BASIC

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TRANSLATIONAL SCIENCE

Exposure to Entry Inhibitors Alters HIV Infectiousness and Sensitivity to Broadly Neutralizing Monoclonal Antibodies Victor G. Kramer, MSc,*† Olivia Varsaneux, BSc,*‡ Maureen Oliviera, BSc,* Susan P. Colby-Germinario, MSc,* Thibault Mesplède, PhD,* and Mark A. Wainberg, PhD*†‡

Background: The development of envelope-specific neutralizing antibodies that can interfere with viral entry into target cells is important for the development of an HIV-1 vaccine. Another means of blocking viral entry is through the use of entry inhibitors such as the CCR5 inhibitor maraviroc (MVC), which can also repel cell-free virus particles from the cell surface. For this reason, we hypothesized that exposure to entry inhibitors might alter viral infectiousness and sensitivity to antibody-mediated neutralization.

Methods: The CCR5-tropic HIV-1 variants BaL, AD8, and CC 1/85 were used to infect PM-1 cells in the presence of 2 entry inhibitors, enfuvirtide and MVC. After 4 hours, culture fluids were ultrafiltered and the infectiousness and susceptibility to broadly neutralizing antibodies (2F5, 4E10, 2G12, b12, VRC01, PG9) of viruses exposed to these entry inhibitors were assessed using TZM-bl cells.

Results: Viruses exposed to the entry inhibitor MVC exhibited lower infectiousness than controls. Enfuvirtide exposure increased AD8 sensitivity to 2F5, 4E10, VRC01, and b12 and increased BaL sensitivity to 4E10 while lowering BaL sensitivity to b12 and VRC01. MVC-exposed BaL became less susceptible to the gp120specific antibodies b12, 2G12, and VRC01. Conclusions: Exposure to entry inhibitors altered HIV-1 infectiousness and sensitivity to gp120-specific neutralizing antibodies. This alteration of entry inhibitor-exposed virus has implications for the development of future entry inhibitors and for vaccine development. Key Words: antibodies, antiretrovirals, HIV, maraviroc, T-20, entry inhibitors (J Acquir Immune Defic Syndr 2014;67:7–14) Received for publication December 13, 2013; accepted May 1, 2014. From the *McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada; †Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada; and ‡Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada. Supported by Canadian Institutes of Health Research. M.A.W. has received grant support from Pfizer, ViiV, Janssen and Merck, Inc. The remaining authors have no funding or have no conflicts of interest to disclose. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.jaids.com). Correspondence to: Mark A. Wainberg, PhD, McGill AIDS Centre, 3999 Côte-Sainte-Catherine Road, F-328, Montrea QC H3T 1E2 Canada (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins

INTRODUCTION The HIV-1 envelope (Env) is the sole protein that the virus expresses on its surface. It consists of 3 copies of gp120/gp41 heterodimers grouped in a trimeric formation. The entry inhibitor class of antiretrovirals targets this protein both directly1–3 and indirectly.4,5 The selective pressure in patients on Env is considerable, as Env must evolve to escape humoral immune responses while maintaining infectiousness in a changing receptor microenvironment. Entry inhibitors can exert additional pressure and this can result in the development of resistance (reviewed in Ref. 6) and changes in entry efficiency.7–11 Selective pressure on Env derives primarily from the humoral immune response. Neutralizing antibodies are those that can prevent virus infection of a target cell. Broadly neutralizing antibodies are a subset of these antibodies that exhibit potent cross-subtype neutralization. Initially, the most potent of these antibodies were 2G12, that binds to gp120 carbohydrates; b12, that binds to the gp120 binding site; and 2F5 and 4E10, that bind to the gp41 membrane-proximal external region (reviewed in Ref. 12). With the development of high-throughput screening techniques, new broadly neutralizing antibodies have been discovered. Recent additions include VRC01, that binds to the CD4 binding site and PG9 that attaches to quaternary epitopes on gp120 (reviewed in Ref. 13). Broadly neutralizing antibodies are rare in patients, with only 1%–3% generating high titers.14 Even in the absence of broadly neutralizing antibodies, individuals living with HIV constantly produce antibodies that can neutralize viruses that were present within an individual during previous months but that are nonactive against contemporaneous HIV.15 Maraviroc (MVC) is unique among Food and Drug Administration–approved antiretrovirals in that it binds to a cellular target rather than a viral one. MVC binding to CCR5 allosterically induces a conformational change that renders the receptor inactive and unusable for entry by HIV.4,16,17 We have previously shown that MVC causes HIV to rebound from the cell surface, where it can rejoin the viral population.18 This phenomenon also occurs to a lesser degree with the fusion inhibitor enfuvirtide (T-20) that targets the third stage of entry (fusion) where it mimics the HR2 portion of gp41 and competitively binds to HR1, thus preventing the formation of the 6-helix bundle required for viral and membrane fusion.19 The likely alteration of the virion surface following MVC-mediated failed entry, coupled with the rebound of virus to the extracellular space prompted us to want to characterize these viruses in regard

