Phenotypic and Immunologic Comparison of Clade B Transmitted/Founder and Chronic HIV-1 Envelope Glycoproteins^▿

Pseudovirus production.

Pseudotyped virus was produced by calcium phosphate cotransfection of 6 μg of pcDNA3.1⁺ containing env with 10 μg of HIV-1 core (pNL43-ΔEnv-vpr⁺-luc⁺ or pNL43-ΔEnv-vpr⁺-eGFP) into 293T17 cells. Virus was harvested at 72 h posttransfection, filtered through a 0.45-μm filter, aliquoted, and stored at −80°C. For primary CD4⁺ T cell infections, pseudovirus was concentrated by ultracentrifugation through a 20% sucrose cushion. Pelleted pseudovirus was then resuspended in phosphate-buffered saline (PBS). All luciferase-encoding pseudoviral stocks were serially diluted and used to infect NP2 cells to define the linear range of the assay. A viral dilution was chosen in the middle of the 5-fold linear range of the assay to maximize sensitivity.

env cloning and sequence analysis.

The derivations of most T/F Env clones used in this study have been described previously (36). THRO.F4.2026, SUMAd5.B2.1713, 9010-09.A1.4924, and PRB959-02.A7.4345 were cloned from SGA amplicons known to contain the nucleotide sequence of the corresponding T/F env sequence into pcDNA3.1 according to the manufacturer's instructions (Invitrogen). The AD17.1 env gene was subcloned from a full-length infectious molecular T/F clone described elsewhere (39). Chronic Envs HEMA.A4.2125 and HEMA.A23.2143 were also cloned in pcDNA3.1. Briefly, viral RNA was extracted from plasma samples from chronically infected patients and amplified using SGA methods. Individual env genes were then either cloned at random or selected to maximize within-patient env sequence diversity. Env clones were sequenced to confirm that they did not contain Taq polymerase errors but represented env genes of viruses circulating in the patient. The nucleotide sequences of all T/F and chronic Envs have previously been reported (Gnanakaran et al., submitted). PNGs were determined with N-glycosite (hiv.lanl.org) (76). To assess lengths of the V1/2, V3, V4, V5, and V1-4 regions, sequences were aligned to HXB2, and then boundaries were identified for each region and nongap residues were counted.

Coreceptor tropism testing and cell line infections.

NP2 cells stably expressing CD4 and either CCR5 (NP2/CD4/CCR5) or CXCR4 (NP2/CD4/CXCR4) were infected with HIV-1 pseudoviruses expressing luciferase by spinoculation in 96-well plates at 450 × g for 90 min at 25°C. Cells were lysed with Brite-Glo (Promega) at 72 h postinfection and analyzed on a Luminoskan Ascent luminometer. Coreceptor tropism was arbitrarily defined by mean relative light units (RLUs) greater than 1 (approximately 100-fold over background). To assess sensitivity to coreceptor inhibitors, NP2/CD4/CCR5 or NP2/CD4/CXCR4 cells were preincubated for 30 min with saturating concentrations of the CCR5 inhibitor maraviroc (MVC) (1 μM), the CXCR4 inhibitor AMD3100 (2 μM), or the fusion inhibitor enfuvirtide (10 μg/ml) prior to infection. To assess sensitivity to broadly neutralizing MAbs, viral pseudotypes were preincubated with 10 μg/ml of antibody for 30 min at 37°C. Virus and antibody mixes were then used to infect NP2/CD4/CCR5 or NP2/CD4/CXCR4 cells. All NP2 cell line infections were done in at least triplicate in at least three independent experiments using R5-tropic JRFL as a positive control and Env-deficient pseudotypes as a negative control.

The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3-deltaE-eGFP (catalog no. 11100) from Haili Zhang, Yan Zhou, and Robert Siliciano (74); bicyclam JM-2987 (hydrobromide salt of AMD-3100; catalog no. 8128) (9, 18, 34); maraviroc (catalog no. 11580) (6, 22, 65), and HIV-1 gp120 MAb IgG1 b12 (catalog no. 2640) from Dennis Burton and Carlos Barbas (5, 11, 12, 55).

