytes of these subjects, and as
Elisa Vicenzi and Guido Poli (eds.), Human Retroviruses: Methods and Protocols, Methods in Molecular Biology, vol. 1087, DOI 10.1007/978-1-62703-670-2_15, © Springer Science+Business Media, LLC 2014
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consequence such cells become susceptible to the growth stimulatory effect of exogenous IL-2 added to the medium during virus isolation. Third, seronegative PBMCs represent target cells for HIV infection. Donor PBMCs may vary in susceptibility to viral infection in vitro, and thus, a mixture of PBMCs obtained from two or more donors is preferable. In addition, the potentially inhibitory effects of the subject’s CD8 positive T cells in the sample are diluted out by using PBMCs in excess. In some cases this may enhance virus isolation. Viral production is determined by p24 antigen (Ag) detection assays either in the culture supernatant or intracellularly by detecting the number of p24 Ag-positive cells. Several sensitive ELISA are commercially available or in-house assay, can be performed to measure the levels of p24 Ag in the culture supernatant (see Note 1). An alternatively approach can be based on the determination of the reverse transcriptase (RT) activity in the culture supernatant. The RT assay measures the activity of the HIV RT enzyme present in supernatants of HIV-infected cultures. As the RT enzyme is virionassociated and it is not found as a free protein, the results of an RT assay are generally assumed to reflect the cellular production of complete virions. Unlike the p24 Ag capture assay with reduced crossclade reactivity, the RT activity can be used to quantify the amount of any lentivirus in culture (e.g., all HIV-1 subtypes and HIV-2). HIV RT is Mg2+ dependent and converts genomic RNA in proviral DNA. Using a detergent, the virion is broken and the RT is released. The reaction mixture consists of a poly(A) RNA template and oligo d(T) primer and (α 32P) 2′-Deoxythymidine 5′-Triphosphate, Sodium (dTTP) as substrate for the action of the HIV RT.
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Materials HIV-1 is a class 3 human pathogen and infectious virus should be handled in a biosafety level 3 laboratory. All waste should be disposed by autoclaving and liquid waste should be inactivated, for example, by detergent treatment (i.e., bleach). All the reagents should be warmed at 37 °C or room temperature before use.
2.1 Reagents and Equipment for PBMCs Isolation
1. Cells from HIV-seropositive individual. 2. Healthy blood donors or buffy coats. 3. Phosphate-buffered saline without calcium and magnesium (PBS; Lonza). 4. Lympholyte-H (Cederlane). 5. Phytohemagglutinine (PHA; concentration 5 μg/ml.
Sigma-Aldrich):
final
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6. Complete medium: RPMI-1640 supplemented with 10 % fetal bovine serum (FBS; Lonza), and 1 % penicillin streptomycin (Pen/Strep; Lonza). 7. Sterile 10 ml pipettes. 8. Sterile 25 ml pipettes. 9. Sterile 50 ml plastic tubes. 10. Electric pipettor. 11. Humidified 37 °C incubator with 5 % CO2. 12. Centrifuge. 13. Sterile flask. 2.2 Reagents for Viral Isolation from PBMCs
1. Three days PHA-stimulated healthy blood donor PBMCs. 2. PBMCs from infected patient. 3. Complete medium: RPMI-1640 supplemented with 10 % FBS and 1 % Pen/Strep. 4. IL2-medium: RPMI-1640 supplemented with 10 % FBS, 1 % Pen/Strep, and 200 U/ml recombinant Interleukin 2 (IL-2; Chiron). 5. Sterile 5 ml pipettes. 6. Sterile T25 flask. 7. Electric pipettor. 8. Humidified 37 °C incubator with 5 % CO2. 9. Centrifuge.