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to both infectiousness and sensitivity to neutralizing antibodies that could potentially be used in immunotherapy,20–26 as well as in vaccine development.27–31 Conformational changes of HIV surface molecules caused by fusion or coreceptor inhibitors may also be helpful in the design of new broadly neutralizing antibodies. In this study, we compared the infectiousness of untreated virus with virus blocked from entry by each of the CCR5 coreceptor inhibitor MVC and the fusion inhibitor enfuvirtide (T-20). We also characterized the sensitivity of viruses repelled by these entry inhibitors to neutralizing antibodies. An ultrafiltration method was used to remove free drug before infection to delineate the effects of entry inhibitors.

METHODS Cells PM-1 cells that express CD4, CCR5, and CXCR4 were obtained through the AIDS Research and Reference Reagent Program (ARRRP)32 and were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (R10 medium) at 37°C and 5% CO2. A total of 293 T cells used for transfection were maintained in Dolbecco’s minimal essential medium (DMEM) in 10% FBS, L-glutamine, and antibiotics (D10 medium) at 37°C and 5% CO2. TZM-bl cells that express CD4, CXCR4, and CCR5 were obtained from the ARRRP33 and were maintained in D10 medium.

Virus Stocks HIV-1 BaL and AD8, both CCR5-tropic T-cell labadapted viruses, were produced by transfecting 293 T cells with the plasmids pWT/BaL and pNL(AD8), respectively. pWT/BaL was obtained through the ARRRP from Dr. Bryan Cullen.34 pNL(AD8) was obtained through the ARRRP from Dr. Eric Freed.35 Lipofectamine 2000 (Invitrogen, San Francisco, CA) was used as a transfection agent. Culture supernatants were collected at 48 hours after transfection, filtered through a 0.45 mm pore filter, and treated with 50 units of benzonase/mL of virus stock for 20 minutes at 37°C to remove contaminating plasmid DNA.36 Virus stocks were frozen at 280°C to halt benzonase activity. The HIV primary isolate CC 1/85 was obtained from Dr. Douglas Richman through the NIH AIDS Reagent program and propagated in cord blood mononuclear cells according to established protocols.37,38 Cord blood mononuclear cells were stimulated for 72 hours with phytohemagglutinin (Gibco; Life Technologies, Montreal, Canada) in R10 medium before infection with the isolate. The viral amplification culture was subsequently grown in R10 supplemented with interleukin 2 (Roche, Montreal, Canada) and incubated at 37°C, 5% CO2.

Infections We exposed PM-1 cells to HIV-1 BaL, AD8, and CC 1/85 in the presence of inhibitory concentrations of MVC (BaL: 50 nM, AD8: 100 nM, CC 1/85: 100 nM) and T-20 (200 nM). PM-1s were used to avoid the donor-to-donor

