Enfuvirtide (ENF) is the most clinically advanced agent in a new class of anti-HIV drugs known as fusion inhibitors, blocking viral entry in target cells . When added to background antiretroviral therapy, ENF significantly decreases viral load and increases CD4 cell count in patients failing with previous treatments [2,3].
The viral entry process begins with the binding of envelope glycoprotein gp120 to host cell surface CD4 and to chemokine receptor CXCR4 (X4 virus) or CCR5 (R5 virus) . This leads the transmembrane glycoprotein gp41 to undergo conformational changes allowing fusion of virus and cell membranes. Since ENF targets this last step [5,6], its use in vivo could influence or be influenced by components of the entry process, which, in turn, impinge on HIV pathogenesis [7–9]. The fact that virus sensitivity to ENF could be influenced by virus–coreceptor interactions is a debated issue [10,11]. It has been shown that coreceptor specificity could modulate in vitro susceptibility to ENF, R5 viruses being less sensitive to inhibition [12,13], and that susceptibility to ENF could be determined by coreceptor affinity, density and fusion kinetics . However, recent investigations have provided evidence that ENF is similarly potent against isolates using either CCR5 or CXCR4 as coreceptor, and that the major determinants of sensitivity to ENF reside in gp41 [15–18].
The state of chronic immune activation induced by HIV infection is thought to be an important factor inducing T cell depletion by increasing the rate of cell death. Blockade of viral replication and reduction of immune activation by highly active antiretroviral therapy (HAART) results in a partial normalization of cell death . Moreover, we have recently shown that fusion inhibitors including ENF also protected bystander CD4 cells from HIV envelope-induced cell death in cell culture [20,21]. This protective effect could contribute to lessening of T cell depletion in vivo, particularly in sites of active viral replication . Therefore, ENF administration should be able to reduce both activation-induced and envelope-induced cell death.
Here, we have assessed the effect of treatment with ENF on several immunological parameters related to entry and pathogenesis and evaluated their influence in the response to treatment.
All patients attending HIV Clinical Unit of Hospital Universitari Germans Trias i Pujol participating in the TORO-2 study  were recruited for this substudy. Written informed consent was obtained from all participants. Virological and immunological status of patients was monitored at different time points up to 32 weeks: quantitative analysis of HIV-1 RNA plasma levels was performed by Amplicor HIV-1 Monitor, version 1.5 (Roche Molecular Systems, Branchburg, New Jersey, USA) at Covance Central Laboratory Services (Geneva, Switzerland). CD4 and CD8 cell counts were assessed by standard flow cytometry techniques.
Patients were HIV-1-infected adults who had been on stable antiretroviral treatment for a minimum of 3 months with each of three classes of antiretroviral drug, documented resistance to all classes of antiviral drug and HIV RNA plasma level of ≥ 5000 copies/ml prior to initiation of the trial. In the substudy, virological failure was defined as a decrease from baseline in plasma HIV-1 RNA level of < 1 log10 copies/ml (log copies/ml) at 32 weeks of follow-up, and patients were classified as responders (RP) or poor-responders (PRP) following this criterion.
Whole blood, plasma and peripheral blood mononuclear cells (PBMC) were obtained and used for determinations. Briefly, 10–20 ml whole blood was collected in ethylenediaminetetraacetic acid-Vacutainer tubes (BD, Madrid, Spain) and processed for staining of cell surface markers and for plasma and PBMC isolation immediately after collection. Plasma was recovered after centrifugation at 1200 × g for 10 min and immediately cryopreserved and stored at −80°C until use. PBMC were obtained by separation on Ficoll–Hypaque (Atom Reactiva, Barcelona) density gradient and used immediately or cryopreserved in liquid nitrogen for further determinations.
