Title: Neutralization resistance of HIV-1 virological synapse-mediated infection is. Running Title: Virological-synapse neutralization resistance

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1 JVI Accepts, published online ahead of print on 2 May 2012 J. Virol. doi: /jvi Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Title: Neutralization resistance of HIV-1 virological synapse-mediated infection is regulated by the gp41 cytoplasmic tail 3 4 Running Title: Virological-synapse neutralization resistance Authors: Natasha D. Durham 1,2, Alice W. Yewdall 2, Ping Chen 2, Rebecca Lee 2, Chati Zony 2, James E. Robinson 3, and Benjamin K. Chen 2 * Affiliations: 1 Microbiology Graduate School Training Program, Department of Microbiology, 2 Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, USA, 3 Department of Pediatrics, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA. * Corresponding author: Benjamin K. Chen, Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, USA. Tel: Fax: ben.chen@mssm.edu Abstract word count: 250 Text word count: 6,801

2 Abstract Human immunodeficiency virus type 1 (HIV-1) infection can spread efficiently from infected to uninfected T-cells through adhesive contacts called virological synapses (VS). In this process, cell-surface envelope glycoprotein (Env) initiates adhesion and viral transfer into an uninfected recipient cell. Previous studies have found some HIV-1 neutralizing patient sera to be less effective at blocking VS-mediated infection than infection with cell-free virus. Here we employ sensitive flow cytometry based infection assays to measure the inhibitory potency of HIV-1 neutralizing monoclonal antibodies (mab) and HIV-1 neutralizing patient sera against cell-free and VS-mediated infection. To varying degrees, anti-env mabs exhibited significantly higher 50% inhibitory concentration (IC 50 ) against VS-mediated infection in comparison to cell-free infection. Notably, the mab 17b, which binds a CD4-induced (CD4i) epitope on gp120, displayed a 72-fold reduced efficacy against VS-mediated inoculums compared to cell-free inoculums. A truncation mutant in the gp41 cytoplasmic tail (CT), which is unable to modulate Env fusogenicity in response to virus particle maturation but can still engage in cell-to-cell infection, was tested for the ability to resist neutralizing antibodies. The CT mutation increased cell surface staining by neutralizing antibodies and significantly enhanced neutralization of VS-mediated infection, and had reduced or no effect on cell-free infection, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of key neutralizing epitopes during cell-to-cell infection and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env 2

3 46 47 conformations exposed on the infected cell surface to enhance protection against VS-mediated HIV-1 spread. 48 3

4 Introduction The ability of HIV-1 to evade neutralizing antibody responses represents a major obstacle to the creation of an effective vaccine. The failure of HIV-1 vaccines is often attributed to the high sequence variability and conformational plasticity of the major neutralizing antigen, envelope glycoprotein (Env) (13, 39). The functional Env subunit is a trimeric spike made of gp120-gp41 heterodimers, which mediate viral entry during infection with both cell-free and cell-associated viral sources (5, 17). Cell-free infection of CD4+ T-cells involves the release of viral particles from a productively infected cell, fluid-phase particle diffusion, viral attachment and entry into an uninfected cell (28). Direct T-cell to T-cell infection occurs through contact between an infected and an uninfected T-cell, resulting in the formation of an infectious cell-cell adhesion referred to as a virological synapse (VS) (17, 18). During VS-mediated infection, it has been proposed that virus particles may bud into the synapse and fuse directly with the plasma membrane at the synaptic space (34). However a number of studies support a model for entry following VS formation involving two steps: (i) co-receptor independent, coordinated transfer of viral particles into the target cell endocytic compartment (cell-to-cell transfer) followed by (ii) co-receptor dependent fusion of the viral and cellular membranes within the endocytic compartment (VSmediated infection) (3, 7, 29, 34). In support of this model, T-cell VS was found to transfer immature HIV-1 particles into target cells, which undergo viral membrane fusion only after proteolytic maturation of the viral core (7). 4

5 In cell-free virus particles, the gp41 cytoplasmic tail (CT) controls Env fusogenicity through inside-out allosteric mechanisms (16, 25, 45). These studies show that during virus particle production, the interaction of gp41 CT with Pr55 Gag maintains Env in a pre-fusogenic conformation. After virus budding, cleavage of Pr55 Gag and subsequent particle maturation relieves the inhibitory function of gp41 to activate Env fusogenicity. Thus, the gp41 CT plays an important role in regulating the fusogenic potential of Env during the virus life cycle. On cell-free HIV-1, the gp41 CT is important in regulating the exposure of both neutralizing and non-neutralizing epitopes on the Env ectodomain of mature virus particles (19). The fusogenicity of Env and the exposure of CD4-induced epitopes are enhanced in gp41 CT truncation mutants when tested with pseudovirion infection assays and cell-cell fusion assays (9, 46). During VSmediated infection, the cell-surface Env glycoprotein functions first as a cell adhesion molecule, then as the viral membrane fusion apparatus (15). In this pathway, Env does not mediate membrane fusion until after the virus particle has undergone maturation (7). While the gp41 CT is not required for VS formation or subsequent infection (5, 10, 23), it does enhance the efficiency of cell-to-cell infection in non-permissive cell types (10). A number of broadly neutralizing monoclonal antibodies (mab) and peptide inhibitors have been tested for their ability to block cell-to-cell HIV-1 transfer or VS-mediated infection (5, 11, 21, 22, 33). To date, only antibodies that block Env-CD4 interaction have been shown to inhibit both cell-to-cell transfer and subsequent VS-mediated infection. Other neutralizing mab and 5