2.3 Reagents for Isolation of Biological Clonal Viral Variants from PBMCs
1. Three days PHA-stimulated healthy blood donor PBMCs. 2. IF medium: IMDM (Iscoves Modified Dulbecco’s Medium, BioWhittaker) supplemented with 10 % FBS and 1 % Pen/ Strep. 3. IL-2 medium: IMDM supplemented with 10 % FBS, 1 % Pen/ Strep, 10 U/ml IL-2, Ciprofloxacin (5 μg/ml), and polybrene (5 μg/ml). 4. Heparin (Leo Pharma). 5. Polybrene (Sigma). 6. Trisodium citrate dihydrate (TNC, Merck). 7. Phosphate-buffered saline (PBS, BioWhittaker). 8. FBS (Hyclone). 9. rIL-2 (Chiron Benelux). 10. Penicillin (Pen) and Streptomycin (Strep) (Gibco BRL). 11. Ciprofloxacin (Bayer).
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12. 0.2 % Triton X-100 (Merck) solution: PBS supplemented with 0.2 % Triton X-100. Addition of a dye makes it easy to recognize Triton inactivated samples. 13. MT-2 cells (ATCC, CRL-9096). 14. Sterile 10 ml pipettes. 15. Sterile 25 ml pipettes. 16. Sterile 15 and 50 ml plastic tubes. 17. Pasteur pipettes. 18. 96 wells flat bottom microtiter plates. 19. Pipette (0.5–10 μl; 2–20 μl; 20–200 μl) 20. Multi channel pipette (50–200 μl). 21. Sterile pipette tips. 22. Humidified 37 °C incubator with 5 % CO2. 23. Centrifuge. 2.4 Reagents for HIV-1 p24 Ag Staining
1. Fix & Perm Kit (Invitrogen). 2. Anti-p24 antibody: FITC-conjugated KC57 monoclonal antibody (Beckman Coulter). 3. Paraformaldehyde solution: 4 % paraformaldehyde (PFA) (Sigma). To prepare 4 % PFA solution, dissolve 1 g of PFA powder in a final volume of 25 ml of PBS. Heat at 56 °C until the powder is completely dissolved. Bring the solution at room temperature and pH to 7.2. Store at 4 °C.
2.5 Reagents for Reverse Transcriptase Detection Assay
1. RT cocktail: 60 mM Tris–HCl pH 7.8, 75 mM KCl, 5 mM MgCl2, 0.1 % Nonidet P40, 1 mM EDTA, 5 μg/ml poly A (Amersham Pharmacia Biotech, Inc.), 0.162 μg/ml oligo dT: pd(T)12–18 (Invitrogen). 2. 0.5 M Dithiothreitol (DTT). 3. (α32P) dTTP (PerkinElmer). 4. DE81 paper (Whatman). 5. SSC 20×: 3 M NaCl and 0.3 M Na Citrate pH 7.0. 6. 1450 MicroBeta PLUS and Cassette for 32P (1450-118).
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Methods
3.1 Isolation of PBMCs from Healthy Blood Donor Buffy Coats
1. PBMCs are isolated from buffy coats from two or more different healthy blood donors. PBMCs from each donor are isolated and stimulated separately. 2. Prepare two 50 ml tubes for each buffy coat and transfer 20 ml of buffy coat in each tube. Dilute the buffy coats 1:1 with PBS.
