Inhibition of Dual/Mixed Tropic HIV-1 Isolates by CCR5-Inhibitors in Primary Lymphocytes and Macrophages

Background Dual/mixed-tropic HIV-1 strains are predominant in a significant proportion of patients, though little information is available regarding their replication-capacity and susceptibility against CCR5-antagonists in-vitro. The aim of the study was to analyze the replication-capacity and susceptibility to maraviroc of HIV-1 clinical isolates with different tropism characteristics in primary monocyte-derived-macrophages (MDM), peripheral-blood-mononuclear-cells (PBMC), and CD4+T-lymphocytes. Methods Twenty-three HIV-1 isolates were phenotipically and genotipically characterized as R5, X4 or dual (discriminated as R5+/X4, R5/X4, R5/X4+). Phenotypic-tropism was evaluated by multiple-cycles-assay on U87MG-CD4+-CCR5+−/CXCR4+-expressing cells. Genotypic-tropism prediction was obtained using Geno2Pheno-algorithm (false-positive-rate [FPR] = 10%). Replication-capacity and susceptibility to maraviroc were investigated in human-primary MDM, PBMC and CD4+T-cells. AMD3100 was used as CXCR4-inhibitor. Infectivity of R5/Dual/X4-viruses in presence/absence of maraviroc was assessed also by total HIV-DNA, quantified by real-time polymerase-chain-reaction. Results Among 23 HIV-1 clinical isolates, phenotypic-tropism-assay distinguished 4, 17 and 2 viruses with R5-tropic, dual/mixed-, and X4-tropic characteristics, respectively. Overall, viruses defined as R5+/X4-tropic were found with the highest prevalence (10/23, 43.5%). The majority of isolates efficiently replicated in both PBMC and CD4+T-cells, regardless of their tropism, while MDM mainly sustained replication of R5- or R5+/X4-tropic isolates; strong correlation between viral-replication and genotypic-FPR-values was observed in MDM (rho = 0.710;p-value = 1.4e-4). In all primary cells, maraviroc inhibited viral-replication of isolates not only with pure R5- but also with dual/mixed tropism (mainly R5+/X4 and, to a lesser extent R5/X4 and R5/X4+). Finally, no main differences by comparing the total HIV-DNA with the p24-production in presence/absence of maraviroc were found. Conclusions Maraviroc is effective in-vitro against viruses with dual-characteristics in both MDM and lymphocytes, despite the potential X4-mediated escape. This suggests that the concept of HIV-entry through one of the two coreceptors “separately” may require revision, and that the use of CCR5-antagonists in patients with dual/mixed-tropic viruses may be a therapeutic-option that deserves further investigations in different clinical settings.


Introduction
The entry of human immunodeficiency virus (HIV) into human primary cells, such as lymphocytes and macrophages, is a complex multistep process that can be targeted for antiretroviral drugs. This process needs the interaction between viral proteins and cellular receptors: in particular the first important interaction occurs between the viral glycoprotein gp120 and the CD4 cellular receptor. In addition, other specific co-receptors, CCR5 and/or CXCR4, interact with the viral gp120 protein, inducing specific conformational changes that facilitate the viral entry into the cell mediated by the fusion protein gp41. These cellular chemokine receptors are G protein characterized by seven-transmembrane domains and are able to determine the cellular tropism of HIV-1. Pure R5 viruses utilize only the CCR5 co-receptor to enter into target cells, while pure X4 tropic viruses utilize only the CXCR4 co-receptor [1][2][3][4][5][6]. Some viruses are able to use both coreceptors to enter into the cell, and are defined dual-tropic [7]. Recent studies highlighted the existence of different types of dual-tropic viruses: those that are more efficient in using the CCR5 co-receptor (R5 + / X4), those that use more efficiently the CXCR4 co-receptor (R5/ X4 + ), and those that can use with the same efficiency both coreceptors (R5/X4) [8]. For historical reasons, R5 viruses are often classified also as macrophage M-tropic viruses because of their propensity to infect these cells. Conversely, X4 strains are named lymphocytic T-tropic, because on their pronounced replication capacity on such type of cells [9,10].