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variability that may arise when using PBMCs. Activated PBMCs were also shown to stimulate the production of CCL3, CCL4, and CCL5, which can interfere with CCR5 and dual-tropic HIV infection through steric hindrance and CCR5 downregulation.39 T cells such as PM-1 do not secrete these cytokines, allowing an unimpeded entry inhibitor– virus interaction to take place. Both ARVs were incubated with PM-1 cells for 1 hour at 37°C before the addition of virus. Two hundred times the 50% tissue culture infectious dose (TCID50) of each virus were incubated together with 5 · 105 cells in 1 mL of D10 medium. After 4-hour incubation at 37°C, the plates were spun for 5 minutes at 1500 rpm. We chose the 4-hour time point because the repelled virus population appears to be largest before 8 hours, most likely because of the rapidity of entry inhibitor engagement. The effects of all classes of entry inhibitors can be quantified by 4 hours following virus exposure in an in vitro system.40 Supernatants were transferred to Centrifree Ultrafiltration devices (30,000 MWC) (EMD Millipore, Billerica, MA) and spun twice for 25 minutes at 2500 rpm. Between spins, 800 mL of fresh D10 medium were added. A virus-only control was ultrafiltered separately. Virus recoveries following ultrafiltration were similar between experimental groups and control recoveries were similar to one another. Virus input was normalized by p24 levels following ultrafiltration. Supernatants were incubated with media containing serially diluted neutralizing antibody (2G12, 2F5, 4E10, b12, VRC01, PG9, and PG16) or untreated medium for 1 hour and transferred to cultures containing 1.5 · 104 TZM-bl cells in a total volume of 200 mL per well. Relative light units were measured after the addition of Brite-Lite Luciferase (Perkin Elmer, Waltham, MA) using a Perkin Elmer Liquid Scintillation Counter. HIV-1 BaL and AD8, 2 CCR5-tropic T-cell laboratory adapted strains, and CC 1/85, a CCR5-tropic clinical isolate, were incubated with U87.CD4.CXCR4, a glioma cell line induced to express CD4 and CXCR4 on its cell surface. This cell line cannot be infected with a CCR5tropic virus and U87.CD4.CXCR4 cells can only be infected by ·4 or dual-tropic viruses such as NL4-3 and 89.6. We exposed U87.CD4.CXCR4 cells to CCR5-tropic HIV-1 strains for 4 hours at 37°C. Two hundred TCID50 of BaL, AD8, and CC 1/85 were added to 5 · 105 cells per well in 1 mL of DMEM at 15% FBS, L-glutamine, and antibiotics. Viruses incubated in medium in the absence of cells served as controls. After 4 hours, supernatants were transferred to TZM-bl cells at 1.5 · 104 cells per well and incubated for 48 hours. Relative light units were measured after 48 hours as described above.

Statistical Analyses Two-way analysis of variance tests with Bonferroni posttest correction were used to analyze infectiousness levels in each treatment and control group. Antibody concentrations that inhibited virus replication by 50% (IC50) values were calculated using nonlinear regression analysis with a least square fit. All data were analyzed using Prism 5 (GraphPad, La Jolla, CA).  2014 Lippincott Williams & Wilkins

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FIGURE 1. Viral infectiousness following exposure to entry inhibitors. Mean levels of infection of (A) HIV-1 BaL, (B) CC 1/85 and (C) AD8 following exposure to entry inhibitors. Viruses exposed to MVC, T-20, and “no cell” controls were subjected to 2 rounds of ultrafiltration. The “No Cell No Ultrafiltration” control was not subjected to ultrafiltration. All points represent serial dilutions following 48 hours of incubation and are expressed in relative light units. Error bars represent SE measurement. Two independent experiments are represented.

RESULTS Changes in Infectiousness Following Exposure to Entry Inhibitors We determined whether exposure of entry inhibitorrepelled viruses to agents acting at different stages of the entry cascade affected infectiousness. For MVC-exposed virus, we observed a nearly 2.5-fold decrease in infectiousness compared with viruses that were unexposed to entry inhibitors or that were exposed to T-20 (Fig. 1). There were no significant differences in infectiousness levels among controls, indicating that ultrafiltration had no inadvertent effects on the viruses. The infectiousness levels of BaL exposed to T-20 did not differ significantly from controls. In contrast, both MVC and T-20 decreased the infectiousness levels of AD8 and CC1/85 compared with controls. We next asked whether entry at the coreceptor stage was responsible for this effect.

Infectiousness of CCR5-Tropic HIV Exposed Only to CXCR4 Coreceptors We next compared the infectiousness of CCR5-tropic virus that was exposed to target cells bearing only CD4 and CXCR4. No significant differences were observed in levels of infectiousness between CCR5-tropic viruses that had engaged

CD4 before being prevented from entry by the absence of CCR5 and virus that had not initiated the entry cascade (Fig. 2). This suggested that CD4 engagement alone was insufficient to alter the infectiousness of HIV-1 BaL, AD8, or CC 1/85 when coincubated with cellular targets expressing only CXCR4. This result is in agreement with previous work18 that showed that dual-tropic virus maintained infectiousness when blocked by MVC.

