Despite a prolonged life expectancy and reduced AIDS-related morbidity with present combination antiretroviral therapies (ARTs), a high risk of cancer persists in HIV-1 infection. The relative risk of hematological malignancies such as Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL) in ART-treated individuals was 26.5 and 9.1-fold, respectively, between 2005 and 2009 [1–3]. Because concerns about the safety of cancer treatment-related immunosuppression in HIV-1-infected patients were alleviated by studies confirming the practicability of standard chemotherapy and of high-dose chemotherapy followed with autologous stem cell transplantation (HDC/ACST) in ART-treated patients, survival improved dramatically [4–8].
In HIV-1-infected cells, reverse-transcribed HIV-1 DNA integrates into the human genome where it persists permanently, forming the so-called viral reservoir [9–11]. HIV-1 DNA quantification in peripheral blood mononuclear cells (PBMCs) or in blood CD4+ T cells is widely used as a surrogate marker of the viral reservoir size [12–15]. The size of the viral reservoir in an infected individual appears to be remarkably stable for over a decade, despite ART [16–19].
To date, a single case of HIV cure was reported. An HIV-1-infected individual with acute myeloid leukemia received two allogeneic stem cell transplantations from a stem cell donor with the exceptional homozygous Δ32 CCR5−/− genotype. CCR5 deficiency renders hematopoietic cells nonpermissive to CCR5-tropic HIV-1 infection, and this individual has lived free of both HIV-1 viremia and viral DNA reservoirs since transplantation [20–22].
At present, a variety of HIV cure strategies aim at viral eradication and/or effective immune control in infected individuals [23–25]. Gene-therapy interventions are designed to genetically modify autologous hematopoietic stem cells or CD4+ T cells at the CCR5 locus, leading to the loss of a functional viral coreceptor at the cell membrane [26–28]. Such trials typically focus on HIV-1-positive individuals who are eligible to nonmyeloablative high-dose chemotherapy (HDC) followed with autologous stem cell transplantation (ASCT) for the treatment of relapsed/refractory lymphoma.
To provide an in-depth characterization of the viral reservoir in the possible participants in HIV-1 gene-therapy trials, we studied the impact of HDC/ASCT on HIV-1 reservoir and CD4+ T-cell subset reconstitution. We describe the fast and consistent post-ASCT reconstitution of a quantitatively and qualitatively unchanged HIV-1 DNA reservoir in blood, despite the dramatic depletion in circulating T cells induced by HDC conditioning.
The retrospective study included successive adult patients treated for HIV-1-related relapsed or refractory lymphoma with HDC/ASCT, from January 2012 to December 2014, in the Department of Clinical Immunology at Saint-Louis Hospital, Paris. ART regimens were selected to be the best balance between HIV drug resistance cumulative history and potential drug–drug interactions with chemotherapy; ART effectiveness was followed by HIV viral loads. In all participants, stem cell-enriched peripheral blood leukocytes were collected by leukapheresis after mobilization with granulocyte-colony stimulating factor, and cryopreserved until transplantation. Thirteen participants were included in the longitudinal quantitative study of reservoir HIV-1 DNA in PBMCs. In addition, we analyzed the viral reservoir composition using deep sequencing approaches in a group of six participants on ART for longer than 12 months before HDC/ASCT, who received the same HDC regimen (carmustine/etoposide/cytarabine/melphalan, i.e. BEAM) and had available samples drawn within the first weeks following ASCT. Clinical and biological data were obtained from medical records .
The study was carried out in accordance with the Declaration of Helsinki. This work was a retrospective noninterventional study with no addition to standard care procedures. After patient information and unless they oppose this use, general reclassification of biological remnants into research material after completion of the ordered virology tests is approved by Saint-Louis Hospital. Data were analyzed using an anonymized database. According to the French Public Health Code (CSP Article L.1121-1.1), such protocols are exempted from individual consent forms.
We included all banked blood samples, from 18 months before to 18 months following HDC/ASCT. The deep sequencing analyzes were conducted on at least four longitudinal blood samples per participant: two drawn in the year before HDC/ASCT, the earliest post-HDC/ASCT HIV-1 DNA-positive sample, and a later post-HDC/ASCT sample drawn in the 6 months following treatment.
