HIV-1 phenotypic properties have been associated with the clinical course of infection. Several studies have demonstrated differences between the biological properties of HIV isolates, such as syncytium-inducing (SI) capacity, cellular tropism, X4/R5 co-receptor usage and replicative rate . HIV cellular tropism is described in terms of the ability of certain viral isolates to grow in macrophages or transformed T-cell lines in vitro. Isolates that can grow in macrophages and peripheral blood mononuclear cells (PBMC) but not in T-cell lines are classified as macrophage tropic (M-tropic), and they are characterized by the use of CCR5 as a co-receptor for viral entry . Isolates that can grow in PBMC and T-cell lines but not in macrophages are called T tropic and are characterized by the use of CXCR4 as a co-receptor [3,4]. HIV-1 primary infection is characterized by viral use of CCR5 as a co-receptor for entry, and non syncytium-inducing (NSI) viral phenotype shown in lymphoid cells [5,6]. SI variants of HIV-1 appear in advanced stages of HIV-1 infection and are characterized by the use of CXCR4 as a co-receptor . SI/X4 variants are thought to be more pathogenic since their emergence has been shown to correlate with accelerated CD4 T-cell decline, highest viral load (VL) and more rapid progression to AIDS . SI/X4 variants are associated with poor prognosis [9,10]. In vitro, SI/X4 variants replicate faster and are generally more cytopathic to T cells than NSI/R5 variants . The presence of SI/X4-tropic isolates may be even more relevant for immune dysfunction in children than in adults because the former have an active thymus, and CXCR4 is the predominant co-receptor expressed on thymocytes which can be therefore infected by the virus .
Antiretroviral therapy (ART), and more specifically highly active ART (HAART), has dramatically altered the course of HIV infection in adults and children, by suppression of viral replication . While ART usually induces a significant reduction of VL in adults, in children it is not common to achieve a sustained descent of VL below detection limits of the ultrasensitive assays [14,15]. After HAART, a concomitant increase in CD4 T-cell counts and clinical improvement takes place, even in children with virologic failure [16–22]. Strong reduction of HIV replication by HAART is the most significant contributor to the decline in HIV morbidity and mortality. Unfortunately, treatment failure is a common, significant problem and as many as 50% of HIV-1 infected patients that maintain detectable plasma RNA despite being on combination ART. Co-formulated lopinavir–ritonavir is a novel protease inhibitor (PI) that, in combination with other antiretroviral agents, suppresses plasma viral load and enhances immunological status in experienced patients with HIV-1 infection . These data led us to test the hypothesis that salvage therapy with lopinavir–ritonavir in heavily treated HIV-1 vertically infected children not only reduces the quantity of virus but also affects the biological phenotype. The study of the temporal relationship between VL and viral phenotype, quantitative virus isolation and biological clonal analysis was performed in PBMC samples obtained from children whose viral phenotype evolution and clinical course were well documented.
The study population includes 20 vertically HIV-1 infected children followed in four Spanish hospitals, in whom lopinavir–ritonavir has been prescribed as a salvage therapy with prior antiretroviral treatment and virological failure. Written informed consent from legal guardians was obtained for all study subjects. Virological failure was defined as a baseline VL > 5000 copies/ml (3.7 log10). Clinical assessment and laboratory determinations were carried out at baseline, at 1 month, 3 months and every 3 months from initiation of lopinavir–ritonavir.