Primary human CD4⁺ T cell tropism assay.

Primary human CD4⁺ T cells, purified by negative selection, were obtained from the University of Pennsylvania's Human Immunology Core. A total of 2 × 10⁶ cells per virus were stimulated with plate-bound anti-CD3 (clone OKT3) (eBiosciences) and anti-CD28 (clone 28.2) (BD Biosciences) and 20 U/ml recombinant interleukin-2 (IL-2) in RPMI containing 10% fetal bovine serum (FBS). At 3 days poststimulation cells were transferred to 96-well V-bottom plates prior to infection. Five microliters/well of concentrated HIV-green fluorescent protein (GFP) was used to infect cells in triplicate. Plates were then spinoculated at 1,200 × g for 2 h. Cells were then transferred to new 24-well plates, and new medium containing 20 U/ml IL-2 was added. At 3 days postinfection, cells were stained for flow cytometry.

Determination of alternative coreceptor use.

Primary human CD4⁺ T cells from two different ccr5Δ32 homozygous donors were obtained and purified as described above. Prior to infection, cells were preincubated with 50 μM AMD3100 for 30 min. Cells were infected as described above. At 2 hours after spinfection, enfuvirtide (1-μg/ml final concentration) was added to all samples to prevent additional fusion prior to transferring cells to a 24-well plate for further incubation. Samples were stained and analyzed as described above.

Flow cytometry.

A total of 1 × 10⁶ to 2 × 10⁶ cells were stained per tube for flow cytometry. All incubations were done at room temperature (RT) and in fluorescence-activated cell sorter (FACS) wash buffer (PBS, 2.5% FBS, 2 mM EDTA), and all antibodies were from BD Biosciences, unless otherwise noted. To stain CD4⁺ T cells, cells were first washed in PBS, and then, live/dead Aqua (Invitrogen) was added and incubated for 10 min. Next, anti-CCR7 IgM in FACS wash buffer was added and incubated for 30 min. Cells were then washed in FACS wash buffer before staining with anti-CD3–Qdot 655 (Invitrogen), anti-CD4–Alexa Fluor 700, anti-CD45RO–phycoerythrin (PE)-Texas Red (Beckman Coulter), and anti-IgM–PE (Invitrogen) for 30 min. Cells were then washed in FACS wash buffer and resuspended in 1% paraformaldehyde (PFA). Samples were run on an LSRII (BD) instrument and analyzed with FlowJo 8.8.6 (Treestar). Cells were gated as follows: singlets (FSC-A by FSC-H), then live cells (SSC-A by live/dead), then lymphocytes (SSC-A by FSC-A), then CD3⁺ cells (SSC-A by CD3), then memory markers (CCR7 by CD45RO).

DC trans-infection assay.

To differentiate DCs, freshly isolated monocytes from the University of Pennsylvania's Human Immunology Core were treated with 50 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D systems) and 100 ng/ml IL-4 (R&D systems) in AIM V serum-free medium (Invitrogen). New medium containing GM-CSF and IL-4 was added on day 3. At 6 days poststimulation, DCs were washed and plated at 3 × 10⁴ cells per well in a V-bottom 96-well plate; 3 × 10⁴ CD4⁺ T cells alone at 3 days poststimulation with plate-bound anti-CD3/anti-CD28 were used as a negative control. Titers of viral stocks were first determined by RLUs on NP2/CD4/CCR5 or CXCR4 cells. Virus sufficient to generate 80 RLUs was added to DCs or a CD4⁺ T cell control and allowed to bind for 2 h at 37°C. Cells were washed three times with fresh medium to remove cell-free virus, and then 3 × 10⁵ stimulated heterologous CD4⁺ T cells were added to each well containing 3 × 10⁴ HIV-bound DCs or CD4⁺ T cells. As an additional control, an equal amount of virus was added to 3 × 10⁵ stimulated CD4⁺ T cells to ensure that there was no differential infection of CD4⁺ T cells. For CD4⁺ T cell luciferase infection, cells were spinoculated at 450 × g for 90 min and then incubated without washing off virus. Cells were then transferred to a flat-bottom 96-well plate for 3 days prior to takedown with Brite-Glo. Experiments for each condition were done in triplicate, and each viral pseudotype was used in at least three independent experiments with cells from different healthy donors.

