Treatment of HIV-1-infected individuals with effective antiretroviral therapy (ART) results in a dramatic decline in viral load and a significant increase in circulating CD4+ T cells in the majority of individuals.1-3 However, a subgroup of treated individuals achieves substantial increases in CD4+ T cells despite persistent detectable viral load (termed a discordant immunologic response).4-7 Even partial viral suppression below baseline is strongly associated with an increase in CD4+ T cells, suggesting that a small reduction in HIV-1 RNA may tip the balance between cytopathicity and cell regeneration in favor of T-cell homeostasis.7 However, in other individuals on ART who are viremic with drug-resistant virus, CD4+ T cells continue to decline, as seen with wild-type virus infection. The pathogenesis of discordant CD4+ T-cell responses has been attributed to reduced thymic pathogenicity of HIV-1 containing mutations in the protease gene,8,9 reduced CD4+ T-cell turnover and activation,10,11 reduced CD4+ T-cell apoptosis,12,13 or enhanced HIV-1-specific T-cell immunity.14-16
We and other investigators have previously reported that although there is a high prevalence of protease mutations associated with a discordant immunologic response, these changes were not exclusive to discordant immunologic responders and were also observed in individuals in whom CD4+ T cells continue to decline (nonresponders).11,14 We concluded that protease inhibitor mutations alone could not explain divergent immunologic outcomes in the setting of drug resistance.11 We therefore undertook this study to identify virologic correlates of enhanced CD4+ T-cell pathogenicity, ie, the ability to deplete CD4+ T cells, in the setting of drug resistance. To achieve this, we characterized virus from discordant responders and nonresponders and evaluated replication capacity in thymus tissue and activated peripheral blood mononuclear cells (PBMCs) together with coreceptor usage.
The clinical details of the study participants are described in Table 1 and have been described previously in greater detail.11 Briefly, this study was a cross-sectional observational institutional review board-approved study of consecutive patients attending 2 large metropolitan HIV outpatient clinics in Melbourne, Australia. All individuals had been compliant with combination ART for at least 12 months prior to study. Briefly, individuals were classified according to degree of viral suppression and rate of change in CD4+ T cells (using least-squares linear regression for loge-transformed CD4+ T-cell counts and referred to as the CD4 slope). Individuals were classified into 2 groups: discordant immunologic responders, those with persistent viral replication (viral load > 400 copies/mL on at least 2 consecutive occasions for >4 months) and an increasing CD4+ T-cell count (CD4 slope > 0); and nonresponders, those with persistent viral replication and a falling CD4+ T-cell count (CD4 slope < 0). In total there were 10 nonresponders and 12 discordant responders included in this study. There was no significant difference between discordant responders and nonresponders in HIV viral load (either prior to initiation of ART or at the time of study); reduction in HIV viral load following ART; duration of ART or duration of viremia (Table 1 and Solomon et al11).
Sequencing of HIV-1 From Patient Plasma
HIV-1 RNA from patient plasma samples was genotyped using the Viroseq HIV-1 Genotyping System kit, version 2.0 (Applied Biosystems, Foster City, CA) as previously described.17 Briefly, a reverse transcriptase polymerase chain reaction (RT-PCR) product (codons 1-99 of protease, 1-320 of RT) was generated using random hexamer primers for RT and specific primers for PCR amplification. The 1.57-kb product was sequenced using BigDye terminator chemistry (Applied Biosystems), assembled, and after minimal editing was compared with the reference sequence of HIV-1 pNL4.3 (HIV-1 Analysis software version 2.2, Applied Biosystems). Interpretation of HIV-1 sequence data for each patient was performed using a standardized algorithm (as defined by the Stanford Database http://hivdb.stanford.edu/hiv/ and Hirsch et al18). All individuals had at least 1 primary mutation in the RT or protease genes (Table 2).