Longitudinal quantification of plasma HIV-1 RNA load and of HIV-1 DNA load in peripheral blood mononuclear cells
Plasma HIV-1 RNA quantification was performed using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0 (limit of quantification: 20 copies/ml = 1.3log10copies/ml). DNA was extracted from blood using the DSP DNA Mini Kit with a QiaSymphony instrument (Qiagen, Courtaboeuf, France). Total HIV-1 DNA quantification was determined as previously described .
Deep-sequencing analysis of the viral envelope gene (gp120)
Given the frequent treatment-induced leukopenia in lymphoma patients and the resulting low number of cell-associated viral DNA templates in samples, we selected an amplicon-based approach. The deep sequencing protocol and bioinformatics analyses are detailed in the Supplemental Digital Dataset 1 (http://links.lww.com/QAD/B384). Briefly, the HIV-1 DNA reservoir quasi-species were studied by targeting the C2V3 hypervariable region in the viral envelope gene (374 bp, HxB2 reference genome nucleotides 7008–7381). Amplicons were generated by nested PCR using generic C2V3 primers available at http://www.hivfrenchresistance.org/. DNA libraries were prepared using the GS Junior Titanium emPCR Lib-A kit, and the pyrosequencing was performed on both strands using the 4.5.4 GS Junior platform (Roche, Basel, Switzerland). Raw output sequences were de-multiplexed, trimmed, and filtered by quality using the open-access pipeline Galaxy . Output reads were aligned to the relevant HIV-1 subtype reference genome. The thousands aligned reads from each sample were used to: compute the average of all pairwise Tamura-Nei 93 (TN93) distances between reads (APD) with at least 100 overlapping base pairs in order to quantify nucleotide diversity ; and generate consensus viral haplotypes using the quasi-species reconstruction software QuRe_v0.99971 . Viral C2V3 haplotypes obtained from serially collected samples were aligned with ClustalW. Phylogenetic trees were inferred by the Maximum Likelihood method with 1000 bootstrap replicates using MEGA6.06 software , based on the Tamura-Nei or the Tamura-3-parameters substitution models with a gamma-distributed substitution rate (four categories) depending on the best model fit for each participant.
Genotypic prediction of coreceptor tropism
C2V3 haplotypes were tested using the Geno2Pheno Coreceptor 2.5 program (Max Planck Institut Informatik, https://coreceptor.geno2pheno.org/) with a significance level of 10% false-positive rate.
Lymphocyte subsets monitoring
Lymphocyte immunophenotyping was performed prospectively on freshly collected EDTA whole blood samples as a part of the standard clinical care of HDC/ASCT patients, using a FACS Canto II flow cytometer and the FACS DIVA software (BD Biosciences, Franklin Lakes, New Jersey, USA). Absolute counts were determined using the TruCount system (BD Biosciences) with anti-CD3 FITC, anti-CD8 PE, anti-CD45 PerCP, and anti-CD4+ APC monoclonal antibodies (mAbs) (BD Multitest, BD Biosciences). Eight-color labeling was performed with the following mAbs (BD Biosciences): anti-CD45 FITC, anti-CD3 V450, anti-CD4+ V500, anti-CD8 PerCP, anti-CD16 APCH7, anti-CD56 PECy7, anti-CD45RA APC, anti-CCR7 BV421, anti-CD27 APCH7.
Standard descriptive statistics were reported as median [interquartile range (IQR)] or number (%). Change in total HIV-1 DNA quantification per million PBMCs was tested using Wilcoxon matched-pairs signed-rank test and multiple Mann–Whitney t tests in participants with more than 1 value per time period. Plasma HIV-1 RNA levels below the quantification threshold were assigned the threshold value. Maximum-likelihood phylogenetic trees were constructed with the bootstrap method (1000 replicates) and the best-fit substitution model according to MEGA6 for each patient. Only bootstrap values higher than 70%, supporting strong reliability of branching, are displayed on the output trees. Lymphocyte counts and phenotypes were compared between periods by the Kuskall–Wallis test. Statistical analyses were conducted using STATA Statistical Software v.14.2 (Stata Corp., College Station, Texas, USA).