Changes in Sensitivity to Broadly Neutralizing Antibodies Following Exposure to Entry Inhibitors The differences in infectiousness between viruses exposed to entry inhibitors prompted us to explore their relative sensitivities to broadly neutralizing monoclonal antibodies. We chose ultrafiltration to clearly delineate the effects that entry inhibitors might have, as excess drug had to be separated from free virus before exposure to TZM-bl cells and antibodies. This process removed most free drug from media. The infectiousness of virus added to MVC or T-20–containing ultrafiltered medium was ;80% of control levels (see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A537). MVC-exposed BaL exhibited a reduced sensitivity to the gp120-specific antibodies 2G12, b12, and VRC01

FIGURE 2. HIV-1 infectiousness does not change following exposure to cellular targets that express alternate coreceptors. Means of levels of infection of (A) HIV-1 BaL, (B) CC 1/85, (C) AD8 in media (“no cell”) or virus exposed to U87.CD4.CXCR4 cells for 4 hours. All points represent serial dilutions following 48-hour incubation and are expressed in relative light units. Error bars represent SE measurements. Two independent experiments are represented.  2014 Lippincott Williams & Wilkins

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FIGURE 3. Broadly neutralizing antibody sensitivity of entry inhibitor-exposed HIV-1. Relative infectiousness of entry inhibitor-exposed HIV-1. BaL (A), CC 1/85 (B), and AD8 (C) in the presence of the serially diluted broadly neutralizing monoclonal antibodies: 2G12, 2F5, 4E10, b12, VRC01 and PG9. Control virus represents baseline neutralization sensitivity of untreated virus stock at 0 hours. Viruses were exposed to either MVC or T-20 for 4 hours and were incubated with serially diluted broadly neutralizing monoclonal antibodies (25 mg/mL to 0.78 mg/mL) for 1 hour before being transferred to TZM-bl cells. All points represent levels of infection relative to “no cells” controls, which were set at 100%. Error bars represent SE measurement. Two independent experiments are represented.

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(Fig. 3A) (Table 1). MVC exposure also reduced sensitivity to glycan-specific 2G12 over the entire dilution range tested, resulting in a decrease in sensitivity of up to 40%. The CD4 binding site antibody b12 exhibited a marked reduction in ability to effect neutralization, between 50% and 60%, at all concentrations tested, and similar findings were obtained with the CD4 binding site antibody VRC01, that is, an 80% change in infectiousness at the lowest concentrations of antibody tested. BaL, which is resistant to neutralization by antibodies PG9 and PG16, remained resistant following MVC exposure (data not shown). Neutralization sensitivity to gp41-specific 2F5 remained relatively unchanged between MVC-exposed virus and controls. T-20- and MVC-exposed CC 1/85 exhibited decreased sensitivity to 2G12 (Fig. 3B). This diminution in sensitivity to 2G12 varied up to 35% for viruses treated with T-20. We did not observe any difference in sensitivity to b12 or VRC01. CC1/85 retained its resistance to 2F5 and 4E10. MVC-exposed AD8 retained resistance to 2G12 and 4E10 (data not shown). We also observed a gain of susceptibility to 2F5 following MVC exposure (Fig. 3C). MVC-exposed AD8 exhibited increased sensitivity to 2G12-mediated neutralization at lower concentrations with differences reaching as high as 40%. This increased sensitivity was also observed for 2F5 and 4E10; AD8 is naturally resistant to the latter. Interestingly, VRC01, PG9, and PG16 exhibited increased sensitivity at lower antibody concentrations, with reductions in infectiousness up to 50%. T-20–exposed BaL exhibited slightly increased sensitivity to 2G12 vs. controls. The CD4 binding site antibodies b12 and VRC01 exhibited reduced potency against T-20– exposed virus, that is, a 50% and 40% increase in infection levels at the lowest antibody concentrations, respectively. There were no significant differences in the sensitivity of