Isolation of HIV-1 From Patient PBMCs and Plasma
PBMCs were isolated by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density centrifugation and stored at −80°C. PBMCs from HIV-1-infected individuals were cocultured with PBMCs from 2 HIV-1-negative blood donors stimulated with 5 μg/mL of phytohemagglutinin (PHA; Murex, Kent, UK). Briefly, cells were cultured in RPMI-1640 medium (Roswell Park Memorial Institute) supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.9 mg/mL L-glutamine (RF-10; Gibco, Grand Island, NY), and 20 U recombinant interleukin-2 (IL-2; Roche, Mannheim, Germany). Cultures were maintained by regular addition of uninfected stimulated PBMCs and fresh media. On the day of peak infection, in most cases following 14 days after coculture, virus supernatant was harvested and filtered through a 0.22-μm filter (Sarstedt, Numbrecht, Germany) and aliquots were stored at −80°C until used. Virus stocks were quantified using a commercially available p24 enzyme-linked immunosorbent assay (Innogenetics-Innotest HIV Antigen mAb kit, Churchill Diagnostics, Gent, Belgium). Viral RNA was isolated from supernatant from PBMCs at day 14 as described for patient plasma. RT and protease were sequenced and compared with the sequence of the virus extracted directly from the patient's plasma. Even though viral isolation from PBMC culture was not performed in the presence of antiretrovirals, there was no difference in any of the primary mutations in RT or protease in viral RNA extracted from patient plasma and virus amplified from PBMCs (data not shown).
For isolation of virus from plasma, patient plasma was diluted 1:2 in phosphate-buffered saline (PBS). The diluted plasma was then layered on 20% (wt/vol) sucrose at a 1:5 volume ratio of diluted plasma to sucrose solution and centrifuged at 17,000 × g for 2 hours at 4°C. The plasma supernatant was removed and the virus pellet resuspended in RF-10 supplemented with IL-2. Concentrated patient plasma was then cocultured with PBMCs, as described previously; however, donor PBMCs were depleted of CD8+ T cells using magnetic beads coated with anti-CD8+ monoclonal antibodies (Dynal Biotech, Oslo, Norway). At day 10 postinfection, culture supernatants were filtered though 0.22-μm filters, aliquoted and stored at −80°C, and then tested for the presence of HIV-1 by p24.
Replication of Primary Isolates in Ex Vivo Thymus Tissue
Thymus tissue was obtained from the Royal Children's Hospital, Melbourne, Australia, from children undergoing elective cardiac corrective surgery (ages 1 week-12 months). Thymus tissue cultures were prepared as previously described with minor modifications.8 Briefly, tissue pieces were cut to 1-2 mm in diameter and infected with virus stock (total virus added was 120 ng p24) or PBMC-conditioned media (for mock infection) for 3-4 hours. This concentration of p24 was chosen to maximize infection efficiency of thymus tissue (data not shown). Thymus pieces were then placed on a 0.8-μm polycarbonate membrane filter (Isopore, Millipore, Bedford, MA) on a square piece of gelatin sponge (Johnson and Johnson, Skipton, UK) in RPMI-1640 supplemented with 1% (vol/vol) human serum, vitamin solution (Gibco), insulin, transferrin, and sodium selenite (Sigma, St. Louis, MO) in a 12-well plate. Ex vivo thymus cultures were maintained for 9 days. At regular time points, single-cell suspensions of thymocytes were obtained by passing thymus blocks through a nylon mesh cloth with a 1-mL syringe plunger. Cells were washed and then lysed in PCR lysis buffer (10 mM trisamine) pH 8, 1 mM ethylenediamine tetra-acetic acid 0.002% (vol/vol), Triton X-100 (Sigma, St. Louis, MO) 0.002% (wt/vol), sodium dodecyl sulfate, and proteinase K 0.8 mg/mL). Virus growth was then assessed using DNA lysates of PBMCs and real-time PCR for quantification of HIV-1 DNA copy number. Input cell numbers were normalized according to quantification of CCR5 as previously described.19,20
Replication of Primary Isolates in Activated PBMCs
PHA-stimulated PBMCs from 2 HIV-1-uninfected donors were infected with virus (30 ng p24) in RF-10 media (described previously). PBMCs and thymus tissue were not obtained from the same donor. Input virus was lower than that used for ex vivo thymus cultures, as previously described.8 Cultures were maintained for 7 days and at regular timepoints cells were harvested, DNA prepared by direct cell lysis, and HIV-1 DNA was quantified as described for the thymus tissue experiments. In some experiments, PHA-stimulated PBMCs were treated with the CXCR4 antagonist AMD3100 at 1 μM (National Institutes of Health AIDS Reagent Repository) 1 hour prior to infection. Cultures were maintained in the presence of the drug for 7 days and cells were harvested at regular intervals for HIV-1 DNA quantification.