Enfuvirtide time-of-addition assay.

To assess the entry kinetics of T/F and chronic Envs, NL43vpr⁺luc⁺ pseudotypes were chilled to 4°C and added to NP2/CD4/CCR5 (or NP2/CD4/CXCR4 for the one X4-tropic Env) cells on metal blocks embedded in ice covered by a moist towel. Cells were then spinoculated at 1,300 rpm for 90 min at 4°C to enhance viral binding. Immediately postspinoculation, cold supernatant was aspirated off and all wells were flooded with 270 μl of prewarmed 37°C medium and transferred to a 37°C incubator. Thirty microliters of 10-μg/ml enfuvirtide (final concentration of 1 μg/ml) was then added at 0, 5, 10, 20, 40, 80, or 160 min postwarming. A no-drug control was also included to normalize percent infection. Cells were then incubated for 72 h and assessed for RLUs. At least three wells per virus per time point were included in each experiment, and all Envs were examined in at least three independent experiments. Data were analyzed with Prism 4.0 (GraphPad Software, Inc.) by fitting a best-fit sigmoidal line to the results from each independent experiment prior to averaging the Hill slopes and time to half-maximal fusion.

Neutralization sensitivity.

Neutralization assays were performed using both NP2 and TZMbl cells in two independent laboratories. To assess sensitivity to MAbs b12, VRC01, PG9, and PG16, viral pseudotypes were preincubated with 10 μg/ml of antibody for 1 h prior to infection of NP2 cells. To assess sensitivity to HIV Ig, pseudotypes were preincubated with 2-fold serial dilutions of clade B HIV Ig from 1,500 to 23 μg/ml. This mix was then added to NP2 cells and spinoculated as described above. For MAbs, neutralization was assessed by determining the maximum percent inhibition (MPI) compared to a no-antibody control. Clade B HIV Ig (lot 12 100158) was obtained from the AIDS Repository.

Neutralization sensitivity was assessed on TZMbl cells as previously described (17, 69). Briefly, 8 × 10³ TZMbl cells were plated overnight. Five-fold dilutions of MAbs (b12, VRC01, PG9, PG16, and clade B HIV Ig) were incubated in the presence of 40 μg/ml DEAE-dextran and 2.000 infectious units (as measured on TZMbl cells) of pseudovirus for 1 h at 37°C. After the medium was removed from TZMbl cells, the virus-MAb dilutions were added to the cells and incubated for 48 h before being analyzed for luciferase expression (Promega). The highest concentration tested for b12, VRC01, PG19, and P16 was 10 μg/ml. The highest concentration of clade B HIV Ig was 1,500 μg/ml. Samples were tested in duplicate, with all experiments repeated at least two times. Fifty percent inhibitory concentrations (IC₅₀s) were calculated as described previously (17).

CELISA.

The binding of MAbs to HIV-1 Env trimers expressed on cells was measured using a cell-based enzyme-linked immunosorbent assay (CELISA) system, as previously described (32). Briefly, COS-1 cells were seeded in 96-well plates (1.8 × 10⁴ cells/well) and transfected the next day with 0.1 μg of a plasmid expressing Env and 0.02 μg of a Rev-expressing plasmid per well using Effectene transfection reagent. Three days later, cells were incubated with the indicated MAb suspended in blocking buffer (35 mg/ml bovine serum albumin [BSA], 10 mg/ml nonfat dry milk, 1.8 mM CaCl₂, 1 mM MgCl₂, 25 mM Tris [pH 7.5], and 140 mM NaCl) for 1 h at room temperature. Cells were then washed four times with blocking buffer and four times with washing buffer (140 mM NaCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 20 mM Tris [pH 7.5]). A horseradish peroxidase (HRP)-conjugated antibody specific for the Fc region of human IgG was then incubated with the samples for 45 min at room temperature. Cells were washed five times with blocking buffer and five times with washing buffer. HRP enzyme activity was determined after the addition of 33 μl per well of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin-Elmer Life Sciences) supplemented with 150 mM NaCl. Light emission was measured with a Mithras LB 940 luminometer (Berthold Technologies). To correct for the level of cell surface expression of each envelope glycoprotein, binding of the antibodies is expressed as percent binding of the CD4-Ig probe at saturating concentrations (5 μg/ml). We decided a priori to exclude all envelope glycoproteins that bound CD4-Ig at less than 20% of the binding measured for the SC05.8H2.3243 control isolate. Five of the 57 Envs were thus not analyzed, including three T/F and two chronic Envs. Measurements of antibody binding and neutralization were performed under code to prevent potential bias.