HIV Infectiousness and Ab Sensitivity

T-20–exposed viruses to PG9 and PG16 vs. controls (not shown). Interestingly, T-20 exposure resulted in a 50% decrease in sensitivity to neutralization by 2F5 at the highest antibody concentration. In contrast, we observed a 50% increased sensitivity to 4E10 at all concentrations tested compared with control virus (Fig. 3A). T-20–exposed CC 1/85 did not exhibit any changes in sensitivity to 2F5 and 4E10 at all antibody concentrations tested and retained resistance toward these antibodies (data not shown). However, 2G12 potency was reduced through the entire range of antibody concentrations, with a 40% increase in infection at lower concentrations of antibody. T-20 exposure did not affect CC 1/85 sensitivity to b12 orVRC01 (Fig. 3B). T-20–exposed AD8 did not exhibit a change in sensitivity to 2G12 or 4E10 and retained resistance to neutralization by these antibodies (data not shown). For 2F5, however, we observed an alteration in sensitivity across the antibody range tested, indicating a loss of resistance. We observed an increase in sensitivity to b12 on a par with MVC exposure, with up to a 60% decrease in infection. Sensitivity to VRC01 and PG9 remained relatively unchanged following exposure to T-20 (Fig. 3C). Altogether, these results indicate that exposure to entry inhibitors alters HIV-1 susceptibility to broadly neutralizing antibodies. Changes in neutralization sensitivity were virus specific, underlying the importance of viral determinants in these observations.

DISCUSSION Our results show that MVC exposure can decrease subsequent HIV infectivity (Fig. 1). Here, we have attempted to establish whether exposure to cellular targets that express the alternate coreceptor required for entry might also affect virus

TABLE 1. Neutralization of Untreated and Entry Inhibitor-Exposed HIV by Broadly Neutralizing Antibodies Baseline BaL 2G12 4E10 2F5 b12 VRC01 CC 1/85 2G12 b12 VRC01 AD8 2F5 b12 VRC01 PG9

IC50

Top

2.3 .25 9.2 ,0.78 ND

87.49 109 2130 109 ND

1.36 4.3 6.9

89.19 102 102

725 7.35 ,0.78 9

91.4 123.4 188 100

MVC Bottom 3.4 258 216.8 258 ND 0.26 24 222 295,360 232.9 25.8 21.4

T-20

IC50

Top

Bottom

IC50

Top

Bottom

10.9 15.24 .25 .25 1.8

79.75 88.79 95.26 88.79 119

19.2 3.6 236,380 3.6 27.4

36.5 8.3 ND 13.2 ,0.78

64.56 72.07 ND 72.07 97.38

262 216.54 ND 216.54 23.6

2.1 1.3 16.47

70.15 108 73

5.43 4.2 6

84.7 107 114

1.4 8.9 23.7

15.71 8.61 ,0.78 .25

77.32 92.55 134.9 74.2

7.39 2.11 ,0.78 6.3

138.2 138.5 305,586 133

14.88 18.8 221.25 222 228.1 1.6 2336

217.46 23.7 2.6 219.38

Monoclonal antibody concentrations (mg/mL) were recorded that inhibit replication of tested virus by 50% in a TZM-bl–based assay. Values at the Top and Bottom represent the % of anticipated or observed plateau of infection. Two independent experiments are represented. ND, not determined.

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FIGURE 4. Model showing the potential effects of entry inhibition because of the absence of coreceptor (A), MVC (B), or enfuvirtide (C) on trimer conformation and infectiousness. A, gp120/gp41 trimer on the virus surface attaches to CD4 on the surface of the cell membrane. Insert: top left: native gp120/gp41 trimer, unbound to CD4. Insert: top right: gp120 conformation on rebounded virus. B, MVC exposure prevents contact between CCR5 and gp120. Insert: top left: native gp120/gp41 trimer, unbound to CD4, unexposed to MVC. Insert: top right: gp120 conformation following exposure to MVC. C, Enfuvirtide (T-20) exposure prevents fusion of viral and cell membranes by preventing formation of the 6-helix bundle. Insert: top left: native gp120/ gp41 trimer, unbound to CD4, unexposed to T-20. Insert: top right: gp120/gp41 conformation following exposure to T-20.