Assessment of CD4 Depletion and Apoptosis
Following HIV-1 infection of activated PBMCs, a change in the proportion of CD4+ T cells was assessed by staining with fluorochrome-conjugated antibodies to CD4 and CD8 (CD4-fluorescein isothiocyanate, CD8-phycoerythrin, Becton Dickinson, San Jose, CA) and flow cytometry (FACSort, Becton Dickinson). Apoptosis of HIV-1-infected cells and HIV-1-uninfected cells was quantified on day 5 following infection. Cells were surface stained with 7-amino-actinomycin D (7AAD; PharMingen, San Diego, CA), then fixed and permeabilized with 0.1% (vol/vol) Triton X-100 and 0.1% NaCitrate (wt/vol). Cells were then stained with anti p24 (KC57-RD1, Coulter, Miami, FL). IgG1-phycoerythrin was used as an isotype matched control. The fixed and permeabilized cells were then incubated in the TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) reaction mix as previously described.21 Cells were analyzed for the proportion of HIV-1-infected and HIV-1-uninfected apoptotic cells by flow cytometry (FACSort, Becton Dickinson). In all experiments, infections with the laboratory isolates NL4.3 and AD8 and cell culture supernatant (mock) were performed in parallel.
Determination of Coreceptor Phenotype
Coreceptor usage by primary HIV-1 isolates isolated from plasma and PBMCs from HIV-1-infected individuals was determined using Cf2-Luc cells expressing CD4 alone or expressing CD4 together with CCR2b, CCR3, CCR5, CCR8, CXCR4, Gpr1, Gpr15, Strl33, or Apj, as previously described.22-24 Entry levels were scored as +++ (>50,000 luciferase activity units), ++ (between 30,000-50,000 luciferase activity units), + (between 10,000-30,000 luciferase activity units), +/− (between 5000-10,000 luciferase activity units), or (<5000 luciferase activity units) as previously described.22
All statistical analyses were performed using SAS software (version 8.0; SAS Institute, Inc., Cary, NC). Nonparametric variables were analyzed by the Mann-Whitney U test. Nominal values were compared using the Fisher Exact test if the sample contained a subpopulation that was <4 or by χ2 test if all subpopulation sizes were not <4.
Efficiency of Isolation of Virus From PBMCs and Plasma
HIV-1 was successfully isolated from PBMCs from 6 of 9 nonresponders and 8 of 12 discordant responders (P = 1.0; summarized in Table 1). Virus was not isolated to sufficient titer (>100 ng p24/mL supernatant) in 1 nonresponder and in 2 discordant responders and therefore these viruses could not be characterized by assessment of replication kinetics or coreceptor usage. Despite previous reports of difficulty in isolating HIV-1 virus from individuals with a discordant immunologic response,14 we were able to isolate virus from PBMCs from 66% of the discordant responders. The ability to isolate virus from PBMCs was unrelated to viral load, CD4+ T-cell count (either nadir or at the time of viral isolation), or CD4 slope (Mann-Whitney, P > 0.05).