Quantification of infectious cellular load and isolation of viral clones
PBMC were obtained and 91 samples of virus were isolated from 20 children. Virus isolation (bulk isolation) from PBMC was performed by a standard co-cultivation technique, as described previously . In addition, we studied the phenotype of biologic clones obtained in samples from four of those HIV-infected children. Isolation of viral clones was performed using the previously described limiting dilution culture system [8,25]. Briefly, serial twofold dilutions of PBMC (from 1 × 105 cells to 781 cells per well, 24 wells for each cell concentration) were cocultured in 96-well plates, with 1 × 105 3-day phytohaemagglutinin (PHA)-stimulated fresh PBMC from healthy blood donor volunteers. Each week for 4 weeks, 100 μl culture supernatants were collected for detection of p24 antigen by a commercially available kit (Innotest HIV Antigen mAB, innogenetics N.V. Haven, Zwijnaarde, Belgium). At the same time, half of the cells were transferred to new 96-well plates and 1 × 105 fresh PHA-stimulated healthy donor PBMC were added to propagate the culture. When fewer than 37% of wells of a given cell input were positive, the viruses in one well are thought to originate from one infected cell . Therefore, virus clones were grown only from those cell concentrations that yielded fewer than 37% positive wells (mean, eight virus clones per time point tested). PBMC from wells testing positive were transferred to 25-ml flasks containing 5 × 106 fresh PHA-stimulated PBMC in 5 ml culture medium to obtain viral stocks.
The phenotype of both bulk and biologic clonal isolates was determined by testing their ability to grow and induce cytopathic effects in MT-2 cells . An isolate was considered SI if both formation of syncytia by light microscopy and p24 Ag production were detected. Moreover, as the MT-2 cell line expresses CXCR4 co-receptor, but not CCR5, it can be used to differentiate CXCR4-using or SI from CCR5-using or NSI strains. X4 but not R5 strains infected the MT-2 cells, with p24 antigen production in the culture supernatants and syncytium formation. Established isolates BaL (R5 or NSI) and pNL3 (X4 or SI) were used as controls. The presence of syncytia in coded samples was repeatedly and independently assessed by two observers. The number of syncytia in a culture was scored in a semi-quantitative manner when indicated .
Infection experiments with cell-free virus
Cell-free virus was obtained by ultracentrifugation (120 000 g, 2 h) of PBMC culture supernatants. For virus stock preparation, pellets were carefully suspended, aliquoted, and stored at −80°C until used. For cell-free infection, 2-day PHA-stimulated PBMC from blood donors were incubated for 2 h at a concentration of 5 × 106 cells/ml at 37°C. Subsequently cells were cultured at 1 × 106 cells/ml in the same medium as used in the virus isolation procedure, without PHA.
Quantification of HIV-1 RNA
Blood samples were collected in EDTA tubes, separated within 4 h and plasma stored at –70°C. Viral load was quantified in plasma by Amplicor (Roche Diagnostic Systems, Indianapolis, Indiana, USA) standard assay and by ultrasensitive assay, if there were < 400 copies/ml.
Quantification of T-cell subsets in peripheral blood
Total counts and percentage of CD4 and CD8 T cells were analyzed by TRUCOUNTTM (Becton-Dickinson Immunocytometry Systems, San José, California, USA) in whole blood .
Demographic and clinical characteristics of the HIV-1 infected children
Twenty antiretroviral experienced HIV-vertically infected children who were on lopinavir–ritonavir were studied over a mean of 16.1 months (range, 9.2–22.5 months) from the initiation of treatment with salvage therapy. These children had a mean ± SD number of therapy changes of 3.8 ± 0.3 (range, 1–5 changes) prior to study entry. Clinical, immunological, virological characteristics and ART at baseline are shown in Table 1. The different ART combinations including lopinavir–ritonavir that the children began to take as a salvage therapy are shown also in Table 1.
Antiretroviral salvage therapy preferentially suppresses SI phenotype
An increase in CD4 T-cell counts and a decrease in VL, sometimes to undetectable levels, after treatment with lopinavir–ritonavir was observed (Fig. 1). Only one of the virus bulk isolates from the study patients had a SI phenotype at the end of the study. To test whether salvage therapy with lopinavir–ritonavir may influence HIV-1 phenotype, we analysed serial viral isolates from the 20 HIV-1 infected children in the study. Bulk isolates obtained from 18 of the 20 (90%) children at baseline were SI (Fig. 1). There was an association between the ability to infect MT-2 cells, as shown by measuring p24 antigen production, and the SI phenotype: a of the NSI bulk isolates were unable to replicate in MT-2 cells. Interestingly, the viral phenotype changed to NSI/R5 in 17 out of the 18 (94.4%) after a mean of 5.7 months (95% confidence interval, 2.14–9.26 months) of salvage therapy with lopinavir–ritonavir. The remaining two of the 20 (10%) children who had NSI/R5 isolates at baseline remained unchanged during the follow-up study.
