Divergent effects of cell environment on HIV entry inhibitor activity
Rusert, Petera; Mann, Axela; Huber, Michaelb; von Wyl, Viktorb; Günthard, Huldrych Fb; Trkola, Alexandraa
aInstitute of Medical Virology, University of Zurich, Switzerland
bDivision of Infectious Diseases, University Hospital Zurich, Zurich, Switzerland.
Received 4 December, 2008
Revised 24 March, 2009
Accepted 30 April, 2009
Correspondence to Alexandra Trkola, Institute of Medical Virology, University of Zurich, Gloriastrasse 30, 8006 Zurich, Switzerland. Tel: +41 44 634 53 80; fax: +41 44 634 49 67; e-mail: firstname.lastname@example.org
Objective: Successful HIV vaccine and entry inhibitor development depends on use of assay systems that closely reflect in-vivo activities. Recent reports suggest that the currently most widely used assay format, which relies on the genetically engineered target cell line TZM-bl, can fail to detect certain neutralization activities detected on primary peripheral blood mononuclear cell (PBMC)-based assay systems. In the present study, we investigate the influence the target cell context bears on HIV entry inhibition.
Design: In a comprehensive survey, the effect of 11 neutralizing antibodies and inhibitors in blocking entry of 30 envelope pseudotyped virus strains in two types of target cells, PBMC and TZM-bl, was evaluated.
Methods: Env-pseudotyped HIV infection of PBMC and TZM-bl cells.
Results: We demonstrate here that depending on the type of inhibitor, relative neutralization potencies are shifted to a variable extent and direction on TZM-bl and PBMC cells. In our assay set up, differences in inhibitor activity were solely effected by the target cell environment and amounted up to 2–3 logs lower activity on TZM-bl cells in several cases. Overall, neutralizing antibodies, 2G12, 2F5 and 4E10, were less active in the TZM-bl system, whereas CD4 binding site directed inhibitor activities were detected equally well on both target cells, raising concerns that the TZM-bl assay may overrate the relevance of CD4 binding site specific responses.
Conclusion: Our data strongly argue that preclinical assessment should not be restricted to a single type of assay, as systematic underestimation or overestimation of activities would be inevitable.
Elicitation of high-titered and broadly cross-reactive antibodies is considered a key component of vaccines against HIV. Although over the past decades immunogen design has struggled to induce potent responses, assessment of the induced activities and comparison between different vaccine approaches was equally complicated by the wide variety of assay systems and viral strains used to evaluate HIV neutralization activity in vitro. Recent years have brought a significant improvement when novel cell line-based assays were introduced that rely on measuring virus entry in genetically engineered reporter cell lines on infection with envelope-pseudotyped luciferase reporter gene virus [1–3]. Uniformity in testing was further enforced by the use of reference panels of virus envelopes from several genetic subgroups [4–8]. Although current TZM-bl-based assay systems are not entirely standardized between laboratories (e.g. different virus backbones, producer cells, incubation periods and detection systems are being used), both inter and intralaboratory variations have been described to overall compare favorably to the peripheral blood mononuclear cell (PBMC)-based systems [4,5,9]. However, recent observations from our laboratory (see accompanying study in this issue by Mann et al.) and from several reports in the literature have brought forward evidence that the TZM-bl-based assay system can fail to detect specific neutralization activities that are recognized in the conventional PBMC-based assay [9–13]. Apart from the type of the target cells, also virus sources and replication systems (single round as opposed to multiple-round infection) differ between these assay types and account altogether for widely different infection environments that complicate direct comparison between these systems. In the present study, we investigate the influence of the target cell environment on HIV entry inhibition.
Material and methods
Reagents were provided by: soluble CD4 (sCD4)  and CD4–immunoglobulin G2 (IgG2)  by W. Olson (Progenics Pharmaceuticals Inc., Tarrytown, New York, USA); IgG1b12 by D. Burton ; 2G12 , 2F5  and 4E10 [19,20] by H. Katinger (Polymun, Vienna, Austria); T-20  by Roche Pharmaceuticals, Nutley, New Jersey, USA; AD101 (SCH-350581) and AMD3100  by J. Stritzki (Schering Plough, Kenilworth, New Jersey, USA) ; and PSC-RANTES by O. Hartely . DARPin 57.2 was produced as described .