The tropism is based upon the presence of selected amino acids in gp120 (particularly within the V3 loop, but not only) and gp41 glycoproteins, that provide greater affinity to use CCR5 or CXCR4 [11]. In the majority of patients, R5 variants predominate during the early stage of infection, but the progression of the disease is associated to an increase of the CXCR4 using virus [5,[12][13][14][15][16][17][18]. This natural shift in viruses using also the CXCR4 coreceptor is fundamental in the emergence of dual-tropic viruses in patient's viral population, that represents approximately the 15%-25% of naïve patients and the 25%-40% of experienced patients carrying HIV-1 B subtype [12,[19][20][21][22]. Conversely, the prevalence of pure X4 tropic viruses is 0.1% in naïve patients and 2-3% in treated patients, always carrying HIV-1 B subtype [12,21,23]. The viral coreceptor usage can be phenotypically determined by the approved tropism test, the Trofile assay (Monogram Biosciences) and its newer iteration, the Enhanced Sensitivity Trofile Assay (ESTA). This test is based on a singlecycle recombinant virus assay that uses pseudovirus with fulllength env-genes from the patient's virus population to infect target cell lines engineered to express CD4 and either the CCR5 or CXCR4 chemokine co-receptor. The genotypic test, also now available for tropism determination in clinical practice, is a rapid method based on the sequence of the third variable (V3) loop of the HIV gp120. The sequence is analyzed by using bio-informatic algorithms, such as PSSM X4/R5 and geno2pheno [coreceptor] (G2P) [24][25][26][27]. Furthermore, with the advent of next-generation deep sequencing, with a much higher sensitivity, today it is possible to detect minority variants of R5 and non-R5 viruses (starting from 0.1% of prevalence) also with a genotypic test [28][29][30].
Maraviroc is a recently Food and Drug Administration (FDA) approved antiretroviral drug with a novel mechanism of action [31,32]. It can be used for the treatment of HIV drug-naïve and drug-experienced patients, and also very important, of patients with multi-resistant viruses to other antiretroviral drugs [32][33][34][35]. It acts by binding to the CCR5 coreceptor within a cavity located among the membrane-spanning helices, and it stabilizes the receptor in a conformation not efficiently recognized by the viral gp120, thus preventing HIV-1 entry [36]. Based on this specific mechanism of action, maraviroc is recommended only in patients infected by pure R5 viruses (http://aidsinfo.nih.gov/contentfiles/ lvguidelines/adultandadolescentgl.pdf).
So far, very few studies have focused on the in vitro and in vivo efficacy of maraviroc against HIV-1 dual-tropic viruses [28,[37][38][39]. Similarly, very few studies also analyzed the replication capacity of HIV-1 dual-tropic viruses in human primary cells, such as CD4+ T-cells and macrophages, that are the two principal targets and sanctuaries of HIV infection [40][41][42][43]. Indeed, macrophages can sustain viral infection for long periods of time, from weeks to months, in vitro and in vivo [44][45][46][47][48][49] and they can efficiently transfer virus to CD4+ T-lymphocytes contributing to their depletion in human cellular compartments [50].
It was recently recognized that R5 HIV-1 isolates can markedly vary in their replication capacity in macrophages and that some X4 viruses are also capable of replication in these cells [51].
For all of these reasons, the innovative aim of the study was to analyze both the replication capacity and the in vitro efficacy of maraviroc against clinical isolates with different tropic characteristics in human primary macrophages, peripheral blood mononuclear cells (PBMC) and lymphocytes. In particular, we tested the activity of entry antagonists against several dual-tropic viruses with a wide range of phenotypic and genotypic tropic preferences.