Statistical analyses.

T/F and chronic Envs were compared by using Mann-Whitney tests, and correlations were assessed by using Spearman tests. P values of less than 0.05 were considered significant. Data were analyzed with Prism 4.0 software.

Ethics statement.

All human cells used in this study were from normal healthy donors who provided written informed consent after approval by the University of Pennsylvania's institutional review board.

Panels of T/F and chronic Envs.

To determine if there are functional differences between T/F Envs and those that predominate during chronic infection, we assembled a panel of 24 clade B T/F Envs previously inferred and cloned from plasma viral RNAs of 24 individuals with acute HIV-1 infection as defined by the Fiebig staging system, in which patients are classified from stage I (viral RNA positive and antibody and antigen negative) to stage VI (ELISA and Western blot positive with multiple bands) (4, 36, 39) (Table 1). Twelve individuals were sampled during Fiebig stage II, five during Fiebig stage III, two during Fiebig stage IV, and five during Fiebig stage V. Acutely infected individuals were predominantly males (22 of 24) from the southeastern United States (18 of 24) with a variety of sexual risk factors, all denying intravenous drug use (IDU). All T/F Envs were inferred from SGA-derived sequences, which are devoid of PCR-induced errors and cloning bias (36). Env clones identical to this inferred T/F sequence were then chosen for phenotypic analysis. Importantly, all T/F Envs were selected from subjects with single-variant transmissions. This was done to increase the likelihood that the viruses encoding these Envs were transmitted across an intact mucosal barrier, thereby maximizing our chances of observing properties required for this process (30, 39).

To generate chronic clade B control Envs, we used SGA to amplify env genes from plasma viral RNAs of two groups of antiretroviral therapy-naïve individuals. The first group consisted of 11 individuals sampled at 14 to 83 months postinfection (mean, 42 months). A test set of 17 Env clones was derived from this group, consisting predominantly of males (8 of 11) from the southeastern United States (10 of 11), all denying IDU (Table 1). An additional 14 clade B control Envs were SGA amplified and cloned from six chronically infected individuals residing in the northwestern United States. This second group of chronic Envs served as a validation set to confirm differences in neutralization sensitivity observed with the first test set. A phylogenetic tree of the 31 chronic Envs is depicted in Fig. 1, along with the 24 T/F Envs. None of the Envs were from epidemiologically linked infections.

Previous studies noticed fewer PNGs in the gp120 region of T/F Envs than in that of chronic Envs (40; Gnanakaran et al., submitted). To determine whether our selected subset of T/F and chronic Envs differed from this much larger group, we compared the variable-loop lengths as well as the numbers and distributions of putative PNGs. There were no differences in V1/2, V3, V4, and V1-4 lengths between T/F and chronic clade B Envs. Further, the median number of gp120 PNGs in T/F Envs was 26.0, compared to 27.0 for the chronic controls (P = 0.16) and 26.0 for clade B Envs in general (74). Thus, the panel of T/F Envs selected for our functional analyses exhibited no statistically significant differences in patient demographics, virus phylogeny, variable-loop length, or PNGs relative to the panel of chronic Envs we assembled or to clade B Envs in general.

Determination of coreceptor tropism.