6 entry inhibitors have been found to block infection from cell-associated HIV-1 after the transfer of virus across the VS. Using an indirect assay to measure increased HIV-1 DNA following co-culture of donor and target cells, one study reported that VS-mediated infection could be inhibited by all neutralizing antibodies tested (21). Other studies have found that sera from HIV-1 positive patients are much less effective at blocking cell-to-cell transfer (5, 7) and VSmediated infection (15) than cell-free HIV-1 infection. These studies on patient sera suggest that quantitative differences in neutralization sensitivity are likely to be found with some neutralizing mab. Here we employ flow cytometry based infectious assays to screen a panel of broadly neutralizing mab and entry inhibitors for their ability to neutralize cellfree and VS-mediated infection. Flow cytometry based assays allow us to directly measure HIV-1 infection specifically in target cells. In contrast to previous studies, we find that most anti-env mab required significantly more antibody to block VS-mediated infection as compared to cell-free virus. A pronounced effect was observed with the mab 17b, which recognizes a CD4- induced (CD4i) epitope on gp120 (42). 17b exhibited the greatest fold difference in IC 50 with 72-fold higher concentration required to block 50% of VS-mediated infection compared to cell-free infection. We further found that the relative resistance of VS-mediated infection to neutralization by both mab and neutralizing sera was partially overcome by truncation of the gp41 CT, while neutralization of cell-free virus was less affected by loss of the gp41 CT. These results support the hypothesis that the gp41 CT regulates conformations of HIV-1 6

7 Env at the cell surface to promote immune evasion during VS-mediated T-cell infection

8 Materials and Methods Viral Constructs. HIV-1 Gag-iGFP is a molecular clone based on pnl4-3 (2) with green fluorescent protein (GFP) inserted between the Gag MA and CA domains (14). Gag-iGFP CT contains a premature stop codon in the Env reading frame causing a deletion of the C-terminal 144 amino acids (5). NL-GI contains GFP in place of nef, and nef is expressed from a downstream internal ribosome entry site (IRES) (6). NL-GI CT was cloned by generating a PCR fragment of the C-terminal Env from the Gag-iGFP CT plasmid. A PCR-based cloning strategy was used to precisely introduce the primary Env sequence into the NL-GI molecular clone. All PCR-amplified sequences were confirmed by sequence analysis. Primary CD4+ T-cells were infected with NL-GI containing the NL4-3 envelope (X4-tropic) or the R5-tropic B-clade primary envelope (20) from prhpa4259 clone 7 (SVPB14), obtained through the AIDS Research and Reference Reagent Program (ARRP), Division of AIDS, NIAID, NIH from Drs. B. H. Hahn and Dr. J. F. Salazar-Gonzalez. Cells and cell culture. Human cell lines Jurkat E6-1 and MT4 (Arthur Weiss and Douglass Richman respectively, ARRP) were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 g/ml streptomycin and 2 mm glutamine (complete RPMI). Primary CD4+ T-cells were obtained from human peripheral blood through the New York Blood Center and isolated by negative selection with Miltenyi CD4+ T-cell isolation kit II (Miltenyi Biotec). Unactivated CD4+ T-cells were maintained in complete RPMI containing 8

9 U/mL interleukin 2 (IL-2; ARRP). Cells were activated on Day 0 by co-culture with irradiated allogeneic peripheral blood mononuclear cells (PBMC) in complete RPMI containing 50 U/mL IL-2 and 2 µg/ml phytohemagglutinin, and used 3 to 4 days post-activation Inhibitors and HIV-1 neutralizing patient sera. The following inhibitors were tested for the ability to block cell-free and VS-mediated infection using 5-fold serial dilutions beginning at the given concentration, unless otherwise stated: 0.5 µg/ml Leu3a, an anti-cd4 antibody (BD Biosciences); 10 µg/ml IgG1 b12, an anti-gp120 mab (b12; Dr. Dennis Burton and Carlos Barbas, ARRP); 30 µg/ml 17b and 120 µg/ml 17b, anti-gp120 mab against the CD4-induced binding site on gp120 (Dr. James E. Robinson, ARRP and Tulane University stocks, respectively); 50 µg/ml 48d, E51, 21c and 412d, anti-gp120 CD4-induced binding site mab (Dr. James E. Robinson, ARRP and Tulane University); 50 µg/ml 4E10 and 2F5, anti-gp41 mab (Dr. Hermann Katinger, ARRP and Polymun Scientific GmbH); 50 µg/ml 2G12, an anti-gp120 glycan-specific mab (Dr. Hermann Katinger, ARRP); 50 µg/ml A32, an anti-gp120 mab (Dr. James E. Robinson, ARRP); 50 µg/ml F425 A1g8, an anti-gp120 CD4-induced binding site mab (Dr. Marshall Posner and Dr. Lisa Cavacini, ARRP); 50 µg/ml T20, a fusion inhibitor (Roche through ARRP); and 10 µg/ml AMD-3100 (bicyclam JM-2987), a CXCR-4 inhibitor (ARRP). HIV-1 Neutralizing sera 1 and 2, and HIV-1 Negative Control Human Serum (Dr. Luba Vujcic, ARRP) were used at a starting dilution of 1:50. 9

10 Cell-free infection assay. Cell-free virus particles were produced in 293T cells (ATCC) by calcium-phosphate transfection (27). Viral supernatants were quantified by p24 ELISA x 10 6 MT4 cells were infected with 15 ng of WT NL-GI, 75 ng (Batch 1) or 255 ng (Batch 2) of NL-GI CT per 1 x 10 6 cells to obtain up to 10% infection after 48 hrs in the absence of inhibitors. Virus supernatant and MT4 cells were pre-incubated separately with equal volumes of inhibitors for 30 min at 37ºC before mixing. After 18 hrs, culture media was replaced with complete RPMI containing 10 µm Zidovudine (AZT; ARRP). 48 hrs after mixing, cells were treated with trypsin-edta to dissociate cell-cell clusters, washed with phosphate-buffered saline (PBS) and fixed in 2% paraformaldehyde x 10 6 activated primary CD4+ T-cells were infected with 27 ng of CXCR4-tropic virus and 20 ng of CCR5-tropic virus per 1 x 10 6 cells. Inhibitors were added at the time of infection. Cells were trypsin-treated and fixed after hrs. VS-mediated infection assay. Jurkat (donor) cells were transfected by nucleofection (Amaxa Biosystems) with 5 µg NL-GI or NL-GI CT DNA as previously described (5), cultured overnight in antibiotic-free medium and purified by Ficoll-Hypaque density gradient centrifugation. Jurkat cells and MT4 (target) cells were dye-labeled with 1 µm CellTrace Far Red DDAO-SE (Molecular Probes) for 10 min at 37 C, or 20 µm CellTracker Blue CMF 2 HC (Molecular Probes) for 45 mins at 37 C, respectively. Fluorescent labeling of both donor and 10