infectiousness. We found that infectiousness of a CCR5-tropic virus was not altered by its exposure to cells that express CD4 and CXCR4 and not CCR5 (Fig. 2). This may seem unexpected, as soluble CD4 engagement can lock gp120 into a functionally incompetent conformation.41 The number of functional trimers on the virion surface may inform these results. Indeed, soluble CD4 binds to all trimers simultaneously, whereas conventional gp120/CD4 engagement occurs in a sequential manner. With an estimated 15–20 gp120/gp41 trimers on the virion surface,42 it is likely that a singular gp120/CD4 interaction may not be sufficient to abrogate infectiousness when other surface proteins remain unaffected. Exposure to cells that do not possess the proper coreceptor despite presenting CD4 is a likely scenario in vivo, because virus is exposed to subsets of cells with differing levels of expression of CD4, CCR5, and CXCR4. In contrast to the absence of the proper coreceptor, MVC exposure decreased BaL infectiousness compared with exposure to T-20 or medium alone (Fig. 1), whereas both T-20 and

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MVC decreased AD8 and CC 1/85 infectiousness. In the case of BaL, the different infectivities of T-20–exposed and MVC-exposed virus may be explained by the possibility that MVC affects more envelope subunits and trimers than T-20 affects fusion proteins (Fig. 4). It is possible that the reduced infectivity of T-20–exposed AD8 and CC 1/85 arises from increased entry efficiency for these viruses that may result in a greater number of envelope subunits than is the case for BaL over the same time period. It has been suggested that increased infectivity may compensate for neutralization sensitivity.43 To shed further light on this question, we tested the neutralizing capacities of wellcharacterized broadly neutralizing monoclonal antibodies that bind to either gp120 or gp41. We observed a decrease in sensitivity to 2G12 in both T-20–exposed and MVC-exposed BaL and CC 1/85 viruses. The similarity in neutralization curves indicate that 2G12 neutralization function is preserved and that the difference we observed likely derives from reduced binding as a result  2014 Lippincott Williams & Wilkins

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of shifting N-glycans on the glycan shield. Previous studies have shown that N-glycan shifting results in a decrease of 2G12 affinity.44 It is possible that MVC and T-20 both induce glycan shifts but the mechanism of action of MVC might allow extra exposures at the cell surface compared with exposure to T-20, which acts later than MVC in the entry cascade. In contrast, AD8 was naturally resistant to 2G12 neutralization (Fig. 3C). As expected, MVC and T-20 exposure did not render AD8 susceptible to this antibody. There was a substantial decrease in BaL sensitivity to CD4 binding site antibodies b12 and VRC01 following entry inhibitor exposure. Structural studies have shown that b12 acts by placing Env into a more open conformation by rotating each gp120 subunit by 20 degrees.45 The decrease in b12 sensitivity of BaL following MVC and T-20 exposure may be caused by a decrease in flexibility of each subunit of gp120 or by a decrease in b12 binding following gp120 engagement with CD4. In contrast, MVC and T-20 exposure did not decrease CC 1/85 susceptibility to b12 and VRC01, although this viral strain was naturally susceptible to neutralization by these antibodies. This suggests that MVC and T-20 exposure did not induce changes in CC 1/85 surface proteins similar to those induced by BaL that resulted in decreased b12 and VRC01 neutralization activities against the latter strain (Fig. 3A). This may be the result of high CD4 binding affinity for CC 1/85.46 In contrast to BaL and CC 1/85, MVC exposure modestly increased AD8 susceptibility to b12 and VRC01, whereas T-20 exposure increased AD8 susceptibility to both antibodies. This may be due to structural differences within the CD4 binding site of AD8 compared with BaL or CC 1/85. Our results demonstrate that both MVC and T-20 exposure decreased the anti-BaL neutralization activity of VRC01, which locks gp120 into a closed conformation.45 This may be because of the fact that both MVC and T-20 allow for CD4 engagement that leaves Env in an open conformation. Exposure to T-20 and MVC did not significantly alter VRC01 activity against CC 1/85 (Fig. 3B). In contrast, both entry inhibitors increased AD8 susceptibility to VRC01 neutralization (Fig. 3C). PG9 binds to strictly trimeric structural epitopes on the envelope that include regions in V2 and V3. BaL and CC 1/85 are resistant to these antibodies and we did not observe any change in sensitivity following exposure to entry inhibitors. Similarly, AD8 was sensitive to PG9 but there was no change in susceptibility following exposure to either MVC or T-20. MVC exposure did not change BaL sensitivity to the gp41specific antibody 2F5. In contrast, T-20 exposure resulted in BaL losing sensitivity to this antibody. It has been shown that 2F5 antagonizes T-20 in synergy studies.47 Our study indicates that this antagonism is not dependent on simultaneous presence, but may rather be the consequence of T-20 exposure before 2F5 administration. However, there was gain of sensitivity to these gp41 antibodies when AD8 was exposed to MVC. The differences between BaL, a Tier 1 neutralization-sensitive virus, and AD8, a Tier 2 neutralization-resistant virus, may be a consequence of intrinsic reactivity defined as the ability to form the required structures for entry following coreceptor or inhibitor engagement.48 Differences in intrinsic reactivity of Tier 1 and Tier 2  2014 Lippincott Williams & Wilkins