Preparation of stimulated primary CD8-depleted peripheral blood mononuclear cell
See supplementary information 1 and .
TZM-bl cells [27–29] were obtained through the National Institutes of Health (NIH) AIDS repository and 293-T cells from American Type Culture Collection (ATCC).
Patient plasma utilized in this study was derived during recently conducted clinical trials as described [30–32]. Written informed consent was obtained from all individuals according to the guidelines of the ethics committee of the University Hospital Zurich.
Generation of env-pseudotyped HIV particles
Origins of plasmids encoding the various HIV-1 envelope genes are listed in supplementary Table 2. 293-T cells were transfected with plasmids carrying the reporter gene expressing the virus backbone, pNLluc-AM  (from A. Marozsan and J. P. Moore) and the functional envelope clone at a ratio of 3: 1 using polyethylenimine (PEI, linear, 25 kDa; Polysciences, Warrington, Pennsylvania, USA)  as described . Viral supernatants were harvested 2 days after transfection, and the 50% tissue culture infective dose (TCID50) was determined on TZM-bl cells by end-point dilution [infection medium: Dulbecco's modified eagle medium (DMEM), 10% fetal calf serum (FCS) and 1% penicillin–streptomycin (BioWhittaker, San Francisco, California, USA) containing 10 μg/ml diethylaminoethyl (DEAE)-Dextran (Amersham Biosciences, Fairfield, Connecticut, USA)]. The use of the vector pNLluc-AM, which carries a luciferase reporter gene under control of the SV40 promotor , allows detection of HIV infection on both, cells that harbor a reporter gene and cells types that do not (e.g. PBMC). On CD8-depleted PBMC, pNLluc-AM pseudotyped virus yields up to 20-fold higher luciferase activity than a conventional reporter vector, pNL4-3.Luc.R-E (data not shown).
Neutralization assay with env-pseudotyped reporter gene viruses on TZM-bl cells
Neutralization activity of entry inhibitors and patient plasma against pseudotyped virus on TZM-bl was determined as described. TZM-bl (1 × 104) cells were seeded in a 96-well, flat bottom plate (Falcon; Becton Dickinsen (BD) Falcon Labware, Franklin Lakes, New Jersey, USA) in DMEM medium containing 10% FCS, antibiotics and 10 μg/ml DEAE-Dextran (Amersham Biosciences) and infected with 200 TCID50/well in presence or absence of inhibitors in a total volume of 200 μl.
When virus-directed agents were tested, viruses were preincubated with the inhibitor for 1 h at 37°C. When cell-directed inhibitors were used, cells were pretreated under the same conditions for 1 h with the inhibitor. Luciferase reporter gene production was quantified 48 h after infection on cell lysis and addition of firefly luciferase substrate (Promega, Madison, Wisconsin, USA). Emitted relative light units (RLUs) were quantified on a Dynex MLX luminometer (Dynex Technologies Inc., Chantilly, Virginia, USA) (medium sensitivity setting). The antibody or inhibitor concentration causing a 70% reduction in luciferase reporter gene production was determined by regression analysis.