Isolation of virus from plasma was successful in 4 of 9 nonresponders and in 7 of 12 discordant responders (P = 0.670, Fisher Exact test). As with isolation of virus from PBMCs, the ability to isolate virus from plasma was unrelated to viral load, CD4+ T-cell count (either nadir or at the time of viral isolation), or CD4 slope. There was some discordance in the capacity to isolate virus from plasma and PBMCs with virus isolated from plasma but not from PBMCs (n = 2) and PBMCs but not plasma (n = 3). Isolation of virus from both PBMCs and plasma was possible in 8 individuals (n = 8).
Coreceptor Usage of Primary HIV-1 Isolated From PBMCs and Plasma
Using a reporter cell line transfected with CD4 and a range of coreceptors, a semiquantitative assessment of coreceptor phenotype was determined for all isolated viruses as previously described (Table 3).22-24 HIV-1 laboratory isolates NL4.3, ADA, and 89.6 were used as positive controls (data not shown). Of the viruses isolated from PBMCs that used CXCR4, all viruses isolated from nonresponders used CXCR4 with intermediate efficiency, whereas the viruses from discordant responders used CXCR4 with low efficiency. Overall, although there was a trend toward a higher prevalence of viruses isolated from PBMCs using CXCR4 in nonresponders as compared with discordant responders, this did not reach statistical significance (Table 3; P = 0.08; Fisher Exact test). There was a significantly increased use of coreceptor Apj in viruses isolated from nonresponders compared with viruses isolated from discordant responders (Table 3; P = 0.03; Fisher Exact test).
Given that virus isolated from PBMCs may also harbor archived forms and therefore may not represent active circulating virus, we also assessed the major coreceptor usage (CCR5 and CXCR4) of virus isolated from plasma. All the viruses isolated from plasma, except 2, used CCR5. In 2 individuals, CXCR4 was also used (Table 1). Of the dual-tropic quasispecies isolated, virus quasispecies isolated from patient 9 (a nonresponder) used CCR5 with greater efficiency than CXCR4, whereas virus quasispecies isolated from patient 15 (a discordant responder) used CXCR4 with greater efficiency than CCR5. There were no viruses isolated from plasma that exclusively used CXCR4. In 8 individuals, virus was isolated from both plasma and PBMCs and the coreceptor use of virus from the 2 compartments could be compared. Concordance in the predominant coreceptor used by virus isolated from PBMCs and virus isolated from plasma was observed in only 3 of 8 individuals. There was no significant difference in the major coreceptor used by plasma virus isolated from discordant responders and nonresponders.
Replication Kinetics in Ex Vivo Thymus Tissue and PBMCs
The kinetics of viral replication in PBMCs and ex vivo thymus tissue of primary isolates from PBMCs from discordant and nonresponders was determined. Replication kinetics of the primary isolates were compared with replication of X4 and R5 laboratory isolates, NL4.3 and AD8, respectively. Overall, we found no significant difference between growth kinetics in thymus tissue from the 11 primary isolates isolated from nonresponders and discordant responders (Fig. 1). As expected, all primary viruses replicated with reduced efficiency compared with laboratory-adapted HIV-1 isolates.
In contrast to replication kinetics within thymus tissue, there was significantly enhanced growth kinetics in activated PBMCs in the viruses from nonresponders compared with discordant responders when comparing HIV-1 DNA per million PBMCs (mean of 3 separate experiments; Fig. 2A) at day 3 (P = 0.045), day 5 (P = 0.013), and day 7 (P = 0.067) after infection. Increased HIV-1 replication in activated PBMCs was also associated with enhanced depletion of CD4+ T cells. The mean percentage of CD4+ T cells at days 5 and 7 after infection was 52% and 43%, respectively, for nonresponders and 62% and 60%, respectively, for discordant responders (P = 0.028). There were increased levels of apoptosis in both infected and uninfected T cells following infection with viruses isolated from nonresponders as compared with discordant responders (P = 0.006; Fig. 3).
