ART-DEX: A novel strategy to monitor broadly neutralizing antibody activity during antiretroviral therapy of HIV-1

Summary Therapeutic use of HIV-1 broadly neutralizing antibodies (bnAbs), passively administered or induced by therapeutic vaccines, is a focus of advanced treatment strategies under development. To enable monitoring of bnAb activity during concurrent antiretroviral therapy (ART), we developed ART-DEX, an analytic strategy that allows high-throughput detection of pure antibody-based neutralizing activity. ART-DEX combines pH-dependent dissociation of antiretrovirals (ARVs) from plasma proteins and size exclusion to effectively remove ARVs from plasma samples, reducing the confounding effects of ARVs on neutralization assays. For complete details on the use and execution of this protocol, please refer to Schwarzmüller et al.1

Note: We typically freeze TZM-bl and HEK 293-T cells at densities of 2-3 3 10 6 .Cells in culture are passaged 2-3 times per week.We recommend a maximum passage number of 20 and 40 for TZM-bl and HEK 293-T cells, respectively.
Note: Cells have to be tested regularly for mycoplasma contamination.
Note: Pseudovirus backbone NLluc-AM 4 and the multidrug-resistant version of NLluc-AM (HIV pv -MDR, Schwarzmu ¨ller et al. 1 ) have been pseudotyped with diverse Env (Murine Leukemia Virus (MuLV) and HIV) and probed in combination with ART-DEX.
Note: It is highly recommended to include pseudoviruses carrying an Env of an unrelated virus (e.g., MuLV) as control.
a. Serially dilute frozen pseudovirus stocks in DMEM-10 starting with undiluted pseudovirus (e.g., 1:4 dilution for 5 dilution steps).b.Add 100 mL of virus dilution to 100 mL TZM-bl cells seeded in 96 well plates (1 3 10 4 cells/well, see steps 1-3, seeding TZM-bl cells).c.Incubate for 48-72 h at 37 C, 5% CO 2 .d. Carefully remove media from cells.e. Add 50 mL Luciferase lysis buffer to each well and incubate for 5 min.f.Add 50 mL Bright-Glo luciferase assay substrate (diluted 1:10 in Luciferase lysis buffer) per well.g.Measure luciferase expression on the PerkinElmer plate reader within 30 min.h.To calculate infectivity of pseudovirus stocks, normalize background-corrected RLU values to virus input and calculate average titer (RLU/mL) over the linear range of the dilution curve.
Pause point: Pseudovirus stocks can be stored at À80 C until use in neutralization assays.

Heat-inactivation of plasma samples
Timing: 1 h 15.All plasma or serum samples irrespective if from people with or without HIV must be incubated for 1 h in a water bath at 56 C to inactivate HIV, potentially other pathogens, complement and coagulation factors.
Note: Prior to use in neutralization assay, store plasma sample for at least 2 h at À80 C.
Pause point: Heat-inactivated plasma can be stored at À80 C until use in neutralization assays.

STEP-BY-STEP METHOD DETAILS
Seeding TZM-bl cells Timing: 1 h on day 1 Assessment of HIV-1 neutralization activity is typically performed using the pseudovirus and TZM-bl cell-based neutralization assay (short TZM-bl assay), considered a standard assay in the HIV-1 field. 2,3In the present protocol TZM-bl cells are kept in continuous culture in T-75 flasks and seeded in 384 well culture plates for the neutralization assay.Antiretrovirals (ARVs) record as virus inhibition in the TZM-bl assay that is indistinguishable from antibody-based inhibition.ART-DEX has been designed to separate ARVs from plasma samples from people with HIV enabling the assessment of pure antibody-based inhibition.Most HIV-1 ARVs are highly bound to plasma proteins. 5ART-DEX builds on this by first releasing ARVs from plasma proteins by alkaline and acidic pH treatment followed by separation by size-exclusion filtration.Note: Exact pH of buffer solution is critical.Check pH before use of the buffer solution for ART-DEX.Troubleshooting 1.

Note:
We have not validated the performance of the ART-DEX step with highly viscous or fatty plasma.As all plasma samples are pre-diluted for the ART-DEX treatment, we expect that most of these plasmas can be processed without problems.We recommend however that the researchers take note of viscous/fatty plasmas at the pre-dilution step to monitor their performance in the following filter step.

Protocol
Note: There may be sample loss (10-15 mL) during centrifugation.
Note: We strongly recommend to directly proceed with neutralization assay after the ART-DEX step.Troubleshooting 3.

Timing: 2.5 h on day 2
To determine the plasma neutralization capacity, ART-DEX-treated plasma samples are in the next steps processed as usual in the TZM-bl neutralization assay. 2,3In brief, samples are serially diluted, pre-incubated with pseudoviruses, then added to TZM-bl cells and incubated until the readout 48-72 h later.9. Serially dilute ART-DEX-treated plasma samples in DMEM-10 containing 25 mM HEPES.a. Start dilution series with the prepared 1:5 dilution of the eluted plasma sample.b.Serially dilute plasma sample 1:4 in DMEM-10 containing 25 mM HEPES for 5 dilution steps.c.Add one well with DMEM-10 containing 25 mM HEPES only to obtain reference value for maximal infectivity of virus strain.10.Add 25 mL pseudovirus (approx.500 RLU/mL, diluted in DMEM-10) to the corresponding 25 mL plasma dilution.Troubleshooting 4. 11.Incubate plasma/virus mix for 1 h at 37 C, 5% CO 2 .12. Transfer 40 mL plasma/virus mix to TZM-bl cells seeded on day 1. 13. Incubate for 48-72 h at 37 C, 5% CO 2 .