Neutralization assay with env-pseudotyped reporter gene viruses on peripheral blood mononuclear cells
For pseudotype virus infection of PBMCs, the protocol was adapted as follows. Infections were carried out in a total volume of 200 μl in RPMI1640 medium (Sigma-Aldrich, St. Louis, Missouri, USA) containing 10% FCS, antibiotics and 2.5 μg/ml polybrene (Sigma-Aldrich). PBMCs (2 × 105) were seeded per well and infected with 1000 TCID50/well (on the basis of titration on TZM-bl cells) in presence or absence of inhibitors in a total volume of 200 μl. Polybrene was used instead of DEAE-Dextran as additive to aid infection, as we noted that PBMC tolerated DEAE-Dextran less well (data not shown). Addition of polybrene was omitted when X4-viruses were probed, as this compound is known to interfere with chemokine (C-X-C motif) receptor 4 (CXCR4) interaction. All other steps, including preincubation time, were carried out as described above. Seventy-two hours after infection, 150 μl supernatant of the total 200 μl assay volume was removed and 50 μl lysis buffer (Promega) added to the cells. After 5 min, luciferase activity was measured in 50 μl of the cell lysate on transfer to a white opaque plate (Costar; Corning Inc., Corning, New York, USA) and addition of 50 μl firefly luciferase substrate (Promega) using a Dynex MLX luminometer (high-sensitivity setting). Background RLU emitted by uninfected PBMC lay between zero and 10 RLUs. We only included virus stocks in our study that yielded at minimum 2000 RLU/well. Calculation of inhibitory activity was performed as described above.
Control for nonspecific inhibitory effects in the neutralization assays
See supplementary information 1.
See supplementary information 1.
Applying HIV pseudotype infection of peripheral blood mononuclear cells to assess neutralization and entry inhibition
To explore to what extent the context of the target cell steers the effect of HIV entry inhibitors, we set out to compare infection of PBMC and TZM-bl cells in a controlled setting. Previous studies from our group (see accompanying study in this issue by Mann et al.) have highlighted the differences between these two assay formats and defined impact of both target and virus producer cell on the efficiency of entry inhibition. To limit the number of deviating factors, we chose here to compare solely envelope pseudotype infection of PBMC and TZM-bl cells. To allow detection of pseudovirus infection of PBMC, we generated viruses using the reporter virus backbone vector pNLluc-AM, which harbors the luciferase gene under control of the SV40 promotor, and thereby generates sufficient amounts of the luciferase reporter for detection of primary cell infection . As we were able to use identical envelope-pseudotyped virus stocks for infection of both TZM-bl and PBMC assays and employed identical assay conditions, the only differences that may be encountered between the two probed assays originate from properties of the target cells.
In a first step, we validated the two assay systems, the pseudotype TZM-bl assay and the pseudotype PBMC assay. To confirm that the TZM-bl system as applied in our laboratory provides the same sensitivity in readout as described by other laboratories, we probed the inhibitory capacity of 2G12, 2F5 and mAb IgG1b12 against a reference panel of 10 pseudotyped viruses (Supplementary Table 1; ). Comparison of in-house and published inhibitory activities against these viruses showed no significant differences (Supplementary Table 1) confirming the validity of the results obtained in our in-house TZM-bl assay.
Next, we verified that env pseudotype virus infection of PBMC gives reproducible results and performs comparable with the TZM-bl assay in terms of assay stability. Although PBMC infection with the reporter gene virus pNLluc-AM yields 1–2 logs lower luciferase activity than infection with the same viruses on TZM-bl, both assays perform comparable and deliver inhibitory dose response curves of high quality (Fig. 1a). Notably, although pseudotype infection of PBMC yields a lower RLU output, these cells also have a 2–3 log lower background activity in the luciferase readout compared with TZM-bl cells, which carry a luciferase gene under control of long terminal repeat (LTR). This low background activity of PBMC allows on average for a two-fold higher signal-to-noise ratio on PBMC compared with TZM-bl (Fig. 1b: mean signal-to-noise ratios per μl virus stock of the 30 probed virus isolates: PBMC, 89.2; TZM-bl, 43.2). Importantly, the emitted RLUs on infection with identical virus stocks correlated tightly between TZM-bl cells and PBMC (Spearman r = 0.79, P < 0.0001; Fig. 1b). We verified that the achieved RLUs on PBMCs allow us to obtain reproducible and robust results by performing neutralizations assays with NAB01pre.39× pseudotyped virus, which had one of the lowest RLU signal/μl virus stock of all tested viruses (mean of five independent assays = 149 RLU/μl virus) and with JR-FL pseudotyped virus, which had one of the highest RLU levels of the probed viruses (mean of five independent assays = 1201 RLU/μl virus) (Fig. 1a). Infectivity and sensitivity to inhibition of these viruses with CD4IgG2 was tested in five independent experiments using five different PBMC donor pools (each pool consisting of three healthy donors PBMCs) and compared with inhibition on TZM-bl cells (Fig. 1a).
