Interleukin-7 (IL-7) is a member of the family of common gamma-chain receptor cytokines that plays an important role in T-cell homeostasis and survival [1–3]. IL-7 has long been considered a potential therapeutic agent for HIV infection, as chronic HIV infection results in progressive CD4 T-cell deficiency and IL-7 can enhance the proliferation and survival of T lymphocytes [4–6]. In 2009, two separate clinical trial studies independently reported that administration of recombinant human IL-7 was well tolerated and led to CD4 and CD8 T-cell expansions in HIV-infected individuals, demonstrating promising capacity for immune reconstitution in that setting [7,8]. In both studies, a proportion of patients treated with recombinant human IL-7 experienced transient increases in plasma HIV-RNA (‘blips’), possibly reflecting ‘purging’ of a quiescent reservoir that provides a barrier to viral eradication. To better understand the short-term effect of recombinant human IL-7 on HIV species in vivo, the current study was undertaken to examine the temporal changes in HIV quasispecies that were found after administration of recombinant human IL-7.
Materials and methods
The study participants were HIV-1-infected adults who had plasma HIV-RNA levels less than 50 copies/ml and CD4 T-cell counts at least 100 cells/μl at screening and had received HAART for a minimum of 12 months. The study was approved by the institutional review boards of all participating sites and written informed consent was obtained from all participants. Participants received a single dose of subcutaneous recombinant human IL-7 on day 0. Details of the clinical trial (AIDS Clinical Trials Group protocol 5241, NCT00099671) have been previously published .
PCR and sequencing
Plasma (1.5–4 ml) and peripheral blood mononuclear cell (PBMCs; 5 × 106) samples were obtained at baseline (day 0 of recombinant human IL-7 therapy), during the episode of viral blips (day 4), and at a time when levels of plasma HIV-RNA had returned to less than 50 copies/ml (day 28). Single molecules of a 425 bp fragment, encompassing the C2-V3 region of the HIV-1 envelope gene, obtained through limiting dilution, were PCR-amplified and cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, California, USA) for sequence analysis of individual clones, as previously described . On average, sequences from eight independent PCR-amplified clones (range: 2–19, Table 1) were obtained for each sample.
Phylogenetic relationships among the HIV envelope sequences were estimated, by use of the neighbor-joining method, with the PAUP* program (Swofford, DL. 2003; PAUP* Phylogenetic analysis using parsimony and other methods; Version 4.0b10; Sinauer Associates, Sunderland, Massachusetts, USA). The sequence data used in this study have been deposited in GenBank and are available under the accession numbers HM118852-HM119132. To elucidate the genealogical relationships among the HIV envelope sequences, a haplotype network was constructed for participants 251536 and 250223 using TCS v1.21 . Genetic diversity was estimated with the use of a phylogenetic approach to estimate nucleotide diversity implemented in LAMARC v.2.1.3 .
Measurement of HIV-1 DNA copy numbers
Levels of HIV-DNA in the PBMC were measured by quantitative real-time PCR as described elsewhere .
Transient ‘blips’ in plasma viremia were detected during the days of observed peak T-cell proliferation (day 4) and increased T-cell counts (day 14) in seven of 15 recombinant human IL-7 recipients with HIV-RNA less than 50 copies/ml at study entry (Table 1) . The magnitude of the blips was low (median: 79 copies/ml, range: 56–154 copies/ml) and plasma HIV levels returned to less than 50 copies/ml by day 28 in all participants except participants 272661 and 250223 in whom suppression to less than 50 copies/ml was achieved subsequently as reported before .