Participants characteristics and outcome of HDC/ASCT
Thirteen patients (Table 1) were treated with HDC/ASCT for relapsed (n = 10) or refractory (n = 3) lymphoma. They were a median age of 46 years (IQR 43–51). Eleven (84%) were male. Five (38%) were treated for Hodgkin's lymphoma and eight (62%) were treated for NHL: diffuse large B-cell lymphoma (five patients) or T-cell lymphoma (three patients).
The median time from initial lymphoma diagnosis to HDC/ASCT was 22.8 months (IQR 7.6–43.9). Salvage chemotherapy was ifosfamide/gemcitabine/vinorelbine (IGEV) for Hodgkin's lymphoma, and it varied with the type of lymphoma for NHL. Salvage chemotherapy was completed 1 month before ASCT (ASCT: day 0), ranging from day (d) −33 to d −25. HDC conditioning was initiated at d −7. At d0, participants were infused with an autologous transplant containing a median of 4.06 × 106 cells/kg CD34+ hematopoietic stem cells (IQR 2.9–4.74 × 106 cells/kg). CD34+ cells accounted for a median of 0.32% (IQR 0.28–0.99%) and lymphocytes for a median of 15.5% (IQR 12–25%) of the collected autologous graft.
The median follow-up was 40.8 months (IQR 25.2–72) following lymphoma diagnosis and 19.3 months (IQR 14.6–19.9) after ASCT. HDC/ASCT outcome was favorable in most participants and 1-year overall survival was 92% [95% confidence interval (CI) 56.6–98.9%]. Two deaths occurred over the first 18 months of follow-up: one patient died from invasive aspergillosis and the other from lymphoma progression. The remaining 11 participants were alive and in complete remission at their last visit to our center, from 2 to 5 years following HDC/ASCT.
History of HIV-1 infection
Lymphoma revealed HIV-1 infection in three participants (C, K, N) who initiated ART after the first course of chemotherapy; the other 10 of 13 (77%) were already treated for HIV-1 infection. At ASCT, the median time from HIV-1 diagnosis was 7.7 years (range 0.25–18). ART generally consisted of two nucleoside reverse transcriptase inhibitors and one protease inhibitor. One participant received a non-nucleoside reverse transcriptase inhibitor and one received an integrase inhibitor. At ASCT, seven participants were virologically suppressed below 20 copies/ml. Six had a low positive viral load (HIV-1 RNA range 55–434 copies/ml): suppression was in progress in the participants with recent ART initiation, patient L displayed a transient blip and patient B had low-level viremia. No ART interruption was planned throughout the study, and adherence was faultless during hospitalization in the week preceding and the weeks following HDC/ASCT. Later, one episode of treatment interruption was reported in patient L who presented as an outpatient at a follow-up visit on d +224 with a viral rebound related to low adherence. The median T CD4+ cell count was 386 cells/μl (IQR 230–590) at lymphoma diagnosis, and was subsequently impacted by recurring chemotherapy courses until HDC/ASCT.
HDC/ASCT does not alter HIV-1 DNA reservoir in peripheral blood mononuclear cells
A median seven longitudinal blood samples per participant (85 samples in total) were assayed for HIV-1 DNA (Fig. 1). Viral DNA was present both before and after HDC/ASCT in all participants. Samples collected within the 2-week post-HDC/ASCT aplasia period were available for 11 participants. HIV-1 DNA was detected as early as from d8–d10 in eight of 11 patients, despite profound lymphopenia. Viral DNA was detected from d16, d19, and d26 in the remaining three cases.
Time-wise, the median HIV-1 DNA load was 2.87 log10copies/106 PBMCs in the 18 months pre-ASCT (IQR 2.52–3.36) and 2.62 log10 copies/106 PBMCs (IQR 2.22–2.90) in the 18 months following ASCT. The number of available samples over both periods varied for each participant. A Mann–Whitney comparison of pre and post-HDC/ASCT HIV-1 DNA levels was practicable in 11 of 13 and did not show any statistical differences (see Fig., Supplemental Digital Content 3, http://links.lww.com/QAD/B384).