PBMC and Monocyte-derived Macrophages (MDM) were obtained from the blood of healthy seronegative donors by separation over Ficoll-Hypaque gradient, as previously described [54,55]. Ethic approval was deemed unnecessary because, under Italian law, biomedical research is subjected to previous approval by ethics committees only in the hypothesis of clinical trials on medicinal products for clinical use (art. 6 and art. 9, leg. decree 211/2003). Also, the research was conducted on blood samples previously anonymized, according to the requirements set by Italian Data Protection Code (leg. decree 196/2003). The research was not conducted outside of the country of residence. PBMC were seeded in T25 flask (Costar) at a density of 1.0610 6 cells/ml in RPMI 1640 (Euroclone) supplemented with 100 U/ml penicillin +100 mg/ml streptomycin, 2 mM L-glutamine, and 10% FBS in addition to 2 mg/ml of phytohemagglutinin (PHA, Sigma) and 50 U/ml recombinant interleukin-2 (IL-2, Peprotech).
In order to prepare MDM, PBMC were resuspended in RPMI 1640 medium supplemented with 20% FBS then seeded into 48well plates (1.8610 6 cells/well). After 5 days, non-adherent cells were carefully removed by repeated washings. MDMs were estimated to be 10 5 /well at the time of infection. Adherent cells obtained using this technique generally consisted of pure differentiated MDM (at cytofluorimetric analysis, more than 95% of cells were CD14 + /CD4 + /CD3 2 ).

Compounds
The CCR5 inhibitor maraviroc (Selzentry, MVC) and Nucleoside Reverse Transcriptase Inhibitor (NRTI) 39-azido-29, 39dideoxythymidine (AZT, used as a control drug) were obtained from NIH AIDS Research and Reference Reagent Program. The CXCR4 inhibitor AMD3100 was synthesized by SIGMA.

Viruses
Twenty-three isolates were obtained expanding primary isolates from CD8 + -depleted PBMC of naïve-or HAART-experienced HIV-infected patients, as previously described [56,57]. All isolates used in the study were previously characterized for the tropism characteristics [8]. The laboratory strains HIV BaL (pure R5-tropic) and HIV IIIB (pure X4-tropic) were used as controls in all in-vitro experiments.

Phenotypic-tropism
Viral tropism was evaluated by a phenotypic assay based on multiple cycles of replication in astroglioma U87MG-CD4 + / CCR5 + and U87MG-CD4 + /CXCR4 + cells [8]. Briefly, U87MG-CD4 + /CCR5 + and U87MG-CD4 + /CXCR4 + cells (2610 5 cells/ ml) were grown in 96-well plates in a final volume of 200 ml/well. After 24 hours, the medium was removed, and the infection was performed by using 100 ml/well of viral suspension (1,200 p24 pg/ w) for 2 hours. The test was performed in quadruplicate. After 14 days of culture, the supernatants were collected and analyzed for p24 production by a commercial ELISA kit, according to the manufacturer (Ag-Genscreen TM HIV-1 Assay, BioRad).

In-vitro Efficacy of Maraviroc
The antiviral activity of maraviroc was evaluated using three different concentrations: 200 nM, 20 nM and 2 nM, corresponding to 100X, 10X and 1X the IC 50 [31], respectively. Zidovudine (AZT) was used as a control at a concentration of 1000 nM, known to inhibit .99% the replication of laboratory strains [58], AMD3100 was used at a concentration of 1300 nM, known to inhibit .99% the replication of laboratory X4-using strain HIV IIIB [59]. Stimulated PBMC and CD4+ T-cells were infected with a standardized input of each virus (16,000 p24 pg/ml/10 6 cells). After 2 hours of adsorption, cells were extensively washed to remove any residual viral particle, re-suspended in complete medium (1 ml) and cultured on 48-well plates (500,000 cells/well) for 7 days. All the drugs were always present in treated wells in 1 hour-pretreatment phase, during infection stage and for the 7 days later. Virus-growth was monitored at the day 7 post infection by measuring supernatant p24 levels, as described above. All assays were performed at least in duplicate wells. Each isolate was tested at least in 3 independent experiments and infected-PBMC were also kept untreated, as a control of viral replication without drugpressure. Primary MDM were infected with a standardized input of each virus (8,000 p24 pg/ml/10 5 cells). After 24 hours of adsorption, cells were extensively washed to remove any residual viral particle, then new complete medium (1 ml) was added and cells were cultured for 7 days. At day 7 post infection cells were washed again and cultured for another 7 days with 1 ml of new complete medium. All the drugs were always present in treated wells in 1 hour-pretreatment phase, during infection stage and for the 14 days later. Virus-growth was monitored at the day 14 post infection by measuring supernatant p24 levels by ELISA kit. All assays were performed in triplicate. Each isolate was tested at least in 3 independent experiments and, infected-MDM were also kept untreated to control for viral replication without drug-pressure.