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3. Prepare four 50 ml tubes (two tubes for each buffy coat) and add 10 ml of Lympholyte-H. 4. Add the diluted buffy coat on top of the Lympholyte-H in each corresponding tube. 5. Centrifuge the tubes for 30 min at 561 × g without brake at room temperature. 6. Remove the ring fraction on top of the Lympholyte-H with a sterile pipet and add two rings together in one clean 50 ml tube. Supplement the ring fractions with PBS to 50 ml. 7. Centrifuge the tubes for 10 min at 389 × g at room temperature with brake. 8. Discard the supernatant and resuspend the cells in 50 ml PBS. 9. Centrifuge the tubes for 10 min at 249 × g at room temperature with brake. 10. Discard the supernatant and resuspend the cells in 50 ml PBS (see Note 2). 11. Centrifuge the tubes for 10 min at 140 × g at room temperature with brake (see Note 3). 12. Discard the supernatant and resuspend the cells in 30 ml complete medium. 13. To activate the cells, count the cells and transfer 1 × 106 PBMC/ml in 25 or 75 ml flask with complete medium + 5 μg/ml PHA. 14. Incubate at 37 °C, 5 % CO2 for 2–4 days (best 3 days). 3.2 Isolation of HIV-1 from Patients’ PBMCs
1. Mix 2 × 106 patient cells with 15 × 106 healthy blood donor PBMCs in the T25 flask in a total culture volume of 8 ml of IL2-medium. 2. Incubate the flask in the incubator at 37 °C with a humidified atmosphere and 5 % CO2 for 3–4 days. 3. On the third or fourth day, discard one-third to two-thirds volume of supernatant and replace fresh IL2-medium. 4. At day 7, test the presence of virus in an aliquot of supernatant using a p24 Ag capture ELISA or the RT activity assay. 5. Remove two-thirds volume of the culture supernatant and add 2–3 × 106 fresh, 2–4 days PHA-stimulated donor PBMCs to the remaining cells in the flask. Add IL2-medium up to 8 ml. 6. Incubate the flask in the incubator at 37 °C with a humidified atmosphere and 5 % CO2 for 3–4 days. 7. Repeat steps 3–5. Test supernatant for HIV productive infection each time prior to medium change or fresh PHAstimulated PBMCs addition (see Note 4). 8. When the culture supernatant is positive, collect the viruscontaining supernatant and aliquot in 1.5 ml cryovials to be stored at −80 °C.
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Fig. 1 Scheme for the isolation of biologically clonal HIV-1 variants
3.3 Isolation of Biological Clonal Viral Variants from PBMCs
1. Dilute 2–3 day PHA-stimulated donor PBMCs in IL-2 medium (see Note 5) to a cell density of 1 × 106/ml. 2. Resuspend patient PBMCs in IL-2 medium at a cell density of 5 × 104, 10 × 104, 20 × 104, and 40 × 104 cells/ml. 3. Seed the patient cell suspension in 96-well microtiter plates at 100 μl/well (32–48 wells per cell concentration). 4. Add 100 μl PHA-stimulated healthy donor PBMCs (1 × 105 cells/well). Wrap culture plates in plastic wrap to reduce evaporation. 5. Culture the cells in an incubator at 37 °C, with a humidified atmosphere and 5–10 % CO2 for 7 days. 6. On the seventh day, transfer one-third volume (65 μl) of culture supernatant (by multichannel pipette) to a new 96-well plate and inactivate the supernatant by addition of 65 μl of 0.2 % Triton X-100 and incubating for at least 30 min (see Fig. 1). 7. Test the supernatant for the presence of virus using an ELISA detecting p24 Ag (see Note 3). Inactivated culture supernatant samples for p24 Ag can be stored overnight at 4 °C or for longer periods of time at −20 °C. 8. Resuspend the culture and transfer one-third volume (65 μl) of culture (by multichannel pipette) to a new 96-well plate.