R5-tropic viruses represent the vast majority of transmitted viruses, with dual (R5X4)-tropic viruses being transmitted less frequently (36, 56, 63, 79). On rare occasions, X4-tropic viruses can be transmitted (3, 47, 66). To determine the coreceptor usage in our panel, we characterized the CCR5 and CXCR4 utilization of the 24 T/F and 17 chronic Envs by producing viral pseudotypes and using these to infect NP2 cell lines expressing CD4 and either CCR5 (NP2/CD4/CCR5) or CXCR4 (NP2/CD4/CXCR4), as well as primary human CD4⁺ T cells. NP2 cells were selected because they provide a 5- to 6-log linear range of infection, approximately 2 to 3 logs greater than that of the TZMbl assay. We found that of the 24 T/F Envs, 21 were R5-tropic and three were R5X4-tropic, while of the 17 chronic Envs, 15 were R5-tropic, one was R5X4-tropic, and one was X4-tropic (Table 1). This is consistent with previous results, with the exception of T/F Envs 1058-11.B11.1550 and CH77.SA2.6559, which were R5X4-tropic on NP2 cells and R5-tropic on TZMbl cells (36) (Gnanakaran et al., submitted). This discrepancy is likely due to differences in CXCR4 expression, as the NP2 cells used stably express high levels of CXCR4 compared to the HeLa-derived TZMbl cells, which express lower endogenous CXCR4 levels. All four R5X4-tropic Envs utilized CCR5 and CXCR4 with approximately equivalent efficiencies as assessed by a less-than-2-fold difference in RLUs between the NP2/CD4/CCR5 and NP2/CD4/CXCR4 cells. To assess coreceptor use on human CD4⁺ T cells, we infected ccr5Δ32 or ccr5wt CD4⁺ T cells in the presence or absence of saturating concentrations of the CXCR4 inhibitor AMD3100. The results paralleled those obtained with the NP2 cell lines. R5-tropic Envs mediated infection of ccr5wt but not ccr5Δ32 CD4⁺ T cells, while R5X4 Envs mediated infection of both cell types. Infection of ccr5Δ32 CD4⁺ T cells by three of the R5X4 Envs was completely inhibited by AMD3100, while Env CRPE.B28.4072 could infect ccr5Δ32 cells in the presence of AMD3100, though with reduced efficiency (data not shown). However, we found that AMD3100 inhibited infection of NP2/CD4/CXCR4 cells by viruses bearing the CRPE.B28.4072 Env by only 50%. In addition, this Env was unable to infect NP2 cells expressing CD4 alone or in combination with any of 17 different putative alternative coreceptors, indicating that this Env can utilize the drug-bound conformation of CXCR4 (data not shown). Several other HIV-1 Env proteins have been shown to exhibit this property (33). In summary, all T/F Envs utilized CCR5, while three were R5X4-tropic. Thus, there were no differences in coreceptor tropism between the T/F and chronic Envs, with the exception of the one X4-tropic chronic Env, and there was no evidence for utilization of coreceptors other than CCR5 or CXCR4 to infect human CD4⁺ T cells.

Sensitivity to coreceptor antagonists and CCR5 utilization efficiency.

Mucosal transmission of HIV-1 is dependent upon CCR5. Hypothesizing that the ability to use low levels of CCR5 may confer a selective advantage to viruses at the moment of transmission, we determined the sensitivity of each Env to the CCR5 inhibitor maraviroc (MVC) as a surrogate for CCR5 utilization efficiency. High MVC IC₅₀s indicate that an Env can mediate infection at low levels of CCR5, while low IC₅₀s suggest that an Env requires high CCR5 expression for viral entry. We found no significant difference in median MVC IC₅₀s between the T/F (2.4 nM) and chronic (2.3 nM) Envs (P = 0.79 by Mann-Whitney test) (Fig. 2A). In addition, we determined the maximal percent inhibition (MPI) of infection by MVC. While they are uncommon, several in vivo-derived MVC-resistant R5-tropic viruses that can utilize the drug-bound form of CCR5 have been identified (67, 70). Such viruses engage the coreceptor differently, relying predominantly upon the N terminus for entry whereas most viruses require the N terminus as well as the extracellular loops of CCR5. Furthermore, determining the MVC sensitivities of T/F viruses has implications for microbicides and preexposure prophylaxis. All 41 Envs examined had MPIs of greater than 85%, with the vast majority greater than 95%. There were no significant differences (P = 0.17 by Mann-Whitney test) between the T/F (median = 99.1%) and chronic (median = 98.3%) Envs (Fig. 2B). Together, these data indicate that the HIV-1 transmission bottleneck does not impose a selection pressure for viruses capable of using low concentrations of CCR5.