To determine the relative contribution of X4 using viruses to overall replicative capacity in activated PBMCs, infection of PBMCs with the panel of primary isolates was performed in the presence of the CXCR4 antagonist AMD3100 (Fig. 2). The presence of AMD3100 inhibited viral replication of 4 of 5 viruses isolated from nonresponders by a median amount of 2.6 log (range 1.2-4.8 log reduction). The only virus not inhibited by AMD3100 was the virus isolated from patient 5 (this virus was a pure CCR5-using virus as demonstrated by the phenotypic assay; Table 3). AMD3100 had little or no effect on the replication kinetics of all viruses isolated from discordant responders (0/6 viruses; P = 0.03, Fisher Exact test). Interestingly, replication of viruses from patients 20 and 23 was only minimally inhibited by AMD3100, despite the low but detectable use of CXCR4 in the phenotypic assay (Table 3; Fig. 2B). In summary, the use of CXCR4 by viruses isolated from nonresponders significantly contributed to their enhanced replicative capacity in activated PBMCs in vitro.
Virologic failure on continuous ART is associated with variable changes in peripheral CD4+ T-cell counts: peripheral CD4+ T-cell counts may decrease in conjunction with a resurgence of plasma virus (nonresponders), or peripheral CD4+ T-cell counts may remain stable or continue to increase despite ongoing virus replication (discordant responders). In this study we found that virus isolated from PBMCs from nonresponders had greater replicative capacity and enhanced ability to deplete both HIV-1-infected and HIV-1-uninfected CD4+ T cells in activated PBMCs. Enhanced replicative capacity in PBMCs of virus isolated from PBMCs from nonresponders was inhibited by AMD3100, a CXCR4 antagonist. Virus quasispecies isolated from PBMCs from nonresponders used either CCR5 or CXCR4 for entry, in contrast to virus isolated from PBMCs from discordant responders, which predominantly used CCR5. In contrast, virus isolated from plasma from both groups predominantly used CCR5.
Virus quasispecies isolated from PBMCs from patients on long-term ART with persistent viremia and declining CD4+ T-cell counts were associated with the use of CXCR4 and enhanced replicative capacity in activated PBMCs. Replicative capacity was significantly reduced in the presence of a CXCR4 antagonist. This is in agreement with a previous study that identified an increased frequency of syncytia-inducing variants from nonresponders and non-syncytia-inducing variants from discordant responders.15 These investigators isolated virus from PBMCs and used the V3 sequence to predict coreceptor usage25 but did not quantify the relative contribution of X4 variants to replicative capacity. We chose to evaluate coreceptor use with a functional assay given the limitations in the use of V3 sequence for the accurate prediction of coreceptor usage, particularly for X4 strains.26,27
We demonstrated significant CD4+ T-cell pathogenicity in vitro using primary HIV-1-containing multiple mutations within protease and RT. The isolation of viruses with high replication capacity and CD4+ T-cell pathogenicity from patients on long-term ART is of particular concern given the recent case report of transmission of a multidrug-resistant, dual-tropic virus leading to rapid CD4+ T-cell depletion in the first year of infection.28 The use of CXCR4 has been demonstrated previously to be associated with enhanced CD4+ T-cell pathogenicity both in vitro and in vivo.29-32 Recent in vitro work with laboratory isolates demonstrated massive HIV-1-induced apoptosis of bystander CD4+ T cells following infection with X4 viruses but little bystander killing following infection with R5 viruses.33-35 In our study, virus isolated from 1 nonresponder used R5 exclusively. Highly pathogenic R5 viruses that cause significant CD4+ T-cell pathogenicity have been described but these were infrequent in our cohort.23,36-39 Given that our study is cross-sectional we were unable to determine whether there was a change in coreceptor usage or pathogenicity on ART or whether the virologic characteristics identified were present prior to initiation of ART.