To achieve sufficient HIV pseudotype virus infection, addition of polycations is required. Although routinely DEAE-Dextran is used for TZM-bl cell infection by R5 viruses, we found that sufficient infection of PBMC with pseudoytpe virus depends on addition of polybrene, as DEAE-Dextran does not yield sufficient entry with all viruses (data not shown). To ascertain that these additives have no confounding effect on the assessment of entry inhibition, we probed their influence on the inhibitory activity of 2G12 against JR-FL under increasing concentrations of DEAE-Dextran and polybrene. As expected, virus entry (as measured by luciferase activity) was the lowest when no polycation was added and increased on addition of DEAE-Dextran and polybrene (Fig. 1c left). Importantly, despite the dramatic impact on the viral infectivity, neither concentration nor type of polycation had a significant impact on the inhibitory activity of 2G12 on TZM-bl cells (Fig. 1c right).
Influence of assay system and entry inhibitor class on antiviral activity
We performed a comprehensive evaluation of inhibitor activities on PBMC and TZM-bl cells on a panel of 30 env-pseudotyped viruses (Supplementary Table 2 and 3). Of note, in both assays, identical pseudotyped virus stocks were used and infectivity determined by quantifying the reporter firefly luciferase activity.
To thoroughly assess, if and to what extent, the potency of different types of entry inhibitors varies depending on the target cell environment, we studied a broad spectrum of inhibitors targeting the CD4-binding site (CD4bs) of gp120 (sCD4, CD4–IgG2 and IgG1b12), a carbohydrate motif in gp120 (2G12), gp41 (2F5, 4E10 and T-20) and the cellular receptors CD4 (DARPin 57.2 ), chemokine receptor 5 (CCR5) (AD101 and PSC-RANTES) and CXCR4 (AMD3100). The comparison was based on the 70% inhibitory concentration (IC70) values as in our experience readout at IC50 can be more prone to pick up low-level nonspecific inhibitory effects. The only exception was sCD4, which due to its low inhibitory activity could only be assessed at the IC50 level. Comparison of the inhibitory activities provided a striking and quite unexpected result: the discrepancy between TZM-bl and PBMC assay can amount up to a 100-fold difference in inhibitory dose, but the effects varied considerably depending on the inhibitor and virus investigated (Fig. 2a–c). The ratios of the IC70 values obtained in the two assays (IC70TZM-bl/IC70PBMC) highlight the observed differences (Fig. 3) ; 2G12, 2F5, 4E10 and the CCR5-targeting inhibitors were significantly less active on TZM-bl cells (Bland–Altmann analyses, Fig. 3). Four viruses (NAB07pre.20, NAB12pre.10, PVO.4 and TRO.11) with mutations in the core epitope of 2F5 (Supplementary Table 3) were resistant to 2F5 in the cell line assay but retained moderate sensitivity to this mAb in the PBMC assay. mAb 2G12 failed to inhibit 12 viruses in the TZM-bl assay, three of which proved to be sensitive in the PBMC assay. Generally, isolates were less sensitive to inhibition by mAbs 2F5, 4E10, 2G12 and the CCR5 inhibitors with up to 100-fold lower sensitivity in the TZM-bl system (Fig. 2a–c and Fig. 3).
The pattern for IgG1b12 was more complex; although in several cases the PBMC assay was also more sensitive for this mAb, the differences between the assays were less pronounced, and assessment of the 95% limits of agreement did not prove relevant differences. AMD3100 showed a markedly enhanced activity in the TZM-bl assay against the four X4 using viruses in our panel. Identical activity in both assay systems was found for the fusion inhibitor T-20, the CD4-targeting compound DARPin 57.2 and the gp120 CD4bs-targeting agent, CD4–IgG2, which mirrors the activity of IgG1b12. Activity of sCD4 was similar on both cell types, although it proved to be less effective in the PBMC assay in some cases. Three viruses were not inhibited at the maximal concentration probed in the PBMC system but were effectively blocked at the same concentration in the TZM-bl assay.