Cell-Associated HIV-1 DNA Quantification
PBMC and MDM, infected and treated as mentioned above, were harvested, after gently washing with warm PBS, counted, dry pelleted and stored at 220uC. Total cell DNA was then extracted from cellular pellets with High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Germany) and stored at 220uC. HIV-1 total DNA was quantified by real-time polymerase chain reaction, using 59 nuclease assay in the long terminal repeat (LTR) region of HIV-1 DNA genome (reference sequence HXB2). The reaction was performed with a LightCycler v3.5 (Roche Molecular Biochemicals, Indianapolis, IN). Briefly, up to 500 ng of DNA was amplified with the sense primer NEC 152 (GCCTCAA-TAAAGCTTGCCTTGA) and the reverse primer NEC 131 (GGCGCCACTGCTAGAGATTTT) producing a 121 bp fragment, in the presence of a dually (FAM and TAMRA) labeled NEC LTR probe (AAGTAGTGTGTGCCCGTCTGTTRTKT-GACT) published by Jean-Paul Viard et al [60]. The first PCR cycle allowing fluorescence detection permitted to quantify HIV-1 DNA by reference to a standard curve signal (sevenfold dilutions from 75,000 to 1.87 copies of 8e5 cell DNA containing one integrated HIV-1 DNA copy per cell [60,61] [60,62]. Results were expressed as number of total HIV-1 DNA copies/10 6 cells.

Statistical Analysis
Comparisons of independent groups were made by Mann-Whitney-Wilcoxon (MWW) test. To correlate the replication capacity (p24 production) and genotypic tropism (values of Geno2Pheno FPR) as well as the p24 production and HIV-DNA content, simple linear regression model and Pearson correlation test have been performed by using the statistical environment R (version 2.15.1; [63]). P-values less than 0.05 were considered statistically significant.

Viral Replication and Maraviroc Activity in PBMC
A goal of the study was to evaluate in different human primary cells the replication capacity of these isolates, according to their tropism preferences in presence and absence of entry-inhibitors. As control, the replication capacity of HIV laboratory strains, BaL (R5) and IIIB (X4), was also analyzed. In PBMC, both laboratory strains showed a similar p24 production at 7 days post infection, with a mean 6 SD of 76,688655,102 pg/ml versus 73,487660,477 pg/ml, respectively. As shown in Figure 1, the majority of isolates, including dual/mixed, regardless of genotypic FPR values, efficiently replicated in PBMC (either in the range of, or more or lower than HIV-1 BaL control strain). Median HIV-1 gag-p24 antigen release in PBMC supernatants at 7 days post infection (compared to HIV-1 BaL ) was 61.4% (34,913 pg/ml; IQR: 18,848-71,949 pg/ml). Statistical analysis showed a direct correlation between viral replication and genotypic FPR values (R 2 = 0.191, p-value = 0.033; rho = 0.437, p-value = 0.033).
We then tested the replication capacity of HIV isolates in PBMC in presence of maraviroc (CCR5-antagonist), or AMD3100 (CXCR4-antagonist). The NRTI AZT has been used as control drug.