11 target cells allowed unambiguous exclusion of donor-target doublets from the infection analyses x 10 6 donor or target cells were pre-incubated separately with inhibitors for 30 mins at 37ºC before mixing at a ratio of approximately 1:1, co-cultured at 37ºC for 30 hrs, treated with trypsin and fixed. In primary cell infection assays, primary activated donor CD4+ T-cells were infected by spinoculation with 8.15 ng of X4-tropic or 6 ng of R5-tropic cell-free virus per 2.5 to 5 x 10 5 cells. After 24 hrs, cells were labeled with 2 µm efluor 670 (ebioscience, Inc.) and mixed at a 1:1 ratio with activated uninfected target cells from autologous donors. Inhibitors were added at the time of mixing. Cells were trypsin-treated and fixed after hrs of co-culture. Cell-to-cell transfer assay. Jurkat cells (donor cells) were transfected by nucleofection with 5 µg HIV-1 Gag-iGFP or Gag-iGFP CT (Amaxa Biosystems) as previously described (5) and cultured overnight in antibiotic-free medium. Unactivated primary CD4+ T-cells (target cells) were resuspended in serum-free RPMI 1640 and stained with 1 µm CellTracker orange CMTMR (Molecular Probes) for 45 mins at 37ºC, washed and cultured overnight in complete RPMI containing 10 U/mL IL-2. Jurkat and CD4+ T-cells were purified by Ficoll- Hypaque density gradient centrifugation. Donor and target cells were mixed at a ratio of approximately 1:1 and co-cultured at 37ºC for 3 hrs before treating with trypsin and fixing, as above. Where inhibitors were used, donor and target cells were pre-incubated separately with equal volumes of inhibitors for 30 mins at 37ºC before mixing. 11

12 Calculations and Statistics. Unless otherwise stated, the percent inhibition was calculated for each well of a given experiment by finding the average of triplicate wells with no inhibitor (i.e. 0% inhibition), and normalizing all other values of the same experiment by the following equation: % inhibition = 100 ( [% infected target cells with inhibitor / average % infected target cells without inhibitor] x 100 ). Statistical analysis and fitting were performed using Prism version 5.0 (GraphPad Software). Titration curves are of pooled data, where error bars represent the standard errors of the mean (SEM) of at least two independent experiments. 50% inhibitory concentration (IC 50 ) values were determined by non-linear regression curve fits of pooled data from at least two independent titration experiments, and statistical significance between Log 10 IC 50 values was determined by unpaired two-tailed Student t test. Statistical significance between individual non-linear regression curve fits for VS-mediated infection experiments with different frequencies of infected MT4 target cells (Figures S1 and S3C, D) was determined by extra sum-of-squares F-test. P- values <0.05 represent statistically significant differences between individual nonlinear regression curve fits for each dataset. P-values >0.05 indicate one nonlinear regression curve adequately fits all datasets. 12

13 Results Relative resistance of VS-mediated infection to neutralization by patient sera and mab We performed antibody neutralization experiments for cell-free and VSmediated infections by infecting a fluorescent-dye labeled T-cell line with a green fluorescent protein (GFP)-expressing molecular clone of HIV-1, NL-GI (5). GFP detection serves as an indicator of de novo early HIV-1 gene expression in productively infected cells. We used flow cytometry to detect infection only in dye-labeled target cells to obviate the need to compensate for the large HIV-1 infection signal derived from input donor cells. Using this assay, we first examined two neutralizing patient sera that can completely neutralize cell-free NL-GI when used to infect the CD4+ T-cell line MT4. When the same sera were titrated against homologous cell-associated HIV-1 challenge, inhibition was not complete at the concentrations capable of neutralizing cell-free infection (Figure 1A). This titration of antisera is consistent with the levels of inhibition that we have previously reported (15). To test whether certain epitopes may be more selective in their ability to distinguish cell-free versus cell-associated HIV-1, we titrated a panel of mab to examine their ability to block infection of the T-cell line MT4 by cell-free or cellassociated wild type (WT) NL-GI. We focused our initial analysis on four mab that target distinct regions of CD4 or Env: 1) Leu3a, an anti-cd4 HIV-1 blocking antibody, 2) b12, an anti-gp120 antibody that binds to the CD4 binding site (CD4bs), blocking CD4-Env interaction and VS formation (31), 3) 17b, an 13

14 antibody which binds to the CD4i bridging sheet epitope in gp120 (42, 44), and 4) 4E10 which binds the membrane proximal external region (MPER) of gp41 (40). Cell-free WT infection (Figure 1B) or VS-mediated WT infection (Figure 1C) were both strongly inhibited in the presence of maximum concentrations of Leu3a (0.5 µg/ml). In contrast, at 30 µg/ml, 17b inhibited cell-free infection with similar efficiency as Leu3a, however it was a poor inhibitor of VS-mediated infection at this same concentration. Titrations of Leu3a, 17b, b12 and 4E10 were performed starting at a maximum mab concentration of 0.5 µg/ml, 30 µg/ml, 10 µg/ml and 50 µg/ml respectively, which blocked >95% of cell-free HIV-1 infection in our assays. The variation in antibody concentration required for high levels of inhibition reflects the differential neutralization potency and binding affinity of these mab. We observed inhibition of both cell-free and VS-mediated infection of MT4 cells (Figure 1D). The inhibition profile of the CD4 binding antibody Leu3a was the same for cell-free and cell-associated HIV-1. In contrast, VSmediated infection required higher concentrations of b12, 17b and 4E10 than cell-free infection to achieve comparable inhibition (Figure 1D, Table S1). Notably, at 30 µg/ml, 17b inhibited 97% of cell-free infection, compared to 30% inhibition of VS-mediated infection. The differences in IC 50 for cell-free and cellassociated viral inoculums were statistically significant (P<0.01 for b12 and 4E10; P<0.001 for 17b). In independent repetitions of the VS-mediated neutralization experiments, variations in the level of WT NL-GI infection in the target cell population did not change the IC 50 for VS-mediated infection (Figure S1). 14