Low-passage primary isolates of HIV-1 were obtained from patient PBMC as previously described . Briefly, 5 × 106 PBMC from infected individuals were cocultured with 5 × 106 healthy donor PBMC activated for 48–72 h with 3 μg/ml phytohaemaglutinin (Sigma, Madrid, Spain) and 25 IU/ml interleukin-2 (IL-2; Roche, Barcelona, Spain). Cocultures were maintained with RPMI supplemented with 20% fetal calf serum (Invitrogen, Barcelona, Spain) and IL-2. Once a week, half of the medium was removed and replaced with fresh IL-2-containing medium with activated donor PBMC. Viral replication was monitored by p24 production using a commercial enzyme-linked immunosorbent assay (ELISA; Innogenetics, Madrid, Spain). Coculture supernatants positive for p24 were collected after centrifugation at 1200 × g for 5 min and stored at −80°C for later use.
Coreceptor use of viral isolates
Primary isolates were evaluated for their use of CXCR4 or CCR5 as previously described  using U87-CD4+CXCR4+ or U87-CD4+CCR5+ cells. Briefly, 5 × 103 cells/well were plated into 96-well plates and 50 μl virus stock was added after 24 h. After a further 24 h, medium was removed and the cells washed and fresh medium added. Five days after infection, cells were observed for syncytium formation and supernatants were collected for p24 antigen ELISA determination. A positive result required syncytium formation and presence of detectable amounts of p24 at 1/1000 dilution. Virus was classified as R5, X4, or dual tropic (R5X4) according to standard criteria. Dual tropic viruses replicated in both cell lines with a difference of < 10-fold.
Ex vivo sensitivity to enfuvirtide
After stimulation of healthy PBMC with phytohaemaglutinin and IL-2 for 48–72 h, cells were washed, resuspended in IL-2-containing medium and plated in 96-well plates (150 × 103 cells/well) containing serially diluted (1:5) ENF concentrations (range, 0.1–10 000 ng/ml). Cells were infected with patient virus isolates or the control R5 HIV-1BaL strain. The tissue culture infectious dose utilized was the minimal virus amount that produced detectable p24 in a 100-fold diluted culture at 7 days post-infection. HIV p24 antigen production, in supernatants collected at day 7, was measured by ELISA.
Cytokines, chemokines and chemokine receptor expression
Cytokines, chemokines and coreceptor expressions were measured as described [25–27]. Plasma stromal cell-derived factor 1 (SDF-1, CXCL12) and interleukin-7 (IL-7) levels were determined by ultrasensitive commercial ELISA assays (Human Quantikine ELISA Kits, R&D Systems, Minneapolis, Minnesota, USA) following the manufacturer instructions. RANTES (CCL5) levels were measured by a commercial ELISA test (Endogen, Barcelona, Spain). Percentages of CD4+ CXCR4+ and CD4+ CCR5+ cells were determined by flow cytometry analysis. Whole blood 50 μl portions were stained with the following monoclonal antibodies: CD4–peridinin–chlorophyll a complex protein (PerCP), CXCR4–phycoerythrin (PE) and CCR5–fluoroscein isothiocyanate (FITC) (BD Biosciences, Madrid, Spain) for 15 min, then, erythrocytes were lysed and samples were washed twice in phosphate-buffered saline (PBS), resuspended in PBS containing 1% formaldehyde and analysed in a FACS calibur flow cytometer (BD Biosciences).
Activation and ex vivo cell death
CD4 and CD8 cell activation was measured in whole blood by flow cytometry as indicated above using the following monoclonal antibodies: CD4–FITC, CD8–PE or CD8–PerCP, CD38–PE and HLA-DR–PerCP (BD Biosciences).
To measure spontaneous ex vivo cell death, freshly isolated PBMC were cultured for 2 days (106 cells/ml) without exogenous stimulation. Cells from duplicate wells were harvested at 24 and 48 h, fixed in 1% formaldehyde PBS and cell death quantified by flow cytometry in forward versus side scatter plots, as previously described . Dead cells show increased side and reduced forward scatter values compared with living cells.
Statistical analysis was performed with the SPSS software 10.0 (SPSS, Chicago, Illinois, USA). Variables were analysed using Kolmogorov–Smirnov test to assess for normal distribution. To compare levels of different parameters, the Student's t test was used. P values shown represent single variable comparisons, that is the probability of statistical significance (95% confidence level) when comparing one variable between two groups. To correct for multiple comparisons, the threshold for statistical significance of the 11 variables tested in this study was calculated to be 0.0047 according to the formula P = 1.00 − 0.95(1/n) where n is the number of parameters studied minus the selection criteria (HIV viral load, viral load). Univariate correlations between different variables were examined with the Pearson correlation coefficient (r).