Quantification of infectious cellular load and isolation of virus clones
For three of the 17 children who had SI/X4 virus at baseline and changed to NSI/R5 variants, and for one of the two who always had NSI/R5 variants, we analysed the phenotypic composition of the virus population at the biological clonal level during the follow-up with salvage therapy with lopinavir–ritonavir. At study entry the three children selected who harboured SI/X4 bulk isolates in PBMC showed a prevalence of SI variants at the clonal level. Thus, PBMC clonal cultures from child 1 allowed the isolation of 27 clones and among those, 23 (85%) were SI/X4 and only four (15%) were NSI (Table 2). Among the 14 clones obtained from child 2, 12 (86%) were SI/X4 and two (14%) were NSI/R5 (Table 2). Three samples were analysed for child 3 during the follow-up period. At entry we observed 24 clones, 21 (87%) SI/X4 and only three (13%) NSI/R5 (Table 2). Interestingly, the clonal composition of PBMC viral population markedly changed after treatment with lopinavir–ritonavir. Globally, after 5.4 ± 0.8 months of treatment with lopinavir–ritonavir the bulk isolates from the all three children changed to NSI/R5 phenotype as mentioned above. As expected, due to the inhibitory effect of salvage therapy on VL, the number of viral clones recovered per child was lower (Table 2). Interestingly, after salvage treatment small syncytia (two nuclei/syncytium) could be detected transiently in only in five of 31 MT-2 cultures with clonal virus; however, p24 antigen detection in those cultures was negative. Therefore we wished to analyse the ability of HIV to grow in MT-2 cells by a more sensitive method, so quantification of the VL in the supernatant of the MT-2 cultures infected with cloned virus was performed. Using this technique there was a detectable VL in two out of 10 (20%) clones for child 1 and three out 21 (14%) clones for child 3 indicative of the presence of clones with the SI/X4 phenotype. Only four clones were isolated from child 4, who had a NSI bulk isolate, and all were NSI at baseline. After salvage therapy, three clones were isolated from child 4 and all of them were NSI.
SI to NSI shift was independent of PBMC donor
To address whether the source of donor PBMC used to support viral replication affects the kinetics of replication, or the phenotype of virus, PBMC from five different volunteer donors were infected with eight viral isolates, three SI/X4 and three NSI/R5 primary isolates (SI before PI treatment: HIV5144I, HIV4849I, HIV4604I, NSI after PI treatment: HIV5559I, HIV5355I, HIV5474I) and two established strains as a control (SI/X4NL4.3 and NSI/R5HIVBaLIII). Although some differences in the kinetics of replication were observed between PBMC cultures from different donors using the same isolate (Fig. 2), the rank order of HIV isolates with respect to replication rate was roughly the same (Fig. 2). Moreover, within 7 days, all four SI isolates produced SI phenotype in cultures from all five donor PBMC, whereas in those cultures infected with the three NSI isolates after treatment with lopinavir–ritonavir the SI phenotype was never observed. Thus, these data indicate that the capacity to induce SI or NSI phenotype in culture is a stable property exhibited by particular HIV strains and is independent of culture isolation conditions.
SI and NSI shift is a replication-independent property of HIV isolates
To study if the SI/X4 to NSI/R5 phenotype change is not just a result of different rates of virus replication between HIV isolates before and after PI treatment, we infected PBMC of two different donors with equal amounts, monitored by p24 antigen, of virus of five different HIV isolates (SI/X4 before PI treatment: HIV5144I, HIV4849I, HIV4604I; NSI/R5 after PI treatment: HIV5559I, HIV5474I). After 2 h of incubation, cells were washed twice to remove free virus. Virus replication, as determined by p24 antigen measured at two time points, did not differ significantly between SI and NSI phenotype. All three isolates, which induced SI in cocultivation experiments, produced high number of large syncytia in MT-2. In contrast no syncytia at all were observed in cultures infected with NSI isolates (Table 3, upper). This experiment indicates that occurrence of SI is not just a result of high virus replication of clones of this phenotype and that between HIV isolates there are obvious differences in SI capacity.