Effects of target cell origin on plasma neutralization activity
Considering the differential detection of activities in the TZM-bl and PBMC assays depending on the type of entry inhibitor and mAb investigated, we reasoned that this may particularly limit the assessment of neutralization activity in a polyclonal setting. To explore this, we performed neutralization assays with NAB01pre.39×, JR-FL and NL4-3-pseudotyped virus and patient plasma derived from chronically infected individuals that we previously characterized extensively [30,37]. We found that the use of the pseudotype PBMC assay for analysis of plasma neutralization activity is possible but needs to take into account the relatively high sensitivity of PBMC to nonspecific (cytostatic or toxic) inhibitory effects of plasma that can occur up to plasma dilutions of 1: 150, in some cases even higher. In comparison, the TZM-bl assay is fairly resistant against nonspecific effects allowing evaluation of neutralization activity at dilutions 1: 40 or lower (data not shown). Inclusion of env-pseudotyped murine leukemia virus (MuLV) infection to determine nonspecific inhibition for each PBMC donor cell pool is therefore pivotal. In our analysis, only plasma samples with MuLV inhibitory activity on PBMC below 1: 150 were assessed for HIV neutralization on PBMC and TZM-bl cells (cutoff 1: 40). The 27 chronic patient plasma samples analyzed (Fig. 4) showed a wide variety of reactivity patterns, reaching from samples with higher activity on TZM-bl (maximum 2.5-fold), identical activities, to higher activity on PBMC (maximum 48.6-fold). Altogether, these analyses support our previous observations and suggest that depending on the composition of the polyclonal response in the respective plasma, activities can be differentially detected in the respective assay systems.
The questions raised by the dichotomy we observed in results between infections of PBMC and TZM-bl cells are evident of high relevance for future assessment of neutralizing activity in vaccine trials: which in-vitro assay system reflects best in-vivo activity? Does the TZM-bl assay underestimates activity in some specific settings, or in contrast does the PBMC assay gives false positive results? Our study provides some answers in this respect but also unravels the complexity of the problem.
In recent years, the use of engineered cell lines for neutralization and entry inhibitor assessment has proven to be superior in terms of inter and intralaboratory performance, which are pivotal features in vaccine development [2–4,12]. The advantages of these methods over systems that depend on infection of stimulated primary PBMC are multifold: an increase in reproducibility as PBMC donor cell variability can account for more than a log of difference in infectivity; usage of standardized virus preparations derived through transfection, which eliminates drifts in virus evolution introduced during long-term culturing; a sensitive readout through reporter gene expression; and the possibility to adapt the assay for high-throughput screening. Discrepancies between the engineered cell line and PBMC-derived assay systems have been noted by some investigators [9–13], and recently, caution in using solely one assay system has been raised . Our present study substantiates this request. We provide compelling evidence that the TZM-bl assay can underestimate certain activities that are reported by PBMC-based assay systems. Our study further clarifies that the discrepancy in results is not due to false positive report in the PBMC-based assays but reveals specific insensitivities of the TZM-bl assay. Considering the wide use of TZM-bl cells and related assay systems in vaccine and entry inhibitor development, unraveling the limitations of this assay system and how these can be overcome are crucial.