For comparison between linear correlations, a model of linear regression was used to compare one variable as independent of the other as controlled variable and taking response to treatment as a second controlled variable where response and poor response were assigned the values of 1 and 0, respectively.
Impact of 32 weeks of enfuvirtide treatment
The 22 patients taking ENF for 32 weeks (P1–P22) were a subgroup of the TORO-2 study . This patient number resulted from 16 individuals initially randomized to take ENF and six in a control group that later switched to take ENF according to the study criteria (a decrease from baseline < 0.5 log copies/ml on two or three measurements after week 8, a decrease < 1.0 log copies/ml after week 14 or a decrease from baseline of > 2 log copies/ml followed by a rebound of > 1 log copies/ml). HIV viral RNA in plasma was 3.73–5.68 log copies/ml at baseline [mean (± SD), 5.11 ± 0.48]. ENF combined with optimized antiretroviral background treatment produced a significant reduction (P < 0.001) in plasma HIV RNA levels (mean decrease, 0.72 log copies/ml) after 32 weeks of treatment. This was accompanied by a significant (P < 0.04) increase in CD4 T cell count (mean increase, 57 × 106 cells/l) (Table 1).
When the 22 individuals were separated according to changes in viral load from baseline to week 32, 14 (64%) could be classified as RP (reduction in plasma HIV RNA > 1.0 log copies/ml) and eight (36%) as PRP (reduction, < 1.0 log RNA copies/ml). The RP group had a mean decrease in viral load of 2.3 log copies/ml (eight patients achieved a viral load < 50 log copies/ml and one patient < 100 log copies/ml) and a mean increase in CD4 and CD8 cells of 88 and 205 × 106 cells/l, respectively. There were no significant changes in viral load, CD4 and CD8 cell count in the PRP group after 32 weeks of treatment (Table 2).
Sensitivity to enfuvirtide of virus isolated at baseline, coreceptor use and response to treatment
Virus isolated from eight patients prior to receiving enfuvirtide were tested for drug sensitivity in activated PBMC infections. As shown in Fig. 1a, all viruses tested were sensitive to inhibition by ENF, with a 90% effective concentration (EC90) < 0.4 μg/ml in all but one HIV isolate. The remaining isolate (P19) had a calculated EC90 of 1.5 μg/ml. Since plasma concentrations of ENF in humans typically exceed 1 μg/ml in vivo [28,29] and data from the phase III studies with 100 mg ENF twice daily indicated steady-state trough plasma concentrations of 2–3 μg/ml, it could be expected that patients carrying these isolates would respond to treatment. However, four out of eight patients in this subset were considered PRP at week 32 (Fig. 1b). Virus isolated at week 32 from three of these four was phenotypically resistant to ENF (EC90 > 10 μg/ml for P18 and P21; EC90 > 2 μg/ml for P22). In addition, five out of eight virus isolates showed the X4 or R5X4 phenotype (P5, P9, P10, P18, P21) while three isolates only used CCR5 as coreceptor (P8, P19 and P22). Therefore, virus sensitivity to ENF and virus coreceptor use at baseline were not predictors of treatment response at week 32. Only the emergence of ENF-resistant strains could be associated with treatment failure.
CD4 T cell activation determines the outcome of enfuvirtide treatment
Immunological variables did not significantly vary at baseline between RP and PRP with the exception of a difference (P < 0.05) in the activation marker HLA-DR and the expression of CCR5 in CD4 T cells (P = 0.05), probably reflecting higher levels of CD4 T cell activation in the PRP group. Consistently, baseline values of the activation marker HLA-DR in CD4 T cells showed a strong correlation (r = 0.650; P = 0.002) with CD4+CCR5+ cells at baseline (Fig. 2a) . Expression of HLA-DR at baseline strongly correlated with the outcome of ENF treatment as measured by viral load (r = 0.595; P = 0.004) (Fig. 2b) and CD4 cell count (r = −0.670; P = 0.001) (Fig. 2c) at week 32. There were weak but significant correlations between CCR5 expression at baseline and viral load (r = 0.494; P = 0.027) (Fig. 2d) or CD4 cell count (r = −0.480; P = 0.032) (Fig. 2e) at week 32 that were not observed for baseline levels of CXCR4 and viral load or CD4 cell count. These data suggest that CD4 T cell activation may be a prognostic marker of response to ENF-containing regimens in HIV infected patients.