Rapid replication kinetics of SI isolates in PBMC
In the above experiments, differences in replication were not found when identical PBMC were infected with high equal viral loads of SI and NSI isolates. Nevertheless, in primary isolates, which start with low number of infected PBMC cells from HIV children, SI isolates grew more rapidly and to higher titers than NSI isolates (Fig. 2). To further study this, PHA pre-stimulated PBMC (0.5 × 106) were infected with equal amounts of the same strains as used in the previous experiments (Table 3, lower). After 2 h of incubation, the cells were washed to remove free virus and added to cultures of 4.5 × 106 PHA pre-stimulated PBMC of the same donor. In agreement with observations in primary isolates, the SI isolates were found to replicate much more rapidly compared to the NSI isolates (Fig. 2; Table 3, lower).
In this study, we investigated the effects of salvage therapy with lopinavir–ritonavir in heavily antiretroviral-experienced HIV-infected children on the biologic clonal composition of viral populations and their relative phenotypic patterns. It has been shown that during HIV-1 infection many different clones may coexist in the same individual . These HIV variants differ in biological properties such as cell tropism, SI-inducing capability and co-receptor usage, which are also associated with different phases of HIV-1 infection and pathogenesis .
The success of antiviral therapy of HIV-1 infected individuals depends on the efficient inhibition of all virus variants present. Of interest, zidovudine (ZDV) monotherapy has been shown to significantly reduce the speed of disease progression in patients with NSI variants only. This might be the clinical consequence of the preferential inhibition of viral replication by ZDV of NSI variants . In contrast with ZDV, didanosine (ddI) montherapy has been reported to result in the preferential loss of SI variants, while in patients on ZDV/ddI combination therapy SI and NSI variant were affected equally. Lamivudine (3TC) monotherapy was also shown to be effective in inhibiting both SI and NSI variants . The antiretroviral nucleoside analogues require phosphorylation to become active and this activation is dependent on cellular kinases whose expression and activity are cell-type specific . Based on this observation, it has been hypothesized that ZDV and ddI are phosphorylated by kinases that are only active in activated cells, the target of NSI variants. In contrast the cellular kinases responsible for phosphorylation/activation of ddI are potent in quiescent cells; the targets of SI/X4 variants whereas zalcitabine and 3TC are phosphorylated in both activated and inactivated cells. These differences in drug activity may explain their differential effects on SI variants.
HIV-1 protease inhibitors (PI) do not need to be phosphorylated to become active . Thus, PI could be expected to affect equally SI/X4 and NSI/R5 variants, as demonstrated for ritonavir monotherapy . Nevertheless, the influence on the phenotype pattern of HIV-1 isolates of combination therapies including multiple types of drugs with different activation mechanism, has not been evaluated yet.
Our study indicates that in HIV-1 infected children with advanced disease and SI isolates, antiretroviral therapy with lopinavir–ritonavir leads to a shift in the predominant viral population from SI to NSI phenotype. This was shown both in the bulk virus population and at clonal level. At the bulk level, virus from in 17 of 18 of the children treated with lopinavir–ritonavir reverted from SI/X4 to NSI/R5 phenotype. Moreover, the HIV-1 clones isolated after treatment showed a much higher percentage of NSI/R5 variants than at baseline. We also observed a reduction in the total number of the clones we were able to grow up from each patient after PI therapy. These data suggest that salvage therapy with lopinavir–ritonavir in heavily treated HIV-1 vertically infected children is not only capable of quantitatively affecting HIV-1 in an infected child but also capable of inducing qualitative changes in viral phenotype.