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9. Add 1 × 105 freshly prepared 2–3 day PHA-stimulated healthy donor PBMCs (steps 12–15) in 135 μl IL-2 medium to each well. Wrap culture plates in plastic wrap to reduce evaporation. 10. Incubate microcultures in an incubator at 37 °C, with a humidified atmosphere and 5–10 % CO2. 11. Add 2 × 104 MT-2 cells in a total volume of 100 μl IF medium per well to the remaining one-third culture volume. Wrap culture plates in plastic wrap to reduce evaporation (see Note 6). 12. Incubate the MT-2 microcultures in an incubator at 37 °C, with a humidified atmosphere and 5–10 % CO2. 13. Check p24 Ag-positive microcultures for syncytia at day 4 and 7 after the onset of the culture by microscopy (Fig. 1). 14. Repeat steps 6–13 every week. Continue cultures for 4–5 weeks. 15. Calculate the frequency of infected PBMCs using the formula for Poisson distribution (u = −lnF0, in which F0 is the fraction of negative cultures). When fewer than 33 % of wells seeded with a certain concentration are positive, the viruses in one well are thought to originate from one infected cell. However, if possible, it is better to pick the virus variants only from concentrations with fewer than 10 % positives. 3.3.1 Anticipated Results
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The number of clonal biological HIV-1 variants that is obtained by the protocol described here is highly variable and depends largely on the viral load of the patient at the moment the PBMC sample was taken. Indeed, a significant association between the viral RNA load in serum and the frequency of productively infected cells present in patient PBMCs has been demonstrated [3]. Also the time between the start of the coculture and detection of the first biological HIV-1 isolates is highly variable and is associated with the stage of the disease of the patient at the moment the PBMC sample was taken. When a PBMC sample from a patient in a late stage of the disease is taken, fast replicating HIV-1 variants can often be detected as early as at day 7. In contrast, isolation of slow replicating HIV-1 or HIV-2 variants from long-term nonprogressors may take 28–35 days of microculture and involve additional steps such as depletion of CD8 cells and/or stimulation of patient PBMCs before virus isolation [4]. SI or CXCR4-using HIV-1 variants develop relatively late in the course of infection in approximately half of the individuals infected with subtype B HIV-1. A relation between the viral load and the virus phenotype has also been demonstrated. The highest viral loads are observed in patients with SI variants.
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The increase in viral load in patients with SI variants is mainly due to the selective expansion of the SI HIV-1 load, although it is important to note that the NSI variants remain present throughout the disease course, even after the emergence of SI variants. In the described procedure the presence of SI or X4-variants in the patient sample is monitored by cocultivation of the primary PBMC cultures with MT-2 cells [5]. 3.4 HIV-1 p24 Ag Staining
1. Resuspend cells suspension with FACS buffer to a final concentration of 10 × 106 cells/ml. Place 100 μl of cell suspension into FACS tubes (Becton Dickinson) (see Note 7). 2. Resuspend the pellet with 100 μl of Buffer A (Fixation Medium from Fix & Perm Kit). Vortex gently to mix, and incubate for 15 min in the dark at room temperature. 3. Wash with 1 ml of FACS buffer. Centrifuge for 5 min at 249 × g and aspirate the supernatant. 4. Resuspend the pellet with 100 μl of Buffer B (Permeabilization Medium from Fix & Perm Kit) containing 0.5 μl of FITCconjugated monoclonal antibody against p24 Ag (KC57). Vortex each tube and incubate for 20 min in the dark at room temperature. 5. Wash with 1 ml of FACS buffer. Centrifuge for 5 min at 249 × g and aspirate the supernatant. 6. Resuspend pellet with 100 μl of FACS buffer for immediate analysis. Vortex gently to mix. 7. It is possible to add 100 μl of storage samples in the dark at 4 °C for maximum 24 h.
3.5 RT Activity Assay
1. Prepare RT cocktail in advance and store in aliquots at −20 °C. ~ 32P) 2. Immediately before use, add 8 μl 0.5 M DTT and 1 μl (◻ dTTP to 1 ml of RT cocktail. 3. Dispense 25 μl/well in a 96-well plate. 4. Add 5 μl of culture supernatant. 5. Incubate the plates at 37 °C in a plexiglass box for 2–3 h. 6. Two hour incubation is generally sufficient for the RT reaction to take place. Radioactivity incorporated into the reaction product will increase linearly with incubation time. Prolonged incubation times may thus be desirable if (α 32P) dTTP is 3 weeks old, however, background counts will increase with prolonged incubation. 7. After incubation with the RT cocktail, any infectious HIV will be rendered noninfectious by the NP-40 detergent in the mix. 8. While the plates are incubating, stamp a sheet of Whatman DE81 paper using an ink stamp that produces a pattern reflecting the format of the 96-well plate.