We conducted a longitudinal analysis of the diversity of HIV-1 reservoir in six participants successfully treated with ART for longer than 12 months before ASCT, who received the same HDC regimen. The average nucleotide pair-wise distance (APD) was estimated between the reads obtained from the deep sequencing of the C2V3 loop. APD ranged from 1 to 7% in the first sample from each participant, consistently with treated chronic HIV-1 infection (Fig. 2a). In all but one patient, viral diversity decreased in conjunction with standard chemotherapy for lymphoma over the months preceding HDC/ASCT. In the weeks following HDC/ASCT, viral diversity rose back to pre-HDC/ASCT levels in four of six patients (B, D, E, L), but was markedly reduced in patient F.
A median of 14 (2 to 40) consensus C2V3 haplotypes were reconstructed from the sequence reads obtained from each blood sample (Table, Supplemental Digital Content 4, http://links.lww.com/QAD/B384). In participant F, the number of viral haplotypes was reduced after HDC/ASCT. In phylogeny trees, the cell-associated C2V3 haplotypes found after HDC/ASCT were similar to the pre-HDC/ASCT haplotypes (see tree topologies with intermingled haplotypes; Fig. 2b). We did not observe significant evidence for a shift or change in the blood reservoir composition following ASCT. In accordance with high bootstrap values, viral haplotypes appeared to segregate by sample in patient D only. In this participant, the d −74 pre-HDC/ASCT viral haplotypes were not seen again in later blood samples, but the viral input into the deep-sequencing analysis was limited and may not account for all circulating viruses.
The genotypic prediction of C2V3 coreceptor tropism did not vary over time. D, E, F and L viral haplotypes were classified CCR5-tropic and G haplotypes were classified CXCR4-tropic. Participant B had both CCR5 and CXCR4-tropic haplotypes.
The prompt reconstitution of the viral reservoir is supported by memory CD4+ T cells in the early phases of lymphoid reconstitution following autologous stem cell transplantation
Following HDC-induced aplasia, the median time required for leukocyte counts to reach the threshold of 109/l was 13 days (IQR 11.5–15 days). The monitoring of post-HDC/ASCT lymphoid reconstitution offered the opportunity to analyze HIV-1 reservoir replenishment in perspective with CD4+ T-cell subsets recovery (see Fig., Supplemental Digital Content 5 for the longitudinal distribution of CD4+ subsets in all participants, http://links.lww.com/QAD/B384).
The retrospective study did not allow a systematic analysis of lymphoid reconstitution at predetermined time-points, and we defined three periods, that is, in the week before HDC initiation, from months 1 to 3 (M1–M3) and from months 12 to 16 (M12–M16) (Fig. 3). Following standard chemotherapy courses, the median CD4+ T-cell count was 117 cells/μl (IQR 58–247). The recovery of CD4+ T cells was progressive after ASCT, and cell counts reached a median of 339 CD4+ cells/μl (IQR 262–409) by M12–M16. Naive CD4+ T-cell counts remained well below normal values at M12–M16 (median 73 cells/μl, IQR 57–84). While HIV-1 DNA levels had replenished in all participants by M1–M3 as described above, the naïve CD4+ T-cell subset was markedly depleted, it was less than 3% of CD4+ T cells in participants C, D, F, I, K, L, and N. In the long term, naive and also central memory cells increased, while transitional memory and effector memory subsets went down.
Detailed immunophenotyping data were available for samples matching the deep sequencing analysis of C2V3 in participants B (d +223), D (d +66), E (d +50 and d +120), F (d −7 and d +64), and L (d +63). In these samples, transitional memory and effector memory CD4+ T cells were prevailing. In participants B and E where all five CD4+ subsets were present, and in participants D and L, who had mostly transitional memory and effector memory cells, viral diversity had returned. In patient E, in particular, the number of recovered viral haplotypes increased dramatically between d +66 and d +114 simultaneously with an expansion of the transitional memory subset. On the contrary, participant F had the highest CD4+ T-cell count by d +64 (480 cells/μl), but viral diversity had dropped, and only two viral haplotypes were found in this sample, where naive (0.1%) and central memory (3.7%) cells were lower than at d −7.