15 Because of the relatively large defect of 17b in blocking VS-mediated infection, we tested a number of other CD4i antibodies for their ability to block cell-associated HIV-1. The CD4i mab 48d showed 30% inhibition of cell-free infection at 50 µg/ml (Figure S2A). Three additional CD4i mab F425 A1g8 (4), E51 (49) and 21c (47), effectively inhibited cell-free infection (>85% inhibition at 50 µg/ml) and were therefore tested for the ability to also block VS-mediated infection. All three CD4i mab showed a statistically significant increase in IC 50 during VS-mediated WT infection, as did the anti-gp41 MPER antibody 2F5 (26) (Figure S2A and Table S1). A small increase in IC 50 of the anti-gp120 carbohydrate antibody 2G12 (35) against VS-mediated infection was also observed, although this did not achieve statistical significance. This shift was less pronounced for the entry inhibitors AMD-3100 (36), a CXCR-4 antagonist, and T20 (43), a peptide fusion inhibitor, and only significant for T20 (P<0.05). The non-neutralizing anti-gp120 mab A32 (44) and the CD4i mab 412d (48) did not block infection by cell-free or cell-associated HIV-1. A side-by-side comparison of the mab shows the largest change in IC 50 value for 17b, with 72.8-fold higher IC 50 for VS-mediated infection compared to cell-free infection (Figure 1E). Two MPER antibodies, 4E10 and 2F5, were intermediate in resistance, exhibiting 19.1 to 20.7-fold higher IC 50 for VSmediated infection compared to cell-free infection. The other CD4i antibodies F425 A1g8, E51 and 21c exhibited moderate resistance indicated by the 10.4 to 13.3-fold higher IC 50 for VS-mediated infection compared to cell-free infection. The mab b12 showed 7.9-fold change in IC 50. Titrations with the carbohydrate 15

16 recognizing 2G12 antibody did not show a statistically significant resistance to neutralization. The small molecule entry inhibitors showed only 2 to 3-fold increase. To varying degrees, neutralization of VS-mediated WT-NLGI infection required a higher IC 50 compared to cell-free infection. The range of resistance suggests that variations in exposure of different epitopes may determine the degree of resistance to neutralization. The neutralization of primary isolates of HIV-1 and infection of primary cells is often more difficult to achieve than neutralization of HIV-1 in cell lines with lab isolates of HIV-1 (37). Therefore, we tested the neutralization resistance of VSmediated infection to 17b, which previously showed the highest fold increase in VS-mediated versus cell-free IC 50, using primary activated CD4+ T-cells as both donor and target cells. We compared NL-GI viruses containing the X4-tropic NL4-3 envelope (Figure 2A, C) or the R5-tropic B-clade envelope (prhpa4259 clone 7) derived from an acute early clinical isolate (Figure 2B, D) (20). The R5- tropic RHPA Env gene was cloned in cis into the GFP-expressing virus, rather than producing pseudovirions by co-transfection of the RHPA expression plasmid. 17b effectively blocked the infection of primary cells with both cell-free X4-tropic or R5-tropic virus by 70-80%. In contrast, the same high antibody concentration inhibited ~30% or less when directed against cell-associated HIV- 1. In comparison, high concentrations (10 µg/ml and 25 µg/ml) of the CD4bs antibody b12 inhibited both modes of infection and both viral clones with similar efficiency ( 80% inhibition). Maximum inhibition of both modes of infection by b12 was achieved at 10 µg/ml. Because lower levels of b12 showed differences 16

17 in neutralization efficiency of cell-free versus cell-associated HIV-1, a 5-fold titration series of b12 was performed in primary cells. Similar to the infection of the MT4 cells, cell-free infection in primary activated CD4+ T-cells is more susceptible to b12 neutralization when compared to VS-mediated infection (Figure S2B). These results confirm our findings in the T-cell line MT4 (Figure 1D) that the relative resistance of VS-mediated infection to neutralization can also be observed when primary activated CD4+ T-cells are infected with a primary isolate R5-tropic virus. The gp41 CT regulates sensitivity to neutralizing mab during VS-mediated infection Because the cytoplasmic tail of gp41 is known to regulate the fusogenic capacity of Env (16, 25, 45, 46), we hypothesized that the exposure of certain epitopes may be regulated on the surface of cells through allosteric interactions with the gp41 CT, which are in turn influenced by the processing status of Pr55 Gag (16, 19, 45). To directly test the role of the gp41 CT in modulating neutralization sensitivity, we performed antibody neutralization assays with an NL-GI viral mutant carrying a truncation of the gp41 CT that deletes the C- terminal 144 amino acids ( CT). When the viral clone carrying CT Env was transfected into Jurkat cells, Western blot showed the expected shift in molecular weight of CT Env (Figure 3A). Since the gp41 CT has been described as playing a role in endocytosis of Env as well as in regulating epitope exposure, we also performed cell-surface staining of the mutant using the neutralizing 17

18 antibodies b12, 17b, 4E10 and polyclonal neutralizing sera from two HIV-1 positive patients (Figure 3B). The mutant showed higher levels of Env surface staining on HIV-1 positive cells expressing CT NL-GI as compared to WT NL- GI. The WT Env expressing cells stained variably and much weaker than CT Env for the antibodies shown in Figure 3B, as well as a number of other neutralizing antibodies examined in this paper (data not shown). The staining of WT Env by polyclonal patient antisera but not many monoclonal antibodies is suggestive of changes in Env epitope exposure on the surface of CT-infected cells. From the antibody staining results, we conclude that the surface binding of most antibodies likely increases as a function of increased expression and/or enhanced epitope exposure on the CT mutant. We next compared the inhibition of cell-free CT infection to the levels of inhibition obtained for cell-free WT infection in Figures 1D and S2A. Because the infectivity of CT viral particles was lower than WT, we tested multiple viral inputs for CT and found that there was no change in the observed IC 50 when the viral input of the CT mutant was increased to infect a similar percentage of target cells as the WT virus (Figure S3A, B). The higher CT viral input that resulted in levels of infection similar to WT was therefore used in cell-free titration experiments. When comparing WT with CT cell-free infections, we observed no significant differences in inhibition by Leu3a, b12, and 17b (Figure 4A, Table S1), as well as 2G12, E51, 21c, AMD-3100 and T20 (Figure S4A). These mab and inhibitors all displayed similar IC 50 values for WT and CT cell-free infection, indicated by a fold change of 1 (Figure 4C, Table S1). For cell-free viral 18