All isolates responded to at least one of the two chemokine coreceptor antagonists, with a dose response modulation. Maraviroc was highly effective in PBMC (Figure 2)  In all these isolates, the median maraviroc IC 50 was #2 nM, and the median IC 90 for each isolate ranged from ,2 nM to 47.2 nM. Somewhat to our surprise, the 2 R5/X4 + isolates with FPR,5% (#1 and #9), tested in the study, were also inhibited by maraviroc, though less efficiently than the previous ones (median inhibition of 89.9% at 200 nM, 63.0% at 20 nM, 42.0% at 2 nM) ( Figure 2). In these two isolates, maraviroc IC 50 was 2.2 nM and 6 nM, respectively, and the IC 90 was always .200 nM. As expected, maraviroc did not inhibit the replication of the pure-X4 isolate (#16). Conversely, AMD3100 at a concentration of 1300 nM was active against the pure-X4 isolate (#16), and also against the dual/mixed isolates, particularly efficient with those having FPR ,5% (median [IQR] inhibition of 92.5% [85.7-96.3] ( Figure 2). As expected, AMD3100 did not consistently inhibit the replication of 2 out of 3 pure-R5 isolates (inhibition activity ,1%), while very poorly inhibited 1 R5-isolate (inhibition activity of 16%). As expected, AZT, used as control, inhibited all strains at the used concentration of 1000 nM.
Thus, the majority of isolates, regardless of their tropism, efficiently replicated in both PBMC and CD4+ T-cells and maraviroc was active in PBMC against also dual/mixed viruses despite the potential X4-mediated escape.

Viral Replication and Maraviroc Activity in MDM
The replication capacity of isolates and control strains was also tested in MDM at 14 days post infection in presence and absence of antiviral compounds. As expected, in MDM, the replication capacity of HIV-1 BaL control strain was very efficient, producing a mean 6 SD of 162,1436127,019 pg/ml, while replication of  HIV-1 IIB was barely detected. By statistical analysis we observed a strong and significant direct correlation between viral replication and genotypic FPR values (R 2 = 0.504, p-value = 1.4e-4; rho = 0.710, p-value = 1.4e-4) (Figure 3). In particular, isolates with FPR.20%, phenotypically defined as pure R5-or R5 + /X4viruses, replicated with higher efficiency compared to isolates with FPR,20%, (median p24 = 8,929 pg/ml, IQR: 3,948-14,124 pg/ ml versus 3,310 pg/ml, IQR: 2,325-5,451 pg/ml, respectively; MWW, p-value = 0.0156), confirming that macrophages sustained mainly the replication of isolates with preferential CCR5-usage ( Figure 3).
The activity of maraviroc, AMD3100 and the AZT was also analyzed in MDM ( Figure 4). As observed with PBMC, maraviroc was highly effective against all tested isolates with FPR.10% Taken together, these results show that macrophages mainly sustain the replication of HIV-1 isolates with pure R5 or R5 + /X4 tropism, and that maraviroc is highly active against all tested isolates.

Evaluation of HIV Total DNA Expression in Presence or Absence of Entry Inhibitors in Human Primary Cells
In order to distinguish HIV infection and viral replication, HIV-DNA of infected cells was also quantified and compared to the p24 production. In PBMC cultures, no main differences were found at 7 days post infection by comparing the total HIV DNA copies with the p24 production (both normalized for 10 6 cells) ( Figure 5, panel  A). Statistical analysis showed a non significant correlation between viral replication and HIV DNA values (R 2 = 0.125, pvalue = 0.149; rho = 0.354, p-value = 0.149).
The majority of isolates that replicated well also contained large amount of total HIV DNA. Isolates that replicated less efficiently were associated with a reduced amount of HIV DNA. Of interest, this rule does not apply for 3 isolates (#28, #9 and #39) that, in T-CD4 + cultures, poorly replicated (p24 production at 7 days post infection ,12.5 pg/ml), but reached high levels of HIV DNA copies (406,714 copies/10 6 cells; 78,869 copies/10 6 cells; 76,049 copies/10 6 cells, respectively ( Figure 5, panel B). This suggests that these isolates were able to enter within CD4 + T cells and were more inclined to produce latent infection than other isolates (where the levels of DNA and p24 were more consistent). Overall, the statistical analysis showed a non significant correlation between viral replication and HIV DNA in CD4+ T cells (R 2 = 0.195, pvalue = 0.131; rho = 0.442, p-value = 0.131). Interestingly, in MDM, no significant differences were found by comparing total HIV DNA copies with the p24 production (both normalized for 10 6 cells) (Figure 6, panel A), while there was a strong and significant direct correlation between viral replication and HIV DNA (R 2 = 0.966, p-value = 6.1e-11; rho = 0.977, p-value = 1.8e-9) (Figure 6, panel B).