CD8+ T-cell, B-cell, and natural killer cell counts were within or above normal values by M12–M16 (Fig. 3). The reconstitution of CD8+ T cells was early and based on the striking expansion of CD8+ memory subsets (Fig., Supplemental Digital Content 6, http://links.lww.com/QAD/B384).
In HIV-1-infected individuals with effective viremia suppression on ART, the lifelong persistence of HIV-1 DNA in blood and tissue-resident CD4+ T cells is the primary obstacle to viral eradication. Most recent HIV-1 cure strategies are designed to destroy or impair the viral reservoir [23–25]. Considering the cytotoxic effect of chemotherapy on hematopoietic cells, this study investigated the potential of HDC/ASCT in altering HIV-1 reservoir in individuals treated for HIV-1-related lymphoma. To our knowledge, this is the first study to report a qualitative analysis of HIV-1 reservoir following HDC/ASCT, and to detail the immune reconstitution in CD4+ T-cell subsets in HIV-infected participants.
Autologous stem cell transplantation following high-dose chemotherapy was effective and well tolerated in this cohort of 13 HIV-1-infected individuals from a reference center, with 92% 1-year overall survival and no AIDS-related mortality. Cell-associated HIV-1 DNA levels were stable over the pre-HDC/ASCT period while participants received multiple standard chemotherapy courses, and over the post-HDC/ASCT period. There was no significant difference between both periods. Observed HIV-1 DNA levels were consistent with published data in ART-treated participants [19,35,36]. These results support previous reports of HDC/ASCT and standard chemotherapy in HIV+ participants, where the HIV-1 DNA reservoir size was found stable [6,37–40]. Based on the available viremia data, we saw no evidence that the replenishment in infected cells following HDC/ASCT was explained by uncontrolled viral replication. All participants were treated with ART and viral suppression was achieved or in progress before ASCT. Moreover, adherence to ART was not questionable during HDC/ASCT-related hospitalization, whereas HIV-1 DNA levels reconstituted in all participants. The persistence of new rounds of infection cannot be completely ruled out, but viral blips were transient and low-level.
The present study investigated whether HDC/ASCT may qualitatively alter the surviving reservoir. We used deep-sequencing approaches to characterize the viral populations before and after HDC/ASCT. Participants were treated with ART for a minimum 12 months before ASCT, alleviating reservoir variations due to ongoing viral replication. We observed a decrease in viral diversity during standard chemotherapy prior to HDC, followed by a rapid increase in viral diversity after HDC/ASCT, and the HIV-1 DNA population sampled in blood before and after ASCT over a median 7.1 months was remarkably conserved.
Because ART was maintained, the resurgence of HIV-1 DNA in blood is unlikely to have been supported by newly infected cells. HDC induced a transient leukopenia period when reservoir and nonreservoir CD4+ T cells were depleted, but HIV-1 DNA re-emerged strikingly fast in conjunction with post-transplantation memory T-cell reconstitution. Peripheral T cells may expand after ASCT from four different sources at least: T cells surviving the conditioning regimen, residual hematopoietic stem cells surviving the conditioning regimen, T cells present in the graft, and/or stem cells present in the graft. In this study, the autologous peripheral transplants contained a large fraction of mature lymphocytes (median 15.5%). Given the HIV-1 DNA levels in pre-HDC peripheral blood, participants were re-infused with a substantial number of mature infected T cells . In addition to the homeostatic proliferation of these graft T cells, the participation of untouched tissue-resident reservoir CD4+ cells to the peripheral T-cell reconstitution is, of course, plausible [41–43]. Studies in HIV-free individuals [44–46] demonstrated that initial CD4+ recovery is earlier with the infusion of mature lymphocytes along with graft CD34+ stem cells [47,48]. De-novo T-cell differentiation from stem cells generates naïve T cells. Here, the naïve CD4+ T-cell subset was dramatically reduced after ASCT in all participants and memory CD4+ subsets prevailed. Of note, memory CD4+ T-cell subsets are known to display 10-fold higher latent HIV-1 infection frequencies compared with naïve T cells, with a key role of TSCM, central memory, and transitional memory subsets [17,49]. In participant E, viral diversity increased simultaneously with the expansion of the transitional memory subset, and in participant F, viral diversity dropped with the depletion of central memory cells, pointing at the major role of both subsets in maintaining HIV reservoir. The naive CD4+ T-cell subset depletion we observed after HDC/ASCT may be attributed to thymus dysfunction related to adult age, cytotoxic therapy, and chronic HIV-1 infection [50–54], and/or to a predominant homeostatic proliferation of memory cells in response to HDC-induced lymphopenia [55–57]. Either way, the role of CD34+ stem cells in the early CD4+ T-cell recovery seems marginal.