19 infections, 4E10 showed the largest fold change of 4.4, followed by F425 A1g8, and 2F5. These three mab required a higher IC 50 (P<0.05) to block infection by WT viral particles compared to CT (Figures 4A and S4A). 48d did not inhibit CT cell-free infection at 10 µg/ml (Figure S4A). Overall we conclude that the gp41 CT truncation has only modest effects on the neutralization of cell-free HIV- 1, which were dependent upon the epitope tested. VS-mediated infection by WT expressing donor cells (from Figure 1D) and CT-expressing donor cells were inhibited equally well by the anti-cd4 antibody Leu3a (Figure 4B, Table S1). In contrast, statistically significant differences in IC 50 were observed during WT and CT VS-mediated infection for b12 (P<0.01), 4E10 (P<0.05) and 17b (P<0.01) (Figure 4B). The other antibodies also showed significantly greater IC 50 against WT VS-mediated neutralization (data from Figure S2A) as compared to CT, with the exception of F425 A1g8, which displayed a sizable shift that did not reach statistical significance in this analysis (Figure S4B, Table S1). In contrast, the entry inhibitors T20 and AMD-3100 did not show a statistically significant change in IC 50. As with cell-free infection, we examined infection conditions that gave rise to different levels of infected target cells expressing CT NL-GI and found that the infection level did not statistically change the IC 50 for VS-mediated infection (Figure S3C, D). Overall, during VSmediated infection, the IC 50 values of CT infection are reduced compared to WT (Table S1) even though more Env epitopes are present at the cell surface (Figure 3). The mab b12 exhibited the largest shift in IC 50 of 29.7-fold increase for WT over CT (Figure 4D). 17b showed an intermediate 7.7-fold increase in IC 50, 19

20 while the remaining mab and inhibitors showed smaller but statistically significant increases of up to 6-fold. When comparing the relative effect of CT on cell-free versus cell-associated inocula, a greater overall effect was observed for most antibodies when testing cell-associated HIV-1 infection (Figure 4C, D). Consistent with this, the fold-change in IC 50 of CT VS-mediated infection over CT cell-free infection (Figure S5) for anti-env mab was smaller than a similar comparison for WT NL-GI (Figure 1E). Given the enhanced staining of CT Env at the cell surface, the enhanced neutralization could be the result of either changes in epitope exposure or increased protein expression at the surface. Our result is supportive of changes in epitope exposure, as increased cell-surface Env would likely enhance mab avidity at the synapse and make VS-mediated infection more difficult to inhibit. We interpret our data to suggest that truncation of gp41 CT generates greater exposure of VS-neutralizing epitopes at the cell surface, and this renders VS-mediated infection by CT more sensitive to neutralizing antibodies. Enhanced sensitivity of CT to 17b occurs after cell-to-cell transfer Because VS-mediated infection of WT NL-GI strongly resisted neutralization by 17b compared to CT NL-GI, we wished to further examine whether this effect was mediated by enhanced inhibition of the first step in the two-step model of VS-mediated HIV-1 entry, namely cell-to-cell transfer. We therefore measured WT and CT Gag-iGFP virus transfer into primary unactivated CD4+ T-cells after 3 hours of co-culture. The Gag-iGFP construct 20

21 allows fluorescent detection of viral transfer across virological synapses (14). Using this assay, Leu3a and b12, two mab that interfere with the CD4-Env interaction, both efficiently inhibited cell-to-cell transfer of both WT and CT GagiGFP into target cells, while inhibition by 17b was minimal at 30 µg/ml (Figure 5A, B). Titration curves show that Leu3a inhibited both WT and CT Gag-iGFP similarly (Figure 5C), confirming that the CT mutation has no effect on the ability of Leu3a to block the gp120 binding site on CD4. The anti-gp120 mab b12 also inhibited transfer of WT and CT Gag igfp at the highest concentration tested, 10 µg/ml (Figure 5A, B), however the CT Gag-iGFP was more sensitive than WT Gag-iGFP to b12 neutralization (Figure 5C). The enhanced sensitivity of CT to b12 during VS-mediated infection (Figure 4B) can be partially attributed to an enhanced ability to block the initial step of cell-to-cell transfer of HIV-1. In contrast, virus transfer levels were unaffected by 17b despite increasing antibody concentrations (Figure 5A, C), and the CT Gag-iGFP mutation had no effect on neutralization by 17b (Figure 5B, C). We conclude that the increased sensitivity of cell-associated CT virus to 17b occurs after cell-to-cell transfer of HIV-1. Given our current model for endosomal uptake of after VS formation, this inhibition is likely to occur within endosomal compartments in the target cell (7). Neutralization properties of HIV-1 neutralizing patient sera We next asked whether the CT mutation could enhance the ability of neutralizing antibodies generated during natural HIV-1 infection to block cell-tocell transfer and VS-mediated infection. Cell-free and VS-mediated infections 21

22 using WT NL-GI in the presence of two well-characterized reference polyclonal sera found that cell-free infection with WT NL-GI could be inhibited up to 100% with increasing sera concentrations (data from Figure 1A also shown in Figure 6A). In contrast, both cell-to-cell transfer (Figure 6B) and VS-mediated infection of WT (data from Figure 1A also shown in Figure 6C), were partially inhibited up to 50% at the maximum concentration tested, a 1:50 dilution. Truncation of gp41 CT had little to no effect on the neutralization of cell-free infection (Figure 6A), however cell-to-cell transfer of CT was inhibited by an additional 25-30% compared to its WT counterpart (Figure 6B). During VS-mediated infection, the patient sera blocked CT by ~30-40% more than WT at maximum concentration (Figure 6C). These results show that the relative resistance of VS-mediated infection to neutralizing patient sera is also partly controlled by the gp41 CT. Downloaded from on April 21, 2018 by guest 22