Chemokines and chemokine receptor expression after enfuvirtide treatment
The effect of ENF treatment on several parameters affecting HIV entry was evaluated, such as plasma levels of chemokines, which may affect HIV coreceptor availability. There were no significant changes in the CCR5 ligand CCL5 (RANTES), the CXCR4 ligand CXCL12 (SDF-1) or IL-7, which is known to induce CXCR4 upregulation [26,31] (Tables 1 and 2).
The effect of ENF treatment on chemokine receptor expression in vivo, as described for HAART [7,9,32], was also examined. The expression of CXCR4 and CCR5 did not change in the PRP group after 32 weeks of treatment. Conversely, CXCR4 and CCR5 expression in CD4 T cells significantly increased (P < 0.05) in the RP group (Fig. 3). The mean percentage of CXCR4 cells in the CD4 cell pool changed from 85 ± 10 to 93 ± 6 and that of CCR5 cells from 34 ± 14 to 47 ± 12 after 32 weeks (Table 2).
T cell activation after enfuvirtide treatment
Chemokine receptor expression is strongly dependent on the circulating CD45RA or CD45RO CD4 subsets and the activation status of CD4 T cells: CCR5 was expressed in the CD45RO+CD26+brightHLA-DR+ CD4 T cells . Therefore, the effect of ENF on the activation status of CD4 and CD8 T cells was evaluated. T cell activation after 32 weeks of treatment was compared for the RP and PRP groups (Fig. 3); neither showed significant changes in the HLA-DR+CD4+ or HLA-DR+CD8+ T cell subsets. HLA-DR expression remained unchanged if analysis was done in the subset of RP with the highest response to treatment (n = 9; viral load < 100 copies/ml). A significant (P < 0.05) decrease only in CD38+CD4+ T cells was noted in the RP group.
Enfuvirtide-containing treatment prevented T cell apoptosis
Spontaneous cell death was measured in 24 h cell cultures without exogenous activation for nine of fourteen RP and five of eight PRP who were available for evaluation. Spontaneous T cell death has been associated with increased T cell activation and HIV disease progression. There was a clear correlation between spontaneous cell death and the expression of HLA-DR+CD4+ cells after ENF treatment in both RP (r = 0.71; P < 0.001) and PRP (r = 0.54; P < 0.001). However, as shown in Fig. 4a, cell death was clearly reduced in the RP group but there were no significant changes in the PRP group. Surprisingly, there was a strong, negative correlation (r = −0.69; P < 0.01) between CD38+CD4+ T cells and spontaneous cell death at week 32 in the RP group that was not seen at baseline (r = 0.08) or in the PRP group. Conversely, the percentage of spontaneous cell death increased with increasing CD38+CD4+ T cells (r = 0.54) in the five patients in the PRP group where measurement were available (Fig. 4b). Given the small number of patients in the PRP group, the statistical comparison between correlation values for the RP and PRP groups was evaluated using a linear regression that considered the mean spontaneous cell death as an independent variable and CD38+CD4+ T cells and response to treatment (where the reference category was PRP) as controlled variables. In this model, baseline data indicated that neither CD38+CD4+ T cells nor classification between RP and PRP could distinguish differences in spontaneous cell death. Conversely, there was a significant difference with 90% confidence (P = 0.093) between the correlations found for the RP and PRP groups despite the relative small sample population in the PRP group.