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9. Using a multichannel pipette and non-sterile pipette tips, pipette 6 μl of the reaction mixture from each well onto the DE81 paper, being careful not to tear the paper by contact with the pipette tips. 10. Dry the paper. 11. Prepare 1× SSC by diluting the 20× SSC stock solution with H2O. 12. Wash the paper four times by placing in a dish containing enough 1× SSC to cover the paper and rocking on a platform for 5 min/wash. 13. Wash again in 95 % ethanol for 5 min. The wash liquid must be discarded in radioactive-liquid waste. 14. Dry the paper in the incubator for 20 min. The paper is very fragile when wet and care must be taken when picking it up. 15. Expose the filter for autoradiography overnight. 16. Develop the film, and count the filter paper in 1450 MicroBeta PLUS using 32P Cassette (1450-118). The advantage of carrying out autoradiography before quantification is that it is possible to visualize the reaction templates and identify any potential irregularity.
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Notes 1. There are several p24 Ag capture ELISA commercially available. Sensitivity of most of them is usually very high and thus, may require extensive dilutions of the culture supernatant to determine the actual value. For the method at point 3.3, HIV-1 p24 Ag capture ELISA used is provided by the AIDS Vaccine Program (NCI-SAIC Frederick). Alternatively for all methods described here in-house equivalent assays can be performed with commercially available antibodies. For example, Aalto Bioreagents supplies antibodies and provides several alternative protocols for the detection (www.aaltobioreagents.ie). 2. When many erythrocytes are present, these can be removed by cell lysis. Resuspend the cells in 1 ml of sterile water before the last washing step, agitate gently for 20 s, add up to 50 ml PBS, and complete the washing. 3. PBMCs can now be cryopreserved in FCS supplemented with 10 % DMSO. Cryopreserved cells can be stored in liquid nitrogen for more than 10 years. 4. Cultures are kept until positive for p24 Ag at increasing levels for three consecutive times (possibly for two consecutive times p24 must be >2 ng/ml). Otherwise cultures are interrupted at day 28–30.
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5. The IL-2 medium contains polybrene, which is known to enhance HIV-1 infection presumably by facilitating the interaction between negatively charged virus particle and cell membrane of the target cell. 6. Working with primary cells from blood donor and HIV-1infected patients may result in frequent introduction of mycoplasma. Precautions should be taken to prevent infection of other cells or cell lines. It is recommended to add ciprofloxacin to the cultures to inhibit replication of mycoplasma. 7. It is important to include a control of uninfected cells. This control should be stained for p24 Ag detection and used to set the background signal. References 1. van 't Wout AB, Schuitemaker H, Kootstra NA (2008) Isolation and propagation of HIV-1 on peripheral blood mononuclear cells. Nat Protoc 3:363–370 2. Sawyer LS, Wrin MT, Crawford-Miksza L, Potts B, Wu Y, Weber PA, Alfonso RD, Hanson CV (1994) Neutralization sensitivity of human immunodeficiency virus type 1 is determined in part by the cell in which the virus is propagated. J Virol 68:1342–1349 3. Blaak H, de Wolf F, van 't Wout AB, Pakker NG, Bakker M, Goudsmit J, Schuitemaker H (1997) Temporal relationship between human
immunodeficiency virus type 1 RNA levels in serum and cellular infectious load in peripheral blood. J Infect Dis 176:1383–1387 4. van Rij RP, Schuitemaker H (2002) Host genetic factors in the clinical course of HIV-1 infection: chemokines and chemokine receptors. Community Genet 5:88–101 5. Koot M, Vos AH, Keet RP, de Goede RE, Dercksen MW, Terpstra FG, Coutinho RA, Miedema F, Tersmette M (1992) HIV-1 biological phenotype in long-term infected individuals evaluated with an MT-2 cocultivation assay. AIDS 6:49–54