23 Discussion Many studies have implicated cell-associated HIV-1 infection as an efficient route of viral spread (5, 8, 38). The results presented here further support that this mode of infection plays a critical role in immune evasion. In vitro neutralization assays have generally found that VS-mediated infection is sensitive to neutralizing antibodies at high antibody concentrations (5, 21, 22, 33). However, studies with patient sera have indicated that VS-mediated HIV-1 can be considerably more difficult to neutralize (5, 15). Using quantitative, flow cytometry based assays, we measured the extent of VS-mediated neutralization by a panel of mab, and find that many mab are less efficient at neutralizing cellassociated HIV-1, as indicated by statistically significant differences in IC 50. The CD4i mab 17b and sera from HIV-1 positive patients were much more potent against cell-free HIV-1 than VS-mediated HIV-1 infection. The differences in the relative efficiencies of various mab to block cell-associated HIV-1 suggest to us that differential epitope exposure plays an important role in neutralization resistance. In addition, we examined a truncation mutant in the gp41 CT and found that cell-associated HIV-1 was particularly sensitive to loss of the gp41 CT relative to cell-free HIV-1. Our recent studies have revealed that particle maturation occurs after viral transfer across VS, and that maturation is required to activate viral membrane fusion (7). Given the enhanced sensitivity of cells infected with a CT virus to be neutralized by some mab, the deletion of the gp41 CT may abrogate the regulation of epitope exposure that occurs during VS-mediated infection. Our 23

24 current model suggests that the VS entry pathway is distinct from cell-free infection in that Env is bound to CD4 prior to particle assembly, well before it has been activated for fusion by the particle maturation process (15). In this model the Env-CD4 interaction persists during viral budding, transfer into the endocytic compartment, and during viral maturation within the endosome. Since viral maturation triggers viral membrane fusion (7), it follows that for antibodies to block this pathway, they must interact with neutralizing epitopes that are well exposed either at the cell-surface or during the maturation process. Conformationally sensitive neutralizing mab or components of polyclonal patient sera may fail to recognize their determinants during VS-mediated infection because: 1) the epitopes are very transiently exposed within the endosomal compartment between Env maturation and membrane fusion, 2) the preformed CD4-Env complex may sterically obstruct access to the epitope or 3) epitopes may be structurally hidden when maturation occurs after CD4-Env engagement. Truncation of gp41 CT may allow cell-surface Env to adopt an alternate conformation that may engage neutralizing antibodies more than the immature Env conformation found on the surface of infected cells. Our studies on the inhibition WT HIV-1 infection by neutralizing antibodies, inhibitors (Figure 1, S2) and neutralizing patient sera (Figure 6) are in agreement with previous studies that conclude antibodies and inhibitors capable of blocking WT cell-free HIV-1 can also inhibit WT cell-associated HIV-1 infection at the highest concentration tested (5, 21, 22, 33). However when specifically examining the IC 50, we find that for most mab there were statistically significant 24

25 increases in IC 50 during VS-mediated infection compared to cell-free infection. In contrast, a previous study by Martin et al. (21) found no statistical difference between cell-free and VS-mediated IC 50 for several mab and entry inhibitors, although a large increase in IC 50 for most mab was noted by the authors for VSmediated infection (21). This discrepancy between findings could be due to differences in assay specificity for the detection of new infection specifically in target cells. In contrast to our study design, Martin et al. use quantitative PCR to detect the relative increase in HIV-1 pol DNA when donor and target cells are mixed (21). This assay does not discriminate between integrated and unintegrated HIV-1 pol DNA in donor or target cells and may result in a larger variability because of a weak signal to noise ratio. Our use of a flow cytometry based assay distinguishes donor and target cells by differential dye labeling, allowing all donor cells to be efficiently excluded from the infection analysis. We also detect de novo early HIV-1 gene expression from newly infected target cells using the NL-GI molecular clone, as opposed to a DNA intermediate that does not directly equate to de novo infection. Prior studies also frequently use chronically infected cell lines (21, 22) that are also likely to carry mutations in HIV-1 accessory genes, such as vpu or nef. Given the known lack of vpu in the IIIB strain (30, 41), it is possible that tethered viruses that accumulate at the cell surface in the absence of vpu display Env in a form that is more similar to that found on cell-free virus. Here we use acutely transfected cell lines or acutely infected primary T-cells to minimize these effects. 25

26 While this work was in review, a publication by Abela and co-workers (1) reported that antibodies and inhibitors directed towards CD4, co-receptor, and the CD4 binding site on gp120 were less effective at neutralizing cell-associated HIV-1 than cell-free virus. While employing different reporter cell-based assays and some flow cytometry based assays, this study generally supports our findings that cell-to-cell transmission is often more difficult to inhibit compared to cell-free infection. However, CD4i mab and HIV-1 neutralizing patient sera were not tested, which show some of the largest differences in our assay (Figure 1). With different viral clones, they observed comparatively small effects for two antibodies against the MPER of gp41, 4E10 and 2F5. Based on this, they conclude that CD4 engagement is more difficult to block during synapse formation compared to post-attachment entry steps that are targeted by antigp41 mab and inhibitors. Here we report a significantly higher IC 50 for the antigp41 MPER mab 4E10 and 2F5 for VS-mediated infection. The fold difference in IC 50 is in fact larger than most of the other mab we tested, including b12 (Figure 1D, E, Figure S2 and Table S1). Additional studies are required to test whether such differences are due to differences in Env genes examined, and if these results are a general property of anti-gp41 mab or are unique to polyreactive MPER mab such as 4E10 and 2F5 (12, 24). The anti-cd4 mab Leu3a and the anti-cxcr4 inhibitor AMD-3100 did not show significant differences between WT cell-free and VS-mediated IC 50 (Figure 1D, E and Figure S2) or any other comparisons in our study. This is indicative of their functional targets CD4 and CXCR4 that are accessible on the surface of the 26

27 uninfected cell and are notably unaffected by conformational changes in Env. In contrast, T20 and all anti-env mab tested except 2G12 displayed a significantly greater IC 50 against WT VS-mediated infection compared to WT cell-free infection. Previous studies have not examined titrations of mab 17b or neutralizing patient sera, which appear to show the strongest phenotypes. Our examination of a panel of CD4i mab suggests that not all CD4i mab are as extreme in differentially neutralizing ability as 17b. By definition, CD4i epitopes are sensitive to conformational changes in Env. The 17b epitope has been noted as one of the most sensitive to conformational changes in gp120 (42, 44), perhaps more so than other CD4i epitopes including those recognized by other CD4i mab we tested. It is also notable that 2G12, which recognizes a glycan based epitope and may be less sensitive to maturation-induced conformational changes in Env (19), is the only anti-env mab which did not display a statistically significant deficit against cell-associated HIV-1, although a 6.7-fold change was still observed (Figure 1E). Overall the data are consistent with our hypothesis that entry through the VS limits the exposure of conformation sensitive epitopes of Env. The data presented also indicate that the CT mutation can enhance the sensitivity of VS-mediated infection at different stages of the viral entry process. Cell-to-cell transfer, the first step in the two-step model of VS-mediated entry, is only sensitive to inhibition by mab that block Env-CD4 interaction and VS formation such as Leu3a and b12 (Figure 5). Consistent with the action of b12, changes in the gp120 ectodomain due to truncation of the gp41 CT enhance b12 27