Proviral DNA was present in all PBMC samples analyzed. Levels of proviral DNA remained stable during the 28-day sampling period: day 0, median 7.3 copies/106 cells (range 2.2–28.5); day 4, median 16.8 copies/106 cells (range 3.8–26.4); day 28, median 15.1 copies/106 cells (range 5.9–35.3; d0 vs. d4, P = 0.50; d4 vs. d28, P = 0.35; d0 vs. d28, P = 0.69, Wilcoxon signed-rank test). Baseline level of provirus did not appear to predict the likelihood of blipping during IL-7 therapy, as the highest level of blips was seen in a patient with the lowest HIV proviral DNA (participant 272661). By use of a nested PCR technique, plasma virus could be detected in the majority of plasma samples, including samples with viral RNA levels less than 50 copies/ml. Overall, the genetic diversity of the plasma virus population appeared to be fairly stable in each participant and did not show a trend toward an increase or decrease during the sampling period: day 0, median θ = 0.014 (range 0.000–0.096); day 4, median θ = 0.037 (range 0.004–0.043); day 28, median θ = 0.010 (range 0.003–0.028) (d0 vs. d4, P = 0.72; d4 vs. d28, P = 0.14; d0 vs. d28, P = 1.00). The genetic diversity was independent of number of sequences obtained (r = −0.33, P = 0.24) and of the magnitude of blips (r = 0.10, P = 0.34). Indeed, the highest genetic diversity was found in participant 251104 who had the lowest level of HIV-RNA in the blips. Additionally, we found no association between increases in cell cycling and viral evolution (the minimum change, 0.76% divergence from day 0, was observed in participant 252400 who had the highest increase in cell cycling) .
To examine the temporal changes in HIV quasispecies that were found after administration of IL-7, phylogenetic relationships between viral quasispecies derived from plasma and PBMCs were examined. Two representative patterns are shown in Fig. 1. The results of phylogenetic analysis of the HIV-1 sequences obtained from the other five patients are shown in Supplemental Fig.1 (http://links.lww.com/QAD/A98). Overall, the plasma virus detected on day 4 was indistinguishable from the viral quasispecies present at day 0 and 28 within an individual patient (Fig. 1b and 1d).
The phylogenetic tree for participant 251536 is segregated into three separate clusterings, supported by 59, 64, and 100 bootstrap values, which are statistical estimates of the reliability of a given cluster in a tree. Approximately, 92% of plasma virus sequences (24 out of 26) of participant 251536 can be found in the main trunk of the tree with the bootstrap value of 59, in which the plasma virus quasispecies are found in an intermingled manner, irrespective of sampling time points (Fig. 1b). Short branch lengths seen for the plasma virus sequences indicate that the virus population is genetically homogeneous. Similarly, all plasma virus sequences derived from participant 250223 are intermixed and found distributed throughout the main trunk of the phylogenetic tree (Fig. 1d). Additionally, all plasma viruses detected in the seven participants were predicted to be CCR5-tropic.
Overall, provirus quasispecies are more genetically heterogeneous than the plasma virus population, as evidenced by the presence of multiple branches within the monophyletic group and the longer branch lengths in both cases. All proviruses are found distributed throughout the phylogenetic tree. The episodic blips did not have any substantial impact on the distribution pattern. Instead, the diverse provirus population present at baseline persisted throughout the sampling period. In both participants, the day 4 virus sequences from the blipping time point clustered with proviral DNA sequences. This indicates that the viruses emerging during the episode of blips were not only similar to the virus quasispecies detected before and after the recombinant human IL-7 therapy but also were genetically indistinguishable from provirus quasispecies detected during the sampling time point.
To further elucidate the genealogical relationships among the plasma virus sequences, a haplotype network was constructed (Fig. 1a and 1c). The genotype network diagram inferred from plasma virus sequences derived from participant 251536 clearly demonstrates that plasma virus population is genetically homogenous (Fig. 1a). Approximately half of the plasma virus sequences (54%) are found to be identical at the nucleotide level, reflected as a hub formation in the center of the diagram. The rest of plasma virus sequences cluster around the main hub, being separated only by 1–2 nucleotide changes. The plasma virus population in participant 250223 is less homogeneous, as reflected by the presence of increased number of steps connecting two virus quasispecies (Fig. 1c). However, all day 4 virus quasispecies from participant 250223 are still found within a maximum of 7-nucleotide substitution away from the day 0 quasispecies. In addition, no shift in the genealogy of plasma virus population was observed during the sampling period.