Primary CD4⁺ T cell tropism.

CD4⁺ T cells, the major target and source of HIV-1 in vivo (37, 77), can be broadly divided into four subsets: naïve (CCR7⁺ CD45RO⁻), central memory (T_CM) (CCR7⁺ CD45RO⁺), effector memory (T_EM) (CCR7-CD45RO⁺), and CD45RA⁺ effector memory (T_EMRA) (CCR7⁻ CD45RO⁻) (61). These subsets are differentially infected due in part to variation in coreceptor expression (50), cellular activation (26), and tissue localization (27). T_EM and T_EMRA cells are found predominantly in effector sites, including the rectal and cervicovaginal mucosae, while naïve and T_CM cells are most abundant in the lymph nodes. T_EM cells, the most abundant subset in mucosal effector sites, are preferentially infected and massively depleted during acute infection (reviewed in reference 42). Since potential target cells in the mucosae may be limiting during transmission, we hypothesized that T/F Envs may infect T_EM and T_EMRA cells preferentially relative to the matched chronic controls.

Peripheral blood CD4⁺ T cells from three normal uninfected donors were purified by negative selection and stimulated with anti-CD3/anti-CD28 and IL-2 for 3 days prior to infection with HIV-1 pseudotypes expressing a GFP reporter. At 3 days postinfection, the viability and expression of CD3, CD4, CCR7, CD45RO, and GFP were assessed by FACS analysis. Productively infected cells were defined as CD3⁺ GFP⁺, since CD4 was downregulated in the majority of infected cells (16). The gating strategy is shown in Fig. 3A. In all three donors, infected cells were predominantly T_EM (∼65%), followed by T_CM (∼30%), T_EMRA (∼3%), and naïve (∼2%) cells (Fig. 3B). As X4-tropic viruses have been previously reported to readily infect naïve CD4⁺ T cells compared to R5-tropic viruses (21, 46, 48), this assay contains an important internal validation: the five viruses that could utilize CXCR4 for entry (one X4-tropic Env shown in cyan and four R5X4-tropic Envs shown in red in Fig. 3B) preferentially infected naïve cells. With the exception of these five Envs, no other pseudotypes were reproducibly outliers in their ability to mediate entry into any of the subsets, and there were no statistically significant differences or trends between the T/F and chronic Envs for any of the four cell subsets in any of the three donors examined (Fig. 3B). In addition, there was no statistically significant difference in overall infectivity between the T/F and chronic Env pseudotypes in any of the three donors examined, suggesting comparable Env fitness in peripheral CD4⁺ T cells (Fig. 3C). Together, these results suggest that transmission and early expansion are not due to differential infection of CD4⁺ T cells or their subsets between T/F and chronic Envs.

DC-mediated trans-infection.