As a confirmation that maraviroc inhibits both viral entry and therefore replication, in PBMC we observed that R5-isolates showed a median inhibition by maraviroc 200 nM of 99.8% for HIV DNA copies and 100% for p24 production (Figure 7). Similarly, in MDM, maraviroc 200 nM was able to completely inhibit HIV DNA copies (.99%) and p24 production (100%) in R5-isolates (data not shown).
According to p24 release, in PBMC maraviroc 200 nM was not able to well inhibit viral entry, in dual R5/X4 + or HIV-1 IIIB X4 tropic strains with FPR ,5% (Figure 7). Taken together, by comparing the total HIV DNA copies with the p24 production, MDM showed a strong and significant direct correlation between viral-replication and HIV-DNA, while in PBMC and CD4 + T-cells the direct correlation observed was not significant. Interestingly, some isolates not replicating in CD4 + T cells, where high levels of HIV DNA were observed, confirming the possibility of these cells to establish a latency phase after HIV-1 entry.

Discussion
This work shows that HIV-1 clinical isolates exhibit a wide range of viral tropism and different rates of replication, and highlights the ability of maraviroc to inhibit not only pure-R5 but also dual/mixed-tropic viruses in human primary cells. These results underline the complexity and the heterogeneity of the viral population circulating in HIV-1 infected individuals, suggesting that using clinical isolates is a good model to appreciate the real viral contribution in mechanisms underlying HIV-1 pathogenesis in vivo (not fully appreciable when recombinant viruses are used).
We found that the majority of HIV-1 isolates efficiently replicated in PBMC and T cells. A weak correlation was observed between the efficiency of replication and viral tropism in both these cell types. In contrast, a stronger and significant correlation was observed in MDM. Consistent with higher expression of CCR5 co-receptor on their membrane, macrophages mainly sustained the replication of HIV-1 isolates with pure R5 or R5 + / X4 tropism. On the other hand, the replication of all dual/mixedtropic viruses with a genotypic FPR ,20% was very poor in these cells.
Notably, in our panel of HIV-1 clinical isolates, viruses showing R5 + /X4 tropism were found with higher prevalence (10/23). R5 + /X4 isolates tended to replicate with comparable or even higher efficiency than isolates with pure R5 tropism. This phenomenon was more evident in macrophages, thus suggesting that dual/mixed-tropic isolates with FPR.20% can be characterized by a selective advantage compared to all other strains [64]. The efficient replication capacity of R5 + /X4 isolates can have important pathogenic implications. Dual/mixed-tropic viruses emerge at later stages of infection in a significant proportion of individuals, differently from R5 viruses that predominate at earlier, asymptomatic stages of HIV-1 infection. Some studies have correlated the HIV-1 ability to replicate in macrophages with a faster disease progression [17,[65][66][67]. For instance, isolates from individuals with advanced HIV-1 infection or AIDS display enhanced replication capacity in macrophages compared to the isolates circulating during the early infection [65,67].
We found that, in all primary cells, maraviroc inhibits viral replication of isolates not only with pure R5 tropism but also with dual/mixed tropism (mainly R5 + /X4 and also to a lesser extent R5/X4 and R5/X4 + ) suggesting that CCR5-inhibitors are highly effective against all viruses with these dual-characteristics. This observation can be explained by a recent study showing a functional interaction of CCR5 and CXCR4 co-receptors in lymphocytes and monocytes, and the ability of CCR5 and CXCR4 antagonists to cross-bind both co-receptors [68].
Thus, the concept of HIV entry through one of the two coreceptors ''separately'' needs to be revised. Indeed, our in vitro results are also consistent with others obtained in vivo: a) from the A4001029-study, showing that maraviroc is effective also in some patients (27%) carrying dual-tropic viruses [69], b) from a recent case-report that demonstrates the ability of maraviroc to inhibit dual-R5 viruses in a dual/mixed HIV-1-infected patient [39].