28 binding and subsequent neutralization of cell-to-cell HIV-1 transfer. In contrast, the mab 17b does not block cell-to-cell transfer (Figure 5) but can inhibit VSmediated infection (Figure 4B), and therefore acts at a step post-vs formation. Given our laboratory s observations that viral membrane fusion is triggered within an internal compartment after cell-to-cell transfer (7), 17b and other mab that do not block cell-to-cell transfer likely function within the endocytic compartment, before membrane fusion. Studies to visualize mab localization within the target cell are ongoing. Given the high plasma viremia sustained during natural infection, and the relatively low levels of Env maintained on the surface of infected cells (32), it is likely that cell-free viral particles act as a dominant antigenic stimulus for B-cell responses. Consistent with this, we find that patient sera have a stronger ability to block cell-free but not VS-mediated infection (Figure 1A). Many antibodies in sera are likely to be directed against cell-free virions, and would therefore neutralize cell-free HIV-1 more effectively than cell-associated HIV-1. We suggest that resistance of cell-associated HIV-1 to certain neutralizing responses depends upon the ability of Env to regulate the exposure of key epitopes during VS-mediated infection. All of the anti-env mab we tested displayed a significant shift in IC 50 between WT and CT VS-mediated infection (Figure 4B and Figure S4B), and the fold-change in IC 50 was greater than when cell-free virions were tested (compare Figure 4C and D). Two antibodies, b12 and 17b showed particularly large enhancement of sensitivity to neutralization during VS-mediated infection 28

29 when the gp41 CT was deleted (Figure 4D). The fold-change in IC 50 between WT and CT VS-mediated infection ranges from 2 to ~29.7-fold, suggesting that truncation of the gp41 CT affects the IC 50 of different mab to varying extents. In the context of our model, this is due to conformational changes in Env, which differentially affect the binding sites of all the anti-env mab antibodies we tested to varying extents. The fact that the IC 50 of the peptide antagonist T20 is less affected by truncation of the gp41 CT during cell-free (Figure 4C) and VSmediated infection (Figure 4D) as compared to 2F5 and 4E10 may suggest that accessibility or steric issues related to the size of the inhibitor may also contribute to the relative differences in IC 50. When comparing WT and CT VS-mediated infection, it is important to note that the levels of Env on the cell surface are considerably higher when the gp41 CT endocytosis YXXL signal is removed. Given a uniform increase in all epitopes on CT Env, one might expect avidity effects to make the CT VS more difficult to inhibit, increasing the IC 50 of all mab equally. We do not see uniform differences across the different antibodies, suggesting that they are at least in part controlled by differences in epitope exposure between the WT and CT states of Env. The small changes in IC 50 we observed during cell-free infection with WT versus CT viral particles suggest that functionally these particles display similar epitopes. However, the two MPER mab 2F5 and 4E10 displayed enhanced sensitivity of CT cell-free virus neutralization over WT cell-free neutralization (Figure 4C), indicating that the absence of gp41 CT can also have a significant impact on cell-free virus. A recent study by Joyner et al. (19) shows increased 29

30 antibody binding of MPER mab 4E10 and 2F5 to immature, protease deficient (PR-) virus particles compared to mature, wild type virus. They conclude that the maturation process may cloak neutralizing epitopes on cell-free virus. In this study, differences in antibody binding could not be correlated with functional changes in neutralization sensitivity because immature particles are noninfectious (19). Furthermore, it remains an open question whether Env on the surface of infected cells during VS-formation is conformationally similar to Env on protease-deficient, immature viral particles. A previous report (9) employed gp41 CT truncations that retain an additional 20 amino acids on the intracytoplasmic side, and observed 2 to 3-fold increases in neutralization sensitivity compared to WT virus to 0.5 µg/ml mab b12, 2G12 and 48d in a luciferase-based pseudovirion infection assay (9). Differences between these observations and our findings could be due to differences in the viral truncation mutant used, or perhaps the expression of Env in trans when producing pseudovirus particles. The changes we observed in IC 50 comparing WT and CT VS-mediated infections were generally larger than those reported previously for cell-free virus. We do see some effects on the neutralization of cell-free HIV-1, however the gp41 CT has a greater effect on epitope exposure and neutralization of VS-mediated infection. These studies and ours together suggest that the gp41 CT can modulate epitope exposure on the surface of both HIV-1 infected cells and virus particles. VS-mediated infection has the potential to contribute significantly to in vivo HIV-1 spread and pathogenesis in lymphoid tissues that contain high numbers of 30

31 susceptible CD4+ T-cells. Current HIV-1 vaccine strategies are largely focused on targeting Env epitopes on cell-free HIV-1 or antigens representing native trimeric Env conformations. Our results provide evidence that unique conformations of cell-surface Env presented during VS-mediated infection should be a critical consideration in immunogen design, to prevent the selective neutralization escape of cell-associated virus at antibody concentrations capable of neutralizing cell-free infection. We also demonstrate a key role for the gp41 CT in controlling epitope exposure during infection through T-cell virological synapses. If an antibody-based vaccine is to be effective at preventing HIV-1 infection, further characterization of the humoral response to cell-surface epitopes that participate in VS-mediated infection is essential. Downloaded from on April 21, 2018 by guest 31

32 645 Acknowledgements We thank members of the Chen Lab and the Flow Cytometry Shared Resource Facility (Mount Sinai School of Medicine, New York, NY) for helpful comments and advice. This work was supported by grants from the NIH, NIAID AI074420, The Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease Award, and the Irma T Hirschl/Monique Weill-Caulier Trust Career Scientist Award to B.K.C. Downloaded from on April 21, 2018 by guest 32