DCs can enhance HIV-1 infection in trans by efficiently capturing virus particles and presenting them to CD4⁺ T cells. In vitro, coculture of monocyte-derived DCs with CD4⁺ T cells results in enhanced virus infection, particularly at low virus inocula (reviewed in reference 51). To assess whether DCs preferentially bind and transfer T/F compared to chronic Env pseudoviruses, we performed DC/CD4⁺ T cell coculture experiments. Viral pseudotype stocks were normalized for infectivity on NP2 cells to control for differences in viral titer. A relatively limiting amount of virus (80 RLUs on NP2 cells) was bound to DCs, which were then washed to remove cell-free virus and cocultured with CD4⁺ T cells. All Envs were assessed in at least three independent experiments, each time using DCs and CD4⁺ T cells from different normal donors. Adding this amount of virus to 3 × 10⁴ CD4⁺ T cells and then washing as with the DCs resulted in infection at background levels. However, adding virus associated with DCs markedly increased infection. Nonetheless, the magnitude of DC/CD4⁺ T cell trans-infection was not different between T/F and chronic Envs (Fig. 4) (P = 0.44 by Mann-Whitney test). In addition, there was no difference in CD4⁺ T cell infectivity in the absence of DCs, and there was no detectable infection of DC control cultures in the absence of CD4⁺ T cells (data not shown). The absence of any difference between T/F and chronic pseudoviruses in this trans-infection assay suggests that, at least when presented with an equal amount of infectious pseudovirus, DCs bind and transfer T/F and chronic Env pseudotypes similarly.

Entry kinetics and enfuvirtide sensitivity.

Productive entry of HIV-1 into cells may occur following internalization and delivery to endosomes, albeit in a pH-independent manner (43). If so, then the rate at which a virus is internalized, fuses, and enters cells could affect viral tropism. In addition, the rate at which a virus fuses is a measure of how well it productively engages CD4 and coreceptor. Hypothesizing that faster-fusing viruses may preferentially overcome mucosal barriers to transmission, we indirectly assessed the entry kinetics of the T/F and chronic pseudoviruses using a time-of-addition experiment with the fusion inhibitor enfuvirtide. As enfuvirtide is not membrane permeative, the time to enfuvirtide escape may reflect the rate of viral endocytosis, fusion, or some combination thereof. HIV-1 pseudotypes were added to NP2 cells on ice. Cells were spinoculated at 4°C to facilitate HIV-1 binding, and then cold medium was removed and replaced immediately with prewarmed medium. Saturating concentrations of enfuvirtide were then added at 0, 5, 10, 20, 40, 80, and 160 min postwarming, and then infectivity was normalized to that of a no-drug control. To control for experimental variation, the prototypic R5-tropic virus JRFL was included in all experiments. There was no significant difference or trend in the rates at which T/F and chronic Env pseudotypes productively entered NP2 cells, thus becoming resistant to enfuvirtide addition. The median times to half-maximal resistance (t_{1/2 max}) postwarming were 32.5 min for the T/F Envs and 31.4 min for the chronic Envs (P = 0.55 by Mann-Whitney test). Interestingly, JRFL became resistant to enfuvirtide significantly faster (t_{1/2 max} = 15.9 min) than all 41 T/F and chronic Envs (Fig. 5A). In addition, we assessed enfuvirtide potency, a measure of pre-hairpin bundle exposure that also reflects the kinetics of CD4/coreceptor engagement and endocytosis (44). There was no difference between the sensitivities of T/F and chronic Envs to enfuvirtide (mean IC₅₀ of 0.10 versus 0.13 μg/ml; P = 0.53) (Fig. 5B). Together, these results suggest that the kinetics of viral entry/endocytosis are comparable for T/F and chronic Envs.

Sensitivity to broadly neutralizing antibodies and HIV Ig.

It has previously been reported that Envs derived from acutely infected individuals may exhibit enhanced sensitivity to antibody-mediated neutralization because of changes in glycosylation and/or variable-loop length (19). This finding raised the possibility that such Envs might be able to bind to CD4 and coreceptor more efficiently. To examine this, we measured the sensitivities of the T/F and chronic Envs to four broadly neutralizing MAbs. MAbs b12 (12) and VRC01 (78) neutralize Env by engaging the CD4 binding site (CD4bs), while the epitopes for PG9 and PG16 (68), distinct germ line variants from the same individual, are glycosylation dependent and include parts of the V1/2 and V3 loops (20). To assess neutralization sensitivity, pseudoviruses were preincubated with a single concentration (10 μg/ml) of each MAb for 60 min prior to infection of NP2 cells. The maximal percent inhibition was then determined by normalizing to a control without antibody. Interestingly, T/F Envs were more sensitive than chronic Envs to both b12 (mean MPI of 66% versus 17%; P = 0.0003) (Fig. 6A) and VRC01 (mean MPI of 89% versus 50%; P = 0.0077) (Fig. 6B) (compare T/F to Chronic 1). There was also a trend toward enhanced sensitivity to neutralization by PG9 (Fig. 6C) and PG16 (Fig. 6D). To confirm these differences, the neutralization sensitivities of T/F Envs were independently examined using a different backbone (SG3) and HIV-1 reporter cell line (TZMbl), with both MPI and IC₅₀s being determined. The results confirmed the NP2 cell data in that the T/F Envs were more sensitive to neutralization by b12 (Fig. 6E) and VRC01 (Fig. 6F). In addition, the T/F Envs exhibited a trend toward increased neutralization sensitivity to both PG9 (Fig. 6G) and PG16 (Fig. 6H). While this did not reach statistical significance, it is consistent with a more neutralization-sensitive phenotype of T/F than of chronic Envs.