The efficiency of maraviroc to inhibit HIV-1 replication in macrophages can have important pathogenic and clinical implications, in particular by reducing the dissemination of HIV in different body compartments and cellular reservoirs. This is particularly relevant in the central nervous system where maraviroc is known to efficiently penetrate [70]. In addition, macrophages at vaginal level have been shown to be permissive to HIV infection after the virus has translocated across the epithelium [71]. The high activity of maraviroc against macrophages might also contribute in controlling mucosal HIV-1 infection in sexual-transmission.
By analysing in vitro activity of CXCR4-antagonist AMD3100, we found the ability of this drug to inhibit the replication of pure-X4 and also some dual/mixed-tropic viruses (in particular R5/ X4 + and R5/X4) in both PBMC and MDM. Again this is in line with another in vivo study, showing the ability of AMD3100 to suppress both X4-tropic and certain dual-tropic variants, that use more efficiently the CXCR4 coreceptor than the CCR5 [72].
Overall, the genotypic tropism testing on population-based V3 sequencing, is a valid tool for tropism determination in clinical practice (as also reported by the European Guidelines 2011 [73]). Indeed, all R5 genotypically predicted viruses were responsive to maraviroc in vitro in primary cells. This methodology has low cost and short turnaround, and has been recently shown to adequately predict virological response to maraviroc in drug-naïve patients of the MERIT Trial [74].
Finally, to better discriminate the viral entry and viral replication, we found no main differences in PBMC and CD4 + T cells by comparing the total HIV DNA copies with the p24 production in presence or absence of maraviroc. Some HIV-1 isolates not replicating in CD4 + T cells, reached high levels of HIV DNA, suggesting a latency phase after HIV-1 entry. The establishment of a latent infection has been generally considered to be dependent on the cell type infected by HIV [75]. Our finding confirms the existence of potential viral mechanisms regulating the entry into a latent phase in lymphocytes. This point deserves further investigation.
In conclusion, this study underlies the existence of complexity and heterogeneity among the HIV-1 population in terms of viral tropism. Our results reinforce the concept that HIV-1 tropism is a phenomenon not only dependent on the coreceptor usage but also on the replication capacity in different cell types. The majority of isolates efficiently replicated in both PBMC and CD4+ T-cells, Figure 6. Comparison of HIV-DNA and viral replication in Macrophages. Panel A: The bars represents the percentage of HIV-1 DNA copies and p24 values (normalized per 10 6 cells) in human primary MDM infected with 8,000 pg per 10 5 cells after 14 days post infection. The percentage of p24 production and DNA copies of each isolate is calculated considering the replication of the control virus HIV BaL as 100 (median p24 production of 499.310 pg/10 6 and 9.3610 6 HIV DNA copies/10 6 ). Panel B. The scatterplot shows the correlation between the replication capacity (p24 production) and HIV-DNA content, by using a simple linear regression model. Pearson correlation coefficient was also used to determine the strength of correlation between p24 production and HIV-DNA copies. R 2 = Coefficient of determination; rho = Pearson correlation coefficient; p, two-tailed p value. The False Positive Rate (FPR) values are based on the V3 sequences and calculated with Geno2Pheno algorithm. The phenotypic tropism was evaluated on astroglioma U87MG-CD4 + -CCR5 + 2/CXCR4 + -expressing cells (DR: R5 + X4; D: R5/X4; DX: R5/X4 + ). doi:10.1371/journal.pone.0068076.g006 regardless of their tropism, while macrophages mainly sustained the replication of HIV-1 isolates with pure R5 or R5 + /X4 tropism. Moreover, the CCR5-antagonist maraviroc was active in both PBMC and macrophages against phenotypically pure R5 or R5 + / X4 viruses, all R5 predicted by genotypic test. This supports the concept of extending the use of CCR5-antagonists to a spectrum of patients potentially larger than only those infected with a pure-R5 virus.