Here, we show that pseudotype virus infection of PBMC gives a very robust readout that mirrors the results of our multiple-round assay, providing a reference for the latter readout. Use of env-pseudotyped virus for PBMC infection experiments restricts the infection to a single round of infection during which neutralizing antibodies have to be active. In contrast, in the traditional multiple-round PBMC assay, neutralizing antibodies have to remain active throughout the entire assay duration and must inhibit consecutive rounds of infection as well as cell–cell transmission of virus elucidating why pseudotype infection of PBMC is more sensitive to inhibition than multiple-round infection of PBMC (see also accompanying manuscript in this issue by Mann et al.). Of particular interest in this respect is the comparatively high activity of mAb 4E10 against psesudotype virus on PBMC, as the mAb was previously described to be less efficiently inhibited on PBMC (using replication competent virus) compared with pseudotype inhibition of TZM-bl [9,12]. All in all, we found that pseudotype infection allows the use of PBMC as target cells for entry inhibitor evaluation under more standardized conditions compared with assays that use primary viruses. We found, however, that employment of this assay system for plasma sample evaluation, although possible, is more restricted as it requires strict monitoring of HIV-1 nonspecific inhibitory effects.
Comparing the results of a panel of 11 entry inhibitors in the TZM-bl and PBMC systems side by side (Figs. 2 and 3) revealed a striking fact: difference in sensitivities of up to 100-fold occurred. The extent and direction of the shifts induced by the use of diverse target cells varied depending on both inhibitor type and virus. The latter is likely a consequence of viral envelope variability and the ensuing effects on recognition by the inhibitors and mAbs. The fact that inhibitor types show overall distinct reactivity patterns independent of the virus investigated is intriguing and suggests that their mode of action is substantially affected by target cell-dependent factors that stir the entry process. This was most evident with CD4bs-directed agents, as these were not (or only marginally) affected by the target cell type, whereas other inhibitors were substantially influenced. This variability imposes strong limitations in detecting or dissecting inhibitory activities directed to different targets in a polyclonal setting as in patient sera. Use of single assay system to evaluate one type of inhibitor – for example, CD4bs-directed activities – is certainly feasible, but comparing these activities to other neutralizing activities may be severely hampered [12,38,39]. Our data highlight the extent of the distortion. Whereas CD4bs-directed activities are detected equally in either system, other inhibitors are 100-fold, in rare cases 1000-fold, less sensitive in the TZM-bl assay. Depending on the overall frequency and concentration of such antibodies in the investigated samples, they may therefore easily be missed.
Apart from the evaluation of the respective assay systems, our analysis provided a detailed insight into the range of activities of the various inhibitors independent of the specific assay system used. Particularly notable was that the three CD4bs-targeting inhibitors showed the highest variability across divergent viruses probed, with activities covering a 4 log range from extremely high (0.057 nmol/l, NDK) to very low sensitivity (241 nmol/l, CAAN5342.A2). mAbs 2G12, 2F5 and 4E10 in comparison were active over a 3 log range. T-20 and the receptor antagonist inhibited over a 1–2 log range (Fig. 2). The lowest variability across isolates paired with a high potency was found for the novel CD4-specific inhibitor DARPin 57.2 and PSC-RANTES, underlining the potential of cell-directed interventions in entry inhibition. We had to limit our study to antibodies and inhibitors for which we could obtain ample amounts to allow for a large, controlled analysis. Several antibodies and inhibitor types of interest (e.g. V3 loop antibodies) could thus not be assessed but clearly should be explored in forthcoming studies aiming to devise assay directives.
Our study provides strong evidence that the sole use of a single assay type could lead to a systematic underestimation or overestimation of specific inhibitor activities. Although our study revealed a necessity to incorporate PBMC-based assay systems into inhibitor and vaccine evaluation, use of PBMC for high-throughput screenings remains difficult. Availability of cells, donor variation and labor intensiveness clearly limits a widespread use. Defining conditions that allow continued use of the TZM-bl assay or development of novel assay systems will be ultimately necessary.
Support was provided by the Swiss National Science Foundation (PP00B-102647 and 310000-120739 to A.T. and 3100A0-103748 to H.F.G. and A.T.), research grants from the Gebert-Rüf foundation (P-041/02) and the EMDO foundation to A.T. and by a research grant of the Kanton Zürich. A.T. is an Elizabeth Glaser Scientist supported by the Elizabeth Glaser Pediatric AIDS Foundation.
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