To assess whether the neutralization-sensitive phenotype of our T/F Envs depended on the particular panel of chronic Envs used, we examined the neutralization sensitivities of 14 clade B control Envs derived from six additional chronically infected individuals (Chronic 2 in Fig. 6A to D). Similarly to the initial test set of chronic Envs (Chronic 1 in Fig. 6A to D), this validation set exhibited increased resistance to b12 compared to T/F Envs (P = 0.0001 by Mann-Whitney test). However, unlike the initial chronic Env panel, the validation Envs were similar to the T/F Envs in their sensitivity to VRC01 (P = 0.14 by Mann-Whitney test). Finally, there were no differences in PG9 and PG16 sensitivity between the T/F and the validation Envs (Fig. 6C and D).

While broadly neutralizing MAbs are useful tools for examining neutralization sensitivity, they are rare in HIV-1-infected individuals and thus may give a biased view of HIV-1 neutralization. Thus, we examined the neutralization sensitivities of the T/F and chronic Envs to pooled sera from patients infected with clade B HIV-1 strains (clade B HIV Ig). The T/F Envs (median IC₅₀, 741 μg/ml) were approximately 2-fold more neutralization sensitive than the chronic test panel (median IC₅₀, 1,179 μg/ml; P = 0.062), the chronic validation panel (median IC₅₀, 1,500 μg/ml; P = 0.0095), and the combined clade B chronic panel (median IC₅₀, 1,324 μg/ml; P = 0.0078) (Fig. 6I).

To examine the basis for the enhanced b12 and VRC01 neutralization sensitivity of T/F Envs, we measured the binding of the two MAbs to both T/F and chronic Envs. Binding to the trimeric form of the Env expressed on the surface of cells was measured using a cell-based ELISA system (32). To obtain an accurate measure of antibody binding affinity, we corrected binding measurements for the levels of cell surface expression of the different Envs. For this purpose, the binding efficiency of b12 and VRC01 was expressed as a fraction of the binding of a CD4-Ig probe added at saturating concentrations. CD4-Ig is a fusion protein that consists of two copies of the two N-terminal domains of CD4 that are linked to the Fc region of human IgG1.

For the entire group of Envs (i.e., T/F and both chronic Envs groups combined), a very strong correlation was observed between the binding of the MAbs to the trimeric Envs and their sensitivity to inhibition. Spearman rank order correlation coefficients of 0.62 (P < 0.0001) and 0.77 (P < 0.0001) were obtained for b12 and VRC01, respectively (Fig. 7A and B). Comparison of MAb binding to the T/F and chronic Envs showed clear differences between the two groups for both b12 and VRC01. Binding of b12 to the T/F Envs was significantly increased relative to that to both groups of chronic Envs (Fig. 7C). Binding of VRC01 to the T/F Envs was increased relative to that to the original group of chronic Envs (P = 0.004) (Fig. 7D). The differential formation/exposure of these epitopes suggests the existence of at least modest structural differences within or near the CD4 binding sites of T/F and chronic Envs. No significant differences were observed between VRC01 binding to the T/F and the Chronic 2 Envs (P = 0.21).