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41 832 Figure Legends Figure 1. Relative neutralization resistance to neutralizing patient sera and mab during WT NL-GI VS-mediated infection. (A) Percent inhibition of the GFP-expressing HIV-1 molecular clone WT NL-GI during cell-free infection (solid lines, solid symbols) of the T-cell line MT4 and VS-mediated infection (dashed lines, solid symbols) of MT4 cells (target cells) by Jurkat cells expressing WT NL-GI (donor cells), in the presence of 5-fold titrations of HIV-1 neutralizing patient sera and HIV-1 negative control human serum. (B) Representative FACS plots of cell-free infection of MT4 cells by WT NL-GI with no inhibitor, the anti-cd4 blocking antibody Leu3a or the CD4i antibody 17b. (C) VS-mediated infection of MT4 cells (target cells) by Jurkat cells expressing NL-GI (donor cells; not shown) with no inhibitor, Leu3a or 17b. (D) Percent inhibition of WT NL-GI during cell-free infection (solid lines, solid symbols) and VS-mediated infection (dashed lines, solid symbols) by 5-fold titrations of mab Leu3a, 17b, b12, or 4E10. Error bars represent the SEM for at least two independent experiments using independently infected target cells or independently transfected donor cells on different days for cell-free or VSmediated infections, respectively. P-values represent statistically significant differences in Log 10 IC 50 derived from non-linear regression curve fits of pooled data. (E) Fold increase in IC 50 of WT VS-mediated infection over WT cell-free infection for mab (light grey) and entry inhibitors (dark grey). An IC 50 ratio of 1 (black line) indicates an equal IC 50 value for a and b. *, P<0.05; **, P<0.01; ***, 41

42 P<0.001 where P-values represent statistically significant differences in Log 10 IC 50 derived from non-linear regression curve fits of pooled data Figure 2. Neutralization resistance of primary activated CD4+ T-cells with CXCR4- and CCR5-tropic HIV-1 during VS-mediated infection. Representative FACS plots of cell-free or VS-mediated infection of primary CD4+ T-cells by HIV-1 containing (A) the CXCR4-tropic NL4-3 envelope or (B) the envelope from a CCR5-tropic acute early isolate (prhpa4259 clone 7) with no inhibitor, the mab b12 or 17b. (C & D) Percent inhibition of cell-free or VSmediated infection by HIV-1 containing the CXCR4-tropic NL4-3 envelope (C), or the envelope from a CCR5-tropic acute early isolate (prhpa4259 clone 7) (D). Error bars represent the SEM for two independent experiments and at least four CD4+ T-cell donors. Figure 3. WT NL-GI and ΔCT NL-GI expression in donor Jurkat cells. HIV-1 protein expression in Jurkat cells nucleofected with WT NL-GI or ΔCT NL- GI, used as donor cells in VS-mediated infection experiments. (A) Western blot of HIV-1 nucleofected Jurkat cell lysate using antibodies to detect gp120, gp41, p24 and actin. (B) Cell-surface immunostaining for HIV-1 envelope. Cells were stained with 15 μg/ml b12, 17b or 4E10, or 1:50 dilution of HIV-1 neutralizing or negative control serum, followed by PE-conjugated donkey anti-human IgG as the secondary antibody. Histogram plots show antibody binding to HIV-1 positive cells for WT NL-GI (blue line) compared to ΔCT NL-GI (red line), as well as WT 42

43 NL-GI and ΔCT NL-GI expressing cells stained with secondary antibody alone (shaded grey histogram and black dotted line, respectively) Figure 4. Truncation of the gp41 CT enhances neutralization sensitivity to mab during VS-mediated infection. Percent inhibition of WT NL-GI (solid lines, solid symbols) and the gp41 cytoplasmic tail truncation mutant ΔCT (dashed lines, open symbols) by 5-fold titrations of mab Leu3a, b12, 4E10 and 17b during (A) cell-free infection and (B) VS-mediated infection. Error bars represent the SEM for at least two independent experiments using independently infected target cells or independently transfected donor cells on different days for cell-free or VSmediated infections, respectively. Percent inhibition for WT infection is from Figure 1D. P-values represent statistically significant differences in Log 10 IC 50 derived from non-linear regression curve fits of pooled data. (C & D) Fold increase in IC 50 of WT cell-free infection over ΔCT cell-free infection (C) or WT VS-mediated infection over ΔCT VS-mediated infection (D) for mab (light grey) and entry inhibitors (dark grey). An IC 50 ratio of 1 (black line) indicates an equal IC 50 value for a and b. *, P<0.05; **, P<0.01; ***, P<0.001 where P-values represent statistically significant differences in Log 10 IC 50 derived from non-linear regression curve fits of pooled data. Figure 5. Effect of the gp41 CT on cell-to-cell transfer of WT and ΔCT HIV-1. Representative FACS plots of cell-to-cell transfer of the fluorescent HIV-1 (A) WT 43

44 Gag-iGFP and (B) ΔCT Gag-iGFP from infected Jurkat cells (donor cells; not shown) to primary CD4+ T-cells (target cells) with no inhibitor, Leu3a, b12 and 17b. (C) Percent inhibition of cell-to-cell transfer of WT Gag-iGFP (solid lines, solid symbols) and ΔCT Gag-iGFP (dashed lines, open symbols) by 5-fold titrations of mab Leu3a, b12 and 17b. Error bars represent the SEM for at least two independent experiments using independently transfected donor cells on different days. Figure 6. Sensitivity of WT and CT HIV-1 to neutralizing patient sera. Percent inhibition of WT HIV-1 (solid lines, solid symbols) and ΔCT HIV-1 (dashed lines, open symbols) during (A) cell-free infection, (B) cell-to-cell transfer and (C) VS-mediated infection by 5-fold titrations of HIV-1 neutralizing patient sera and HIV-1 negative control human serum. Error bars represent the SEM for at least two independent experiments using independently infected target cells for cell-free infection, or independently transfected donor cells on different days for cell-to-cell transfer or VS-mediated infections. Percent inhibition for WT infection in (A) and (C) is from Figure 1A. 44

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