Luciferase readout
Timing: 30 min on day 5 Infection of TZM-bl cells by the pseudoviruses can be quantified through the expression of the firefly luciferase reporter protein expressed upon infection. 2,3.Lyse TZM-bl cells.
a. Carefully remove media from cells.b.Add 25 mL Bright-Glo luciferase assay substrate (diluted 1:10 in Luciferase lysis buffer) per well.15.Measure luciferase expression on the PerkinElmer plate reader within 30 min.Troubleshooting 5.

EXPECTED OUTCOMES
ART-DEX is designed to separate ARVs from plasma samples, so that confounding effects of the ARVs on antibody neutralization measured in pseudovirus-based neutralization assays can be minimized.In this way, ART-DEX provides means to accurately measure plasma neutralization activity of ART-treated PWH.
To verify if removal of ARVs by ART-DEX was successful, neutralization against Murine Leukemia Virus (MuLV) Env pseudoviruses (or other non-HIV Env pseudovirus) should be measured.MuLV is not inhibited by plasma from PWH unless the plasma contains ARVs. Figure 1 shows an example of a successful ART-DEX treatment of a plasma sampled from a PWH on ART (Emtricitabine + Rilpivirine + Tenofovir, data from Schwarzmu ¨ller et al., 1 ; Figure 6).Without ART-DEX the plasma inhibits both MuLV pseudovirus (black) and the HIV-1 pseudovirus CNE40 (blue).With ART-DEX, ARVs are fully removed, no MuLV inhibition occurs.The inhibition measured against CNE40 is therefore solely due to HIV-1 specific antibodies in the plasma.Note in Figure 1 the wild-type pseudovirus backbone was used.

LIMITATIONS
ART-DEX treatment is highly efficient in removing ARVs from plasma but, as we show in Schwarzmu ¨ller et al., 1 some residual ARV activity may be retained, depending on concentration and potency of the drug.It is thus important to verify if full ARV removal was achieved by probing the inhibitory activity of ART-DEX treated plasma against MuLV.Should residual inhibitory activity against MuLV be detected, combining ART-DEX with a multi-drug resistant pseudovirus as introduced in Schwarzmu ¨ller et al. 1 is highly recommended.

TROUBLESHOOTING Problem 1
Unstable pH for phosphate buffer solution (pH 10.0).

Potential solution
As the pH is outside of the buffer range of phosphate buffers, the pH of the solution may be unstable.We recommend to adjust pH of the buffer solution first to pH 9.5 and adjust to pH 10.0 the next day.Re-measure pH each time before using it in the ART-DEX protocol.

Potential solution
We strongly recommend to re-measure pH each time before using the buffer in the ART-DEX protocol.

Problem 3
We did not evaluate the impact of long-term storing pH-treated plasma samples on plasma antibodies.

Potential solution
We strongly recommend to directly proceed with neutralization assays after ART-DEX without storing plasma samples for longer time as the pH treatment may otherwise impact plasma antibodies.

Problem 4
Low infectivity of pseudoviruses in combination with some Envs (e.g., infectivity of pseudoviruses less than 10-fold over background signal of TZM-bl cells).

4 .
Treatment of plasma sample with basic pH.a. Dilute plasma sample 1:2.5 in phosphate buffer, pH 10.0.b.Incubate sample for 2 h at room temperature.

Figure 1 .
Figure 1.Expected outcomes after ART-DEX The data show the inhibitory activity of plasma from a person with HIV containing ARVs (Emtricitabine + Rilpivirine + Tenofovir) against MuLV pseudovirus (black) and the HIV-1 Env pseudovirus CNE40 (blue) in the standard TZM-bl based neutralization assay (left).As ARVs are present MuLV is inhibited.Analysis of the same plasma sample after the ART-DEX step in the TZM-bl assay (right) shows that ARVs are removed effectively from the plasma samples, as no MuLV inhibition is observed.The inhibition measured against CNE40 is therefore HIV-1 antibody specific.

Figure 2 .
Figure 2. Use of multidrug-resistant backbone to overcome residual inhibitory activity of ARVs after ART-DEX The data show the inhibitory activity of plasma from a person with HIV containing ARVs (Emtricitabine + Elvitegravir + Tenofovir) against MuLV pseudovirus either in the standard TZM-bl based neutralization assay (black), after ART-DEX (red) or after ART-DEX in combination with use of the multidrug-resistant backbone HIV pv -MDR (orange).

TABLE MATERIALS
(Continued on next page) Media supplemented with FCS (DMEM-10): DMEM with 10% of FCS + Penicillin/Streptomycin [Store at 4 C for several weeks]