Autologous stem cell transplantation following high-dose chemotherapy is an uncommon therapy in HIV-1-infected individuals, and alike other groups, we could not retrospectively characterize HIV-1 reservoir in the grafts. To maximize hematopoietic reconstitution and in accordance with treatment-induced lymphopenia, all collected mononuclear cells were transplanted, and there was no CD4+ T-cell collection following HDC/ASCT, forbidding cell sorting and functional reservoir assays in this study. Low CD4+ T-cell counts resulted in low numbers of cell-associated viral DNA templates, and some sampling bias cannot be ruled out from both the quantification and the qualitative analysis. Future prospective studies with increased sampling may allow single genome analysis of HIV proviruses, investigating the contribution of clonal CD4+ T-cell expansions to the post-ASCT reservoir shaping, similarly to poststandard chemotherapy reconstitution .
The success of gene therapy strategies for HIV cure will rely on the perpetuation of HIV-resistant cells [58,59]. Several studies in nonhuman primates have investigated the impact of ASCT on simian/human immunodeficiency virus (SHIV) infection and the persistence of tissue reservoirs , the successful engraftment and persistence of CCR5-modified hematopoietic stem cells, and the outcome of ART interruption [60,61]. However, these models did not always involve SHIV-infected stem cell transplants, and most importantly, ASCT was delivered following myeloablative total body irradiation conditioning, which may not be the case in human cure trials. To enhance gene-therapy engraftment in humans, modified cells are typically delivered after nonmyeloablative chemotherapy conditioning, minimizing complications. The infusion of mature lymphocytes within cellular transplants has long been considered crucial to enhance immune recovery, and T-cell depletion is not recommended in ASCT [47,48]. The present study underlines the role of transplanted infected autologous T-cells in maintaining the viral reservoir following current standard HDC/ASCT for high-risk lymphoma. Yet, few HIV cure trials (ACTRN12615000763549, NCT01734850) involve the modification of both stem cells and CD4+ T cells.
In conclusion, our study indicates that the circulating HIV-1 reservoir is both quantitatively and qualitatively unchanged by HDC/ASCT. Given the delayed onset of naïve CD4+ T-cell recovery following HDC/ASCT, the infused peripheral memory T cells appeared to be critical to the early reconstitution of the circulating infected CD4+ T-cell pool. Apart from these encouraging results in terms of immune reconstitution, the transferred autologous T cells concurrently supported the resurgence of a conserved HIV-1 reservoir, questioning the ability of HDC/ASCT to reset and/or control the pool of virus-infected cells.
Contributions: H.M.D., L.Ge., and C.D. designed the study; L.Ge., L.Ga., and E.O. provided medical care to the participants and collected clinical and biological data; L.D. and H.M. performed and analyzed T-cell phenotypes; H.M.D. performed experiments; H.M.D., A.C., L.Ge., M.R., M.S., and H.M. analyzed results and made the figures; L.Ge. performed statistical analyses; H.M.D. and C.D. wrote the paper. All authors reviewed and accepted the final version of the manuscript.
The work was supported by the Fondation Groupe Pasteur Mutualité (Bourse de recherche 2015 to H.M.D) and the Assistance Publique – Hôpitaux de Paris.
Conflicts of interest
C.D. receives financial support as an adviser for Gilead Sciences, Merck, Bristol-Myers Squib, and ViiV healthcare, and research grants from Merck and ViiV Healthcare. This work was supported by the Fondation Groupe Pasteur Mutualité (Bourse de recherche 2015 to H.M.D). Other authors have no conflicts of interest to disclose.
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* Antoine Chaillon and Marie Roelens contributed equally to the writing of this article.
† Eric Oksenhendler, Hélène Moins-Teisserenc, and Constance Delaugerre also contributed equally to the writing of this article.