Interestingly, a greater proportion of viruses able to use alternative coreceptors, particularly Apj, for entry were isolated from PBMCs from nonresponders compared with viruses isolated from the discordant responders. A previous study demonstrated an association between expanded usage of alternative coreceptors and X4 tropism.40 However, the clinical significance of alternative coreceptors in mediating HIV-1 infection in vivo is uncertain.
In contrast to our findings for viruses isolated from PBMCs, we found that viruses isolated from plasma in both nonresponders and discordant responders were predominantly R5-using viruses. This finding is in agreement with a previous study that used V3 sequence from plasma virus to predict coreceptor usage of viruses isolated from nonresponders and discordant responders.14 Although assessment of coreceptor phenotype is the gold standard, viral isolation may induce some selection bias with amplification of a “fitter” minority quasispecies and possible selection for X4 quasispecies due to the downregulation of CCR5 and maintained expression of CXCR4 in activated PBMCs.41,42 In this study, given that viral isolation was performed for both PBMC- and plasma-derived viruses, we believe direct comparisons between the 2 compartments can be performed. One potential explanation for the differing coreceptor usage of viruses isolated from PBMCs and plasma could be that both defective and latent proviruses can persist for extended periods in PBMCs in both the setting of undetectable viral load43,44 and drug-resistant viremia.45 Therefore virus isolated from PBMCs may provide a record of archived X4 or pathogenic viruses rather than current active replication of X4 viruses.27 We are unable to exclude this as a possible explanation for our findings.
In contrast, a difference in replication kinetics in thymus tissue of viruses from discordant responders and nonresponders was not identified. Studies using an ex vivo severe combined immunodeficiency hu-Thy-Liv mouse model showed that laboratory strains of X4 viruses replicate to higher levels and induce a marked degree of thymocyte depletion as compared with laboratory strains of R5 viruses.46-48 The difference in replication of X4 and R5 viruses in the thymus has previously been explained by the high expression of CXCR4 on immature thymocytes, whereas CCR5 is expressed on a limited number of mature thymocytes.49-51 It is possible that replication of primary R5 and X4 isolates, as opposed to laboratory strains, differs, eg, in their dependence on the quantity of CCR5 required for entry into target cells as recently described.52 However, in agreement with our study, another study using primary isolates of HIV-1 from HIV-1-infected children showed similar replication kinetics of primary X4 and R5 viruses in thymocytes and also showed that replication capacity in thymus tissue was unrelated to clinical progression.53 Similarly in tonsil, R5 and X4 viruses are equally cytopathic for cells expressing the appropriate coreceptors,54 suggesting that replication capacity differs in different target tissues.
In summary, although drug resistance may lead to impaired viral fitness, the capacity of virus from PBMCs to use CXCR4 has significant consequences for viral replicative capacity and potentially for clinical outcome. We identified differences in coreceptor use of virus isolated from PBMCs and plasma and this may be important in evaluating the use of newly developed coreceptor antagonists for salvage therapy. Finally, the potential for transmission of multidrug-resistant HIV-1 with high replicative capacity may have broader public health implications as more individuals receive and develop resistance to long-term ART.
The authors wish to acknowledge Mr. Andrew Cochrane, Royal Childrens Hospital, Parkville, Australia for providing thymus tissue from children undergoing elective cardiac surgery; Mandy Dunne for sequencing and Tracy Middleton for the provision of plasma specimens; Dr. Jennifer Hoy, Dr. Alan Street, and Sally Algar for recruitment of individuals to the study; Jane Howard and Anne Ellett for assistance with cloning; Judy Chang for assistance with graphics and statistical analysis; and Paul Cameron for helpful discussion. We are also grateful to J. Sodroski for providing Cf2-Luc cells, and J. Sodroski, R. Doms, S. Peiper, and D. Gabuzda for CD4 and coreceptor plasmids.
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