Although during the course of therapy, inhibition of both NSI/R5 and SI/X4 variants was observed, it seems that NSI/R5 strains might be less affected by the therapy with lopinavir–ritonavir than SI/X4 variants. The persistence and the higher percentage of NSI clones during the treatment suggest that these variants could ‘escape’ those antiretroviral drugs. Thus NSI/R5 HIV-isolates obtained after therapy might represent ‘latent’ viral variants, not replicating in vivo at the time of the blood sampling.
The reason for the preferential suppression of SI isolates and shift in HIV co-receptor usage caused by lopinavir–ritonavir treatment is not clear, but several mechanisms are possible. There is a relationship among virologic, host target cells and microcellular environmental (cytokines and chemokines) factors . Thus, those immune parameters may contribute to the selective suppression of SI/X4 isolates over time. On the other hand, the decrease of CD4 T-cells in the presence of SI/X4 viruses could be due to a lower production of new CD4 T-cells as a consequence of the inhibitory effect of SI/X4 isolates on thymic function . This effect depends on the ability of SI/X4 viruses to infect T-cell precursors using CXCR4 receptors, which are highly expressed in immature thymocytes . Thus, the change to NSI/R5 isolates after salvage therapy may lead to a decreased thymocyte destruction, which may allow the reconstitution of early progenitor cells that will be released into the peripheral blood. However, more detailed studies are necessary to determine whether a shift in viral phenotype may predict which patients are likely to achieve a durable response to salvage therapy with lopinavir–ritonavir.
From cell-free transmission experiments, it can be observed that the capacity to induce SI in culture is a stable property exhibited by particular HIV strains. When PBMC cultures were infected with equal amounts of individual cell-free HIV isolates, no differences in kinetics of replication were observed between SI/X4 and NSI/R5 isolates from heavily treated children. On the other hand, experiments with cellular inocula, containing comparable numbers of infected cells, revealed clear differences in kinetics of replication in PBMC culture between the two types of isolates. Thus, SI isolates have a high infectivity, resulting in a more rapid spread, leading to higher replication rates as detected by p24 antigen ELISA in the culture of the supernatant. The early and high p24 antigen production by SI isolates in this experiment is in agreement with the early and high p24 antigen production observed during primary isolation of SI isolates (Table 3, lower; Fig. 2). Despite this higher capacity of SI clones to expand under low inoculum conditions, NSI clones and NSI bulk isolates were preferentially recovered from infected PBMC from the lopinavir–ritonavir treated children.
Slowing of HIV-1 disease progression and prevention of the emergence of resistance has been associated with suppression of VL, making the elimination of detectable VL a major goal of treatment. However, we have found that some children treated by salvage therapy with lopinavir–ritonavir did not experience sustained suppression of VL, even though this treatment resulted in immunological and clinical benefits, in agreement with other reports in adults [16,17]. Thus, the clinical benefits of salvage therapy with lopinavir–ritonavir may stem from two effects on the virus: suppression of VL and a shift in the viral phenotype toward less pathogenic R5-using strains as shown in this report. In conclusion, this is the first report indicating that salvage therapy with lopinavir–ritonavir can have a dramatic impact on the phenotypic properties of HIV-1. Our data suggest that HIV-1 clonal composition and the relative phenotype pattern undergo different changes not only during the natural course of HIV-1 infection but also while patients are on antiretroviral combination therapies. It has been generally accepted that once the pathogenic SI variants emerge no reversion to the less pathogenic NSI phenotype is possible. Our data indicate that such a reversion is possible under certain circumstances.
Sponsorship: Supported by grants from Abbott Laboratorios, the Red Temática Cooperativa de Investigación en SIDA ‘(RIS G03/173) del FIS, the Fondo de Investigación Sanitaria (00/0207), Fundación para la Investigación y la Prevención del SIDA en España (36365/02), Programa Nacional del Salud, and Comunidad de Madrid (08.5/0034/2001).
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