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viruses may be exacerbated by exposure to entry inhibitors that interfere with initial antibody–virus binding or envelope reactivity after antibody binding. We observed a significant increase in 4E10 sensitivity of BaL following T-20 exposure that was not observed with MVC exposure. 4E10 and T-20 have been shown to have synergistic effects.47 Our temporal separation of 4E10 and T-20 reveals that simultaneous exposure is unnecessary to achieve this effect; it is rather likely that T-20 exposes the 4E10 epitope, resulting in increased binding for BaL. Our results also show that complexities in envelope conformation can translate into various conformational changes that are induced by entry inhibitors. Recent new strategies in HIV therapy have included the administration of broadly neutralizing antibodies, possibly in combination with antiretroviral therapy.23,49 Neutralizing antibodies may also become an important component of an effective HIV vaccine. Our results suggest that HIV may be less susceptible to some of these neutralizing antibodies in patients who are treated with entry inhibitors. Our results also demonstrate that the method by which virus is blocked from entry impacts envelope function and sensitivity to neutralizing antibodies. MVC and T-20 act at different points of the entry cascade and this difference accounts for the number and extent of trimers affected, directly impacting infectiousness through conformational changes and neutralizing antibody sensitivity through epitope occlusion and exposure. This work provides proof-ofconcept that different entry inhibitors possess distinctive means of interacting with cell-free virus and that this may have implications for the future use and development of entry inhibitors and neutralizing antibodies. REFERENCES 1. Cervia JS, Smith MA. Enfuvirtide (T-20): a novel human immunodeficiency virus type 1 fusion inhibitor. Clin Infect Dis. 2003;37:1102–1106. 2. Hanna GJ, Lalezari J, Hellinger JA, et al. Antiviral activity, pharmacokinetics, and safety of BMS-488043, a novel oral small-molecule HIV-1 attachment inhibitor, in HIV-1-infected subjects. Antimicrob Agents Chemother. 2011;55:722–728. 3. Nettles RE, Schurmann D, Zhu L, et al. Pharmacodynamics, safety, and pharmacokinetics of BMS-663068, an oral HIV-1 attachment inhibitor in HIV-1-infected subjects. J Infect Dis. 2012;206:1002–1011. 4. Dorr P, Westby M, Dobbs S, et al. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother. 2005;49:4721–4732. 5. Donzella GA, Schols D, Lin SW, et al. AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nat Med. 1998;4: 72–77. 6. De Feo CJ, Weiss CD. Escape from human immunodeficiency virus type 1 (HIV-1) entry inhibitors. Viruses. 2012;4:3859–3911. 7. Grupping K, Selhorst P, Michiels J, et al. Mini CD4 protein resistance mutations affect binding to the HIV-1 gp120 CD4 binding site and decrease entry efficiency. Retrovirology. 2012;9:36. 8. Putcharoen O, Lee SH, Henrich TJ, et al. HIV-1 clinical isolates resistant to CCR5 antagonists exhibit delayed entry kinetics that are corrected in the presence of drug. J Virol. 2012;86:1119–1128. 9. Tsibris AM, Hu Z, Paredes R, et al. Vicriviroc resistance decay and relative replicative fitness in HIV-1 clinical isolates under sequential drug selection pressures. J Virol. 2012;86:6416–6426. 10. Roche M, Jakobsen MR, Sterjovski J, et al. HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism. J Virol. 2011;85:4330–4342.

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Exposure to entry inhibitors alters HIV infectiousness and sensitivity to broadly neutralizing monoclonal antibodies.

The development of envelope-specific neutralizing antibodies that can interfere with viral entry into target cells is important for the development of...
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