ENF is the first HIV fusion inhibitor to be approved for antiretroviral treatment. HIV fusion is linked to a myriad of viral characteristics: entry into the host cell, transmission, coreceptor usage, tropism and viral fitness. As such, the immunological and virological benefits of enfuvirtide treatment, beyond classical markers of disease progression (i.e., viral load and CD4 cell count), need to be evaluated.
In agreement with a previous report , the sensitivity of virus isolated at baseline was not predictive of virological response at week 32. Response to an optimized treatment including ENF may depend on a number of factors, such as pharmacokinetics, adherence to treatment or synergism with other antiretroviral drugs that act independent of HIV gp41-dependent fusion.
Here, we show that the expression of HIV coreceptors at baseline appears not to have influenced virus susceptibility to ENF ex vivo. The natural evolution of HIV during the course of infection points to a switch in coreceptor use from R5 to X4 as the infection progresses to AIDS in a majority of patients. Indeed, a relatively high number of individuals in our study carried X4 or dual-trophic strains of HIV, reflecting the advanced disease stage of patients included in this study group. However, there was no correlation between CCR5 or CXCR4 expression and coreceptor use at baseline or between coreceptor use at baseline and susceptibility to ENF of isolated virus strains. The levels of RANTES, a ligand for CCR5, SDF-1, the natural ligand of CXCR4, and IL-7 did not vary significantly before and after ENF-containing treatment. These parameters, which may affect coreceptor expression [25–27], did not have an effect on the treatment outcome at week 32. Taken together, the results on susceptibility of virus isolates to ENF, HIV coreceptor use and factors affecting coreceptor expression suggest that antiviral activity of ENF did not alter and was not significantly altered by factors affecting HIV entry prior to gp41 function.
A marker of treatment response that could be associated in our study was the degree of CD4 T cell activation at baseline. As previously shown [34–36], patients with increased T cell activation had a poorer response to antiretroviral treatment. It was expected that 32 weeks of effective treatment (as seen by a > 2.0 log copies/ml decrease in HIV RNA viral load) would be followed by a decrease in T cell activation. However, there were no changes in the mean HLA-DR+CD4+ T cell number in the RP or PRP groups including in the subset of patients who responded best to treatment (viral load < 100 copies/ml). A decrease in the percentage of CD38+CD4+ T cells and a clear reduction in apoptotic (spontaneous) cell death suggest that effective treatment has led to a decrease in T cell activation. Surprisingly, in the RP group, a strong negative correlation was noted between CD38 T cell numbers and spontaneous cell death at week 32 that was not observed for the PRP group. A possible explanation for this observation may be that the increased T cell number after effective treatment may be the consequence of replenishment of T cells from recent emigrants from the thymus, which express high levels of CD38 . Thymic output fails to explain the increase in CCR5+CD4+ cells and unchanged HLA-DR expression (Fig. 3), two parameters known to be reduced by HAART. Whether the dual protective effect of ENF against HIV infection and bystander CD4 T cell death induced by HIV envelope  contributes to this observation remains to be elucidated but cannot be excluded.
Consequently, most of the observations that have been made with HAART may be extrapolated to ENF-containing regimens. That is, viral suppression will induce a reduction in T cell activation, as measured by CD38 expression, and there is a concomitant reduction in apoptotic cell death. Taken together, our results show that virus sensitivity and virological and immunological outcome after treatment with ENF were not altered by the coreceptor used at baseline but were determined by CD4 T cell activation, which may be considered as a marker of response to treatment.
We thank Arantxa Gutiérrez for technical assistance, Nuria Perez-Alvarez for statistical analysis and revision and Margarita Bofill for critical review of the manuscript. U87.CD4 cells were obtained through the AIDS Research and Reference Program, Division of AIDS, NIAID, NIH, USA.
Sponsorship: This study was partially supported by grants from the Spanish Ministerio de Ciencia y Tecnología project BFI-2003-00405, the Fundació La Marató de TV3 project 020930, the FIS Red Cooperativa de Investigación en SIDA (RIS) and the European TRIoH Consortium (LSHG-2003-503480). J. Blanco is a researcher of the Fundació per a la Recerca Biomèdica Germans Trias i Pujol, J. Barretina and A. Llano hold predoctoral scholarships from FIS.
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