In the present study, we have clearly demonstrated that plasma viruses detected during episodic HIV viremia following administration of IL-7 resemble the viruses present in the plasma immediately before and after recombinant human IL-7 therapy. By examining the temporal relationship among plasma virus quasispecies derived from baseline (when plasma HIV-RNA levels were <50 copies/ml), during the episode of blips, and the time when levels of plasma HIV-RNA returned to less than 50 copies/ml, we were able to demonstrate that the viruses detected at the times of episodic HIV viremia were derived from the same source(s) giving rise to viruses in plasma before and after recombinant human IL-7 therapy.
Plasma viruses detected during HIV viral blips were not only similar to the viral quasispecies present before and after the initiation of recombinant human IL-7 therapy, but also genetically indistinguishable from provirus quasispecies detected during the sampling period. Levels of genetic diversity for the plasma virus quasispecies population remained fairly stable within a given patient. In addition, no shift in the genealogical relationships among the plasma virus quasispecies was associated with the initiation of recombinant human IL-7 therapy. Given these observations, it is likely that recombinant human IL-7 is inducing a transient viral burst primarily by amplifying virus present before IL-7 therapy, rather than inducing production from previously silent reservoir(s) [13,14].
Had silent reservoir(s) contributed to these transient viral bursts, an increased genetic diversity would be anticipated following the initiation of recombinant human IL-7 therapy. However, the genetic diversity of the plasma virus quasispecies population remained fairly stable within a given patient with no particular trend toward an increase or decrease during the sampling period. In addition, unusual formation of sequence cluster that consists only of the blipping viruses was not observed in the phylogenetic trees presented in this study, further supporting the notion that activation of previously silent quasispecies played a minimal, if any, role in the HIV blips induced by recombinant human IL-7. However, our observations are limited, as the sampling was only from peripheral blood and CD4 subsets were not studied due to limited sample availability.
Transient HIV viremia raises concerns that these episodes could reflect activation and further seeding of cellular reservoirs. In the present study, plasma virus returned to the baseline level by day 28 in five out of seven patients and within no more than 2 months in the other two who had experienced blips after recombinant human IL-7 administration. In addition, there was no significant increase in levels of proviral HIV-DNA in PBMC after recombinant human IL-7 in any studied participant. It is, therefore, unlikely that the recombinant human IL-7-induced HIV blips led to substantial re-seeding or spread of viral reservoirs, particularly in so far as these patients were receiving suppressive antiretroviral therapy.
Taken together, these results indicate that transient low level increases in plasma HIV-RNA in response to a single dose of recombinant human IL-7 do not result in substantial changes in viral quasispecies. Additional long-term follow-up study on patients receiving multiple doses of recombinant human IL-7 may be necessary to evaluate the long-term impact of recombinant human IL-7 administration on HIV quasispecies.
The present study was supported in part by the AIDS Clinical Trials Group funded by the National Institute of Allergy and Infectious Diseases and the individual AIDS Clinical Trials Units at Case Western Reserve University (AI 25879), Rush University (AI 68636), Northwestern University (AI 25915), University of California, Davis Medical Center (AI 38858), and University of Miami (AI 27675). This work was also supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The authors thank all study participants and the ACTG5214 study team.
H.I. performed the experiments, analyzed the data, and wrote the paper. G.D. performed experiments. D.V.A., M.A.F., and A.L.L. contributed in study planning, data interpretation, and paper preparation. M.M.L. and I.S. contributed in study planning, data interpretation, and wrote parts of the paper. All the authors have read and approved the text as submitted to AIDS.
M.M.L. has received research support from Cytheris. All other authors have declared no conflict of interest.
This project has been funded in part by federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E, the National institute of Allergy and infectious Disease (NIAID), National Institutes of Health, under contract HHSN261200800001E and by the Intramural Research Program of NIAID. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
None of this material has been published or is under consideration for publication elsewhere.
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